53rd Symposium of the Society for Histochemistry … › fileadmin › 01...Welcome Message 2 Dear Members of the Society for Histochemistry, Dear Scientists and Colleagues, On behalf

53rd Symposium of the Society for Histochemistry

Current Role of Histochemistry

in Preclinical and Clinical Research

12 – 15 October 2011 · Munich · Germany

Table of Contents 1

Table of Contents

Table of Contents................................................................................... 1

Welcome Message ................................................................................. 2

Society for Histochemistry .................................................................... 3

Scientific Program Overview .............................................................. 13

Scientific Program ............................................................................... 17

Abstracts of Plenary Lectures .............................................................. 21

Posters .................................................................................................. 66

Abstracts of Poster Presentation .......................................................... 71

General Information .......................................................................... 133

Access to Venues ............................................................................... 136

Social Events ..................................................................................... 139

Exhibitors and Sponsors .................................................................... 140

Map of Exhibition Area ..................................................................... 142

Welcome Message

2

Dear Members of the Society for Histochemistry,

Dear Scientists and Colleagues,

On behalf of the Organizing Committee we are delighted to welcome you to the 53rd

Symposium of the Society for Histochemistry 2011. The Symposium will bring

together leading international experts and provide a critical mass in the field of

molecular histology, molecular medicine and cell biology. Join us to enjoy great

science and Munich, one of the most beautiful cities in Germany.

The 53rd Symposium of the Society for Histochemistry will provide insight into the

Current role of Histochemistry in Preclinical and Clinical Research. The “tissue is the

issue” remains an important principle for further progress in a frontier research in

molecular medicine, molecular and cell biology and pathology. The conference will

focus on latest morphology-based methodological developments as well as state-of-

the-art technology and applications in research and diagnostics. Particular emphasis

will be on Imaging Mass Spectrometry in biomedical imaging and in life science

research.

We are confident that you will enjoy the Scientific Program of this international

symposium.

Just enjoy it!

Axel Walch Ioannis Mylonas

Local organizer Local organizer

Treasurer of the Society for Histochemistry

Society for Histochemistry

3

The Society for Histochemistry is an international association of scientists which was

founded in 1952. Membership of the Society is open to scientists of all countries. The

Society organizes annual scientific symposia, sponsors the Robert-Feulgen Lecture

and awards the Robert-Feulgen Prize. The activities of the Society are connected with

histochemistry and all related fields, especially cell and tissue biology, molecular

biology, pathology, anatomy, and microscopy. The official journal of the Society is

Histochemistry and Cell Biology.

President: Marco Biggiogera

Symposium Scientific Committee

Local Organizers

Axel Walch

Ioannis Mylonas

Local Organizing Committee

Ansgar Brüning

Annette Feuchtinger

Heinz Höfler

Ioannis Mylonas

Sandra Rauser

Axel Walch

http://histochemistry.eu/index.html

Marco Biggiogera Pavia, Italy

Paul Debbage Innsbruck, Austria

Heinz Höfler Munich, Germany

Pavel Hozák Prague, Czech Republic

Ioannis Mylonas Munich, Germany

Axel Walch Munich, Germany

http://histochemistry.eu/index.html


4

Why to become a Member of the Society

Prestigious prizes, awards and stipends as well as competitive student packages

for attending symposia of the Society.

Personal subscription to the Society’s official "Histochemistry and Cell

Biology" at only ten percent of the regular rate. Based on the ISI-impact factor,

"Histochemistry and Cell Biology" is the leading journal in the field.

Interactions with senior scientists resulting in multiple career opportunities.

Selected top scientists in the field can be met and contacts and discussions with

them are encouraged in the informal atmosphere of the symposia.

Symposia are conveniently scheduled (usually in September) at reasonable

prices.

Besides oral presentations by top scientists in the field, the program of the

symposia allows for selected presentations on the basis of submitted abstracts.

"Free communications" and presentation of posters receive ample attention at

the symposia and are considered to be important elements of the symposia.

Access to the "members only" section of the Society´s highly informative

homepage (http://www.histochemistry.eu).

Annual Newsletter for members only.

An annual membership fee of only 25 EUR, which entitles a reduction of the

registration fee for the symposia of the Society.

http://www.histochemistry.eu/


5

List of Robert Feulgen Lectures

2011 Munich, Germany

Richard M. Caprioli, Nashville, TN, USA

Molecular imaging of tissue sections by mass spectrometry: Providing information

beyond the microscope

2010 Prague, Czech Republic

Stefan W. Hell, Göttingen, Germany

Nanoscopy with focused light

2009 Fulpmes, Austria

P. J. Peters, Amsterdam, The Netherlands

Cellular organelles as nanomachines

2008 Interlaken, Switzerland

K. Takata, Maebashi, Japan

Localization and trafficking of aquaporin 2 in the kidney

2007 Freiburg, Germany

M. Frotscher, Freiburg, Germany

New ways of looking at synapses

2006 Stresa, Lake Maggiore Italy

D. Hernandet-Verdun, Paris, France

The nucleolus: a model for the organization of nuclear functions

2005 Noordwijkerhout, Netherland

M. Dahan, Paris, France

From analog to digital: exploring cell dynamics with single quantum dots


6

2004 Prague, Czech Republic

St. Fakan, Lausanne, Switzerland

The functional architecture of the nucleus as analyzed by ultrastructural cytochemistry

2003 Les Diablerets, Switzerland

A. Engel, Basel, Switzerland

Structure and function of membrane channels

2002 Vlissingen, The Netherlands

T. Misteli, Bethesda, MD, USA

New views of the cell: Genomics, proteomics and dynamic networks

2001 Vienna, Austria

R. G. W. Anderson, Dallas, TX, USA

Caveolae spatially organize signal transduction at the cell surface

2000 Les Diablerets, Switzerland

J. Lippincott-Schwartz, Bethesda, MD, USA

Dynamic fluorescence imaging of living cells

1999 Gargellen, Austria

A. Willie, Cambridge, UK

Apoptosis in the genesis and treatment of cancer

1998 Giessen, Germany

D. Vestweber, Münster, Germany

Molecular mechanisms that control leukocyte extravasation

1997 Jena, Germany

K. Simons, Heidelberg, Germany

Biogenesis of a polarized cell surface in epithelial cells


7


M. Trendelenburg, Heidelberg, Germany

Novel insights into the nucleolar structural complexity and function

1995 Rigi-Kaltbad, Switzerland

D. Shotton, Oxford, UK

Electronic light microscopy: Past, present, future

1994 Heidelberg, Germany

M. J. Karnovsky, Boston, MA, USA

Cytochemistry and oxy radicals


S. Rosen, San Francisco, CA, USA

L-selection and its endogenous ligands

1992 Munich, Germany

G. Klein, Stockholm, Sweden

The contribution of encogenes and tumor supressor genes to the multistep

development of cancer

1991 Ghent, Belgium

J. E. Dumont, Brusels, Belgium

The surface receptors in the model of the thyroid cell


M. N. Moore, Plymouth, UK

Environmental distress signals: cellular reactions to marine pollution


W. W. franke, Heidelberg, Germany

The intermediate filament cytoskeleton and its association with other structures


8


L. I. Larsson, Copenhagen, Denmark

Cytochemical detection of regulatory peptides and of mRNA molecules coding for

peptide precursors

1987 Basel, Switzerland

W. J. Gehring, Basel, Switzerland

The generation of the body plan as studied by in situ hybridization in the developing

embryo


G. C. Bennet, Montreal, Canada

Radioautographic and cytochemical studies of the synthesis and intracellular transport

of glycoproteins

1985 Göttingen, Germany

K. Weber, Göttingen, Germany

Cytoskeletal proteins: structure, function, pathology

1984 Maastricht, The Netherlands

I. B. Black, New York, USA

Phenotypic plasticity in the nervous system


A. G. E. Pearse, London, UK

The phylogeny of the diffuse neuroendocrine system


W. E. Stumpf, Chapel Hill, NC, USA

Histochemical characteristics and significance of cell receptors in biology and

pathology


9

1981 Münster, Germany

G. Pfefferkon, Münster, Germany

Histochemische Analyse mit Licht - und Elektronenstrahlen

1980 Würzburg, Germany

L. A. Sternberger, Rochester, NY, USA

Immunocytochemistry - Past, present, future


O. Eränkö, Helsinki, Finland

Histochemical observations on the distribution of catecholamines and catecholamine-

synthesizing enzymes in the nerve cells and SIF cells of the sympathetic ganglion


10

List of Robert Feulgen Prize Laureates

2010

H. de Wit

Amsterdam, The Netherlands

2009

P. F. Lenne

Marseille, France

2008

B.N.G. Giepmans

Groningen, The Netherlands

2007

A. Pombo

London, United Kingdom

2006

P. J. Verschure


M. Nilsson

Uppsala, Sweden

2005

Th. Misgeld

Cambridge, USA

2003

J. Priller

Berlin, Germany

2002

F.J. Iborra

Oxford, UK

2001

J. Lippincott-Schwartz

Bethesda, MD, USA

2000

K. König

Jena, Germany

1999

E.J. Speel

Zürich, Switzerland

1997

Eveline Baumgart

Heidelberg, Germany

1996

R.W. Dirks

Leiden, The Netherlands

1995

M. Thiry

Liege, Belgium

1994

J. Oberdick

Columbus, OH, USA

1993

J.-L. Carpentier

Genève, Switzerland


11

1992

C.R. Green

London, UK

N.J. Severs

London, UK

1991

M.P. Bachmann

Mainz, Germany

1990

W.J. Streit

Gainsville, FL, USA

1989

R. Gebhardt

Tübingen, Germany

D.J. Taatjes

Burlington, VT, USA

1988

S. Thanos

Tübingen, Germany

1987

J.E. Scott

Manchester, UK

C.F.J. Van Noorden


R.G. Butcher

London, UK

1986

J. Gerdes

Berlin, Germany

1985

P. Brandtzaeg

Oslo, Norway

1984

M. Bendayan

Montreal, Canada

1983

J.G.J. Bauman

Leiden, The Netherlands

1982

J. Roth

Genève, Switzerland

1981

A.C. Cuello

Oxford, UK

1980

H.F. Teutsch

Freiburg/Br., Germany

1979

H. Korr

Würzburg, Germany


12

1978

G. Moyne

Villejuif, France

1977

P.T. Kjellstrand

Lund, Sweden

1976

W.D. Kuhlmann

Heidelberg, Germany

1975

D. Grube

Heidelberg, Germany

1974

J. Winckler

Frankfurt a. M., Germany

1973

G. Thiessen

Hannover, Germany

1972

J. Nolte

Freiburg/Br., Germany

D. Pette

Konstanz, Germany

Scientific Program Overview

13


Wed

nesd

ay

14:00 - 18:30 Registration desk open

17:30 - 18:30 Welcome drink

18:30 - 19:00 Symposium opening, awarding the Young Histochemist Travel Awards

19:00 - 19:45 The Robert Feulgen lecture: Richard M. Caprioli (USA)

19:45 Get Together

Th

ursd

ay

08:30 - 10:10 Workshop I: MALDI Imaging for protein analysis of tissues - Part 1

08:30 -08:50 Bernhard Spengler (Germany)

08:50 - 09:10 Michael Volný (Czech Republic)

09:10 - 09:30 Fred Hamprecht (Germany)

09:30 - 09:50 Peter Maaß (Germany)

09:50 - 10:10 Andreas Römpp (Germany)

10:10 - 10:25 Coffee break

10:25 - 12:05 Workshop II: Drug imaging in tissues and living systems

10:25 - 10:45 Jens Siveke (Germany)

10:45 - 11:05 Jonathan Stauber (France)

11:05 - 11:25 Peter Marshall (United Kingdom)

11:25 - 11:45 Brendan Prideaux (Switzerland)

11:45 - 12:05 Walter Stumpf (USA)

12:05 - 13:00 Lunch break


13:00 - 13:20 Isabelle Fournier (France)

13:20 - 13:40 Mareike Elsner (Germany)

13:40 - 14:00 Liam McDonnell (The Netherlands)

14:00 - 14:20 Malcolm Clench (United Kingdom)

14:20 - 16:00 Poster session with refreshments

16:00 - 17:00 Industry Workshop: Bruker Daltonik GmbH

16:00 - 16:15 Sören-Oliver Deininger (Bruker Daltonik GmbH)

16:15 - 16:30 Kristina Schwamborn (Germany)

16:30 - 16:45 Benjamin Balluff (Germany)

16:45 - 17:00 Detlev Suckau (Bruker Daltonik GmbH)

17:00 - 17:45 Keynote Lecture: Gerd Binnig (Nobel Laureate, Germany)

17:45 - 18:45 Business meeting


14

09:00 - 11:45 Workshop III: Ultrastructural studies in biomedicine

Frid

ay

09:00 - 09:20 Margit Pavelka (Austria)

09:20 - 09:40 Pavel Hozák (Czech Republic)

09:40 - 10:00 Marco Biggiogera (Italy)

10:00 - 10:20 Jürgen Roth (South Korea)


10:45- 11:05 Marlene Thaler (Carl Zeiss NTS GmbH)

11:05 - 11:25 Paul Debbage (Austria)

11:25 - 11:45 Ben Giepmans (The Netherlands)


13:00 Transfer to Helmholtz Zentrum München - Registration obligatory! - Meeting point: Registration desk Frauenklinik Maistraße

14:00 - 17:00 parallel sessions

Guided tour at Helmholtz Zentrum München

- Registration obligatory! -

Industry Workshop (Carl Zeiss NTS GmbH)


17:00 Transfer to Frauenklinik Maistraße Meeting point: Lecture room 052 / building 57

19:00 Symposium banquet - Ratskeller München

09:00 - 10:40 Workshop IV: Novel techniques in histochemistry, pathology and microscopy

Satu

rd

ay

09:00 - 09:20 Vasilis Ntziachristos (Germany)

09:20 -09:40 Olga Ilina (The Netherlands)

09:40 - 10:00 Savvas Damaskinos (Intas Science Imaging & HuronTechnologies)

10:00 - 10:20 Ralf Schönmeyer (Definiens AG)

10:20 - 10:40 Jan-Erik Heil (Carl Zeiss NTS GmbH)


11:00 - 12:20

parallel sessions Workshop V: Microscopy and

image analysis - DNA/RNA/protein analyses of tissues

Industry Hands-On Workshop

(Intas Science Imaging & Huron Technologies) lecture hall 1+2

Immunhistochemistry or Fluorescence

probes can be scanned in that workshop


11:00 - 11:20 Niels Grabe (Germany)

11:20 - 11:40 Stefanie Hauck (Germany)

11:40 - 12:00 Michaela Aubele (Germany)

12:00 - 12:20 Sibylle Gündisch (Germany)

12:20 - 13:00 Lunch

13:00 - 14:20 Free topics communication

13:00 - 13:20 Stefan Maier (Germany)

13:20 - 13:40 Julia Hess (Germany)

13:40 - 14:00 Gudrun C. Thurner (Austria,Young Histochemist Travel Award)

14:00 - 14:20 Rémi Longuespée (France, Young Histochemist Travel Award)

14:20 - 14:40 Closing of the Symposium and Poster Awards

Scientific Program

15

Scientific Program

Wednesday, 12 October 2011

14:00 - 18:30 Registration desk open

17:30 - 18:30 Welcome drink

18:30 - 19:00 Symposium opening, awarding the Young Histochemist Travel

Awards

19:00 - 19:45 The Robert Feulgen lecture

(Chair: Marco Biggiogera)

Richard M. Caprioli (USA): Molecular imaging of tissue sections by mass spectrometry: Providing information beyond

the microscope

19:45 Get Together

Thursday, 13 October 2011


(Chair: Bernhard Spengler)

08:30 - 08:50 Bernhard Spengler (Germany): High resolution in mass and

space: AP-MALDI FTMS imaging of biological tissue

08:50 - 09:10 Michael Volný (Czech Republic): Visualization of Small Molecules in Tissues by Laser Desorption Ionization Mass

Spectrometry Imaging

09:10 - 09:30 Fred Hamprecht (Germany): Automated analysis of (MS)

images

09:30 - 09:50 Peter Maaß (Germany): MALDI imaging: clustering, super-

resolution and applications

09:50 - 10:10 Andreas Römpp (Germany): A new dimension of histological information: Highly specific molecular imaging at cellular

resolution


Scientific Program

16

10:25 - 12:05 Workshop II: Drug imaging in tissues and living

systems (Chairs: Isabelle Fournier / Walter Stumpf)

10:25 - 10:45 Jens Siveke (Germany): MALDI molecular imaging for translational approaches in pancreatic cancer

10:45 - 11:05 Jonathan Stauber (France): Quantification of drug candidates by Label free Mass Spectrometry Imaging

11:05 - 11:25 Peter Marshall (United Kingdom): Combining Histology and

MALDI-Mass Spectrometry Imaging in Drug Discovery

11:25 - 11:45 Brendan Prideaux (Switzerland): Mass spectrometry imaging: A powerful tool in drug discovery and development

11:45 - 12:05 Walter Stumpf (USA): Drugs in the brain - cellular imaging and discoveries with receptor microscopic autoradiography


13:00 - 14:20 Workshop I: MALDI Imaging for protein analysis of

tissues - Part 2

(Chair: Richard M. Caprioli)

13:00 - 13:20 Isabelle Fournier (France): MALDI imaging mass

spectrometry for studying cancer diseases

13:20 - 13:40 Mareike Elsner (Germany): MALDI imaging reveals mitochondrial dysfunction as strong predictor for response to

neoadjuvant chemotherapy in Barrett's Cancer

13:40 - 14:00 Liam McDonnell (The Netherlands): Differentiating between morphologically identical tumors using imaging MS-based

molecular histology

14:00 - 14:20 Malcolm Clench (United Kingdom): MALDI-MS imaging and

profiling of pancreatic and stomach cancer tissue microarrays

14:20 - 16:00 Poster session with refreshments

16:00 - 17:00 Industry Workshop: Bruker Daltonik GmbH

16:00 - 16:15 Sören-Oliver Deininger (Bruker Daltonik GmbH): Tools for MALDI imaging based histology and data interpretation

16:15 - 16:30 Kristina Schwamborn (Germany): Prostate Cancer - What

can we learn from MALDI imaging

Scientific Program

17

16:30 - 16:45 Benjamin Balluff (Germany): MALDI imaging mass

spectrometry in gastric cancer

16:45 - 17:00 Detlev Suckau (Bruker Daltonik GmbH): Strategies for

identification and characterization of Protein biomarkers for MALDI imaging studies

17:00 - 17:45 Keynote Lecture

(Chairs: Axel Walch / Ioannis Mylonas)

Gerd Binnig (Nobel Laureate, Germany): Present and Future Impact of Image Analysis and Image Mining on Histochemistry

17:45 - 18:45 Business meeting

Friday, 14 October 2011

09:00 - 11:45 Workshop III: Ultrastructural studies in biomedicine

(Chairs: Margit Pavelka / Jürgen Roth)

09:00 - 09:20 Margit Pavelka (Austria): Fine structural 3D-analyses of

cellular reorganizations induced by metabolic stress

09:20 - 09:40 Pavel Hozák (Czech Republic): Nanoparticle-based

Immunocytochemistry reveals PIP2-containing microarchitecture of the cell nucleus

09:40 - 10:00 Marco Biggiogera (Italy): Tracing the movement of mRNA towards the nuclear pore

10:00 - 10:20 Jürgen Roth (South Korea): Microscopic analysis of protein misfolding diseases and the effect of therapeutic synthetic

chaperones


10:45- 11:05 Marlene Thaler (Carl Zeiss NTS GmbH): Energy Filtering TEM

applications in medical diagnostics

11:05 - 11:25 Paul Debbage (Austria): Ultrastructure and drug delivery:

gateways and barriers

11:25 - 11:45 Ben Giepmans (The Netherlands): Correlated microscopy and

nanotomy to analyze Islets of Langerhans in Type I diabetes


Scientific Program

18

13:00 Transfer to Helmholtz Zentrum München Meeting point: Registration desk Frauenklinik Maistraße - Registration obligatory! -

14:00 - 14:30 Lecture room 052

Introduction to the Helmholtz Zentrum München (Christian Langebartels, Department Program Planning and

Management) - Registration obligatory! -

14:30 - 17:00 - Registration obligatory! -

parallel sessions

Guided tour at Helmholtz Zentrum

München

Industry Workshop

(Carl Zeiss NTS GmbH)

German Mouse Clinic (Helmut Fuchs) Introduction to Energy Filtering

TEM" - Live practical session on the microscope

Research Unit Medical Radiation Physics and

Diagnostics (Christoph Hoeschen)

Genome Analysis Center (Jerzy Adamski)

17:00 Transfer to Frauenklinik Maistraße Meeting point: Lecture building 57/room 052 (Helmholtz Zentrum

München)

19:00 Symposium banquet - Ratskeller München

Saturday, 15 October 2011

09:00 - 10:40 Workshop IV: Novel techniques in histochemistry, pathology and microscopy

(Chairs: Vasilis Ntziachristos / Pavel Hozák)

09:00 - 09:20 Vasilis Ntziachristos (Germany): Illuminating biomedical discovery with advanced photonic imaging

09:20 - 09:40 Olga Ilina (The Netherlands): Tissue structures guiding

collective breast carcinoma invasion

09:40 - 10:00 Savvas Damaskinos (Intas Science Imaging & Huron Technologies): Scanning Laser Imaging of Macroscopic

Samples

10:00 - 10:20 Ralf Schönmeyer (Definiens AG): Automated co-analysis of MALDI- and H&E-images of retinal tissue for an

improved spatial MALDI-resolution

10:20 - 10:40 Jan-Erik Heil (Carl Zeiss NTS GmbH): Superresolution

Technologies from Carl Zeiss: Fluorescence Imaging beyond the Resolution Limit

Scientific Program

19


11:00 - 12:20 Workshop V Industry Hands-On

Workshop parallel sessions

11:00 - 12:20 Workshop V: Microscopy and image analysis - DNA/RNA/protein analyses of tissues

(Chair: Heinz Höfler)

11:00 - 11:20 Niels Grabe (Germany): Towards systems-pathology by integrating whole-slide imaging, high-dimensional data and

biological networks

11:20 - 11:40 Stefanie Hauck (Germany): Quantitative Tissue Proteomics for Deciphering Molecular Processes in

Autoimmune Uveitis

11:40 - 12:00 Michaela Aubele (Germany): In situ quantification of protein-protein complexes in paraffin-sections using

Proximity Ligation Assay (PLA)-technique

12:00 - 12:20 Sibylle Gündisch (Germany): Evaluation of PAXgene-fixed, paraffin-embedded tissues for morphological and

molecular analysis

11:00 - 12:20 parallel in lecture

hall 1+2

Industry Hands-On Workshop (Intas Science Imaging & Huron Technologies):

“Laser Confocal Slide Scanning System”

Scan your small or big Tissue section with the unique TissueScope4000 - Laser Confocal Slide Scanning System.

Slide Dimensions: 25x75mm up to 150x200mm Immunhistochemistry or Fluorescence probes can be

scanned in that workshop. - Registration obligatory! -

12:20 - 13:00 Lunch

Scientific Program

20

13:00 - 14:20 Free topics communication

(Chair: Marco Biggiogera)

13:00 - 13:20 Stefan Maier (Germany): Building the MALDI Imaging Protein Biomarker List from the Bottom Up

13:20 - 13:40 Julia Hess (Germany): Gain of chromosome band 7q11 in papillary thyroid carcinomas of young patients is associated

with exposure to low-dose irradiation

13:40 - 14:00 Gudrun C. Thurner (Austria, Young Histochemist Travel

Award): MRI molecular imaging with targeted albumin-based nanoparticles: conceptual design strategies to create

the Magic Bullet

14:00 - 14:20 Rémi Longuespée (France, Young Histochemist Travel Award): MALDI MSI for ovarian cancer biomarkers

research: latest developments of the technology for screening and tracking

14:20 - 14:40 Closing of the Symposium and Poster Awards

Abstracts of Plenary Lectures

21


Molecular imaging of tissue sections by mass spectrometry: Providing information beyond the microscope Caprioli R.M.

1

1Vanderbilt University, Biochemistry, Nashville, United States

Imaging MALDI MS (matrix-assisted laser desorption ionization mass spectrometry)

produces molecular images of peptides, proteins, lipids and metabolites present in

intact tissue sections. It employs desorption of molecules by direct laser irradiation to

map the location of specific molecules from fresh frozen and formalin fixed tissue

sections without the need of target specific reagents such as antibodies. Molecular

maps can be directly correlated to known histological regions within the tissue. A high

density of spots (pixels) ablated by the laser over the entire tissue produces many

hundreds of molecular images or density maps with spatial resolutions from 5-200

microns. Images are produced in specific m/z (mass-to-charge) values, or ranges of

values, typically covering the MW range 1000-100,000. Individual m/z values derived

from each pixel can then be assembled to produce selected molecular images.

Similarly, the approach has also been applied to a protocol termed histology-directed

molecular analysis whereby only selected areas of cells in the tissue are of interest are

ablated and analyzed based on studies performed by microscopy and other histology

protocols. Both fresh frozen and formalin fixed tissues can be analyzed. The

technology is extraordinarily high throughput with high molecular specificity, easily

lending itself to the analysis of tissue microarrays. Sections obtained from any tissue

type can be imaged, including sections through whole organs or animals.

We have employed Imaging MS in studies of a variety of diseases, including several

types of cancers, neurodegenerative diseases and kidney diseases, comparing proteins

differentially expressed in diseased tissue with those in the corresponding normal

tissue. From such comparisons, molecular signatures are developed that differentiate

these tissues, typically consisting of 10-20 or more different proteins. Imaging MS has

been applied to drug targeting and metabolic studies following drug administration.

Whole animal sagittal sections have been imaged to measure molecular changes in


22

proteins in multiple organs and correlating this with drug concentrations in these same

organs. 3-D images may also be generated from serial tissue sections after registration

and volume rendering.

This presentation will focus on recent technological advances both in sample

preparation and instrumental performance to achieve images at high spatial resolution

and at high speeds so that a typical sample tissue (e.g., a whole mouse section) can be

imaged in less than 10 min. Some selected examples will include studies of tumor

bearing tissues and normal developmental processes in mouse. Other aspects of the

technology, such as 3-D imaging, will also be discussed.

High resolution in mass and space: AP-MALDI FTMS imaging of

biological tissue Spengler B.

1, Römpp A.

1, Günther S.

1, Schulz O.

1, Köstler M.

1, Bouschen W.

1, Leisner

A.1, Hinz K.-P.

1

1Justus Liebig University, Analytical Chemistry, Giessen, Germany

MALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1],

has only recently turned into a routine method of highest performance for the

molecular histology of biological tissue [2]. The method, providing high resolution in

mass and space, has been developed for a reliable identification and localization of

individual tissue components, and has been applied to a number of research areas in

pathology, cancer diagnostics, metabolic pathway analysis and plant research.

Targeted compound classes include phospholipids, peptides, proteins, drug compounds

and metabolites.

The home-built atmospheric pressure ion source is based on a dedicated microoptical

setup, designed for highest optical resolution under mass analytical sampling

conditions, providing a spatial resolution on tissue of 3 to 5 micrometer. The ion

source is coupled to a Fourier transform mass spectrometer (ion cyclotron resonance or

orbital trapping), at a mass accuracy of better than 1 ppm RMS. Ion images can be

acquired with an acquisition speed of more than 2 pixels per second. The presentation

will describe technical principles of the new instrumentation and its availability,

including examples for imaging biomolecules in mammalian tissue and plant tissue at


23

high spatial resolution in relation to classical histological staining exeriments.

Combining high spatial resolution down to 3 µm with high mass resolution, high mass

accuracy, high imaging selectivity, MSn capability and an improved sample

preparation technology resulted in an unprecedented quality of imaging data in our

experiments. It shows that imaging mass spectrometry in particular requires highly

accurate raw data from FT instruments, while low accuracy data contain a high risk of

creating non-reliable, invalid, non-authentic or ambiguous information in imaging

experiments of complex biological tissue samples. Fundamental and technical aspects

of high performance UV/IR MALDI imaging, its properties and its prospects will be

described, including optical focusing, ion formation and transfer at atmospheric

pressure, image acquisition, data analysis and substance identification.

The high specificity in mass and space of the method results in an unprecedented

information quality and depth which can be advantegeous in a large number of

clinical, pharmacological or fundamental applications in the future.

References:

[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion

Imaging with a new Scanning UV-Laser Microprobe, Proceedings of the 42nd Annual

Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3,

1994, pp 1041.

[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B.

Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization

Obtained from High-Accuracy Bioanalytical Imaging. Angewandte Chemie

International Edition. 49(22): p. 3834-3838.


24

Visualization of small molecules in tissues by laser desorption

ionization mass spectrometry imaging Strohalm M.

1, Pól J.

1, Faltýsková H.

1, Novák P.

1, Havlíček V.

1, Vidová V.

1, Volný

M.1,2

1Academy of Sciences of the Czech Republic, Institute of Microbiology, Prague, Czech

Republic, 2University of Washington, Department of Chemistry, Seattle, United States

Laser Desorption Ionization (LDI) Mass Spectrometry Imaging (MSI) can be used to

investigate the spatial distribution of lipids and other small molecules in tissue

sections. In LDI MSI, compounds are ionized directly from the tissue slice, which is

attached on a conductive surface moving in set raster steps under a laser beam. Mass

spectra collected from each point within the raster are used to assemble the molecular

images in correlation with their x- and y- positions. In matrix assisted LDI (MALDI),

which is the most common variant of the LDI experiment, the tissue surface is

uniformly coated by the deposition of a matrix aerosol, which facilitates desorption

and ionization. The coating process is computer controlled to avoid excessive over-

wetting, which would deteriorate the spatial distribution of the molecules in the tissue.

While the presence of matrix is essential for ionization of proteins, it can be sometimes

avoided for small molecules if specially designed surfaces and/or special sample

treatment procedures are used. Ions desorbed from the tissue are accelerated into the

mass spectrometer for separation based on the mass-to-charge ratios (m/z) of the ions

and detected. Sampling of the ions directly from the tissue into the mass spectrometer

provides very rich mass spectra with respect to the number of ions with different m/z,

so a high resolution mass analyzer is often an advantage.

In the presented work LDI MSI images were acquired using APEX Ultra 9.4 T FT-

ICR mass spectrometer or Ultraflex III TOF/TOF mass spectrometer (both Bruker

Daltonics, Germany). Both standard mass spectrometers were upgraded to be equipped

with a Smart beam laser (200 Hz). The molecular images of the analytes were

analyzed using an open source program mMass (www.mMass.org) and visualized by

the FlexImaging software. Lipid Maps database (www.lipidmaps.org ) was used for

identification


25

Three main examples of LDI MSI applications for tissue imaging are presented and

discussed 1) In situ detection of globotriaosylceramide (GL3) by LDI MSI in a murine

lysosomal alpha-Gal A gene knock-out (Fabry disease model). The nature of the

accumulation in the mouse model is investigated by the determination of the spatial

distribution of GL3 in kidney tissue sections from wild-type and Fabry mice.

Individual isoforms can be distinguished by MSI and overall distribution is compared

with the antibody staining. 2) Visualization of spatial distribution of

glycerophospholipids and sphingomyelins in the ocular lenses 3) Imaging of

phospholipids and other small molecules in tissue imprints by LDI-MSI without

matrix addition.

Acknowledgment: The work was supported by the Czech Science Foundation

(Project P206/10/P018). M.V.´s research was supported by a MC IR Grant within the

7th EC Framework Program. Other support was provided by the Ministry of

Education, Youth, and Sports of the Czech Republic (MSMT LC07017, LC545 and

ME10013).

Automated analysis of (MS) images

Hamprecht F.A.1

1Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany

I will present ilastik, the Interactive Learning and Segmentation Toolkit. ilastik is a

user-friendly tool for image classification and segmentation in up to three spatial and

one spectral dimension which requires no experience in image processing.

In its basic form, ilastik uses local image or spectral features along with user

annotations to train a nonlinear classifier that allows to distinguish and predict an

arbitrary number of classes. The program provides real-time feedback of the current

classifier predictions and thus allows for targeted training and overall reduced labeling

time. Once the classifier has been trained on a representative subset of the data, it can

be exported and used to automatically process a very large number of images.

Recent developments include a framework that allows to combine modularized

operators to sophisticated workflows. Functionality such as unsupervised learning


26

(probabilistic latent semantic analysis, pLSA) are particularly useful for MS images

which can now be read in the Analyze 7.5 format.

Besides MS images, I will demonstrate application to high throughput screening data

and to the segmentation of 3D neural tissue data.

ilastik is open source and available from http://ilastik.org

MALDI imaging: clustering, super-resolution and applications Alexandrov T.

1, Maass P.

2, Thiele H.

3, Trede D.

1

1Steinbeis Innovation Center for Scientific Computing in Life Sciences, Bremen,

Germany, 2University of Bremen, Center for Industrial Mathematics, ZeTeM, Bremen,

Germany, 3Bruker Daltonik GmbH, Bremen, Germany

Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry

(MALDI-imaging) is a label-free technology used for spatial molecular analysis. Over

the last decade MALDI Imaging has enjoyed a tremendous development. By now

MALDI Imaging is regarded as a most promising innovative measurement technology

in biochemistry, pharmaceutical research and medical applications.

Nevertheless, the information content of a MALDI Imaging data set has not yet been

fully exploited. This is particularly the case for 3D MALDI Imaging applications. Due

to the high experimental costs and due to the enormous computational challenges

posed by the shear size of full 3D MALDI Imaging data sets, this field of application

has not seen much progress beyond a recent proof-of-principle, where a 3D model

corresponding to a single m/z-value was reconstructed from consecutive slices and co-

registered.

Even the most basic task of discriminating different sample areas with distinctly

different molecular structure requires highly efficient and robust computational

methods, which moreover need to be adapted to the medical task under investigation.

We will discuss the potential and limitations of advanced computational methods for

spatial segmentation of MALDI-imaging data sets. In addition we propose a

computational tool for improving the resolution in visualizing MALDI Imaging

results. We will highlight the procedures with 2D applications (rat brain coronal


27

section highlighting anatomical and functional structures of the brain; neuroendocrine

tumor invading the small intestine) as well as first results for 3D spatial segmentation

of a real-life 3D MALDI-imaging data set .

A new dimension of histological information: Highly specific

molecular imaging at cellular resolution Roempp A.

1, Guenther S.

1, Schober Y.

1, Takats Z.

1, Spengler B.

1

1Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry,

Giessen, Germany

Mass spectrometry imaging (MS imaging) has become a method of outstanding

importance in life sciences. The information content of the generated MS images

greatly depends on the quality of the underlying mass spectral data. Mass

spectrometers with high mass resolution are routinely used in many bioanalytical

areas, but only few applications were reported for MS imaging so far. Here we applied

a mass spectrometry imaging method that combines accurate mass measurements and

spatial resolution in the low micrometer range for the analysis of biological tissue.

These experiments were performed with a home-built atmospheric-pressure imaging

source attached to a 'LTQ Orbitrap' or 'Exactive Orbitrap' mass spectrometer (Thermo

Scientific GmbH, Bremen) [1]. All measurements were done with a mass accuracy

better than 3 ppm (root mean square), i.e. compounds were identified with high

confidence. MS images were generated with a bin size of Δm/z = 0.01, which largely

eliminates interferences from neighboring peaks.

Phospholipids, peptides and drug compounds were imaged in a wide range of

biological samples at an effective spatial resolution of 5 to 10 µm. Tissue types of

mouse urinary bladder were discriminated at the cellular level based on molecular

information. The internal structure and surrounding area of metastases in human brain

sections were characterized by their phospholipid pattern. This provided structural

features which were not visible in the histological staining experiments.

Lysophospholipids were specifically detected in the necrotic zone of lung carcinoma.

Phospholipids are less specific than proteins, but their pattern can be used to

distinguish different tumor types and also to determine primary tumors form


28

measurement of the metastases. Tissue sections were stained (H&E or toluidine) after

MS analysis. MS image analysis for all these cases showed excellent agreement with

histological structures.

MS imaging of proteins remains a challenging task. Direct detection and identification

of proteins on tissue is limited by a number of factors including limited mass range

and fragmentation efficiency as well as incompatibility with formalin-fixed samples.

On-tissue digestion of proteins and detection of the resulting peptides can overcome

some of these limitations. In the current study we used a spraying device to apply

trypsin on tissue sections. With this setup we were able to obtain a spatial resolution of

50 µm for tryptic peptides. Initial experiments were done on mouse brain sections, but

the method has also been applied to human tumor tissue in the meantime.

In all experiments the high resolution and mass accuracy proved to be essential for

specific image generation and reliable identification of analytes. This significantly

improves the quality of molecular and structural information that can be obtained from

tissue sections.

[1] Römpp et al. (2010) Angewandte Chemie International Edition 49(22): 3834.

MALDI imaging mass spectrometry for marker discovery and drug

imaging in pancreatic cancer

Siveke J.1, Grüner B.

1, Winkelmann I.

2, Hahne H.

3, Esposito I.

4, Maier S.

3,

Feuchtinger A.2, Trajkovic-Arsic M.

1, Mazur P.

1, Rauser S.

2, Schmid R.

1, Küster B.

3,

Walch A.2

1II. Med. Klinik, Klinikum rechts der Isar der TU München, Munich, Germany,

2Institute of Pathology, Helmholtz Center Munich, Helmholtz Center Munich, Munich,

Germany, 3Department of Bioanalytics, Technical University of Munich, Munich,

Germany, 4Institute of Pathology, Technische Universität München, Munich, Germany

Pancreatic ductal adenocarcinoma (PDAC) remains a fatal disease despite tremendous

therapeutic efforts. The devastating clinical course is due to late detection and high

chemoresistance of PDAC. Recent evidence points to inefficient drug delivery as an

important contributor to the insensitivity of PDAC to therapy. Here we demonstrate


29

the applicability of MALDI IMS for (i) diagnostic marker discovery in preneoplastic

lesions and (ii) drug distribution imaging using genetically engineered mice (GEM)

with endogenous PDAC.

For detection of potentially meaningful biomarkers of early neoplastic disease, various

GEM of PDAC were analyzed by MALDI IMS to investigate the peptide/protein-

expression pattern of preneoplastic lesions in comparison to normal pancreas, chronic

pancreatitis (CP) and PDAC with cellular resolution. Statistical analysis revealed

several discriminative m/z-species between normal and diseased tissue. Proteins from

candidate m/z-species were identified using liquid chromatography and tandem mass

spectrometry (LC-MS/MS). Further validation of two proteins was performed in

murine and human tissue and serum. In conclusion we show that GEM of endogenous

PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS

analysis, allowing in situ analysis of small precursor lesions and identification of

potentially meaningful preneoplastic biomarkers.

In a second approach we utilized MALDI IMS for drug imaging in PDAC. In MALDI

imaging of drug molecules and metabolites one faces the challenge to measure the

intensity of targeted drug signals in a complex background of matrix signals and

endogenous metabolites. The necessary specificity in such an analysis is usually

achieved by the measurement of a specific fragment of the targeted drug in MS/MS

mode (single reaction monitoring SRM). For in tissue analysis of drug molecules and

metabolites, we established and evaluated a novel sensitive and robust MS-based assay

(FAST-SRM) for the small molecule tyrosine kinase inhibitor erlotinib, which targets

the EGF receptor and is an approved therapy in pancreatic and lung cancer. FAST-

SRM measurement of erlotinib and des-methyl-erlotinib demonstrated detection of

both drugs with spatial resolution and an unambiguous correlation of the signal

intensity with the administered dose. Interestingly, signal intensity was lower in PDAC

than in normal exocrine tissue and preneoplastic lesions. In conclusion, the FAST-

SRM mode described here is suitable to analyze drugs and metabolites in tissue. It is

easy to set up and turns fast reflectron MALDI-TOF instruments such as the autoflex

speed into universal imaging instruments. The obtained results support the hypothesis


30

that poor drug delivery may be an important contributor to poor chemotherapeutic

efficacy in PDAC.

Quantification of drug candidates by Label free Mass Spectrometry

Imaging

Hamm G.1, Bonnel D.

1, Piveteau C.

2, Willand N.

2, Deprez B.

2, Delbos J.M.

3, Bouzom

F.3, Stauber J.R.

1

1IMABIOTECH, Villeneuve d'Ascq, France,

2INSERM U761, Biostructures and Drug

Discovery, Lille, France, 3SERVIER TECHNOLOGIES, Orléans, France

Unlike traditional imaging techniques such as autoradiography, magnetic resonance

imaging or positron emission tomography, mass spectrometry imaging (MSI) permits

the label-free study of several compounds of interest simultaneously on the same tissue

section. However, the difficulty of obtaining an absolute quantification of

experimental data remains one of MSI's major disadvantages. This quantification

requires reproducible and homogenous measurements (signal), as well as a calibration

curve with minimal variability and increased linearity. Several methods addressing this

issue are described in literature, but none have universal applications.

Using different examples of drug distribution studies on whole-body samples, we

report a new methodology intended to respond to the main obstacles in quantification

through MALDI imaging. ANd we compare the results of quantitative mass

spectrometry to others gold standards methods such as QWBA (quantitative Whole

body autoradiography) and LC-MS².

Combining histology and MALDI-Mass spectrometry imaging in drug discovery Marshall P.S.

GlaxoSmithKline, Stevenage, United Kingdom

The use of MALDI-MS Imaging in the drug discovery process serves as a valuable

tool to investigate normal and diseased tissues and allows for the visualisation of


31

compound distribution in tissue. The technique is routinely used to determine the

spatial distribution of small molecules (drugs and metabolites) and larger

biopharmaceuticals molecules. The information obtained can add an extra dimension

of understanding to the location of compound in the selected tissue.

However, drug distribution profiles remain “just a pretty picture” unless the data is

correlated with the histological features of the tissue section examined. To demonstrate

this important aspect examples will be shown of the direct overlay of histology data

with MALDI MS imaging data performed on the same section. The presentation will

illustrate how this combined data forms an integral part of the drug discovery process

and provides a greater insight into pharmacological and toxicological findings of a

compound or drug.

Mass spectrometry imaging: A powerful tool in drug discovery and

development Prideaux B.

1, Staab D.

1, Morandi G.

1, Ehrhard N.

1, Stoeckli M.

1

1Novartis Institutes for BioMedical Research, Analytical Sciences, Basel, Switzerland

Mass spectrometric imaging (MSI) technologies enable label-free spatial analysis of

biological tissue samples. Since MSI bases the detection of the analytes on the

molecular weight and/or specific fragmentation pattern, it offers the possibility to

simultaneously measure compound and metabolite distribution without the

requirement to label the drug. In addition, the potential to simultaneously acquire

thousands of endogenous species of interest during the same experiment such as lipids,

peptides and proteins opens up the possibility of simultaneous monitoring of drug,

metabolites and markers of both disease and therapeutic response.

Application of MSI technologies to biomedical research will be discussed with a

particular focus upon contributions to drug discovery and development. Examples

from applications including absorption, distribution, metabolism and excretion

(ADME), pharmacokinetics, toxicology and biotherapy will be presented highlighting

the unique value offered to drug discovery in disease research areas such as

tuberculosis, oncology, acne, phototoxicity and animal health.


32

Drugs in the brain - cellular imaging and discoveries with receptor

microscopic autoradiography

Stumpf W.E.

University of North Carolina, Chapel Hill, NC, United States

For cell and tissue localization of drugs, receptor microscopic autoradiography is

reviewed, including its development history, multiple testing, extensive applications

and significant discoveries. This sensitive high-resolution imaging method is based on

the use of radiolabeled compounds (esp. tagged with 3H or

125I), preservation through

freezing of in vivo localization of tissue constituents, cutting thin frozen sections, and

close contact with the recording nuclear emulsion.

After extensive testing of the utility of this method, the distribution of radiolabeled

compounds has been identified and characterized for estradiol, progestagens, adrenal

steroids, thyroid hormone, ecdysteroids, vitamin D, retinoic acid, metabolic indicators

glucose and 2-deoxyglucose, as well as extracellular space indicators. Target cells and

associated tissues have been characterized with special stains, fluorescing compounds,

or combined autoradiography-immunocytochemistry with antibodies to dopamine-

beta-hydroxylase, GABA, enkephalin, specific receptor proteins, or other cellular

products. Blood-brain barrier and brain entries via capillary endothelium, ependyma,

or circumventricular recess organs have been visualized for 3H-dexamethasone,

210Pb

lead, and 3

H-1,25(OH)2 vitamin D3.

Through this histopharmacologic approach, cellular details and tissue integrative

overviews can be assessed at the same time in the same preparation. As a result,

information has been gained that would have been difficult or impossible otherwise.

Maps of brain drug distribution have been developed and relevant target circuits have

been recognized. Examples include, the stria terminalis that links septal-amygdaloid-

thalamic-hypothalamic structures and telencephalic limbic system components which

extend as the periventricular neuroendocrine system into the mid- and hindbrain.

Discoveries challenged existing paradigms, engendering new concepts and providing

seminal incentives for further research toward understanding drug actions. Most

notable are discoveries made during the 1980s of over 50 target tissues of vitamin D

(reviewed 1995 in Histochem. Cell Biol.) that went beyond systemic calcium


33

regulation and revealed strong nuclear receptor binding in specific cell populations in

the brain and spinal cord (rendered negative in biochemical radioassays), pituitary,

adrenal, skin, and multiple other tissues.

Such unexpected findings have been instrumental in recognizing the life sustaining

role of this 'sunshine hormone' (soltriol), beyond effects on bone growth and repair.

The vitamin D nuclear receptor data, reviewed in specific brain maps and in a

systemic-holistic 'Drug Homunculus ', are a source of important information for

biochemical and clinical follow-up in drug development. This methodology has been

crucial in enabling vitamin D-related CNS prophylaxis and therapies for multiple

sclerosis, parkinsonism, depression, memory loss, sleeping problems, neuroendocrine

disorders and more.

MALDI imaging mass spectrometry for studying cancer diseases Longuespée R.

1, Boyon C.

1, Vinatier D.

2, Salzet M.

1, Fournier I.

1

1University Lille, Fundamental and Applied Biological Mass Spectrometry - EA 4550,

Villeneuve d'Ascq Cedex, France, 2Hôpital Jeanne de Flandre, Clinique

Gynécologique, Lille, France

Early diagnostic and disease management is one of the most important challenges

facing modern medicine, which is particularly relevant in cancer. The lack of effective

assays measuring multiple blood-based biomarkers is absent in many types of cancer.

Moreover, transforming a biomarker into a useful clinical diagnostic test is a complex

process, which starts with identification, proceeds through validation. Identification

can be carried out by various means (gene arrays, purification procedures, proteomics),

that focus on observed changes of the marker correlated with the disease progression,

either in the solid tumour or in a body fluid. As the markers are identified within

extracts or in a non-spatial context, further validation is always required. Several

choices are then available, such as establishing specific antibodies, using protein

microarrays or including more refined techniques such as tissue laser micro-dissection.

A major new alternative that combines both biomarker identification and validation in

a single step is now possible at the tissue level with the development of MALDI Mass


34

Spectrometry Imaging. In a single experiment, molecular information on hundreds of

molecules can be retrieved. By automation of this method and data processing,

molecular maps of compounds can be generated from tissue sections. The obvious

advantage of having the spatial localization of identified compounds is the predictive

potential of which markers are most likely to be successful at the clinical level.

MALDI-MSI is a non-targeted analysis, but due to its high data acquisition, permits

the establishment of a classification of cell phenotypic changes at the molecular level,

which can be used in complement to histology techniques. The correlation between

molecular images obtained by MALDI-MSI and the ones obtained by pathologists

using classical histochemistry can be inclusive of all grades, stages, cancer types, and

cell types. However, differently from classical histochemistry.

In this context, we present here data obtained on ovarian cancer using such a

technology. Many questions are being raised about the potential mechanisms of

ovarian cancer origin and progression. Our findings reflect that a global molecular

profile is more associated with the pathological states observed in endometrioid or

serous ovarian cancers compared to the benign stage, based on principal component

analysis (PCA) and Hierarchical clustering (HC) analyses of MALDI MS profiling

studies. We also identified several biomarkers related to immune response modulation

at early stages of the disease like the Cter part of PA28, “Reg Alpha” and HLA-G.

Based on our data we investigate more deeply the serous ovarian cancer origin, its

escape and invasion strategies. These data open the door of therapeutic strategy based

on collected data obtains by MALDI MSI.


35

MALDI imaging reveals mitochondrial dysfunction as strong

predictor for response to neoadjuvant chemotherapy in Barrett's

Cancer

Elsner M.1, Rauser S.

1, Aichler M.

1, Ludyga N.

1, Maier S.

1, Balluff B.

1, Schöne C.

1,

Meding S.1, Sarioglu H.

2, Feuchtinger A.

1, Langer R.

3, Feith M.

4, Küster B.

5, Ueffing

M.2, Höfler H.

1,3, Walch A.

1

1Helmholtz Zentrum München, Institute of Pathology, Neuherberg, Germany,

2Helmholtz Zentrum München, Department of Protein Science, Neuherberg, Germany,

3Technische Universität München, Institute of Pathology, München, Germany,

4Klinikum rechts der Isar, Technische Universität München, Department of Surgery,

München, Germany, 5Technische Universität München, Chair of Proteomics and

Bioanalytics, München, Germany

Aims: In esophageal adenocarcinoma (Barrett's Cancer), neoadjuvant chemotherapy

improves survival in a subgroup of patients. To avoid unnecessary therapy, there is a

need for markers that predict therapy response. In order to find proteomic markers,

MALDI imaging was performed on pretherapeutic Barrett's Cancer biopsies.

Methods: Pretherapeutic endoscopic Barrett's Cancer biopsies (n=23) were obtained

during primary staging. After two cycles of cisplatin and 5-fluoroouracil therapy,

response to treatment was assessed in each patient. The tissue samples were measured

by MALDI imaging and proteomic profiles were correlated with the response data.

Candidate species discriminating between responders and non-responders were

identified by LC-MS/MS. Predictive impact of the mitochondrial protein COX7A2

was validated on an independent set of pretherapeutic, endoscopic biopsies (n=54).

Subsequently, the correlation between COX7A2 expression and mitochondria was

evaluated by electron microscopy.

Results: MALDI imaging revealed 22 m/z species correlating with therapy response in

Barrett's Cancer (p< 0.05). Hierarchical clustering showed that MALDI imaging

profiles could be used to accurately define responders to neoadjuvant therapy from

non-responders. By LC-MS/MS, a signature of 4 mitochondrial proteins with reduced

expression in responders was identified. Immunohistochemistry of the most

discriminating protein COX7A2 in the validation set confirmed the MALDI imaging


36

results and revealed the predictive impact of COX7A2 (p=0.003). Electron microscopy

showed a destruction of mitochondria in cases with reduced-expression of COX7A2.

Conclusion: By MALDI imaging, a novel signature of mitochondrial proteins was

found, which plays a role in response to neoadjuvant chemotherapy in Barrett's

Cancer. Furthermore, a correlation between reduced expression of mitochondrial

proteins, mitochondrial dysfunction and therapy response could be shown for the first

time.

Differentiating between morphologically identical tumors using imaging MS-based molecular histology Jones E.A.

1, Waaijer C.

2, Schmitz N.

2, van Remoortere A.

1, van Zeijl R.J.

1, Deelder

A.M.1, Bovee J.V.M.G.

2, Mc Donnell L.A.

1

1Leiden University Medical Center, Department of Parasitology, Leiden, Netherlands,

2Leiden University Medical Center, Department of Pathology, Leiden, Netherlands

Introduction: Matrix assisted laser desorption/ionization mass spectrometry

(MALDI-MS) can generate profiles directly from tissue that contain hundreds of

distinct biomolecular ions. Spatially-correlated analysis, imaging MS, can

simultaneously reveal how the intensity of each of these biomolecular ions varies in

tissue samples. There is growing evidence that imaging MS is having an impact in

disease detection, particularly cancer. The differential profiles found in tumors can be

used to identify specific candidate biomarkers. One of the advantages of imaging MS

is that it can differentiate regions of tissue based on their MS profiles and thereby

distinguish biomolecularly different regions even if they are not distinct using

established histological tools.

Here we demonstrate how imaging MS-based molecular histology can help

differentiate between morphologically identical chondrosarcomas. Chondrosarcoma

are a heterogeneous group of bone tumors. The most common subtypes, conventional

central and secondary peripheral, are morphologically identical but exhibit different

clinical behavior, have different genetic backgrounds and different treatment targets

have been identified. They are largely insensitive to conventional chemo- and radio-


37

therapy, and so surgery is the mainstay of treatment. It is thus vital to identify markers

that confidently differentiate central from peripheral chondrosarcoma.

Methods: MALDI-Imaging MS of proteins was performed using an Autoflex

MALDI-ToF and sinapinic acid as matrix. Automated feature identification and

extraction reduced the data load by >300X enabling a series of multivariate analyses to

be performed to identify the regions of tissue consistently highlighted as molecularly

distinct.

Results: Five central and five peripheral grade II chondrosarcoma samples, identical

morphology and grade, were analyzed. Pair-wise analyses revealed that the tumors

could be differentiated using a variety of multivariate methods. However, significant

intratumor heterogeneity led to variability in the MS features that differentiated

between the tumors.

Automated data reduction of the entire ten-tissue dataset enabled, for the first time,

molecular histology of the patient series, and thus allowed three sources of variance to

be distinguished.

intratumor variability within each patient tissue

inter-patient variability within each tumor class

variability between central and peripheral chondrosarcoma.

Variable stroma and intense blood-related protein signals from a subset of tissues

undermined initial unsupervised separation. Reducing the weight of these signals

enabled the entire patient series to be correctly assigned, and highlighted twelve

peptide and proteins biomarkers.

Conclusion: Imaging MS based molecular histology helped identify the first

biomarkers that distinguish between morphologically identical chondrosarcomas.


38

MALDI-MS imaging and profiling of pancreatic and stomach cancer

tissue microarrays Djidja M.-C.

1, Turner A.J.

1, Scriven P.

2, Claude E.

3, Cole L.M.

1, Clench M.

1

1Sheffield Hallam University, Biomedical Research Centre, Sheffield, United Kingdom,

2The University of Sheffield, Academic Surgical Oncology Unit, University of

Sheffield, Sheffield, United Kingdom, 3Waters Corporation, Manchester, United

Kingdom

The development of tissue micro array (TMA) technologies provides insights into

high-throughput analysis of proteomics patterns from a large numbers of archived

tumour samples. In the work reported here, matrix assisted laser desorption/ionisation-

ion mobility separation-mass spectrometry (MALDI-IMS-MS) profiling and imaging

methodology has been used to visualise the distribution of several peptides and

identify them directly from TMA sections after on-tissue tryptic digestion. A novel

approach that combines MALDI-IMS-MSI and principal component analysis-

discriminant analysis (PCA-DA) is described, which has the aim of generating tumour

classification models based on protein profile patterns. The molecular classification

models obtained by PCA-DA have been validated by applying the same statistical

analysis to other tissue cores and patient samples. The ability to correlate proteomic

information obtained from samples with known and/or unknown clinical outcome by

statistical analysis is of great importance, since it may lead to a better understanding of

tumour progression and aggressiveness and hence improve diagnosis, prognosis as

well as therapeutic treatments. The selectivity, robustness and current limitations of

the methodology are discussed.

Tools for MALDI imaging based histology and data interpretation:

Deininger S.

Bruker Daltronik GmbH, Bremen, Germany

Often times in powerpoint presentations or papers on MALDI imaging the correlation

of some histological data with MALDI images is shown. What does not become clear

in this context is how the correlation is actually done: We will show in a live demo


39

how the flexImaging software handles the integration of microscopic data into the

MALDI imaging workflow on selected examples. We will also show the benefit of the

implementation of new statistical methods for simplifying the analysis of MALDI

imaging data.

Prostate Cancer - What can we learn from MALDI imaging

Schwamborn K.1,2

, Wild P.3, Caprioli R.

2

1Technische Universität München, Institute of Pathology, Munich, Germany,

2Vanderbilt University, Department of Biochemistry, Nashville, United States,

3University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland

Prostate cancer (PCa) is the most common cancer in men in the United States and

Germany. Although highly curable at an early stage the overall death toll remains high

due to recurrence of “cured” cases and progression to hormone-refractory and/ or

metastatic disease. Notwithstanding the sizeable number of cancer-related deaths, only

a small proportion of cancers will progress to a life-threatening disease. Elucidating

changes at the protein level involved in PCa progression would provide invaluable

information. Imaging mass spectrometry (IMS) applied to prostate cancer tissue

biopsies enables the visualization of the spatial distribution of cancer specific

protein/peptide expression profiles in correlation with histological features that allow

disease staging and prediction of outcome.

Formalin-fixed and paraffin-embedded prostate samples (normal, 69; PCa, 506;

hormone-refractory PCa, 49 and metastases, 23) were subjected to on-tissue tryptic

digestion. Briefly, sections were mounted onto conductive glass slides and underwent

paraffin removal well as antigen retrieval. On-tissue digestion was achieved by

spotting trypsin onto the tissue in an array pattern using a Portrait 630 reagent multi-

spotter. Following digestion, CHCA was spotted directly onto the array of tryptic

spots. Samples were analyzed utilizing an Ultraflextreme MALDI-TOF/TOF mass

spectrometer. Additionally, MS/MS measurements of selected peptides were acquired.

Data analysis was performed by using the ClinProTools 2.2 and FlexImaging 2.1

software.


40

On-tissue tryptic digestion of prostate tissue revealed on average 210 high abundance

peptides in the mass range from m/z 900-4000. When analyzing spectra from cancer

regions in comparison to spectra from regions without cancer distinctive differences in

peak patterns could be identified. For example, from the cancerous prostate regions

peptides detected at m/z 1515.64, 1570.82 and 2798.11 were at significantly higher

expression. Similarly, peptides from the non-cancer regions at m/z 2087.88 and

2402.79 were also seen with significantly higher abundances. Combining 23 peaks in a

support vector machine based model resulted in an overall cross validation of 95%

with a sensitivity of 99.3% and a specificity of 90.6% in the training set. Classification

of spectra from localized PCa and hormone-refractory PCa samples could be achieved

by combining 6 peaks in a genetic algorithm based model resulting in an overall cross

validation of 84.9% with a sensitivity of 79.3% and a specificity of 90.4%. The tryptic

peptide at m/z 1515.64 could be identified as a peptide from actin. Another trptic

peptide at m/z 2798.11 was sequenced as a peptide from tubulin beta. Further

identification of differentially expressed peptides and validation of these initial

promising findings on an independent second sample set are ongoing and could

facilitate the discovery of proteins involved in the progression of PCa.

MALDI imaging mass spectrometry in gastric cancer

Balluff B.1,2

, Ebert M.3, Walch A.

1

1Institute of Pathology, Helmholtz Zentrum München - German Research Center for

Environmental Health, Neuherberg, Germany; 2Department of Medicine II, Klinikum

rechts der Isar, Technische Universität München, Munich, Germany, 3 Department of

Medicine II, Universitätsklinikum Mannheim, University of Heidelberg, Mannheim,

Germany

Histology-driven matrix-assisted laser desorption/ionization (MALDI) imaging mass

spectrometry allows measuring the molecular content of tissues. We used MALDI

imaging in pre-clinical studies for the identification of clinical relevant proteins in

terms of prognosis and therapy response in gastric cancer patients. The results


41

highlight the usefulness of MALDI imaging for providing novel and clinical relevant

information from tumor tissues, as well as its potential for tissue diagnostics.

Strategies for identification and characterization of Protein

biomarkers for MALDI imaging studies.

Suckau D.

Bruker Daltronik GmbH, Bremen, Germany

To identify the marker proteins that were discovered in clinical MALDI imaging

studies is crucial for their validation, e.g., by IHC, and for the further clinical use. We

present established procedures that provide access to protein identities that can be used

for both a) intact protein identification from fresh frozen tissue, and b) digest based

protein identification compatible with the use of FFPE tissue

Present and future impact of image analysis and image mining on

histochemistry Binnig G.

Definiens AG, München, Germany

The increasing spread of digital image acquisition hardware such as whole tissue slide

scanners provides the basis for a growing relevance of image analysis. Through

advanced context based image analysis complex images can be evaluated and objects

and structures therein can be quantified. Some of those derived data are not or hardly

accessible through the human visual system. Examples are multispectral

investigations, comprehensive analyses of a bundle of different or of multidimensional

images and the quantification of a large number of relevant objects. In particular the

local co-expression analysis of several immunohistochemical slides is nearly

impossible by visual inspection but conveniently achievable through image analysis of

co-registered tissue slides. The co-registration by itself is of high value and only

feasible in digital pathology in a meaningful way and enriched by sophisticated image

analysis opens the door for novel kind of investigations.


42

Whereas image analysis provides rich useful information, image mining goes a step

further by extracting knowledge from large collections of images. Image analysis is

part of image mining and the more sophisticated the image analysis is the more

valuable the derived knowledge will be. The amount of data statistically analyzed in

image mining can be huge and therefore can only be handled by a computer. Very

valuable are correlations of health-related data of individuals with image based data.

Fine structural 3D-analyses of cellular reorganizations induced by metabolic stress Meisslitzer-Ruppitsch C.

1, Ranftler C.

1, Vetterlein M.

1, Ellinger A.

1, Neumüller J.

1,

Pavelka M.2

1Medical University of Vienna, Vienna, Austria,

2Medical University of Vienna, Cell

Biology and Ultrastructure Research, Vienna, Austria

Aims: The work presents 3D-analyses of fine structural changes in cells responding to

ATP-depletion and aims to receive improved insight into mechanisms cells use to

manage metabolic stress.

Methods: In cultured HepG2 hepatoma and endothelial cells, a reduction of the ATP-

pool was achieved by replacing in the culture media D-glucose by its non-

hydrolyzable analogue 2-deoxy-D-glucose (1,2). Peroxidase-labelled wheat germ

agglutinin (WGA) and BODIPY-ceramide (Cer) internalized into the cells prior to or

after ATP-depletion were visualized by oxidation and photooxidation of

diaminobenzidine (DAB), respectively. Three-dimensional analyses were performed

by electron tomography of 200-300nm thick sections of the Epon-embedded cell

cultures in a 200kV transmission electron microscope (Tecnai-20, FEI). For

reconstruction, the Inspect 3D software (FEI), the IMOD software, or the

“Tom_Release_2008 toolbox” software (kindly provided by W.Baumeister and

J.Plitzko, MPIB Martinsried) were used. The models were drawn with help of the

Amira 4.1 software (Mercury Computer Systems, Merignac Cedex, France).

Results: The results revealed that ATP-depletion leads to reversible reorganizations of

compartments of the secretory and endocytic systems. The Golgi apparatus stacks

loose their regular organization and become replaced by tubulo-glomerular bodies and


43

networks. Initial membrane changes were apparent as early as 10 min after start of the

experiments. The 3D-reconstructions demonstrated that the regular stack organization

is already broken up at this time; hairpin bend-connections and twisted cisternae

occurred. After 45min ATP-depletion, regular Golgi stacks were almost absent and the

glomerular Golgi bodies and networks dominated. The combined endocytosis studies

showed that both endocytic and non-endocytic parts of the Golgi apparatus become

reorganized. The Golgi bodies formed in response to ATP-depletion contained

ceramide-concentrating subcompartments, supposed to be sites of synthesis of higher

lipids. Most interestingly, endocytosed Bodipy-ceramide was taken up and

concentrated in compartments of the Golgi bodies even, when they were formed prior

to the start of Cer-internalization indicating retained functionalities in the reorganized

Golgi apparatus.

Conclusions: The results showed that cells do not become damaged by metabolic

stress induced by ATP-depletion but respond with reversible reorganizations. It

became evident that the cells react rapidly to the changes of the ATP-levels; the

findings with both endocytosed WGA- and ceramide indicate maintained

functionalities, which may be significant for the cells´ management of metabolic stress

and their survival.

References:

1. M. Del Valle et al., J.Cell Sci. 112 (1999) p4017.

2. C. Meisslitzer-Ruppitsch, et al. Histochem. Cell Biol. 135 (2011) p15

The authors thankfully acknowledge the technical work and help of Mag.Beatrix

Mallinger, Mrs.Regina Wegscheider, Mr.Peter Auinger, Mr.Thomas Nardelli and

Mr.Ulrich Kaindl.


44

Nanoparticle-based Immunocytochemistry reveals PIP2-

containing microarchitecture of the cell nucleus Hozak P.

1

1Institute of Molecular Genetics ASCR, Prague, Czech Republic

While fluorescent microscopy allows for simultaneous detection of multiple antigens,

the electron microscopy (EM) sensitive immunodetection is limited to only two

antigens. I will summarize the current possibilities of single molecule visualization

inside of cells and tissues, and discuss future needs of researches in biomedicine.

In order to overcome the current limitations of immunodetection, we prepared a set of

novel nanoparticles (NPs) which fulfill several criteria: size in the frame of 5-12 nm,

small size distribution, good contrast and stability in the electron microscope, stability

of colloidal solution during conjugation, and surface properties allowing for

conjugation with antibodies. The colloids were prepared from soluble metal salts by

controlled chemical reduction. Subsequently, conditions for conjugation of NPs with

antibodies were optimized and obtained conjugates were probed for the ability of

immunolabelling on ultrathin resin sections of cells. With the use of novel NPs,

various combinations with commercial gold NPs can be made to obtain a set for

simultaneous labelling. For the first time in ultrastructural histochemistry, up to five

molecular targets can be identified simultaneously. For conventional transmission

electron microscopy (TEM), NPs of different shapes can be included in addition to

spherical gold NPs. Different elemental composition of NPs can be used for their

discrimination by EFTEM, STEM/HAADF, TEM/EDX, or FESEM. Using our

previously developed tools of spatial statistics one could then map the regions of

distribution of multiple molecular targets within the cell, as well as to analyze a high

number of individual molecular interactions.

These methods allowed us to progress with understanding some novel molecular

interactions in the cell nucleus. I will discuss interactions of phosphatidylinositol-4,5-

bisphosphate (PIP2) and nuclear myosin I (NMI) which are involved in regulation of

gene expression. PIP2 resides in the nucleus in a different form than the classical

bilayer membrane, apparently forming specific nuclear protein complexes. Our data


45

suggest that nucleolar PIP2 might serve as a transcription factor for ribosomal genes.

We therefore also investigated PIP distribution in cell nuclei with a special attention to

nucleoli by ultrastructural tomography, and mapped PIP colocalization with various

factor involved in RNA pol I transcription. We also showed that NM1 binds to PIP2 in

the cell nucleus, and this was further confirmed by electron microscopy. The results

will be discussed in the frame of the current nucleolar model and lipid functions in the

cell nucleus.

Acknowledgement: This research was supported by the Grant Agency of Czech

Republic (P305/11/2232, 204/09/H084), MSMT (LC545, LC06063 and 2B06063),

Academy of Sciences of the Czech Republic (KAN200520704) and by the IMG grant

AV0Z50390512.

Tracing the movement of mRNA towards the nuclear pore Basello D.

1, Cisterna B.

1, Biggiogera M.

1

1Università di Pavia, Dip. Biologia Animale - Lab. Biologia Cellulare, Pavia, Italy

Extranucleolar RNA synthesis takes place in the so called Perichromatin Area, a 100-

200 nm zone at the periphery of condensed chromatin (Albiez et al., 2006). There the

perichromatin fibrils (PF) are formed by the association of nascent transcripts with

hnRNPs and snRNPs. PF are cotranscriptionally spliced, RNA is cleaved and

polyadenylated and the pre-mRNA/mRNA moves towards the nuclear pore. In some

cases and for reasons so far not yet clarified, PF coil and form a Perichromatin Granule

(PG) which can be stored for some time in the nucleus and then can be exported into

the cytoplasm. PG are possibly a storage form for some mRNAs the cell would need in

time. Transcription, splicing and the movement of these structures involve several

proteins and factors, including actin and Nuclear Myosin I (see Visa and Percipalle,

2010) which are not only involved in transcription but also accompany PF in their

movements. So far, nuclear export of RNAs has been considered to follow a diffusion

mechanism through the interchromatin space, i.e. moving within a space physically

limited by macromolecular structures, and consequently undergoing an anomalous

diffusion process. In recent years, however, it has been proved that at least one


46

macromolecular structure, the small ribosomal subunit, can be exported also by an

active mechanism which represent about 15% of the total export (Cisterna et al., 2006,

2009).

In this view, we have investigated by high resolution EM the movement of pre-

mRNA/mRNA by following the PF as well as the PG inside the cell nucleus after

immunolabelling of actin or nuclear myosin I in different experimental conditions on

HeLa cells. We have analyzed the number of transcripts present at the nuclear pore in

control cells, in cells after in vivo incorporation of anti-actin or anti-myosin antibodies,

or after blockade of de novo ATP synthesis. Immunolabelling with marker antibodies

as well as selective RNA staining with terbium has allowed a semiquantitative analysis

of the transcripts near the nuclear pore. In control cells, the presence of RNA in the

close vicinity of the pore is about 1.8% of the number of trascripts visible inside the

nuclear section, while after actin or ATP blockade the number rises over 6%. These

data suggest that a part of the movements of the non nucleolar transcripts seems to be

linked to an active mechanism. Moreover, PF contain a single RNA molecule which is

never coiled and clearly recognizable from the RNA contained inside PG after terbium

staining. Our preliminary data indicate that the (partial) blockade of energy-requiring

mechanism influence the transfer of one form of transcript more than the other. It is

tempting to hypothesize that cells may use diffusion for one RNP structure and ATP-

dependent movement for the other, thus adding a sort of priority to some mRNAs

instead of others.

Microscopic analysis of protein misfolding diseases and the effect of therapeutic synthetic chaperones Roth J.

1

1Yonsei University, World Class University Program of Yonsei University Graduate

School, Seoul, Korea, Republic of

Protein misfolding has been recognized as the cause of various human diseases. Non-

native proteins will be recognized and retained in the endoplasmic reticulum (ER) by a

sophisticated quality control mechanism and assisted by ER chaperones to achieve


47

proper folding. Those proteins failing to reach a native conformation will become

dislocated to the cytosol for ER-associated degradation (ERAD) involving degradation

by proteasomes or macroautophagy (1, 2). Fabry disease is a lysosomal storage disease

caused by alpha-galactosidase A deficiency. Private point mutations cause enzyme

misfolding and retention in the ER, resulting in progressive multi-systemic

glycosphingolipid deposition, mainly globotriosylceramide Gb3. Fabry disease is the

prototype of a “loss-of-function” disease. Primary open-angle glaucoma represents the

prototype of a “pathological gain-of-function” disease and is caused by point

mutations of myocilin. Misfolded mutant myocilin forms heteromeric complexes with

wild-type myocilin in the ER, which result in Mallory body formation and apoptotic

cell death. Aspects of the molecular pathogenesis of both protein misfolding diseases

as revealed by microscopy will be presented to illustrate through which different

mechanisms cells manage the presence of misfolded proteins in the ER. Furthermore,

the use of synthetic chaperones to rescue the mutant proteins from the ER protein

quality control and to reverse the disease phenotype will be discussed.

Supported by the World Class University program through the National Research

Foundation of Korea funded by the Ministry of Education, Science and Technology

(R31-2008-000-10086-0), a grant from the National Research Foundation of Korea by

the Ministry of Education, Science and Technology (2010-0027736) and the Swiss

National Science Foundation.

1) Roth, J., Cho, J.W., Zuber, C., Park, S., Jang, I., Lee, Y., Gaplovska Kysela, K., Le

Fourn, V., Santimaria, R., Guhl, B.: Protein N-glycosylation, protein folding, and

protein quality control. Mol Cells 30: 497-506, 2010

2) Roth, J., Yam, G.H-F., Fan, J., Hirano, K., Gaplovska-Kysela, K., Le Fourn, V.,

Guhl, B., Santimaria, R., Torossi, T., Ziak, M. and Zuber, C.: Protein quality control:

the who's who, the where's and therapeutic escapes. Histochem Cell Biol 129, 163-

177, 2008


48

Ultrastructure and drug delivery: gateways and barriers Thurner G.C.

1, Wallnöfer E.A.

1, Soelder E.

2, Kremser C.

1, Talasz H.

3, Helbok A.

4,

Klammsteiner N.5, Dietrich H.

6, Jaschke W.

1, Debbage P.

5

1Medical University of Innsbruck, Clinic for Radiology, Innsbruck, Austria,

2Medical

University of Innsbruck, Department of Obstetrics and Gynaecology, Innsbruck,

Austria, 3Medical University Innsbruck, Sektion für Klinische Biochemie, Biozentrum,

Innsbruck, Austria, 4Medical University of Innsbruck, Department of Nuclear

Medicine, Innsbruck, Austria, 5Medical University Innsbruck, Department of Anatomy,

Histology & Embryology, Innsbruck, Austria, 6Medical University Innsbruck, Central

Animal Research Facility, Innsbruck, Austria

Introduction: Size, charge and aqueous solubility are the major factors determining

whether a drug can access its target molecules in pathophysiologically altered tissue.

Focussing on size, most drugs even now are small, 1-5 nm in size. Most cellular

substructures are an order of magnitude larger, so by adjusting charge and solubility of

the drug it has generally been possible to access the target molecules. However, such

drug molecules also access a wide range of other (non-target) molecules because they

enter most functional compartments of the body. The development of macromolecule-

sized drugs has brought size relations at the nanoscale into prominence: these drugs

are excluded from accessing most potential targets by cellular structures which are

about the same size as the drug itself. Development of nanoparticles as drug vehicles

brings nanoscale size relationships into the very foreground of pharmaceutical interest,

and provides presently the most significant of several Grand Challenges in

Nanomedicine. In Innsbruck we examined these issues by synthesizing

macromolecules for use as drug vehicles, and by using these macromolecules to create

nanoparticles. Systemic application of these materials in laboratory animals revealed

barriers to their distribution in the body.

Methods: Albumin-DTPA-gadolinium conjugates were synthesized and injected

intravenously into mice and rats, and also into human placental cotyledons ex situ.

Albumin-DTPA-gadolinium or albumin-DTPA-indium111

conjugates were emulsified

with polylactic acid to create nanoparticles, which also were injected intravenously

into the test animals. The distribution of the albumin component was followed by


49

immunohistochemistry of fixed and embedded organs, mainly using light microscopy.

The nanoparticles were visualized by use of transmission electron microscopy. The

gadolinium component was tracked by use of atomic absorption spectrophotometry.

Indium111

was tracked by PET/SPECT and by scintigraphy.

Results: Both nanoparticles and the conjugated protein localised rapidly into the liver,

spleen and kidneys. The conjugated protein failed to cross endothelial barriers and

therefore did not enter organ compartments. The stabilized albumin nanoparticles

crossed endothelial barriers but not the underlying epithelial barriers, and therefore

also did not enter most organ compartments.

Discussion: Our data reveal the numerous blood-tissue barriers, the test materials

being excluded from most compartments and localizing into unspecific uptake systems

(liver, spleen) or into an organ with albumin-uptake mechanisms (kidney). The data

also show significant differences in pharmacokinetics between the macromolecular

material (7-10 nm in size) and the nanoparticles (30 nm diameter). These results

elucidate the recent impressive failure of several major industrial research efforts to

develop nanoparticle-based formulations for delivery of highly promising new drugs.

Correlated microscopy and nanotomy to analyze Islets of Langerhans in Type I diabetes Giepmans B.N.G.

1, Kalicharan R.

1, Sjollema K.A.

1, Dijk F.

1, Visser J.T.

1, Algra A.

1,

Avramut M.C.2, Koster A.J.

2, Faas F.G.

2, Ravelli R.B.

2

1University Medical Center Groningen (UMCG), Dept. Cell Biology, Groningen,

Netherlands, 2Leiden University Medical Center (LUMC), Dept. Molecular Cell

Biology, Leiden, Netherlands

Electron microscopy (EM) is the method to visualize tissue composition, cellular

interactions and physiological conditions. However, EM only covers a small area and

usually lacks the context of the tissue. Moreover, EM precludes live-cell imaging and

protein-identification with ultrastructural preservation remains a hurdle. Here, we

present (1) novel probes for correlated microscopy; and (2) large-area EM imaging

that allows to characterize large cross-sections (up to millimeters) at unprecedented

resolution, called “nanotomy”. Islets of Langerhans during Type I diabetes onset have


50

been studied with nanotomy. Our data not only shows the progressive destruction of

insulin-producing cells, but also small electron-dense particles that seem to be

associated with diabetes progression. We will discuss our data and present the

workflow of nanotomy, that will be of general use to other tissues and research

questions.

Illuminating biomedical discovery with advanced photonic imaging

Ntziachristos V.1

1Technische Universität München / Helmholtz Zentrum München, Neuherberg,

Germany

Optical imaging is unequivocally the most versatile and widely used visualization

modality in clinical practice and life sciences research. In recent years, advances in

photonic technologies and image formation methods have received particular attention

in biological research and the drug discovery process for non-invasively revealing

information on the molecular basis of disease and treatment. An increasing availability

of endogenous reporters such as fluorescent proteins and probes with physiological

and molecular specificity enable insights to cellular and sub-cellular processes through

entire small animals, embryos, fish and insects and have revolutionized the role of

imaging on the laboratory bench, well beyond the capability of conventional

microscopy. This talk describes current progress with instruments and methods for in-

vivo photonic tomography of whole intact animals and model biological organisms.

We show how new tomographic concepts are necessary for accurate and quantitative

molecular investigations in tissues and why it could be potentially a valuable tool for

accelerated investigations of therapeutic efficacy and outcome. We further

demonstrate that cellular function and bio-chemical changes can be detected in-vivo,

through intact tissues at high sensitivity and molecular specificity. Examples of

imaging enzyme up-regulation, carcinogenesis and gene-expression are given. The

potential for clinical translation is further outlined. Limitations of the method and

future directions are also discussed.


51

Tissue structures guiding collective breast carcinoma invasion Ilina O.

1, Bakker G.-J.

1, Bult P.

2, van Krieken H.

2, Friedl P.

1

1Radboud University Nijmegen Medical Centre, NCMLS, Department of Cell Biology,

Nijmegen, Netherlands, 2Radboud University Nijmegen Medical Centre, Department

of Pathology, Nijmegen, Netherlands

Tumor cell invasion through surrounding tissue occurs by diverse modes, including

single-cell or collective migration patterns. Collective invasion results from the

motility of cell groups that retain cadherin-based cell-cell junctions, multicellular

front-rear polarity and coordinated acto-myosin contractility. Here we address how

pre-existing tissue structures guide cancer cell invasion and regulate a transition

between different invasion modes. To identify pro-invasive extracellular matrix

(ECM) patterns, 3D immunofluorescence and second harmonic generation imaging

from the tumor-stroma interface of thick sections from human ductal breast carcinoma

samples was performed. Fibrous tissue adjacent to normal lactiferous ducts was mainly

composed of a random network of collagen type I fibers that formed barrier-free tracks

with interfibre clefts measuring up to 20 µm in cross-section. Collagen fibers of the

peritumoral region were realigned to form densely packed parallel bundles laterally

bordering extensive tracks filled by single-cell files or multicellular carcinoma masses.

Invading carcinoma cells retained E-cadherin and beta-catenin along cell-cell

junctions, the functional role of which was confirmed using RNA interference against

E-cadherin in breast cancer cells collectively migrating within 3D organotypic

cultures. To validate the impact of barrier-free tracks formed by bundled collagen

fibres as guiding structures, cell-cell junctions, ECM conditions and the capacity to

proteolytically degrade ECM were altered in context. In loose or experimentally

patterned 3D collagen matrices, downregulation of E-cadherin dependent cell-cell

junctions was permissive for single-cell dissociation, whereas dense collagen

conditions imposed collective invasion irrespective of E-cadherin expression level.

Likewise, invasion into patterned 3D collagen mimicking realigned regions in vivo

supported MMP-independent collective invasion along fibrillar collagen interfaces,

whereas random collagen structures required MMP-dependent pericellular

collagenolysis and cell-generated path formation. These results suggest that the


52

alignment and density of the ECM has major impact on the mechanisms of cell-cell

cohesion and MMP-dependence of breast cancer invasion.

Scanning Laser Imaging of Macroscopic Samples Damaskinos S

1

1Intas Science Imaging & Huron Technologies, Waterloo, Canada

Modern high resolution digital slide imagers compete well at resolutions once

dominated by traditional microscopes. Laser based point scanning through a high

numerical aperture lens is one means of creating such a device. These devices have

several advantages over other means of image acquisition, along with some draw

backs. They excel at producing confocal images in fluorescence and are also capable

of generating high resolution transmitted light images.

The TISSUEscope is a laser point scanning system based on patented imaging

technology. It uses an f-theta, telecentric laser scan lens instead of a standard

microscope objective lens in an imaging configuration. In this presentation the optical

arrangement of the TISSUEscope technology will be described and its

advantages/drawbacks relative to other imaging technologies will be discussed.

Applications of the technology in the field of large whole mount imaging will be

presented.

Automated co-analysis of MALDI- and H&E-images of retinal tissue for an improved spatial MALDI-resolution Schönmeyer R.

1, Schmidt G.

1, Meding S.

2, Walch A.

2, Binnig G.

1

1Definiens AG, München, Germany,

2Helmholtz Zentrum München, Institute of

Pathology, Neuherberg, Germany

MALDI imaging is a powerful technology to gain information with high mass

spectroscopic resolution for tissue slides. As its spatial resolution is lower than that of

optical microscopy, methods for improvements are desired. In this contribution we

present an approach to virtually improve MALDI's spatial resolution where the

relevant structures approximately exhibit a linear shape.


53

Therefore a research prototype of a system based on Definiens Cognition Network

Technology fully automatically segments and classifies different regions of interest in

registered high resolution H&E-images. On an upper hierarchical analysis level tissue

types such as inner and outer nuclear layer as well as the layer of rods and cones are

found. On a lower analysis level the system also is capable of detecting individual

nuclei. We use the fact that cross-sections of retinal tissue show a defined sequence of

retinal layers. They can be seen as a one-dimensional system where centers of MALDI

spots have a certain distance to a reference line; in our case the border line between

outer nuclear layer and the layer of rods and cones. This line and all those

corresponding distances of all MALDI spots are automatically calculated and

projected into a one-dimensional system where the reference line represents the origin.

It is beneficial if the variation of distances is relatively large in contrast to them being

clustered around to a few values. Even for a regular sampling of MALDI spots this is

given for the retina due to its moderate curvature. In the one-dimensional system we

consider a range from -75µm to +75µm covering the layers of interest with enough

samples for binning with ~10µm resolution. For each bin ten to fifteen spectra are

available and averaged. This way spectral information with a five times higher spatial

resolution compared to MALDI and additionally an improved signal to noise ratio is

achieved.

Together with other quantitative measures from image analysis, like e.g. density of

nuclei in distinct nuclear layers, this constitutes valuable input data for an extended

MALDI analysis that will help to model the retinal function using a comprehensive

systems biology approach.

Parts of the work presented here are funded by the German Federal Ministry for

Education and Research (SysTec, grant 0315508).


54

Towards systems-pathology by integrating whole-slide imaging,

high-dimensional data and biological networks Grabe N.

1

1Institute of Medical Biometry and Informatics and Hamamatsu Tissue Imaging and

Analysis Center, BIOQUANT University Hospital Heidelberg, Heidelberg, Germany

The paradigm of systems biology is currently transforming the search for mechanistic

explanations of complex biological processes. This is caused simply by technological

advances resulting in increasing amounts of quantitative and high-dimensional cell and

tissue data.

Such complex data sets can only be interpreted by network reaction models

comprising the RNA-, the protein- and other cellular levels.

Systems biology is thus not a scientific trend but the result of a technological

evolution. For pathology this creates the challenge to integrate spatial and quantitative

tissue data with systems biological methods into real systems pathological approaches.

Therefore, first the essential importance of histological, microscopic whole-slide

imaging and computational quantitative image analysis is outlined at examples from

our tumor and wound healing projects. From this it gets clear that the automatic and

objective quantitative spatial analysis of histological slides is the key stone of systems

pathology. It is then shown how quantitative tissue data could be linked to the

intracellular level via growth-factor-receptor network models. Such multi-scale models

will, when embedded in quantitative tissue data,form the conceptional basis of systems

pathology.


55

Quantitative Tissue Proteomics for Deciphering Molecular

Processes in Autoimmune Uveitis Hauck S.M.

1, Deeg C.A.

2, Ueffing M.

1,3

1Helmholtz Zentrum München, Department of Protein Science, Neuherberg, Germany,

2Ludwig-Maximilians University, Institute of Animal Physiology, Department of

Veterinary Sciences, Munich, Germany, 3Centre of Ophthalmology, University

Medical Centre, Tübingen, Germany

Autoimmune Uveitis is an inflammatory disease of the eye, which is caused by

autoimmune processes and occurs in recurrent relapses resulting in irreversible

blindness. Autoreactive T-cells (CD4) transgress the blood-retinal barrier and enter the

inner eye where they destroy the normal tissue structures. However, the molecular

targets as well as the destructive mechanisms and factors triggering the relapses have

remained largely unknown. For elucidating molecular pathomechanisms of the

disease, we use the only available spontaneous animal model of this disease: the horse.

Horses develop equine recurrent uveitis (ERU) with a very high incidence and the

disease progression closely resembles the human form since it also occurs in remitting-

relapsing episodes.

Quantitative proteomics methods offer the unique possibility to directly assess changes

in disease tissues related to differential protein expression as well as identifying the

primary target molecules of autoagressive T-cells. Autoantigen identification was

achieved by mass spectrometric identification of proteins which are selectively

recognised by circulating antibodies from ERU horses and revealed CRALBP as a

novel target. CRALBP was then confirmed as disease-causative in two different

animal models (Lewis rats, horses: EAU) and was additionally shown to be an

important auto-antigen in human patients.

In a 2D-gel-based initial quantitative comparison of ERU and healthy retinal tissue, we

found increase of intermediate filaments, GFAP and vimentin, both indicating

reactivity of retinal Müller glial cells. Furthermore, in uveitic retina, Müller cells

expressed IFNg and lost expression of PEDF. However, disease progression is also

accompanied with a break-down of the blood-retinal barrier and consequently serum-

derived proteins mask the potential target tissue-related changes. To overcome this


56

limitation, we recently used membrane-enriched retina fractions prepared from horses

suffering from equine recurrent uveitis (ERU), and compared expression levels by a

label-free LC-MSMS-based strategy to healthy control samples. We could readily

identify a total of 893 equine proteins with 57% attributed to the GO term

“membrane”. Of these, 179 proteins were found differentially expressed in ERU

tissue. Pathway enrichment analyses indicated an increase in proteins related to

antigen processing and presentation, TNF receptor signaling, integrin cell surface

interactions and focal adhesions. Additionally, loss of retina-specific proteins

reflecting decrease of vision was observed as well as an increase in Müller glial cell-

specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1,

integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry

and tissue staining patterns pointed to a significant increase of these proteins at the

level of the outer limiting membrane which is part of the outer blood-retinal barrier.

Taken together, quantitative proteomics, especially the membrane enrichment in

combination with LC-MSMS-based label-free quantification greatly increased the

sensitivity of the comparative tissue profiling and resulted in detection of novel

molecular pathways related to equine recurrent uveitis.

In situ quantification of protein-protein complexes in paraffin-

sections using Proximity Ligation Assay (PLA)-technique Aubele M.

1, Ludyga N.

1, Braselmann H.

2, Feuchtinger A.

1, Schmitt M.

3, Bartlett J.M.

4


2Helmholtz Zentrum München, Department of Radiation Cytogenetics, Neuherberg,

Germany, 3Technische Universität München, Department of Obstetrics and

Gynecology, Munich, Germany, 4University of Edinburgh, Western General Hospital,

Edinburgh, United Kingdom

Nearest neigbor-analysis for the determination of protein-protein complexes are of

high clinical and therapeutical impact in breast cancer. The fairly new method of PLA

(proximity ligation assay technique) allows visualization and quantification of protein-

protein complexes in sections from formalin-fixed, paraffin-embedded (FFPE) human

tumor tissues.


57

We employ this PLA technique on FFPE tissue sections from invasive breast

carcinomas to locate and analyse several different protein-protein complexes.

Examples will be shown for PTK6 (protein tyrosine kinase 6) - HER2, PTK6 - HER3,

for the HER2 - HER3 heterodimers, and for markers of the uPA system like uPA -

uPA-receptor and uPA - PAI-1. Images from the fluorescence labelled PLA-slides are

captered using a confocal laser scanning microscope (AxioImager, Zeiss, Jena,

Germany), and signals from protein complexes are evaluated using the Definiens

Enterprise Image Intelligence Suite software (Definiens, Munich, Germany). Protein

expression levels are then statistically analysed for their association with

histopathological parameters and the clinical follow-up of the disease. As an example,

PTK6 - HER2 complexes showed significant correlation with tumor size, and a low

PTK6 - HER2 signal frequency was associated with better prognosis of patients.

The PLA technique enables accurate visualization, subcellular localization and

quantitative evaluation of protein-protein-complexes in FFPE tissue.

Evaluation of PAXgene-fixed, paraffin-embedded tissues for

morphological and molecular analysis

Gündisch S.1, Schott C.

1, Reischauer B.

1, Meding S.

2, Langer R.

1, Kap M.

3, Viertler

C.4, Ferch U.

5, Riegman P.

3, Zatloukal K.

4, Walch A.

2, Becker K.-F.

1

1Institute of Pathology, Technische Universität München, München, Germany,

2Institute of Pathology, Helmholtz Center Munich, Munich, Germany,

3Department of

Pathology, Josephine Nefkens Institute, Rotterdam, Netherlands, 4Institute of

Pathology, Medical University of Graz, Graz, Austria, 5Third Medical Department,

Technische Universität München, Munich, Germany

Aims: For molecular diagnostics and personalized medicine protein biomarkers need

to be precisely measured in clinical tissue samples. In formalin-fixed and paraffin

embedded (FFPE) tissues protein analysis is still challenging. Within the European

project SPIDIA we evaluated the novel formalin-free tissue fixative “PAXgene Tissue

System” for better integration of morphological and molecular analysis, focussing on

protein approaches.


58

Methods: Different murine and human tissue samples were either snap-frozen, fixed

with the novel tissue fixative, PAXgene tissue fixation and stabilization reagents, or

with formalin before paraffin-embedding. Proteins were analyzed by Coomassie

staining, two-dimensional gel electrophoresis, Western blotting, reverse phase protein

microarrays (RPPA) and matrix-assisted laser desorption/ionization imaging mass

spectrometry (MALDI-IMS). The preservation of the phosphoproteome was

investigated in a large-scale comparative study with 16 different non-malignant and 4

different malignant tissue entities with 11 phosphorylation-specific antibodies by

Western blot. Morphology and immunohistochemistry were evaluated and RNA

quality was assessed by PCR amplification assays.

Results: We were successful in extraction of non-degraded and immunoreactive

proteins from PAXgene-fixed tissue specimens. We analyzed for example E-cadherin,

Hsp70 and beta-actin and phosphorylated proteins, including p-Akt, p-Erk-1/2, and p-

NFkB. Recovered proteins showed very similar properties when compared to

cryopreserved samples by Western blotting or RPPA and were superior to proteins

from FFPE samples. Furthermore, the spectra of MALDI-IMS analysis were similar to

cryopreserved samples which were visualized by insulin and glucagon expression in

pancreatic tissue. Finally, morphology was comparable to FFPE samples whereas

RNA was far better preserved in PAXgene-fixed samples.

Conclusion: The PAXgene Tissue System preserves not only the proteome and most

importantly the phosphoproteome of a tissue sample but as well morphology and

nucleic acids. Thus it has great potential to serve as a novel multimodal fixative for

modern pathology, enabling widespread biomarker studies on clinical tissue samples.


59

Building the MALDI Imaging Protein Biomarker List from the

Bottom Up Maier S.

1, Balluff B.

2, Elsner M.

2, Englert S.

2, Meding S.

2, Schöne C.

2, Walch A.

2,

Küster B.3

Institute(s):

1Technische Universität München, Department of Proteomics and Bioanalytics,

Freising, Germany, 2Helmholtz Zentrum München, Institute of Pathology, Neuherberg,

Germany, 3Technische Universität München, Department of Proteomics and

Bioanalytics, München, Germany

Introduction: MALDI imaging-MS allows merging morphological information with

the distribution of many different molecular features in-situ. In MALDI imaging

studies of proteins, many m/z values of candidate biomarkers remain unidentified but

the identification of the underlying proteins is mandatory as a starting point for further

biological and clinical assessment of these biomarkers. Here we used a bottom-up

approach to identify proteins from sinapinic acid coated tissue sections using trypsin

digestion and tandem mass spectrometry. While this bottom up strategy breaks the link

between the identified proteins and the m/z values of the intact species in the tissue,

the list of identified proteins serves as the pool of proteins from which all potential

biomarkers must be derived.

Methods: MALDI IMS samples were prepared from 10 different fresh frozen tissues.

The sinapinic acid layer was extracted first by 7.5% and second by 60% acetonitrile in

0.1% TFA. The remaining tissue slice was excised from the glass slide. All three

samples were, separated by SDS-PAGE, digested with trypsin, analyzed by LC-

MS/MS and proteins identified by sequence database searching.

Results: The rationale of our bottom up analysis of tissue sections is as follows: Only

proteins that are in the tissue can be embedded into the MALDI matrix layer and only

those proteins that are embedded into the layer can be ionized and detected in the

MALDI MS. Therefore, the bottom up list of proteins must necessarily represent the

pool of all potential protein markers from that tissue. From a single tissue, we

identified 2,100 proteins on average 400 of which were found in the low acetonitrile

and 700 in the high acetonitrile matrix extracts. Across the 10 analyzed tissues, 3738


60

non redundant proteins were identified. With very few exceptions, the proteins

identified in the matrix extracts were almost always also identified in the total tissue

extract. Small proteins are enriched in the matrix layer, whereas structural proteins are

underrepresented. Most of the successfully identified m/z-species from the literature

are observed in our dataset with a high frequency. This shows the validity of this

approach and therefore focuses the number of possible markers down to a few hundred

that have to be considered further. Identification of the matrix proteome can also be

used to reduce the database search space for protein identification. Taken together, our

data shows that the task of identifying putative protein biomarkers from MALDI

imaging data can be greatly simplified and also lead to more confidence in the results

of targeted top- or middle down identification approaches.

Gain of chromosome band 7q11 in papillary thyroid carcinomas of

young patients is associated with exposure to low-dose irradiation Heß J.

1, Braselmann H.

1, Bogdanova T.

2, Thomas G.

3, Zitzelsberger H.

1, Unger K.

1,3

1Helmholtz Zentrum München, Research Unit of Radiation Cytogenetics, Neuherberg,

Germany, 2Institute of Endocrinology and Metabolism, Kiev, Ukraine,

3Imperial

College London, Human Cancer Studies Group, Department of Surgery and Cancer,

London, United Kingdom

The main consequence of the Chernobyl accident has been an increase in childhood

papillary thyroid carcinomas (PTC) in the contaminated areas. Although RET/PTC

rearrangements are prevalent in PTC it has been shown in previous studies that this

rearrangement does not reflect exposure to radiation. Consequently, this study aimed

to identify genomic radiation biomarkers.

We analysed a main (n=52) and a validation cohort (n=28) of PTC from patients that

were young at the time of exposure (median 1.5 years). DNA and RNA from the

tumours were provided by the Chernobyl Tissue Bank

(www.chernobyltissuebank.com). Both cohorts were matched for age at diagnosis, sex

and residence, and consisted of patients exposed (born before the Chernobyl reactor

accident) and not exposed (control group, born at least one year after the accident) to


61

radioiodine fallout. Array CGH was performed to detect copy number alterations in

the tumours.

We found that gain of the chromosome band 7q11.22-11.23 was associated

(FDR=0.035) with exposure to radioiodine fallout. 39% from the exposed group and

none from the unexposed group showed the alteration. This finding was confirmed in

the validation set.

Since only a subgroup of cases in the exposed group exclusively showed gain of

chromosome band 7q11, it is likely that different molecular subgroups and routes of

radiation-induced carcinogenesis exist. The mRNA expression of the genes PMS2L11,

PMS2L3, and STAG3L3 correlated with gain of 7q11.22-11.23. The candidate gene

CLIP2 was specifically overexpressed in the exposed cases at the mRNA and protein

level (IHC). Candidate genes (n=56) from the gained region showed enrichment of GO

(Gene Ontology) terms associated with “DNA repair” (PMS2L3, PMS2L5), “response

to DNA damage stimulus” (BAZ1B, PMS2L3, PMS2L5, RFC2), and “cell-cell

adhesion” (CLDN3, CLDN4).

This study that shows a genomic radiation marker provides novel insights into the

radiation-related carcinogenesis of young onset PTC.


62

MRI molecular imaging with targeted albumin-based

nanoparticles: conceptual design strategies to create the Magic

Bullet Thurner G.C.

1, Wallnöfer E.A.

1, Rohr I.

2, Talasz H.

3, Kremser C.

1, Abdelmoez A.A.

2,4,


5, Matuszczak B.

6, Jaschke W.

1, Debbage P.

2


2Medical

University Innsbruck, Department of Anatomy, Histology & Embryology, Innsbruck,


Innsbruck, Austria, 4Assiut University, Department of Pharmaceutical Organic

Chemistry, Assiut, Egypt, 5Medical University Innsbruck, Central Animal Research

Facility, Innsbruck, Austria, 6University of Innsbruck, Institute of Pharmaceutical

Chemistry, Innsbruck, Austria

Introduction: Paul Ehrlich in 1900 [1] described the idea of a targeted vehicle

carrying a drug. His notion of a „Magic Bullet“ changed the whole concept of medical

treatment. Richard Feynmann in 1959 [2] challenged scientists to create

nanomachines. Since then Nanomedicine has promised improved drug delivery,

reduced dosages and side-effects: "personalized medicine". Why has Nanomedicine

not delivered on this promise? Two major challenges hinder realisation of this dream:

1. identification of suitable biomarkers; 2. poor availability of nanoparticles capable of

homing to biomarker molecules hiding behind intact tissue barriers. We review the

reasons for these difficulties and suggest some approaches to overcome them.

Materials and methods: Albumin-based nanoparticles bearing gadolinium were

developed and extensively characterised. The lectin LEA was attached to the particles

to target oligolactosamines. During MR Imaging of living rats both imaging and

quantitative approaches were applied. Coordinated with the MRI, chemical and

immunohistochemical analyses tracked components of the nanoparticles in

longitudinal time series after injection, 15 minutes to 6 weeks, obtaining large runs of

quantitative data.

Results: Our nanoparticles were ~30 nm diameter. They were pure, contained no

starting materials and had good imaging properties with relaxivities ~1 • 107 1/Ms.

They were stable in various in vitro testings though not in SDS-gel-electrophoresis. In


63

haemagglutination tests they agglutinated red blood cells; after intravenous injection

into living rats they gave high-resolution MR imaging of the vascular wall lasting >2

hours. The numbers of (lectin) targeting groups required for molecular imaging and of

gadolinium ions per nanoparticle necessary for high-resolution MRI were assayed in

relation to particle size and type of crosslinking.

Discussion: We determined critical parameters for MR Molecular Imaging by use of

nanoparticles. These particles require a second type of targeting group to migrate

across vascular walls and access subendothelial interstitial compartments, for

Molecular Imaging and Molecular Targeting of disease sites behind intact tissue

barriers. The concept of multiple targeting is new in Nanomedicine and represents the

hurdle that limits present-day techniques [3]. Assuming that quantitative aspects of

targeting will be similar for each of the multiple targeting groups, we already know

how to design nanoparticles for targeting both drugs and contrast agents to disease

lesions hiding behind blood-tissue barriers. Multiply-targeted nanoparticles are

Ehrlich´s "magic bullets".

Acknowledgements: Austrian Nano-Initiative (Project N201-NAN); Austrian

National Bank Jubilee Program (Projects 9273, 10844, 11574)

References:

1. Ehrlich, P. (1900) Proc R Soc Lond 66: 424 - 448.

2. Feynman RP. (1959) Miniaturization. Horace D. Gilbert, Ed. © Van Nostrand

Reinhold. New York

3. Debbage P., Thurner GC. (2010) Pharmaceuticals 3: 332 – 3416


64

MALDI MSI for ovarian cancer biomarkers research: latest

developments of the technology for screening and tracking. Longuespée R.

1,2, Boyon C.

1,3, Kerdraon O.

4, Desmons A.

1, Vinatier D.

3, Fournier I.

1,

Day R.2, Salzet M.

1

1Laboratoire de Spectrométrie de Masse Biologique Fondamentale et

Appliquée/Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France,

2Institut de Pharmacologie de Sherbrooke/Université de Sherbrooke, Sherbrooke,

Canada, 3Service de Chirurgie Gynécologique/Hôpital Jeanne De Flandre/CHRU

Lille, Lille, France, 4Centre d'Anatomie et de Cytologie Pathologiques/CHRU Lille,

Lille, France

Ovarian cancer is the second major cause of gynecological death in Europe and USA.

Ovarian cancer has been studied at a proteomic level for biomarkers hunting with the

emergence of diverse techniques such as 2D electrophoresis and SELDI TOF MS. At

this time, CA125 is still the only biomarker used for the monitoring of the pathology.

Its sensitivity is 80% for stage III and stage IV cancers but only 30% for early stages

with high rates of false positives. Since its introduction in the early 90's, MALDI

Imaging Mass Spectrometry (IMS) has widely been used for biomarkers discovery in

many pathologies thanks to the possibility to discriminate the localization of

compounds in tissues of interest. Using this technology, we found the Cter part of

PA28, “Reg Alpha” present in the cancerous regions of ovarian biopsies. Recently, we

found this peptide in stage I epithelial cancer tissues, making it a potential marker for

wide screenings among women populations. Then, our recent developments in the

field of IMS allowed us to associate Histo Immuno Chemistry and IMS for the specific

tracking of this molecule in cancerous tissues. We indeed propose to incubate tissues

of interest with known anti-markers antibodies, then digest the surface of the tissues

with a solution of trypsin and finally perform the MALDI Image of the prepared

tissue, to reveal the presence of the tryptic peptides of the antibodies. It's now possible

to proceed to the quantitative detection of markers by directly analyzing the presence

of tryptic peptides of the antibodies targeting Reg Alpha. This procedure allows the

use of Immuno Histo Chemistry with the Mass Spectrometric devices as revealers

instead of a microscope, and would be strongly relevant for the tracking of cytotoxic


65

antibodies in tumors. These developments also pave the way for new pharmacologic

applications such as the monitoring of the effect of treatments of the pathology by

screening the evolution of the presence of the compound in malignant tissues.

Posters

66

Poster

General Information

The poster session will be on Thursday at 14:20. The poster exhibition will take place

at the Frauenklinik Maistraße, lecture hall 1+2. Every poster board carries the poster

number given in the program on the upper left corner. Please refer to the program for

your poster number and ask at the registration desk for the exact placement of your

poster board. The material for mounting the posters will be supplied by the local

organizers. The posters should be mounted until Saturday. Please note that posters

which have not removed at the end of the meeting cannot be returned.

The presenting authors should be at their poster during the poster session on Thursday

for personal presentation.

Poster Prizes of the Society for Histochemistry

Three best student posters will be awarded during closing of the symposium with

100 € (conditions: First author, PhD student).

Poster Index

01 Balluff B. MALDI imaging identifies prognostic seven-protein

signature of novel tissue markers in intestinal-type

gastric cancer

02 Balluff B. Classification of HER2/neu status in gastric cancer using

a breast-cancer derived proteome classifier

03 Claude E. The advantages of coupling high efficiency ion mobility

separation with MALDI MS imaging of on-tissue

digested proteins

04 Crecelius A.C. Combining lipidomics and proteomics by MALDI-MSI

05 Deininger S.O. Normalization in MALDI-TOF imaging of proteins

Posters

67

06 Enthaler B. MALDI imaging of endogenous compounds directly in

human skin-tissue sections

07 Gerbig S. Desorption Electrospray Ionization (DESI) imaging of

tumor samples - using mass spectrometry to support

classical histology

08 Kobarg J.H. Spatial segmentation of hyperspectral 3D volume data

09 Krásný L. pHPMA-based tissue embedding medium compatible

with MALDI mass spectrometry imaging experiments

10 Longuespée R. MALDI MSI for ovarian cancer biomarkers research:

latest developments of the technology for screening and

tracking.

11 Meding S. Proteomic markers for regional lymph node metastasis in

primary colon cancer tissues

12 Nipp M. S100-A10, Thioredoxin, and S100-A6 as biomarkers of

papillary thyroid carcinoma with lymph node metastasis

identified by MALDI Imaging

13 Oetjen J. The MALDI-imaging/ MULTI-ARRAY core facility: A

new service platform at the University of Bremen

14 Schöne C. MALDI Imaging as a novel approach for the

systematical study of intratumoral heterogeneity

15 Strohalm M. In-tissue lipid profiling by mass spectrometry imaging

16 Suckau D. Spatial proteomics: A new LC-MS/MS tissue imaging

workflow providing protein identities and their

distribution in tissue

17 Trede D. Software for 3D MALDI imaging data: Visualization and

3D spatial segmentation

18 Wurlitzer M. MALDI-Imaging of frozen prostate tissue micro arrays

Posters

68

19 Acar N. The evaluation of expressions of FKBP52 and PRDX6

proteins and ultrastructure of wild type and Fkbp52

knockout mice uterus during pregnancy

20 Aunapuu M. Analysis of endometrial receptivity based on

immunohistochemical study

21 Bauer L. Differential expression of stem cell related genes in

neoadjuvant treated gastric cancer.

22 Bonin S. Cell mobility and metastatic spreading: a study on

human breast cancer cells using the nano-mechanical

approach

23 Brüning A. The HIV reverse transcriptase inhibitor efavirenz

selectively kills human cancer cells

24 Buchwalow I. Non-specific binding of antibodies in immunoassays:

fakes and facts

25 Buchwalow I. Signal amplification in immunoassays: heteropolymeric

HRP conjugates vs. polymer backbone conjugates

26 Csoka L. Polarization optical and histochemical characterization

and supramolecular structure of carbohydrate-protein

fibrils

27 Ebach K. Immunohistochemical investigations of keratin

expression for a characterization of epidermal stem cell

niches in bovine fetal skin

28 Emody L. Topo-optical follow up of phenothiazine induced charge

transfer reactions in the yeast cell wall

29 Epis S. The intramitochondrial bacterium Midichloria: novel

tools for its detection

30 Giagnacovo M. Ribonucleoprotein-containing foci increase in size upon

cell-cycle exit in cell nuclei from patients affected by

myotonic dystrophy type 2

Posters

69

31 Han K.-H. Renal ischemia-reperfusion injury causes intercalated

cell-specific disruption of occludin in the collecting duct

32 Heindl S. E-cadherin mutations contribute to gastric carcinogenesis

by modulating multiple EGFR-dependent downstream

signalling pathways

33 Houtkamp M.A. Immunohistochemical detection and non-invasive U-

SPECT imaging reveal distinct tumor tissue distribution

patterns of anti-EGFr antibody zalutumumab in a tumor

xenograft model

34 Kaneko T. Immune laser capture microdissection of macrophages in

engineered dental pulp tissues

35 Klappan A. Autophagosome formation by quercetin

36 Lee H.J. Cordycepin induces double strand breaks and the DNA

damage response in breast cancer cells

37 Luber B. Biomarker analysis of cetuximab plus

oxaliplatin/leucovorin/5-fluorouracil in first-line

metastatic gastric and oesophago-gastric junction cancer:

results from a phase II trial of the Arbeitsgemeinschaft

Internistische Onkologie (AIO) v

38 Makovitzky J. The topo-optical investigation of Alzheimer Amyloid

39 Makovitzky J. Late gastrointestinal metastases of invasive lobular

breast carcinoma mimicking Crohn´s disease

40 Markelic M. Role of macrophages in brown adipose tissue remodeling

in hyperinsulinemic rats

41 Matsingou C. Selective Induction of Inhibin beta E by the ER stress

reaction

42 Rodler D. Glycohistochemical characterization of the avian inner

perivitelline layer

43 Salovska J. Thyroid gland-commonness of hyperthyreodism and

hypothyreodism in the female and male population

Posters

70

44 Sinowatz F. Immunohistochemical localization of transit cells,

putative stem cells and stem cells in benign prostate

hyperplasia (BPH) and adenocarcinoma of the prostate

45 Stoemmer P. Cell adhesion molecules in melanocytic tumors

46 Strelkova O.S. Nuclear lamina in organization of DNA replication.

47 Thurner G.C. MRI molecular imaging with targeted albumin-based

nanoparticles: conceptual design strategies to create the

Magic Bullet

48 Timme S. EGFR, HER2 and HER3 expression and dimerization in

esophageal cancer

49 Ustunel I. Zonal distribution of CD105+/CD166+ cells in growing

rat humerus proximal epiphyseal cartilage

50 Weber E. Is CRIP1 a target gene of HER2?

51 Weipoltshammer K. Class I HDACs in the developing heart and limb of

chicken embryos

Abstracts of Poster Presentation

71


The evaluation of expressions of FKBP52 and PRDX6 proteins and ultrastructure of wild type and Fkbp52 knockout mice uterus

during pregnancy Acar N.

1, Hirota Y.

2, Daikoku T.

2, Dey S.K.

2, Ustunel I.

1

1Akdeniz University, School of Medicine, Histology and Embryology, Antalya, Turkey,

2University of Cincinnati College of Medicine Cincinnati Children's Hospital, Division

of Reproductive Sciences, Cincinnati, United States

Implantation is the nidation of blastocyst into mother's uterus. It starts on day 4 night.

An immunophilin FKBP52 (FK506 binding protein) serves as a cochaperone to govern

normal progesterone (P4)-P4 receptor (PR) signaling in mouse uterus. Previous studies

reported that Fkbp52 knock out (KO) females with reduced P4-PR signalling have

implantation failure, which is rescued by P4 supplementation in genetic background

and pregnant stage dependent manners (Tranguch S et al, J Clin Invest, 117 (2007)

1824; Tranguch S et al, Proc Nat Acad Sci USA, 102 (2005) 14326).

Our previous study showed that peroxiredoxin6 (PRDX6) is downregulated in Fkbp52

KO uterus. PRDX6 is an antioxidant molecule. Therefore we aimed to determine the

location and expression intensities of FKBP52 and PRDX6 proteins and to investigate

the ultrastructural characteristics of wild type and Fkbp52 KO mice uterus on certain

days of pregnancy.

For our purpose one male and two female mice were retained together in the same

cage overnight and next morning female mice with vaginal plug were accepted on day

1 of pregnancy. Wild type mice were sacrified on days 1,4,5,8 and 14 of pregnancy.

Fkbp52 KO mice were injected P4 until their sacrificatin on days 4,5,8 and 14. Uterus

horns or implantation sites were removed, processed for immunohistochemistry,

immunoflorescence and electron microscopic evaluation.

On days 1,4,5 of pregnancy in control group PRDX6 expression was observed

especially in apical side of lumen epithelium of endometrium, gland epithelium and in

stroma. On day 8 of pregnancy PRDX6 was expressed in antimesometrium and

primary decidual zone. On day 14 of pregnancy PRDX6 was expressed in labyrinth

and especially spongiotrophoblast of placenta and expression was weaker compared to


72

previous days. FKBP52 expression was colocalised with PRDX6 in the cytoplasm of

lumen epithelium on day 1; in cytoplasm of gland epithelium on day 4; in the stroma

of endometrium on day 5; in antimesometrium of uterus and primary decidual zone on

day 8; in labyrinth of placenta on day 14. On fourth day of pregnancy uterus

morphology of Fkbp52 KO mice without P4 injection was abnormal. PRDX6

expression was not continuous through the apical side of lumen epithelium. On the

same day in P4 injected Fkbp52 KO group uterus morphology was similar to control

group and PRDX6 expression was all through the apical side of lumen epithelium.

PRDX6 expression of Fkbp52 KO mice with P4 injection was similar to wild type

mice on other studied days

Electron microscopic observations done on day 4 of pregnancy showed although there

were differences between wild type and Fkbp52 KO mice without P4 injection;

ultrastructure of P4 injected Fkbp52 KO mice was similar to wild type mice.

By means of P4 injection lumen epithelium and stromal cells of Fkbp52 KO mice

become similar to wild type so implantation is succeeded. In addition to its

cochaperone function FKBP52 may cooperate with PRDX6 to overcome oxidative

stres.

Analysis of endometrial receptivity based on immunohistochemical

study Aunapuu M.

1, Arend A.

2

1University of Tartu, Anatomy, Tartu, Estonia,

2University of North CarolinaTartu,

Anatomy, Tartu, Estonia

Introduction: Infertility is a condition that affects a couple and is defined as the lack

of conception after an arbitrary period of 12 months with regular sexual intercourse

and without using any contraception. Infertility is a common medical problem present

in about 10% of couples in reproductive age. Tubal factor infertility (TFI) is one of the

leading causes of infertility in the world including Estonia. The most common method

treating infertility due to TFI is In Vitro Fertilization (IVF). In the IVF-procedure

oocytes are fertilized in in vitro conditions followed by incubation for 2-5 days.

Finally 1 to 3 embryos will be transplanted into the uterus. Occurrence of TFI


73

decreases receptivity of the uterine mucosa by lowering the expression of ανβ3

integrins, trophinin on the endometrial epithelium, which reduces the possibility of

implantation.

Materials and methods: Endometrial biopsies of patients with infertility problems were

carried out in Nova Vita Clinic (Tallinn, Estonia). Patients, who have had several

unsuccessful IVF procedures, were divided into two groups: Group I - younger than 34

years and Group II - older than 35 years. Material for light microscopy and

immunohistochemistry (IHC) were fixed in 10% buffered formalin solution and

embedded in paraffin with a vacuum processor Tissue-Tek®

VIPTM

5 Jr (Sakura,

USA). Specimens were cut with microtome Ergostar HM 200 (Microm, Germany) at

three-mm thickness and stained using H&E and van Gieson methods for general

orientation to sections. Histological methods were used to specify the stage of

menstrual cycle. IHC staining was used to detect integrin beta3 (CD61) and trophinin

expression in the endometrium. Slides were observed and ptohographed by a Zeiss

Axiophot 2 microscope (Zeiss, Germany).

Results: Light microscopy studies of biopsies revealed no pathologic changes in

endometric morphology. The ratio between glandular and stromal components varied

from 1:1 to 2:1. IHC staining with trophinin showed a weak reaction (grade 2) in two

patient's luminal epithelium and minimal (grade 1) in 4 patient's luminal epithelium

and two patient`s glandular epithelium. IHC staining with CD61 showed a very intense

reaction only in one patient biopsy. This result showed decreased receptivity of the

uterine mucosa by lowering the expression of β3 integrins on the endometrial

epithelium.

Conclusion: Although there are well characterised morphological and molecular

markers of implantation, the complete dynamics of the process as well as the

importance of each and every marker is still vague. Morphological studies showed that

TFI patients have decreased expression of β3 integrins and trophinin.

This research was supported by Estonian Science Foundation grant No. 7301.


74

MALDI imaging identifies prognostic seven-protein signature of

novel tissue markers in intestinal-type gastric cancer Balluff B.

1,2, Rauser S.

1, Meding S.

1, Elsner M.

1, Schöne C.

1, Feuchtinger A.

1,

Schuhmacher C.3, Novotny A.

3, Jütting U.

4, Maccarrone G.

5, Sarioglu H.

6, Ueffing

M.6, Braselmann H.

7, Zitzelsberger H.

7, Schmid R.M.

2, Höfler H.

1,8, Ebert M.P.

9,

Walch A.1


2Klinikum rechts der Isar der TU München, 2. Medizinische Klinik und Poliklinik,

München, Germany, 3Klinikum rechts der Isar der TU München, Department of

Surgery, München, Germany, 4Helmholtz Zentrum München, Institute of

Biomathematics and Biometry, Neuherberg, Germany, 5Max Planck Institute of

Psychiatry, Department of Proteomics, München, Germany, 6Helmholtz Zentrum

München, Department of Protein Science, Neuherberg, Germany, 7Helmholtz Zentrum

München, Department of Radiation Cytogenetics, Neuherberg, Germany, 8Technische

Universität München, Institute of Pathology, München, Germany,

9Universitätsklinikum Mannheim, Department of Medicine II, Mannheim, Germany

Proteomics-based approaches allow us to investigate the biology of cancer beyond

genomic initiatives. We used histology-based matrix-assisted laser

desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that

predict disease outcome in gastric cancer after surgical resection.

A total of 181 intestinal-type primary resected gastric cancer tissues from two

independent patient cohorts were analyzed. Protein profiles of the discovery cohort

(n=63) were directly obtained from tumor tissue sections by MALDI imaging. A

seven-protein signature was found to be associated with an unfavorable overall

survival independent of major clinical covariates (HR=4.03; 95% CI: 1.69 - 9.61;

P=0.002). The prognostic significance of three individual proteins identified (CRIP1,

HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of

an independent validation cohort (n=118). While HNP-1 and S100-A6 were found to

further subdivide early (UICC-I) and late stage (UICC-II-III) patients into different

prognostic groups (P=0.024, P=0.013), CRIP1, a protein previously unknown in


75

gastric cancer, was confirmed as a novel and independent prognostic factor for all

patients in the validation cohort (HR=1.57; 95% CI: 1.01-2.44; P=0.044).

The protein pattern described here serves as a new independent indicator of patient

survival complementing the previously known clinical parameters in terms of

prognostic relevance. These results show that this tissue-based proteomic approach

may provide clinically relevant information that might be beneficial in improving risk

stratification for gastric cancer patients.

Classification of HER2/neu status in gastric cancer using a breast-cancer derived proteome classifier Balluff B.

1,2, Elsner M.

2, Kowarsch A.

3, Rauser S.

2, Meding S.

2, Schuhmacher C.

4,

Feith M.4, Herrmann K.

5, Röcken C.

6, Schmid R.M.

1, Höfler H.

2,7, Walch A.

2, Ebert

M.P.8

1Klinikum rechts der Isar der TU München, 2. Medizinische Klinik und Poliklinik,

München, Germany, 2Helmholtz Zentrum München, Institute of Pathology,

Neuherberg, Germany, 3Helmholtz Zentrum München, Institute of Bioinformatics and

Systems Biology, Neuherberg, Germany, 4Klinikum rechts der Isar der TU München,

Department of Surgery, München, Germany, 5Klinikum rechts der Isar der TU

München, Department of Nuclear Medicine, München, Germany, 6Christian-

Albrechts-University, Institute of Pathology, Kiel, Germany, 7Technische Universität

München, Institute of Pathology, München, Germany, 8Universitätsklinikum

Mannheim, Department of Medicine II, Mannheim, Germany

Testing for HER2-status (human epidermal growth factor receptor 2) in breast and

gastric cancers is mandatory for the treatment with trastuzumab. We hypothesized that

imaging mass spectrometry (IMS) of breast cancers may be useful for generating a

classifier that may determine HER2-status in other cancer entities irrespective of

primary tumor site.

A total of 107 breast (n=48) and gastric (n=59) cryo tissue samples were analyzed by

IMS (HER2 was present in 29 cases). The obtained proteomic profiles were used to

create HER2 prediction models using different classification algorithms.


76

A breast cancer proteome derived classifier, with HER2 present in 15 cases, correctly

predicted HER2-status in gastric cancers with a sensitivity of 65% and a specificity of

92%. In order to create a universal classifier for HER2-status, breast and non-breast

cancer samples were combined, which increased sensitivity to 78%, specificity was

88% respectively.

Our proof of principle study provides evidence that HER2-status can be identified on a

proteomic level across different cancer types suggesting that HER2 overexpression

may constitute a unique molecular event independent of the tumor site. Furthermore,

these results indicate that IMS may be useful for the determination of potential

drugable targets, as it offers a quicker, cheaper and more objective analysis than the

standard HER-testing procedures immunohistochemistry and fluorescence in situ

hybridization.

Differential expression of stem cell related genes in neoadjuvant treated gastric cancer. Bauer L.

1, Langer R.

1, Becker K.

1, Ott K.

2, Novotny A.

3, Hapfelmeier A.

4, Höfler H.

1,5,

Keller G.1

1Institut für Pathologie der TU München, München, Germany,

2Cirurgische Klinik der

Universität Heidelberg, Heidelberg, Germany, 3Cirurgische Klinik der TU München,

München, Germany, 4Institut für Medizinische Statistik und Epidemiologie der TU

München, München, Germany, 5Institut für Pathologie, Helmholtz Zentrum, München,

Germany

Neoadjuvant treatment of gastric cancer offers the opportunity to investigate residual

tumor cells after chemotherapy (CTX). According to the cancer stem cell (CSC)

hypothesis, there might be an enrichment of cells expressing CSC related genes within

the residual tumor. We analyzed the expression of CSC related genes in residual

gastric tumors for an association with overall survival and compared corresponding

pretherapeutic biopsies and resected tumors for CTX associated expression changes.

An initial screening of 44 genes selected according to their relevance for

differentiation and development or as putative CSC markers was performed. Resected

specimens from 63 gastric cancer patients treated with neoadjuvant platinum/5-FU


77

based CTX that demonstrated a partial response based on histopathological tumor

regression (i.e. 10-50% residual tumor cells) were included. mRNA was isolated from

macrodissected FFPE tissues and gene expression was quantified by real time PCR

using TaqMan®

low density arrays. Data were analyzed for correlation with overall

survival and clinicopathological parameters. Selected genes (n=12) were compared

between corresponding biopsies and resected specimens from patients with partial

(n=22) and minimal/no tumor regression (i.e. >50% residual tumor cells, n=22).

Genes involved in Wnt and notch signaling pathways, such as GSK3B, β-catenin and

Notch2, were among the genes demonstrating a prominent association with overall

survival (p=.006, p=.043 and p=.072, respectively) in the initial screening.

Comparison between biopsies and resected specimens revealed an increase of Notch2

and LGR5 expression in tumors with partial response (p=.002 and .017) and of

POU5F1 in both partial and minimal/non responding tumors (p=.028 and .002).

Notch1 expression was significantly decreased in minimal/non responding tumors

(p=.001) and DNMT1 expression was significantly down regulated in both groups

(p=.009 and .002).

In tumors with partial regression after neoadjuvant CTX, the expression of genes

involved in CSC associated signaling pathways, such as the Wnt and notch pathway,

showed a prognostic significance, which may be used for risk stratification in this

patient group. The comparison of expression levels between corresponding biopsies

and resected specimens revealed gene expression alterations that might be a result of

enrichment of chemotherapy resistant residual tumor cells.

Cell mobility and metastatic spreading: a study on human breast

cancer cells using the nano-mechanical approach Tavano F.

1, Stanta G.

1, Cojoc D.

2, Pinato G.

2, Migliorini E.

1, D'Este E.

1, Bonin S.

1

1University of Trieste, Trieste, Italy,

2CNR-IOM, National Laboratory TASC, Trieste,

Italy

Background: The primary causes of death in cancer patients are local invasion and

metastasis. The common classification of cancers according to tumor stage and grade

is not sufficient to predict the clinical outcome. The mechanism that characterises


78

tumor progression from the primary to the metastatic sites is not completely

unravelled. Metastatization is accompanied by alterations of the cytoskeleton and

membrane structure leading to changes in their biomechanical properties 1. Recently

mechanical tests have demonstrated that tumor cells are softer than their normal

counterpart 2.

Methods: In this study we analyzed, by means of Optical Tweezers (OT) 3,4

and

atomic force microscopy (AFM), the mechanical properties of two different breast

carcinoma cell lines corresponding to different metastatic potential (MDA-MB231 and

MCF7). Then we compared this value with those obtained by the analysis of a breast

line, assembling the normal breast tissue, HBL-100 (mamma lactans). The data gained

by OT and AFM were compared with the expression of proteins obtained by Western

Blot (WB). As for mechanical data we also determined the Young's Modulus for the

Integrin-Fibronectin bound by AFM. The proteins we analyzed by WB were involved

in epithelial-mesenchimal transition (Vimentin), in cell adhesion (Integrinαvβ3,

Viculin and E-Cadherin) and in breast cancer differentiation (Cytokeratin 8).

Experimental data were also implemented by the confocal images of the Actin

cytoskeleton obtained with the Actin Cytoskeleton and Focal Adhesion Staining Kit

(Millipore).

Results: The rigidity of the membrane and the tether stiffness obtained by OT were

significantly different among the analysed breast cell lines (p=0.01 and p=0.02

respectively), displaying higher values for the not-cancerous HBL-100. The Young's

modulus also significantly differed among the breast cell lines (p=0.000). Regarding

the MCF-7, associated to a low metastatic potential, and the MDA-MB 231, poorly

differentiated with a high metastatic potential, they showed different tether stiffness

with a value four times lower for the MDA-MB231. The Actin cytoskeleton resulted

highly organized in HBL-100 and MCF7 in comparison to MDA-MB231. The

mechanical data were confirmed by WB analyses, which demonstrated that the

molecules associated with poorly outcome in breast Ca, such as Vimentin, were

positive in MDA-MB231, but not in MCF7. On the other hand E-Cadherin was

positive in MCF-7 but not in MDA-MB231.


79

Conclusions: Our results seems to confirm the hypotesis that metastasis prone cells

are softer than less aggressive cancer cells, and support the use of OT for these

measurements for its sub-pN force resolution and because cells are manipulated

without damage.

References:

1 Suresh S. Acta biomaterialia 2007; 3: 413-38.

2 Suresh S. Nature nanotechnology 2007; 2: 748-9.

3 Tavano F, Bonin S, Pinato G et al. Int. J. Optomechatronics In Press.

4 Schmitz J, Benoit M, Gottschalk KE. Biophysical J 2008; 95: 1448-59.

The HIV reverse transcriptase inhibitor efavirenz selectively kills

human cancer cells Brüning A.

1, Burger P.

1, Vogel M.

1, Kost B.

1, Burges A.

1, Friese K.

1

1University Hospital Munich, OB/GYN, Munich, Germany

Aims: Several HIV drugs have recently been described to exert anti-tumour effects.

We have investigated the effect of efavirenz on the cell survival of human cancer cells.

Methods: A variety of human cancer cell types, including breast cancer, ovarian

cancer, and leukemia cells were analysed by MTT assays, clonogenic assays and

FACScan analysis for the occurrence of cell cycle arrest and apoptosis. Immunoblot

analyses were performed to investigate the effect of efavirenz on intracellular

signalling pathways.

Results: Efavirenz selectively induces cell death in human cancer cells, but not in non-

malignant human epithelial cells or fibroblasts. Efavirenz-induced cell death was

associated with the induction of apoptosis, as revealed by caspase activation and p53

phosphorylation. In breast cancer cells, the cytotoxic effect of efavirenz could be

enhanced by tamoxifen.

Conclusions: Efavirenz, although a non-nucleosidic reverse transcriptase inhibitor,

selectively induces apoptosis in human cancer cells with little effects on

untransformed human cells, and could thus be of high interest for cancer treatment

purposes.


80

Non-specific binding of antibodies in immunoassays: fakes and

facts Buchwalow I.

1, Samoilova V.

1, Boecker W.

1, Gergs U.

2, Mohamed S.A.

3, Tiemann

M.1

1Institut für Hämatopathologie, Hamburg, Germany,

2Martin-Luther-Universität

Halle-Wittenberg, Institut für Pharmakologie und Toxikologie, Halle (Saale),

Germany, 3Universitätsklinikum, Schleswig-Holstein, Klinik für Herzchirurgie,

Lübeck, Germany

The current protocols for blocking background staining in immunohistochemistry are

based on conflicting reports. Background staining is thought to occur as a result of

either non-specific antibody (Ab) binding to endogenous Fc receptors (FcRs) or a

combination of ionic and hydrophobic interactions. This concept has been mentioned

in all publications regarding immunohistochemistry since its inception half a century

ago, but we have been unable to find the original source of the idea. This prompted us

to explore whether commercially available Abs have a propensity for random non-

specific binding in the immunolabelling of routinely fixed cell and tissue samples.

In this study, cell and tissue samples were processed according to routine protocols

either with or without a blocking step (goat serum or BSA). In view of the fact that

FcRs are expressed primarily on monocytes, macrophages, B cells, dendritic cells,

neutrophils and platelets, we paid special attention to cell and tissue samples where

these cells are abundant, specifically bone marrow, spleen, tonsils and blood cell

smears. Surprisingly, no Abs in samples processed without a blocking step showed any

propensity for non-specific binding leading to background staining, implying that

endogenous FcRs do not retain their ability to bind the Fc portion of Abs after standard

fixation. Likewise, we did not find any non-specific Ab binding ascribable to either

ionic or hydrophobic interactions. After performing immunostaining using

fluorophore-conjugated Abs, we also found that the omission of the protein blocking

step did not lead to non-specific background staining in single or multiple fluorescence

immunolabelling with the use of either fluorophore-conjugated Ab or streptavidin.

This was a clinically oriented study focused on human patient tissue samples.

However, many researchers perform immunohistochemistry on tissues from


81

experimental animals, particularly rodent tissues. Similar to immunostaining of human

cell and tissue samples, omission of the protein blocking step did not lead to unwanted

background staining in histological samples from experimental and farmed animals.

In contrast to the commonly accepted view, we found that the protein blocking step

traditionally used in immunohistochemistry is unnecessary in the immunostaining of

routinely fixed cell and tissue samples taken both from human patients and from

nonhuman species. Omission of the traditional protein blocking step may save

substantial reagent costs and preparation time both in research and

immunohistopathology.

Signal amplification in immunoassays: heteropolymeric HRP

conjugates vs. polymer backbone conjugates Buchwalow I.

1, Samoilova V.

1, Boecker W.

1, Wolf E.

1, Tiemann M.

1

1Institut für Hämatopathologie, Hamburg, Germany

The peroxidase-labeled antibody (Ab) method, introduced in 1968, was the first

practical application of Abs to paraffin-embedded tissues for most clinical and

research studies (Nakane, 1968). During the past few decades, improvements in the

reagents and protocols used for immunohistopathology have led to increased

sensitivity of detection systems. A significant level of signal amplification was

achieved by the peroxidase-antiperoxidase complex system (PAP). This technique uses

large complexes which contain many detection agents bound together. The binding of

one of these complexes to each secondary antibody amplifies the signal manyfold. For

many years, the PAP procedure represented the most sensitive and hence most popular

techniques in many pathology laboratories. However, today these techniques are only

rarely used being substituted by modern more sensitive methods - heteropolymeric

HRP conjugates and polymer backbone conjugates.

In this study, we compared the level of signal amplification achieved with the use of

heteropolymeric AmpliStain™-HRP conjugate (SDT GmbH) and EnVision™+-HRP

backbone polymer conjugate (DAKO Corporation). Primary Abs were applied for 1h

at room temperature in series of several dilutions of twofold increments. Detection of

bound primary Abs with AmpliStain™ and EnVision™+ was performed pairwise on


82

two immediately adjacent sections for each Ab dilution. Chromogen development after

application of the polymeric HRP conjugates was monitored under microscopic

control until reaching the optimal staining and stopped simultaneously for the entire

series of primary Ab dilutions.

Immunohistochemical staining with various anti-mouse and anti-rabbit primary Abs

revealed a significantly higher level of signal amplification with the use of AmpliStain

for detection of bound primary Abs. In order to quantify the immunostaining

efficiency of AmpliStain™ and EnVision™+, we run ELISA tests for specific Rabbit

and Mouse IgG Abs. Quantitative ELISA readout data show that, compared with

EnVision, anti-Mouse AmpliStain enables at least three times more sensitive detection

of mouse Abs, whereas anti-Rabbit AmpliStain is on the average ten times stronger

than anti-Rabbit EnVision.

Our results show that the AmpliStain™ HRP system is a more sensitive method that

allows higher dilutions of the primary Abs. Based on the German market pricing, price

per test of the EnVision™+ system is markedly higher than that of the AmpliStain™

system. Moreover, the aggregate cost per test with AmpliStain™ HRP can be further

reduced thanks to the much higher dilution of the primary Abs. Apart of allowing

cheaper immunohistochemical assays, higher dilutions of the primary Abs with

AmpliStain™ HRP system also enable obtaining more reliable results since higher Ab

dilutions prevent unwanted background staining.

The advantages of coupling high efficiency ion mobility separation with MALDI MS imaging of on-tissue digested proteins Claude E.

1, Langridge J.

1

1Waters Corporation, Manchester, United Kingdom

Aims: The main advantages of MALDI imaging mass spectrometry (MSI) introduced

in 1997 of by Caprioli et al are the unnecessecity to label the molecules (i.e. proteins)

to image them but also the ability to image multiple molecules within one MS

experiment. The disadvantages of this technique when analyzing proteins is the lack of

identification of the proteins of interest which help with the understanding of the

biological processes. Indeed to identify a protein by MSI, one needs to fragment the


83

protein to get the full or part sequence of the amino acid sequence that constitute the

protein due to the lack of collision energy but also to perform tandem mass

spectrometry, the precursor ion needs to be selected and it becomes problematic after a

certain m/z regardless of the mean of selection. By performing a form of enzymatic

digestion of the proteins (typically trypsin) directly from tissue section, one can have

access to its peptides which have a lower m/z and therefore are more suitable to

MS/MS identification. A means of increasing the separating power of a MALDI

imaging experiment is the use of high efficiency ion mobility separation (IMS),

coupled with time-of-flight mass spectrometry which offers a new dimension of

separation. Using this technique it is possible to separate different compound

classes.However in this study, high efficiency ion mobility separation (IMS) was used

for the analysis of tryptic digestion on-tissue sample as a mean of selectivity to

decongest the MS imaging data but also to differentiate isobaric species.

Methods: The sample under investigation was a 12 µm section of rat brain. The tissue

section was washed with ethanol and chloroform. Trypsin solution was sprayed onto

the tissue section using the SunCollect (SunChrom, Germany) and incubated for 5

hours at 37°. 5 mg/mL of CHCA mixed with aniline solution was subsequently

sprayed onto the tissue using the SunChrom. Data were acquired on a MALDI G2

HDMS system (Waters Corporation, Manchester, UK) operated in HDMS mode over

the m/z range of 700 to 3000 with a laser repetition rate of 1000 Hz. After acquisition

HDMS data were processed and visualised using High Definition Imaging (HDI)

software (Waters Corporation, Manchester, UK) where ion mobility dimension is fully

integrated in the software. Subsequently, MS/MS experiments were carried out on an

adjacent tissue section for identification of the tryptic peptides, directly from tissue.

Results: Here we show how ion mobility separation can be used to provide a

dimension of separation that can be used post ionisation and hence can be utilised in a

MALDI imaging experiment. Using the new HDI software, example of ions images

from tryptic peptides and lipids that have been separated by ion mobility will be

presented.

Furthermore, identification by MS/MS of tryptic peptides directly from tissue will be

demonstrated.


84

Combining lipidomics and proteomics by MALDI-MSI Crecelius A.C.

1,2, von Eggeling F.

3, Schubert U.S.

1,2

1Friedrich-Schiller-University Jena, Laboratory of Organic and Macromolecular

Chemistry (IOMC), Jena, Germany, 2Friedrich-Schiller-University Jena, Jena Center

for Soft Matter (JCSM), Jena, Germany, 3Friedrich-Schiller-University Jena, Institut

für Humangenetik und Anthropologie Universitätsklinikum Jena, Jena, Germany

Introduction: Matrix-assisted laser desorption/ionization mass spectrometric imaging

(MALDI-MSI) is a method that allows the investigation of the molecular content of

tissues within its morphological context.[1] Recently, the use of multiple matrices by

applying inkjet-printing on a single tissue surface has been presented.[2] The aim of

this contribution is to show its useful application by analyzing two different compound

classes, lipids and proteins, in a variety of different tissue organs. This developed

methodology is in particular interesting when precious tissue samples are employed.

Experimental part: Organs, such as liver, brain, and kidney were rapidly extracted

from a C57BL/6J mouse and snap frozen in liquid nitrogen and stored at -80 °C. For

the MALDI-MSI analysis 12 µm thick sections were cut in a microcryotome and thaw

mounted on ITO glass slides. Prior to MALDI-MSI analysis the sections were dried in

a vacuum desiccator for 10 minutes. For the analysis of lipids a-cyano-4-hydroxy

cinnamic acid and for proteins sinapinic acid were used. The matrices were applied

with a spatial resolution of 500 x 500 µm across the tissue section with an offset of

250 µm x 250 µm using an inkjet-printer. A volume of 3 drops per spot during each

pass and 12 passes of matrix application were applied. Mass spectra were acquired on

an Ultraflex III MALDI-TOF/TOF instrument (Bruker Daltonics) running in the

positive mode. A m/z range of 500-1,600 for lipids and of 2,000-23,000 for proteins

were analyzed, respectively. A total sum of 500 shots per spot was acquired in steps of

50 shots in a random pattern. MS acquisition and visualization was performed using

FlexImaging and FlexControl software (v. 2.0, Bruker Daltonics).

Results and discussion: The methodology was first tested on mouse liver tissue, since

this is the most homogeneously available tissue. As expected the distribution of lipids

as well as proteins was more or less homogeneously as determined by a series of

MALDI-MSI images. The next investigated tissue type was mouse brain. Since the


85

limit of resolution is currently 500 µm for the application of two matrices on a single

tissue slice, some anatomical features of the mouse brain are presented by only a few

pixels in the recorded MALDI-MSI images, however a lower resolution caused the

generation of a homogeneous film instead of discrete spots. Further improvements are

still ongoing. Finally, mouse kidney was analyzed and the distributions of lipid and

protein signals were compared. The developed methodology shows the simple

combination of two compound classes or areas (lipidomics and proteomics) in the

emerging technique MALDI-MSI.

References:

[1] B. Balluff, C. Schöne, H. Höfler, A. Walch, Histochem. Cell Biol. 2011, DOI

10.1007/s00418-011-0843x.

[2] J. T. Delaney, A. Urbanek, L. Wehder, J. Perelaer, A. C. Crecelius, F. von

Eggeling, U. S. Schubert, ACS Comb. Sci. 2011, 13, 218.

Polarization optical and histochemical characterization and

supramolecular structure of carbohydrate-protein fibrils Csoka L.

1, Makovitzky J.

2, Eitner A.

2, Jirikowski G.

2

1University of West Hungary, Institute of Wood and Paper Technology, Sopron,

Hungary, 2Friedrich Schiller University, Institute of Anatomy II, Jena, Germany

Topooptical staining reactions were used to investigate chemically and structurally the

carbohydrate-protein structures of bacterial cellulose, chitosan and alginic acid from

brown algae. Chitosan and alginic acid are associated with glucosaminoglycans

(GaGs), which appear as a continuous sheath enveloping the axial layer or periodically

distributed along the fibrils. The results obtained with polarization microscopy indicate

that reactive carbohydrates and proteoglycans contain chemical end-groups similar to

the ones found in human amyloid fibrils. The introduced staining sequences can be

used as a reliable method for the histochemistry with light and polarization microscopy

of GaGs.


86

Normalization in MALDI-TOF imaging of proteins Deininger S.O.

1, Cornett D.S.

2, Paape R.

1, Becker M.

1, Pineau C.

3, Rauser S.

4, Walch

A.4, Wolski E.

1

1Bruker Daltonik GmbH, Bremen, Germany,

2Bruker Daltonics, Billerica, United

States, 3INSERM U625, Proteomics Core Facility Biogenouest, Rennes, France,

4Helmholtz Zentrum München - Institute of Pathology, Munich, Germany

Introduction: In MALDI imaging normalization is often used to improve the quality

of the images, but there is still discussion in the field whether this procedure creates

artifacts and is therefore generally applicable. In protein imaging, the normalization to

total ion count (TIC) is often used, but other procedures such as Median or noise level

have also been investigated. In this work, we have used a kidney dataset to do a

quantitative comparison of the effects of different normalization procedures. While

this kidney dataset showed no artifacts due to normalization, we used a testis dataset

that did lead to artifacts after normalization to TIC. We investigated how potential

artifacts can be detected and avoided by correlation of different normalization

methods.

We also show data that stress the importance of normalization in the comparsion

across multiple datasets and how normalization can remove the effect of varying laser

intensites and instrument settings.

Results: On the kidney data, the normalization on TIC, median, noise, and vector

norm did not change the ratio of the average intensities in the different regions of the

kidney, which did indicate that there is no artifact introduced by normalization. The

variance of the selected signal in the individual regions was decreased after all

normalizations, indicating a positive effect of the normalization. The improvement in

the variance was stronger for the TIC and vector norm as opposed to the median or

noise level normalization. This could be confirmed by the respective images.

In the testis dataset, a peak with an unusually large area in some regions led to an

artifact in normalization to TIC and the vector norm, which could be evaluated by the

comparison with the non-normalized dataset. Normalization was necessary though for

the display of certain histological features. Here, the normalization to median and

noise level was able to improve the image with respect to histology without creating


87

artifacts. The biggest improvement was achieved by normalization to TIC with the

exclusion of the aberrant peak (TICx).

On another kidney dataset we performed a measurement with deliberately different

instrument settings, such as detector gain and laser energy. A principal component

analysis with and without normalization clearly shows how the effect of different

instrument settings is removed by the normalization. This observation is of crucial

importance for the analysis of clinical imaging data, where a study can be ongoing for

years.

Immunohistochemical investigations of keratin expression for a

characterization of epidermal stem cell niches in bovine fetal skin Ebach K.

1, Kenngott R.A.-M.

1, Sinowatz F.

1

1Institute of Anatomy, Histology and Embryology, Department of Veterinary Sciences,

LMU, Munich, Germany

Purpose: The pattern of keratin expression characterizes different cell types of

ectodermal origin. Especially cell populations with a potential to regenerate the

epidermis, hair follicles or glands show an expression pattern that is useable as a

marker for those cell types. During the bovine fetal skin development it is possible to

differentiate and observe changes in those cell niches by analysis of expression of

keratin 8, 15, 18 and 19.

Results: Results that give an impression of the diversity of epidermal cells are for

example the strong expression of keratin 8 and 15 which is confined to the outermost

cell layer of the hair bulge, whereas keratin 18 and 19 can be detected in inner and

outer cell layers of this compartment. An interesting finding is the expression of

keratin 18 around the bulge region, which appears strongly positive in its main part

and negative in its upper part. The keratin 18 negative area of the upper hair bulge

shows a strong staining with the Periodic Acid Schiff reaction (PAS), whereas the

major part of the hair bulge is PAS-negative.

Conclusions: These findings support the observations of other authors that these two

areas of stem cells can be distinguished by their characteristic expression pattern of

keratin 15, nestin and Lgr6 in murine and human tissue. The upper part of this stem


88

cell niche serves as a reservoir for pluripotent stem cells with the potential to

differentiate also into the neuronal cell lineages, whereas the larger lower part contains

multipotent cells that serve as source for all epidermal cell types. The comparison of

markers in human, murine and bovine skin provides a closer inspection of those

important cell niches and provides a basis for further studies dealing with the

regeneration of epidermal skin compartments.

Topo-optical follow up of phenothiazine induced charge transfer reactions in the yeast cell wall Tigyi Z.

1, Emody L.

1, Molnar J.

2, Jozsi M.

3, Makovitzky J.

4

1University of Pecs, Department of Medical Microbiology and Immunology, Pecs,

Hungary, 2University of Szeged, Department of Medical Microbiology and

Immunobiology, Szeged, Hungary, 3Hans Knöll Institute, Leibniz Institute for Natural

Product Research and Infection Biology, Jena, Germany, 4University of Heidelberg,

Department of Neuropathology, Heidelberg, Germany

In this paper phenothiazine induced charge transfer reactions are studied on various

yeasts, i.e. Candida species, Cryptococcus neoformans and Saccharomyces cerevisiae.

Pre-treatment of the fungal smears with any of eleven phenothiazine compounds

resulted in a weak linear positive birefringence with respect to the cell surface even

without subsequent addition of dyes. Completing the CT reaction by staining the

preparations with Eosin, Erythrosin or Rose Bengal, respectively, did not change the

character of birefringence but strongly increased its intensity. Further polarization

optical analysis revealed that the phenothiazine and xanthen dye complexes are bound

in parallel orientation to the fungal cell wall in a sterically oriented manner. Pre-

treatment with 1% aqueous periodic acid abolished the charge-transfer reactions while

pre-treatment with lipid solvents did not affect them. These results suggest that

oxidation of the hydroxyl (OH) groups in the sugars to aldehyde groups could inhibit

either their interaction with the phenothiazine compound or the complex formation of

the xanthen dye with the fungal cell surface component. Methylation of the different

fungal cell wall sugars entirely ceased the charge transfer reactions. The individual

fungal species required different incubation times of methylation to abolish their


89

charge transfer reactions, i.e. C. krusei, C. tropicalis 6 hours, C. glabrata 12 hours and

C. albicans 18-24 hours, respectively. These results suggest that methylation of the OH

groups in the sugar compounds could alter the interactions with phenothiazine

molecules, and consequently their complex formation with eosin molecules in fungal

cell surface structures. However, treatment with periodic acid-bisuphite after the

methylation reaction reconstituted the chlorpromazine-eosin CT reaction resulting in

an intensive deep green birefringence. Taking together our results point to a pivotal

role of yeast cell wall sugar OH groups in the phenotiazine induced topo-optical

phenomena. Enzymatic reactions and extraction procedures are planned to disclose the

fine molecular mechanisms of these charge transfer interactions.

MALDI imaging of endogenous compounds directly in human skin-

tissue sections Enthaler B.

1,2, Pruns J.

1, Wessel S.

1, Rapp C.

1, Fischer M.

2, Wittern K.-P.

1

Institute(s):

1Beiersdorf AG, Hamburg, Germany,

2University of Hamburg, Hamburg School of

Food Science, Hamburg, Germany

Since introduction of matrix-assisted laser desorption/ionization imaging mass

spectrometry (MALDI-IMS) by Caprioli et al. in 1997, there has been an increasing

number of publications in the last few years. However, only a few groups have dealt

with skin tissue and subsequent MALDI-IMS measurements up to now. The

localization of endogenous and exogenous compounds directly in tissue sections,

including the investigation of biological processes within tissue, is a challenging task

in skin research.

In this manuscript, we report the possibilities of MALDI-IMS in localization of

endogenous compounds directly in human skin-tissue sections. In general, handling

and properties of skin tissue differ from that of other organs, e.g. the kidney.

According to the practical procedure one main difficulty is caused by the low adhesion

of skin-tissue sections to commercially available indium-tin-oxide-coated (ITO) glass

slides, resulting in detachment of tissue sections while matrix coating or hematoxylin

and eosin (HE) staining. In this work, corona-discharge treatment was used to modify


90

the glass-slide surface. As a result, adhesion of skin-tissue sections to ITO-coated glass

slides was improved. Surface modifications were investigated by electron

spectroscopy for chemical analysis (ESCA). This represents an improved sample

preparation procedure for MALDI-IMS measurements of skin-tissue sections (1).

Furthermore, we describe the development of an approach that performs MALDI-IMS

on skin-tissue sections followed by removal of the matrix by washing steps and

subsequent histological staining. Corresponding to the size of some specific skin areas,

e.g. sweat glands, the use of serial sections is not advisable, because of their non-

identical character. It cannot be assumed that specific skin areas are present in both

serial sections or rather located at the identical area. Hence, it is advisable to use one

single skin-tissue section.

For localization of endogenous skin compounds by MALDI-IMS, different matrices

were applied. Using 9-aminoacridine and the negative-ion mode, the distribution of

primary metabolites like ATP within tissue sections were acquired. The assignment to

specific skin areas was enabled trough co-registering of MALDI results and the

corresponding HE-staining image. The compounds were identified by post-source

decay.

Additionally, the poster employs the possibilities of MALDI-IMS in mapping proteins

and tryptic peptides directly in skin-tissue sections.

(1) Enthaler et al. (2011), Improved sample preparation for MALDI-IMS and mapping

of endogenous and exogenous compounds directly in skin-tissue sections, Analytical

and Bioanalytical Chemistry (submitted)


91

The intramitochondrial bacterium Midichloria: novel tools for its

detection Mariconti M.

1, Epis S.

1,2, Sacchi L.

3, Biggiogera M.

3, Sassera D.

1, Bandi C.

1,

Bazzocchi C.1

1University of Milan, Milano, Italy,

2University of Camerino, Camerino, Italy,

3University of Pavia, Pavia, Italy

Midichloria mitochondrii is a fascinating intramitochondrial bacterium symbiont of

Ixodes ricinus, an arthropod of medical and veterinary interest. Midichloria is present

in several cell types (luminal cells, funicular cells, and oocytes) of the reproductive

tissues of I. ricinus females. Electron microscopy shows that these bacteria multiply

inside the tick mitochondria, leading to the consumption of the mitochondrial matrix.

Current data indicate 100% prevalence in female I. ricinus and approximately 50% in

males. Available information suggests that Midichloria bacteria circulate among ticks,

likely through the infection of their vertebrate hosts. It is thus important to develop

tools for the specific detection and staining of these bacteria. Recently, the sequencing

of the Midichloria genome showed the presence of 26 genes coding for proteins of the

flagellar apparatus. This would be the first evidence for the presence of flagellar genes

in a bacterium of the order Rickettsiales. We thus decided to produce in recombinant

form the flagellar protein FLID of Midichloria, to be used as an antigen for serological

analysis, and for the production of polyclonal antibodies, with the aim of developing a

tool for the staining of these bacteria and their flagellum. Ticks ovaries were fixed and

examined by transmission electron microscopy (TEM), indirect immunofluorescence

assay and immunogold staining. Preliminary results indicate that anti-FLID antibodies

react with structures inside the Midichloria cells, but also at their surface and in the

surrounding cytoplasm and mitochondria of infected cells. This staining pattern appear

specific, since no staining was observed in oocytes of a closely related ticks, not

infected by Midichloria. The staining of Midichloria and its cellular structures might

have important implication in the study of tick symbiosis and tick-borne diseases, and

to investigate the possibility that these bacteria infect humans and animals.


92

Desorption Electrospray Ionization (DESI) imaging of tumor

samples - using mass spectrometry to support classical histology Gerbig S.

1, Golf O.

1, Schaefer K.C.

1, Balog J.

2, Takáts Z.

1

1Justus-Liebig-University, Analytical Chemistry, Gießen, Germany,

2Medimass Ltd.,

Budapest, Hungary

Aims: Identification of tissue samples based on their specific phospholipid profile

using desorption electrospray ionization mass spectrometry

Methods: Desorption Electrospray Ionization (DESI) is a mass spectrometric

ionization method described in 2004. DESI is suitable for the analysis of various types

of molecules and it is capable for imaging mass spectrometric investigation of intact

tissue samples. Tissue samples are sectioned using a cryotome and analyzed in their

native state without further sample preparation. The phospholipid profile of tissue

samples was investigated by scanning the surface with DESI. For the identification of

the detected ion signals, high resolution and tandem mass spectrometry was used.

Results: DESI imaging experiments included investigation of different healthy and

cancerous tissue samples. For comparison of the detected structures with histological

findings, H&E stained sections of the samples were produced and tissue areas on the

sections were assigned by pathologists.

Summarized spectra of the different tissue areas visible in the ion images were

extracted from raw MS data and stored in an SQL-type database. Data was processed

using multivariate statistical analysis scheme, comprising Principal Component

Analysis (PCA) and Linear Discriminant Analysis (LDA) which classify spectra

according to their similarities and differences. The results showed that the spectra of

similar tissues are grouped together, while distinct separation of data points was

observed for different histological types.

Construction of a spectral library using reference samples enables the mass-

spectrometry based identification of unknown samples. This identification of unknown

samples was performed in particular for every single pixel, resulting in an artificial

histological map of the tissue section visualizing tissue structures of 100-200 µm. This

extends the potential of automated tissue recognition and leads to a mass-spectrometric

histology-like approach.


93

Conclusions: DESI imaging allows the mass spectrometric characterization of tissue

samples based on the phospholipid profile. Clinical applications of this approach

include the rapid identification of tissues during surgical interventions. Using

electrosurgical tools coupled to mass spectrometric analysis (e.g. REIMS), spectra are

collected in-situ. Real-time analysis of these spectra based on the spectral database can

provide assignment of tissue in-vivo and in case of tumor resection indicate whether

the surgeons keep sufficient margin or already dissect tumor-infiltrated tissue.

Ribonucleoprotein-containing foci increase in size upon cell-cycle exit in cell nuclei from patients affected by myotonic dystrophy

type 2 Giagnacovo M.

1, Malatesta M.

2, Cardani R.

3,4, Meola G.

5, Pellicciari C.

1

1University of Pavia, Department of Animal Biology, Pavia, Italy,

2University of

Verona, Department of Neurological, Neuropsycological, Morphological and Motor

Sciences, Verona, Italy, 3University of Milan, Department of Molecular Biology and

Biotechnology, Milan, Italy, 4Centre for the Study of Neuromoscular Diseases - CNM,

Milan, Italy, 5University of Milan, Department of Neurology, IRCCS Policlinico San

Donato, Milan, Italy

Myotonic dystrophies (DM) are genetically-based neuromuscular disorder with

multisystemic traits among which muscle hyperexcitability (myotonia), muscular

dystrophy with increased number of clumped or centrally located nuclei in skeletal

muscle fibres, dilated cardiomyopathy and cardiac conduction defects (1)

. Two DM

forms exist, called DM1-Steinert's disease (OMIM 160900) and DM2 (OMIM

602688). DM1 is more severe, and depends on the expansion of a (CTG)n

trinucleotide sequence in the 3' untranslated region of the Dystrophia Myotonic Protein

Kinase gene (OMIM 605377). DM2 displays a milder clinical phenotype and is caused

by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc

Finger Protein (ZNF)-9 gene (OMIM 116955). DM pathogenesis depends on the

accumulation of the expanded RNAs in the nucleus resulting in the ectopic

sequestration of several RNA-binding proteins (CUGBP1, MBLN1) and some

ribonucleoprotein (RNP) splicing factors (2)

into peculiar intranuclear foci. The


94

functional deregulation of these protein factors results in the general alteration of the

mRNA pathways in the cells of different tissues (3)

. The cytochemical detection of

expanded RNAs or MBNL1 in the nuclear foci is an accepted diagnostic marker for

DMs (4)

. It is apparent that in DM patients the most significant pathological features

are found in the skeletal muscle, heart and the central nervous system where non-

cycling cell populations (i.e., myofibres, cardiomyocytes or neurons) are mainly

found; on the contrary, cells from self-renewing tissues (such as skin fibroblasts or

layering epithelial cells) appear much less affected.

In this investigation, we aimed to elucidate whether in DM tissues the exit of cells

from the cell cycle may lead to the increase in size of intranuclear foci due to the

progressive sequestration of protein factors (namely MBNL1); to do this, we

investigated by immunocytochemical and morphometric techniques the fate of

MBNL1-containing foci in proliferating cells during the cell cycle, and in non-cycling

cells. Cultured skin fibroblasts from DM2 patients were chosen as a model cell system.

We found that nuclear MBNL1-containing foci do not associate with chromosomes at

mitosis, and remain in the cytoplasm at cytodieresis, being disassembled in early G1

and re-formed in the nucleus, at each cell cycle. After cells had spontaneously stopped

dividing in senescing fibroblast cultures, the nuclear foci were observed to increase in

number and size. Interestingly (and consistently), morphometric measurements on

sections of muscle biopsies taken from the same DM2 patients at different ages

demonstrated that in myonuclei MBNL1-containing foci become larger with

increasing patient's age.

References:

1 - Meola G and Moxley RT 3rd

, J Neurol 251:1173-82, 2004

2 - Malatesta M et al., Histochem Cell Biol 135, 419-25 , 2011

3 - Schoser B and Timchenko L, Curr Genomics 11:77-90, 2010

4 - Cardani R et al., Eur J Histochem 50:177-82, 2006


95

Renal ischemia-reperfusion injury causes intercalated cell-specific

disruption of occludin in the collecting duct Lee S.-Y.

1, Shin J.-A.

1, Kwon H.M.

2, Weiner I.D.

3, Han K.-H.

1

1Ewha Womans University School of Medicine, Department of Anatomy, Seoul, Korea,

Republic of, 2University of Maryland School of Medicine, Division of Nephrology,

Baltimore, United States, 3University of Florida College of Medicine, Division of

Nephrology, Gainesville, United States

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose

of this study was to examine the effects of ischemia-reperfusion (IR) injury on

expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat

kidney. IR injury was induced by clamping both renal pedicles for 30 minutes and

animals were sacrificed at 6 hours after the reperfusion. IR injury decreased blood

bicarbonate level but did not persistently alter pH, Na+, K

+, or Cl

−. The control kidneys

showed strong occludin immunoreactivity in the tight junctions of the thick ascending

limb, distal convoluted tubule, and collecting duct. Occludin staining was moderate in

the thin limbs of the loop of Henle, and was not detected in the proximal tubule,

glomerulus, and blood vessels. ZO-1 was expressed in the same sites as occludin, and

in addition was also expressed in the proximal tubule, glomerulus, and vascular

endothelial cells. In the IR injury kidneys, many collecting duct cells, as well as

proximal tubule cells, in the outer medulla were damaged. In the collecting duct,

intercalated cells lost their polarity and occludin was localized diffusely in the

cytoplasm after IR injury, whereas principal cells were unaffected. The ZO-1 patterns

showed abnormal cellular distribution in both the proximal tubule and collecting duct.

IR injury did not detectably alter the occludin and ZO-1 distribution in the thick

ascending limb and collecting duct principal cells. The abundance of occludin protein

expression was not changed with IR injury. In summary, renal IR injury causes

cellular damages not only in the proximal tubule but also in the collecting duct and

that the disruption of tight junctions, particularly occludin, is cell-specific to

intercalated cells in the collecting duct. The collecting duct is composed of two

histologically different cell types, the principal cells and intercalated cells. The

principal cells reabsorb water, whereas the intercalated cells are involved in acid-base


96

homeostasis. The results may have significant value in understanding clinical

manifestations of renal ischemic injury such as metabolic acidosis.

Key words: Collecting duct, ischemia/reperfusion injury, kidney, occludin, tight

junction

This work was supported by National Research Foundation of Korea (2009-0073733,

2011-0016068).

E-cadherin mutations contribute to gastric carcinogenesis by modulating multiple EGFR-dependent downstream signalling

pathways Heindl S.

1, Malinowsky K.

1, Wolff C.

1, Luber B.

1

1TU München, Pathology, München, Germany

Mutations in the gene encoding the cell adhesion molecule E-cadherin and

overexpression of the epidermal growth factor receptor (EGFR) represent fundamental

genetic alterations associated with diffuse-type gastric cancer. Mutations, e.g. in frame

deletion of exon 8 (del 8), frequently affect putative extracellular Ca2+

-binding sites of

E-cadherin, thereby impairing its functionality. Several studies have shown a

bidirectional crosstalk between E-cadherin and EGFR. Functional wild-type (wt) E-

cadherin inhibits ligand induced EGFR activation, but mutations lead to a loss of its

suppressive function, resulting in increased EGFR activation and recruitment of

downstream signalling components. The phenotype of cells harboring such mutations

is characterized by increased proliferation and elevated migratory and invasive

potential.

Our project aims at studying the impact of somatic mutations of E-cadherin on EGFR-

mediated signalling pathways, in order to identify signalling molecules of these

pathways as molecular targets for selective therapies in gastric cancer.

Cell lines expressing wt and mutant E-cadherin (del 8) were EGF-stimulated and

cellular lysates were analyzed by commercial proteome profiler antibody array (R&D

Systems). The array allowed detection of the phosphorylation status of 48 signalling

molecules and facilitated identification of differential activation. We found differential

response to EGF stimulation of proteins involved in DNA damage and repair. Whereas


97

activity of tumorsuppressors p53 and CHK2 increased with EGF-stimulation for

different time spans in cells expressing wt E-cadherin, it remained low or even

decreased in cells with mutations of E-cadherin. As upon DNA damage both proteins

are needed for induction for DNA repair and initiation of cell cycle arrest or cellular

apoptosis, a loss of function of these molecules can lead to uncontrolled cell cycle

progression and increased proliferation promoting tumor progression. Furthermore,

Src-family kinases (SFKs) like Src, Yes, Fgr, Lck and Lyn exhibited similar activation

profiles in response to EGF-stimulation as described above. As SFKs are key players

in multiple cellular processes like cell growth, differentiation, cellular adhesion,

migration and invasion, the impact of their differential activation on the cellular

phenotype is subject of ongoing experiments. To determine the role of the identified

targets in gastric carcinogenesis and the mechanisms of crosstalk between E-cadherin

and EGFR a protein microarray platform was established, allowing analysis of

multiple samples under the same experimental conditions. This approach will reveal

new insights in EGFR-mediated signalling pathways contributing to gastric

carcinogenesis and complete our data describing how tumor associated mutations of E-

cadherin affect cellular signalling.

Immunohistochemical detection and non-invasive U-SPECT

imaging reveal distinct tumor tissue distribution patterns of anti-EGFr antibody zalutumumab in a tumor xenograft model Houtkamp M.A.

1, Branderhorst W.

2, Blezer E.L.A.

2, Witteveen H.

1, Gerritsen A.F.

1,

Parren P.W.H.I.1, van Dongen G.A.M.S.

3, Beekman F.J.

4, Bleeker W.K.

1

1Genmab, Utrecht, Netherlands,

2University Medical Center Utrecht, Image Sciences

Institute, Utrecht, Netherlands, 3VU University Medical Center, Nuclear Medicine &

PET Research, Amsterdam, Netherlands, 4MILabs B.V., Utrecht, Netherlands

Therapeutic antibodies (Abs) for cancer treatment must penetrate solid tumors and

bind to a tumor-specific target in order to be effective. To gain further insight in these

aspects for zalutumumab, a human anti-EGFr monoclonal Ab (mAb), we have studied

an A431 xenograft model with high EGFr expression and intratumoral necrotic areas.

111In-labeled zalutumumab was administered at a non-saturating dose and total body


98

111In distribution was determined in vivo at different time points using ultra-high-

resolution focused pinhole SPECT imaging. After 48h, the A431 tumor was removed

for ex vivo SPECT imaging and immunohistochemical (IHC) analysis of zalutumumab

biodistribution and EGFr (target) expression . Consecutive whole tissue IHC images

were microscopically captured and co-registered to the SPECT images at equidistant

reference points. With IHC, the in vivo administered zalutumumab showed a diffuse

distribution throughout the A431 tumor, which appeared to be exclusively localized

within areas of high EGFr expressing tumor cells, but was absent in other tumor cells

with lower EGFr expression. Interestingly, ex vivo SPECT imaging after 48h showed

overlapping 111

In tumor distribution within all EGFr target expressing tumor cells but

also showed hotspots just outside of the tumor area. Both IHC and SPECT imaging

were negative for intratumoral necrotic areas. An explanation for the observed

differences between IHC and U-SPECT could be that IHC mainly detects intact tumor

cell-associated zalutumumab, whereas 111

In imaging also detects accumulated 111

In

derived from internalized and degraded zalutumumab. The hotspots might then be

indicative of uptake of 111

In-labeled tumor cell debris by tissue macrophages which

appear in clusters adjacent to the tumor. We conclude that SPECT imaging is a

suitable technique for non-invasive assessment of Ab penetration into the tumor,

which provides insight in target localization. When evaluating U-SPECT images it

should be kept in mind that part of the signal may be related to residual 111

In derived

from antibody uptake and subsequent degradation.

Immune laser capture microdissection of macrophages in engineered dental pulp tissues Kaneko T.

1, Yamanaka Y.

1, Yoshiba K.

1, Okiji T.

1

1Niigata University Graduate School of Medical and Dental Sciences, Division of

Cariology, Operative Dentistry and Endodontics, Niigata, Japan

We have recently developed a method of engineering dental pulp tissues from stem

cells from human exfoliated deciduous teeth, by culturing these cells in a poly-L-lactic

acid scaffold placed in the canal space of human tooth slices for 14 days. In the

engineered tissue, macrophages may play some roles in absorbing the scaffold by their


99

phagocytic ability or maintaining the tissue homeostasis by acting as antigen

presenting cells. To examine the roles of macrophages in the engineered dental

pulp tissue, we performed immune-laser capture microdissection (LCM) of CD68

(anti-human macrophages)-immunoreactive cells from formaldehyde-fixed (24 h),

formic acid-demineralized (7 days) and paraffin-embedded tissues, followed by a real-

time PCR analysis on the mRNA expression of class II MHC, which is highly

expressed in activated antigen presenting cells. Results demonstrated that class II

MHC mRNA expression levels were significantly higher in macrophages retrieved

from the area where most of the scaffold was absorbed, as compared with

macrophages in the area where most of the scaffold was still present. The results

suggested that macrophages show different activation levels in different area of

engineered tissues. The immune-LCM method presented here allows for the

quantitative analysis of gene expression in paraffin-embedded tissue sections from

demineralized specimens, suits for the analysis of relatively rare cell types within a

tissue, and thus may constitute a new approach for histochemistry of mineralized

tissues, Also, the immune-LCM method may open the door for the acquisition of new

data from archived specimens, and may improve our ability to perform differential

diagnosis of pathologies.

Autophagosome formation by quercetin Klappan A.

1, Mylonas I.

1, Friese K.

1, Brüning A.

1


Aims: Many studies have shown that quercetin, a bioflavonoid, causes anti-cancer

effects. However, the mechanism behind this remained unknown. We have performed

cell biological studies to analyse the effect of quercetin on human cancer cells.

Methods: The occurrence of autophagy was shown in viable cells by using fluorescent

autophagy markers and acidotropic dyes. Proteasome activity was analysed by a

bioluminescence assay using synthetic substrates. Western blot analysis was

performed to investigate the mTOR signalling pathway.


100

Results: Application of quercetin to epithelial human cancer cells induced strong

intracellular vacuolation that caused cell cycle arrest and apoptosis. These vacuoles

could be characterized as phagolysosomes. Immediately after application of quercetin,

inhibition of mTOR activity, an autophagy-controlling pathway, occurred in quercetin-

treated cancer cells, as revealed by reduced phosphorylation of 4E-BP1 and p70S6

kinase, all major mTOR substrates. Evaluation of cellular proteasome activity in

quercetin-treated cancer cells showed an effective and immediate inhibition of all three

enzymatic activities of the proteasome and, as a result, accumulation of

polyubiquitinated proteins and protein aggregates.

Conclusions: Proteasome inhibition by quercetin and triggering of massive autophagy

due to the accumulation of polyubiquitinated proteins can be seen as a major

contributor to quercetin-induced cancer cell death. Since proteasome inhibitors

represent a highly effective group of anti-cancer drugs, these results suggest potential

new applications for quercetin in cancer treatment.

Spatial segmentation of hyperspectral 3D volume data Kobarg J.H.

1, Alexandrov T.

1,2

1University of Bremen, Zentrum für Technomathematik, Bremen, Germany,

2Steinbeis

Innovation Center SCiLS (Scientific Computing in Life Sciences), Bremen, Germany

Segmentation of hyper-spectral imaging data using clustering requires special

algorithms, which consider spatial relations between the pixels. This strategy can

improve the clustering of noisy data, since neighbor pixels should usually be clustered

into one group. However, in the case of the spectral dimension p being large, cluster

algorithms already suffer from the curse of dimensionality and have high memory

needs as well as long runtimes.

We propose to incorporate neighboring pixels from a window of w×w pixels to define

a feature space of size npw², then apply a clustering method to the projected points.

The effect of improvement is controlled by weights depending on the spatial distance

between the pixels to be clustered. We propose a data-adaptive way to define weights

based on the similarity of pixels. Any vectorial clustering algorithm like k-means can


101

be directly applied to the projected points. In addition, we employ FastMap, to find a

Euclidean space of dimension nq corresponding to the projection.

The proposed algorithm is well suited for hyper-spectral imaging data as found in

imaging mass spectrometry where the number of pixels is relative high (n≈104).

Literature:

Alexandrov, T. and Kobarg, J.H. (2011). Efficient spatial segmentation of large

imaging mass spectrometry datasets with spatially aware clustering. Bioinformatics,

27, i230-i238.

Faloutsos, C. and Lin, K.-I. (1995). FastMap: a fast algorithm for indexing, data-

mining and visualization of traditional and multimedia datasets. ACM SIGMOD, May

1995, San Jose, CA, pp. 163--174.

Tomasi, C. and Manduchi, R. (1998). Bilateral filtering for gray and color images. Intl.

Conf. Computer Vision, Jan. 1998, Bombay, India, pp. 839--846.

pHPMA-based tissue embedding medium compatible with MALDI

mass spectrometry imaging experiments Strohalm M.

1, Strohalm J.

2, Kaftan F.

3,4, Krásný L.

1,5, Volný M.

1, Novák P.

1, Ulbrich

K.2, Havlíček V.

1

1Institute of Microbiology, Academy of Science of Czech Republic, Laboratory of

Molecular Structure Characterization, Prague, Czech Republic, 2Institute of

Macromolecular Chemistry, Academy of Science of Czech Republic, Prague, Czech

Republic, 3Institute of Organic Chemistry and Biochemistry, Academy of Science of

Czech Republic, Prague, Czech Republic, 4Charles University, Department of

Analytical Chemistry, Faculty of Science, Prague, Czech Republic, 5Institute of

Chemical Technology, Department of Biochemistry and Microbiology, Prague, Czech

Republic

Due to the ion suppression effect in MALDI ionization and number of background

peaks in the low-mass region, traditional tissue-sectioning embedding media are not

suitable for mass spectrometry imaging (MSI) experiments. Droplets of water, that

does not affect MALDI ionization, are often used to mount tissue on a sectioning


102

target. But, the ice block formed around the tissue does not provide a good support for

sectioning of fragile samples. In this work, we propose a novel embedding medium

based on the poly[N-(2-hydroxypropyl)methacrylamide], providing smooth cutting

surface and fully compatible with MSI experiments and conventional histological

haematoxylin-eosin staining.

Cordycepin induces double strand breaks and the DNA damage response in breast cancer cells Lee H.J.

1, Burger P.

1, Vogel M.

2, Brüning A.

1, Friese K.

1

1University Hospital Munich, OB/GYN, Munich, Germany,

2University Hospital

Munich, Munich, Germany

Aims: Cordycepin (3-deoxyadenosine) is a fungal metabolite known to exert anti-

inflammatory effects and anti-tumor activities. We have investigated the effect of

cordycepin on growth and cell survival of human breast cancer cells.

Methods: Cell survival and apoptosis of human breast cancer cells was analyzed by

the MTT assay and FACScan analysis. Activation of intracellular signalling pathways

was investigated by immunoblot and immunofluorescence analysis.

Results: Treatment of human breast cancer cells with cordycepin reduced cell

proliferation and clonal growth. Cordycepin treatment was associated with marked

morphological changes, resulting in a multi-nuclear and differentiation-like phenotype.

At the molecular level, a strong induction of the DNA damage response (DDR), which

included phosphorylation of ATM, ATR, and histone gammaH2AX, could be

observed. This indicates induction of DNA double strand breaks induced by

cordycepin in proliferating breast cancer cells, resulting either in apoptosis or

persistent cell cycle arrest.

Conclusions: The genotoxic effect of cordycepin on proliferating breast cancer cells

indicates a new mechanism of cordycepin-induced cancer cell death, and its

preferential activity against highly proliferative and undifferentiated breast cancer cells

indicates new perspectives for cordycepin in the treatment of advanced breast cancer.


103

MALDI MSI for ovarian cancer biomarkers research: latest

developments of the technology for screening and tracking. Longuespée R.

1,2, Boyon C.

1,3, Kerdraon O.

4, Desmons A.

1, Vinatier D.

3, Fournier I.

1,

Day R.2, Salzet M.

1

1Laboratoire de Spectrométrie de Masse Biologique Fondamentale et

Appliquée/Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France,

2Institut de Pharmacologie de Sherbrooke/Université de Sherbrooke, Sherbrooke,

Canada, 3Service de Chirurgie Gynécologique/Hôpital Jeanne De Flandre/CHRU

Lille, Lille, France, 4Centre d'Anatomie et de Cytologie Pathologiques/CHRU Lille,

Lille, France

Ovarian cancer is the second major cause of gynecological death in Europe and USA.

Ovarian cancer has been studied at a proteomic level for biomarkers hunting with the

emergence of diverse techniques such as 2D electrophoresis and SELDI TOF MS. At

this time, CA125 is still the only biomarker used for the monitoring of the pathology.

Its sensitivity is 80% for stage III and stage IV cancers but only 30% for early stages

with high rates of false positives. Since its introduction in the early 90's, MALDI

Imaging Mass Spectrometry (IMS) has widely been used for biomarkers discovery in

many pathologies thanks to the possibility to discriminate the localization of

compounds in tissues of interest. Using this technology, we found the Cter part of

PA28, “Reg Alpha” present in the cancerous regions of ovarian biopsies. Recently, we

found this peptide in stage I epithelial cancer tissues, making it a potential marker for

wide screenings among women populations. Then, our recent developments in the

field of IMS allowed us to associate Histo Immuno Chemistry and IMS for the specific

tracking of this molecule in cancerous tissues. We indeed propose to incubate tissues

of interest with known anti-markers antibodies, then digest the surface of the tissues

with a solution of trypsin and finally perform the MALDI Image of the prepared

tissue, to reveal the presence of the tryptic peptides of the antibodies. It's now possible

to proceed to the quantitative detection of markers by directly analyzing the presence

of tryptic peptides of the antibodies targeting Reg Alpha. This procedure allows the

use of Immuno Histo Chemistry with the Mass Spectrometric devices as revealers

instead of a microscope, and would be strongly relevant for the tracking of cytotoxic


104

antibodies in tumors. These developments also pave the way for new pharmacologic

applications such as the monitoring of the effect of treatments of the pathology by

screening the evolution of the presence of the compound in malignant tissues.

Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-

fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the

Arbeitsgemeinschaft Internistische Onkologie (AIO) v Luber B.

1, Deplazes J.

1, Keller G.

1, Walch A.

2, Rauser S.

2, Langer R.

1, Höfler H.

1,

Fend F.3, Peschel C.

4, Lordick F.

5

1Technische Universität München, Pathology, München, Germany,

2Helmholtz

Zentrum München, Pathology, Neuherberg, Germany, 3Universität Tübingen,

Pathology, Tübingen, Germany, 4Technische Universität München, 3rd Medical

Department, München, Germany, 5Klinikum Braunschweig, 3rd Medical Department,

Braunschweig, Germany

Aims: The activity of the therapeutic antibody cetuximab against the epidermal

growth factor receptor (EGFR) and chemotherapy was assessed in first-line metastatic

gastric and gastro-esophageal junction cancer in a phase II study of the

Arbeitsgemeinschaft Internistische Onkologie (AIO) with objective tumor responses in

65% of patients and a low mutation frequency of KRAS (3%). The aim of the

correlative tumour tissue studies was to investigate the molecular mechanisms

underlying clinical response to treatment.

Methods: Patients were a subset from the phase II trial of cetuximab plus weekly

oxaliplatin, 5-fluorouracil and folinic acid (n=39). We performed FISH analysis,

immunohistochemistry and mutation analysis.

Results: We analysed the association of clinical outcome with (1) EGFR gene copy

number, (2) expression levels of EGFR, phosphorylated EGFR (pEGFR),

phosphorylated mitogen-activated protein kinase (pMAPK), phosphorylated Akt

(pAkt) and E-cadherin and (3) the mutation profile of selected exons of the E-cadherin

gene CDH1, KRAS and BRAF.


105

Our study showed a significant association between increased EGFR gene copy

number (≥4.0) and OS in gastric and OGJ cancer, indicating the possibility that

patients may be selected for treatment on a genetic basis. Furthermore, a significant

correlation was shown between activated EGFR and shorter TTP and ORR, but not

between activated EGFR and OS. No V600E BRAF mutations were identified. On the

other hand, an interesting trend between high E-cadherin expression levels and better

OS was observed and two missense mutations in exon 9 of the E-cadherin gene

(CDH1) (A408V and D402H) were detected.

Conclusions: Our data suggest that the EGFR gene copy status, activated EGFR and

E-cadherin are clinically important factors that may help to identify subgroups of

patients who are likely to benefit from EGFR-targeted therapy in combination with

chemotherapy.

The topo-optical investigation of Alzheimer Amyloid Makovitzky J.

1, Kovacs G.

1, Appel T.

1

1Niedersächsisches Internatsgymnasium, Bad Bederkesa, Germany

In 2010 we presented the examination of human amyloid fibrils with various topo-

optical staining reactions. We showed that different ex vivo amyloid fibrils (fibrils

isolated from post-mortem tissue) all contain lipids, chondroitin sulfate

glycosaminoglycans and sialic acid. The carbohydrates are accessible from outside of

the fibril, and highly ordered.

In the current study formalin-fixed paraffin-embedded brain samples of 10 Alzheimer

patients were investigated with topo-optical reactions. Neurofibrillary tangles, amyloid

plaques and vessels gave positiv staining reactions with Congo red, PSI, Pinacyanol,

Toluidine blue and Rivanol.

The amyloid deposits are resistent to potassium permanganate / trypsin and to

permanganate / pronase treatment. We found similar results after performic acid /

trypsin and performic acid / pronase treatment. The carbohydrate (ABT) and sialic

acid specific reactions were negative. We selectively demonstrated

glycosaminoglycans with the critical electrolyte concentration method (CEC). After


106

GAG-specific enzymatic treatment the binding of Congo red to amyloid was

increased.

As a control, in vitro amyloid beta (1-40) and amyloid beta (1-42) fibrils are also

positiv with Congo red, PSI, Pinacyanol, Toluidine blue and Rivanol. In vitro fibrils

are negative with carbohydrate and sialic acid specific reactions and the critical

electrolyte concentration method (CEC).

Using topo-optical staining reactions we could differentiate between aggregated

protein, glycosaminoglycans, non-acidic carbohydrate and carbohydrates containing

sialic acid in Alzheimer tissue deposits. The staining reactions followed by microscopy

with polarised light provide strong analytical evidence for molecular components of

amyloid in a native (tissue) setting. Topo-optical reactions should be applied to other

kinds of amyloid tissue and studied in more detail.

Late gastrointestinal metastases of invasive lobular breast carcinoma mimicking Crohn´s disease Baranyay F.

1, Szabó J.

1, Makovitzky J.

2

1Kanizsai Dorottya Hospital, Pathology, Nagykanizsa, Hungary,

2University of

Heidelberg, Department of Neuropathology, Heidelberg, Germany

Introduction: Invasive lobular carcinoma - comprising approximatelly 10 percent of

breast cancers - is considered to be a histologically, molecular genetically distinct

entity metastatizing mainly the gastrointestinal tract. They manifest after 3-20 years

from the recognition of the primary tumor and they appear to be inflammatory disease

or a secundary tumor

Case report: The female patient with breast cancer died at the age of 53 years. 8 years

after tumor-free state upper abdominal spastic pain emerged irradiating into the back

with belt-like pattern. Radiologically Crohn´s disease was diagnosed. Ileum biopsy

was negative. Patient was treated ex juvantibus with methylprednisolon. In the

background of mechanic ileus the resection of the terminal ileum and partly the

ascended colon was surgically removed. The patient died in 3 weeks after operation.

Histology, immunohistochemistry: We compared the immunohistochemical reactivity

of the primary breast cancer, the axillary lymph glands, the terminal ileum, liver and


107

spleen metastatic cells with ABH blood group specific lectins, antibodies to

cytokeratis, breast carcinoma antigen (BRCA), CA15-3, estrogen-progesteron

receptors, C-erbB-2, EMA, CEA, Ki 67 and ABH blood group antigens.

Autopsy findings: The whole stomach wall and the initial section of the duodenal wall

was diffusely thickened 4-5 mm in linitis plastica-like emergence. The lobular

structure of the liver was blured, firm palpation and diffusely with miliary reddish

nodules. The cut surface of the spleen appeare to be like a ham blured structure and

pale reddish colour.

Results: The sections of the terminal ileum showed intensive fibrotic, desmoplastic

reaction with lobular carcinoma cells infiltratring from the serosa to the mucosa in

"indian file" pattern.The carcinomatous cells in the primary tumor didn´t possess

estrogen receptors, however progesteron receptors could be detected in breast

carcinoma cells and in the metastatic cells of the axillary lymph glands. The

gastrointestinal tumor infiltrates, the cells of the liver and splenic metastases didn´t

react with anti-progesteron, nor with cerbB-2. All the applied antibodies were reacting

with cells of the primary tumor, the tumor cells of axillary lymph nodes, gastrintestinal

and hepato-lienar metastatic cells. The Ki-67 proliferation antigen detected with MIB-

1 labelled only 10% of metastatic carcinoma cells. The patient according to her red

blood cell phenotype belonged to AB blood group and the ABH specific lectins and

mabs reacted with both the primary carcinoma and the metastatic cells intensively.

Conclusion: Late invasive lobular carcinoma metastasis may mimic wide scale of

gastrointestinal disease or may simulate Crohn´s disease. They may cause linitis

plastica of the gastroduodenal wall and may bring about pseudo-cirrhosis and special

splenic metastasis. In our case all these listed gastrointestinal metastases occured in

one patient.


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Role of macrophages in brown adipose tissue remodeling in

hyperinsulinemic rats Markelic M.

1, Velickovic K.

1, Golic I.

1, Otasevic V.

2, Stancic A.

2, Jankovic A.

2,

Vucetic M.2, Buzadzic B.

2, Korac B.

2, Korac A.

1

1University of Belgrade, Faculty of Biology, Belgrade, Serbia,

2University of Belgrade,

Institute for Biological Research 'Sinisa Stankovic', Belgrade, Serbia

There are two types of fat in mammals including humans - white (WAT) and brown

adipose tissue (BAT). WAT is an energy-storage, endocrine organ while BAT

functions as an energy-dissipating organ through adaptive thermogenesis. Previous

studies have suggested that macrophage (MA) functions and phenotypes vary

considerably in different fat depots. Evidence has accumulated indicating that obesity

is associated with a WAT chronic, low-grade inflammation (INF) with presence of

MAs proinflammatory phenotype, suggesting that INF may be a potential mechanism,

whereby obesity leads to insulin resistance (IR). Given the fact that little is known

about BAT-MA interactions, we investigated their possible role in BAT under

hyperinsulinaemia (HI) which is known to precede IR and represents one of the

metabolic syndrome symptoms.

The effects of different doses of insulin (0.4 IU or 4.0 IU/kg, i.p., 1 or 3 days) were

studied on Wistar rats. Saline-treated animals served as physiological controls. 3 hrs

after the last injection animals were sacrificed.

In BAT, MAs of all groups were mostly sparsely distributed. Generally they had a

round appearance, more alike to resident than to proinflammatory phenotype. In some

cases, massive MA infiltration around capillaries was visible, with an activated

phenotype (with secondary lysosomes, indicators of prominent phagocytic activity)

even in control animals. MAs were also present in the regions of erythrophagocytic

activity, which is common for BAs, still increased by insulin. In these regions, they

showed heme oxygenase 1 (HO-1) and HO-2 immunopositivity, indicating their

assistant role to BAs in hemoglobin degradation. Even monocytes inside of capillary

lumen near erythrophagocytic region were HO-1/HO-2 positive. MAs in other regions

showed no HOs positivity.


109

Although it is not clear what the responsible molecular mechanisms are, we have

shown that there are significant interactions between BAs and MAs that affect their

functions and probably differentiation. MAs in BAT of HI rats have no

proinflammatory phenotype, but rather serve in tissue remodeling. It is possible that

HOs expression in BAT MAs contributes to suppression of apoptosis, necrosis, INF

and oxidative stress, since it is known that CO and bilirubin suppress these processes

while the iron formed enhances the synthesis of the antioxidant ferritin. Also, HO

system has been shown to modulate both the metabolic and inflammatory systems

suppressing IR and INF. In this study, we have seen no signs of INF in BAT, and MAs

appeared quiescent, with exception of those in erytrophagocytic and apoptotic regions,

and some cases of their infiltrations around capillaries. Previously showed increased

angiogenic, adipogenic and hypertrophic effects of HI allow us to postulate MAs role

mainly in BAT remodeling, maintenance of insulin sensitivity and anti-inflammatory

state, even in condition of extreme HI.

Supported by Serbian Ministry for Education and Science, Grant 173055.

Selective Induction of Inhibin beta E by the ER stress reaction Matsingou C.

1, Brüning A.

1, Friese K.

1, Mylonas I.

1


Aims: The endoplasmic reticulum (ER) is highly sensitive to changes in the pH value,

redox state, and calcium content. Especially in cancer cells, its ability to respond to

changes in homeostasis is of crucial importance for cancer cell survival under

conditions of hypoxia, interstitial acidification, and nutrient deprival. We have

investigated factors that could contribute to the survival of cancer cells under these

conditions.

Methods: ER stress was induced by various means, including the use of fungal

antibiotics, such as tunicamycin and calcimycin (A23187), and chemotherapeutics

such as nelfinavir and bortezomib. Induction of the ER stress reaction was verified by

XBP1 splicing analysis, and expression of ER stress target genes was monitored by

RT-PCR and immunofluorescence analysis.


110

Results: In addition to the induction of the canonical downstream targets of the ER

stress response, a strong induction of the peptide hormone Inhibin beta E could be

observed. Inhibin beta E expression was in direct association with the induction of the

ER stress response, independent of the inducing agent. Ectopic overexpression of

ATF4, a key transcription factor induced by the ER stress response, enhanced the

expression of Inhibin beta E. Other inhibin subunits remained unaffected by the ER

stress response.

Conclusions: The selective and strong induction of Inhibin beta E by the ER stress

reaction, mediated by activation of the transcription factor ATF4, provides a new link

of the ER stress response to the expression of inhibin beta E and its involvement in

cancer cell survival.

Proteomic markers for regional lymph node metastasis in primary

colon cancer tissues Meding S.

1, Balluff B.

1, Elsner M.

1, Schöne C.

1, Rauser S.

1, Nitsche U.

2, Maak M.

2,

Schäfer A.3, Hauck S.

3, Ueffing M.

3,4, Langer R.

5, Höfler H.

1,5, Friess H.

2, Rosenberg

R.2,6

, Walch A.1


2Klinikum rechts der Isar, Technische Universität München, Department of Surgery,

München, Germany, 3Helmholtz Zentrum München, Research Unit Protein Science,

Neuherberg, Germany, 4Institute for Ophthalmic Research, Center for Ophthalmology,

University of Tübingen, Germany, 5Technische Universität München, Institute of

Pathology, München, Germany, 6Kantonsspital Baden, Klinik für Allgemein-, Viszeral-

und Gefässchirugie, Baden, Switzerland

Regional lymph node metastasis negatively affects prognosis in colon cancer patients.

Patients without metastases can often be fully cured by resection of the primary tumor.

For a priori determination of the metastatic potential of primary tumors, reliable

markers, especially proteomic ones, are still lacking. In this study, two complementary

proteomic methods, MALDI Imaging and label-free, quantitative proteomics, have

been employed in order to identify proteomic markers for regional lymph node

metastasis. A tissue cohort comprising of primary colon tumors which were either


111

without metastasis (UICC II, pN0, n = 21) or with lymph node metastasis (UICC III,

pN2, n = 33) was analyzed for proteomic marker identification. Validation by

immunohistochemistry was performed on an independent tissue cohort consisting of

primary colon tumors specimens (n = 168). MALDI Imaging yielded 10

discriminating m/z species and label-free, quantitative proteomics 28 proteins. Two

MALDI Imaging derived candidate proteins (FXYD3 and S100A11) and one coming

from label-free, quantitative proteomics (GSTM3) were validated on the independent

tissue cohort. FXYD3 (p = 0.0110), S100A11 (p = 0.0071) and GSTM3 (p = 0.0173)

had a significant correlation with lymph node metastasis. With our complementary

approaches it was possible to identify proteomic markers for regional lymph node

metastasis directly in primary colon cancer tissues.

S100-A10, Thioredoxin, and S100-A6 as biomarkers of papillary thyroid carcinoma with lymph node metastasis identified by

MALDI Imaging Nipp M.

1, Elsner M.

2, Balluff B.

2,3, Meding S.

2, Sarioglu H.

4, Ueffing M.

4, Rauser S.

2,

Unger K.5, Höfler H.

2,6, Walch A.

2, Zitzelsberger H.

1

1Helmholtz Zentrum München - Research Unit of Radiation Cytogenetics, Department

of Radiation Sciences, Neuherberg, Germany, 2Helmholtz Zentrum München - Institute

of Pathology, Neuherberg, Germany, 3Technische Universität München - Klinikum

rechts der Isar, Department of Medicine II, Munich, Germany, 4Helmholtz Zentrum

München - Core Facility Proteomics, Department of Protein Science, Neuherberg,

Germany, 5Imperial College London, Hammersmith Hospital, Human Cancer Studies

Group, Department of Surgery and Cancer, London, United Kingdom, 6Technische

Universität München - Institute of Pathology, Munich, Germany

In papillary thyroid carcinoma (PTC), metastasis is a feature of an aggressive tumor

phenotype. To identify protein biomarkers that distinguish patients with an aggressive

tumor behavior, proteomic signatures in metastatic and non-metastatic tumors were

investigated comparatively. In particular, matrix-assisted laser desorption/ionization

(MALDI) imaging mass spectrometry (IMS) was used to analyze primary tumor

samples. We investigated a tumor cohort of PTC (n=118) that were matched for age,


112

tumor stage and gender. Proteomic screening by MALDI-IMS was performed for a

discovery set (n=29). Proteins related to the discriminating mass peaks were identified

by 1D-gel-electrophoresis followed by mass spectrometry. The candidate proteins

were subsequently validated by immunohistochemistry (IHC) using a tissue

microarray for an independent PTC validation set (n = 89). In this study, we found 36

m/z (mass-to-charge-ratio) species that specifically distinguished metastatic from non-

metastatic tumors, among which m/z 11,608 was identified as thioredoxin, m/z 11,184

as S100-A10, and m/z 10,094 as S100-A6. Furthermore, using IHC on the validation

set, we showed that the overexpression of these three proteins was highly associated

with lymph node metastasis in PTC (p < 0.005). For functional analysis of the

metastasis specific proteins we performed an Ingenuity Pathway Analysis (IPA) and

discovered a strong relationship of all candidates with the TGF- β dependent EMT-

pathway. Our results demonstrated the potential application of the MALDI-IMS

proteomic approach in identifying protein markers of metastasis in PTC. The novel

protein markers identified in this study may be used for risk stratification regarding

metastatic potential in PTC.

The MALDI-imaging/ MULTI-ARRAY core facility: A new service

platform at the University of Bremen Oetjen J.

1,2, Maass P.

1,3, Maedler K.

2,4, Alexandrov T.

1,2,3

1University of Bremen, Center for Industrial Mathematics, Bremen, Germany,

2University of Bremen, MALDI-imaging lab, Bremen, Germany,

3Steinbeis Innovation

Center SCiLS (Scientific Computing in Life Sciences), Bremen, Germany, 4University

of Bremen, Islet Biology Laboratory, Centre for Biomolecular Interactions, Bremen,

Germany

The MALDI-imaging/ MULTI-ARRAY Core Facility at the University of Bremen

(http://www.maldi.uni-bremen.de) is a newly established institution providing

expertise and service in matrix assisted laser desorption/ ionization (MALDI)-imaging

mass spectrometry, also called MALDI-imaging, and MULTI-ARRAY® technology

combined with computational solutions for data analysis. MALDI-imaging is a

powerful tool for the analysis and visualization of (bio)molecules in their spatial


113

proximity directly from tissue sections, plant samples and bio- or polymer films.

Biomarkers can be detected in a molecular discovery process and validated by

exploitation of the MULTI-ARRAY® electrochemiluminescence-based platform for

high throughput analysis of proteins in serum-, cell and tissue lysates.

The MALDI-imaging/ MULTI-ARRAY Core Facility holds the Autoflex SpeedTM

MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany)

equipped with the smartbeamTM

-II laser technology providing a repetition rate of 1000

Hz for a maximum performance and guaranteed spatial resolution down to 50 µm for

linear and reflector analysis both in positive and negative modes. Our service model

envisages access to the technology for internal and external users by the

straightforward 'sample in - data out' approach. The MALDI-imaging workflow will

be executed by our specialists and encompasses sectioning, tissue and requirement-

specific sample processing, matrix application by ImagePrep® sample preparation

device (Bruker Daltonik), measurement, staining and data analysis in a user-tailored

fashion. Complimentary data analysis and evaluation can be achieved by specialized

and up-to-date developed computational methods provided by long-standing experts in

the analysis of MALDI-imaging data.

Here, we aim to introduce the MALDI-imaging/ MULTI-ARRAY Core Facility and

the service we are providing. We present the organization structure and the

instrumentation of the Core Facility. The power of the MALDI-Imaging technology

will be demonstrated exemplified by own executed tissue samples pointing out the

importance of data analysis methods for improved information extraction from

MALDI-imaging datasets using our own computational pipelines.


114

Glycohistochemical characterization of the avian inner perivitelline

layer Rodler D.

1, Sinowatz F.

1

1Ludwig-Maximilans-University, Department of Veterinary Sciences, Munich,

Germany

The avian inner perivitelline layer (IPVL), is a homologous structure to the

mammalian zona pellucida. Is is deposited between the granulosa cells and the huge

polylecithal oocyte during folliculogenesis. In our study, a panel of FITC-labelled

lectins was used to characterize and localize the oligosaccharide sequences of the

IPVL glycoproteins at different stages of follicular development in the ovary of two

bird species, the small quail (Coturnix japonica) and the huge ostrich (Struthio

camelus). Control reactions using competivite inhibition with the corresponding

sugars, deacetylation and sialidase digestion were also performed. Contrary to

mammals, where the topographical distribution of the investigated carbohydrate

residues is not uniform throughout the zona pellucida, indicating the regionalization of

oligosaccharide chains, we found a homogenous lectin staining of the comparatively

thin IPVL of birds. In the quail, variations in the presence and distribution of the

carbohydrate residues in the IPVL during different stages of follicular growth could be

demonstrated. The IPVL of previtelline follicles distinctly stained with WGA, sWGA

and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α−D-GalNac in the

glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-

D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc), VAA

(Gal), DBA (α-DGAlNAc (1-3)-GalNAc) and UEA1 (α-L-Fuc). With continuing

follicular growth of the oocyte and the follicle, this staining pattern changed. PNA-

and LCA-staining in the IPVL now became distinctly positive. The IPVL of the ostrich

(Struthio camelus) showed a much simpler staining pattern. During oocyte growth, its

IPVL which covers the huge oocyte was only distinctly labelled with WGA and

sWGA, whereas all other lectins are only weakly bound to the IPVL or were

completely negative. The differences in the lectin staining pattern between these

different avian species are shortly discussed.


115

Thyroid gland-commonness of hyperthyreodism and

hypothyreodism in the female and male population Salovska J.

1, biochemistry

1Institute of Biology, Faculty of Natural Sciences and Mathematics, Biology, skopje,

Macedonia, the Former Yugoslav Republic of

Introduction: The hormones of the thyroid gland are essential for normal growth and

development of the organism. In order to keep the metabolic processes in normal, the

thyroid hormones must be constantly secreted in the proper amount. The reduced or

excessive secretion of the thyroid hormones in the adult population causes serious

problems. The diseases which develop on the thyroid gland due to lack or excessive

secretion of the thyroid hormones are hyperthyreodism and hypothyreodism.

Hyperthyreodism appears as a result of hyperactivity of the thyroid gland, i.e.

excessive secretion of hormones in the gland , and their release in the bloodstream ,

and they appear during the maturity, and more in the woman population.

Hypothyreodism is defined as lack of biologically active hormones in the tissue or

incapability of the tissues to use the thyroid hormones, condition which is due to

subnormal concentration of thyroid hormones in the blood, it appears mostly due to

lack of iodine in the food.

Materials and methods: For the analysis of the serum at the patients, the apparatus

VIDAS had been used, which is automatic analysis for the Vidas system, which

enables for the human serum to be quantitatively measured, and the principle of

analysis combines the method of enzyme imuno-analysis and final fluorescent

detection, for the detection of the free FT3 and FT4, and for the detection of the human

thyrotropin in the human serum TSH, by using the ELFA technique (Enzyme Linked

Fluorescent Assay). The serum of 220 patients from Gostivar and the region has been

divided in 6 age groups: ≤ 20 years, 21-31 years, 31-40 years, 41-50 years, 51-60 years

and above 60 years.

Discussion: Out of 220 patients taken for analysis, 26 are male, while 194 are females

divided by age, and of all 100 % of the patients taken for analysis, 5.6 % are patients

with Hyperthyreodism, 10.4 % are patients with Hypothyreodism and 84 % of the

patients are in good health condition.


116

Through the aspect of sex, hypothyreodism is the most common among the female

population with 88 %, while males have 12 %, and as for the hyperthyreodism it is the

most common in the female population with 79 percent while the male has only 21 %.

According to the results from 250 patient, we can see that 79 % are female patients

with hyperthyreodism in the age of 41-50 years, while 11 % are males in the age of 51-

60.

On the other hand, 88 % of the female patients between the age 41-50 have

hypothyreodism, while males are 12 % in the age 41-60.

Conclusion

1) The most common population with hypothyreodism is the female with 88.46 %

2) The most common population with hyperthyreodism is the female with 78.58%

MALDI Imaging as a novel approach for the systematical study of

intratumoral heterogeneity Schöne C.

1, Rauser S.

1, Balluff B.

1, Elsner M.

1, Meding S.

1, Feuchtinger A.

1, Schmitt

M.2, Aubele M.

1, Walch A.

1

1Helmholtz Center Munich, Neuherberg, Germany,

2Klinikum rechts der Isar der TU

München, Munich, Germany

Aims: Tumors are complex. Cancer cells within a tumor may vary in their protein

expression and their therapy response. While this intratumoral heterogeneity is well

known, its systematic analysis has proven to be difficult so far. Here we want to

present MALDI Imaging as an innovative tool for the analysis of this phenomenon. Its

capability to perform label-free, multimodal proteomic measurements, while retaining

the morphoplogical information of the tissue, makes it very well suited to tackle this

problem.

Methods: A total of 40 invasive-ductal breast cancer samples were measured using

MALDI Imaging. Tumor specific proteomic profiles where then selected by virtual

microdissection. After selection hierarchical clustering was applied to these spectra, to

discern internal differences between the tumor cell proteomes. The resulting clusters

were then superimposed onto the histological staining of the respective tissue in order

to correlate them with the morphological information of the tumors.


117

Results: Using this approach we were able to identify intratumoral heterogeneity of

varying degrees in about 20% of the analyzed samples. In addition, we could perform

comparisons between the proteomes of the different cancer cell populations allowing

us to find masses with expression levels varying between these populations that might

be of special interest for identification.

Conclusion: We could show that MALDI Imaging is a method very well suited for the

study of intratumoral heterogeneity. It allows the combination of proteomic

measurements with morphological analysis. This could allow the systematical study of

intratumoral heterogeneity and the gain further insights in the clonal variation within

cancers.

Immunohistochemical localization of transit cells, putative stem

cells and stem cells in benign prostate hyperplasia (BPH) and adenocarcinoma of the prostate Sinowatz F.

1, Albanaw A.

2, Rodler D.

1, Kenngott R.

3

1Ludwig-Maximilians-University, Department of Veterinary Sciences, Munich,

Germany, 2Kuwait University, Department of Medical Laboratory Sciences, Faculty of

Allied Health, Kuwait, Kuwait, 3Ludwig-Maximilians-University, Department of

Veterinary Science, Munich, Germany

Understanding the processes leading to benign and malign prostate tumour is of great

importance for the assessment of disease progression and the choice of treatment.

Recent gene expression studies have demonstrated that processes that regulate the

development of the prostate gland are also relevant for prostatic tumours. The

transcription factor SOX9 has been shown to be essential for normal prostate

development. Oct3/4 also plays a role during embryogenesis of the human prostate. In

our investigation, we studied the expression of SOX9 and Oct3/4 in 20 cases of BPH

and 36 cases of prostatic carcinoma of different Gleason grades using

immunohistochemistry. Additionally the localization of keratin 14 (marker for

“putative stem cells) and keratin 18 (marker for “transit cells”) were investigated. Our

results show that in BPH and prostate cancer a nuclear localization of Oct3/4 and

SOX9 was found. Using keratin-immunophenotyping we could demonstrate a


118

significant number of transitional cells, both in BPH and malignant prostate tumours.

If these results could indicate an etiological relationship between the two diseases

appears possible, but certainly needs more extensive studies.

Cell adhesion molecules in melanocytic tumors Stoemmer P.

1, Torres-Galea P.

1

1Forschungslabor und Gemeinschaftspraxis Pathologie, Augsburg, Germany

Cell adhesion molecules are very important for the intercellular connections, the

resulting architecture and the biological behaviour of tumours in general and

particulary in melanocytic proliferations.They are the basis of the functional

communications with other epithelial and mesenchymal cells.

In contrast to other proteins, usually tested in malignant and not-so-malignant

melanocytic tumours such as S-100, HMB-45 or MALT-1, their expression is not a

feature of melanoma or naevus in general, but an affair of a peculiar cell in a specific

surrounding, and thus more a functional than a histogenic marker in the tumour.

Therefore we found great local differences of their expression in the various

melanocytic tumours.

Materials and methods: we analysed 10 formalin-fixed paraffin-embedded (FFPE)

tissue sections of each junctional, compound and dermal nevi, blue nevi and different

melanomas (SSM, LM and NMM and our sole case of nevoid fungating melanoma) in

respect of the expression and location of E-, N-, P-cadherin, alpha- and beta-catenin

and H-CAM/CD44v. We tested FFPE blocks with different antibodies in a qualitative

and semiquantitative evaluation of their expression in respect of the architectural

structure in the tumours.

Results and discussion: Cell adhesion molecules seem to be of great importance for

the connection of cells in their environment. They contribute to the architecture of a

tumour and by shifting from one type of CAM to another, the tumour changes not only

the morphological appearance, but also -probably and in concert with other factors- its

ability for infiltrative growth pattern and formation of metastases.


119

According to conventional ideas of nevi, intraepidermal nevomelanocytes may

proliferate, penetrate the basal lamina and invade the dermis; the interaction with this

microenvironment drives them to maturation and senscence. Melanocytes type A have

E-cadherin, beta-catenin and CD44v as main adhesion complex molecules; no N- and

P-cadherin. Type B-melanocytes lose their E-cadherin without expriming N-cadherin

and blue nevi have never E-cadherin, only N-cadherin in their type C melanocytes.

In malignant melanomas and their precursors there is a shift from E-cadherin to N- and

P-cadherin at first, in the in situ tumour (cells resembling type A melanocytes) there is

a coexpression of E-cadherin and N-cadherin; in massively invasive cells

(monocellular infiltration, invasionfront, spindle cells) E-cadherin is no more

exprimed but N- and P-cadherin; DC44v surrounds only the mesenchymal border of

tumor complexes. This may enable these tumour cells for the better invasive growth

and metastasis.

Nuclear lamina in organization of DNA replication. Strelkova O.S.

1, Dianova V.D.

1, Kurchashova S.Y.

1, Abramchuk S.S.

2, Alieva I.B.

1,3,

Kireev I.I.1,4

1A.N.Belozersky Inst. of Phys-Chem Biology, Moscow, Russian Federation,

2Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,

Moscow, Russian Federation, 3People's Friendship University of Russia, Department

of Histology, Сytology and Embryology, Moscow, Russian Federation, 4Moscow State

University, Dept. of cell biology and Histology, School of Biology, Moscow, Russian

Federation

Nuclear lamina is a main structural component of nuclear skeleton, which provides

mechanical rigidity of the cell nucleus. It also takes part in the chromosome

organization by linking special chromosome loci to nuclear lamina. Association of

chromosomes with nuclear lamina is correlated with transcription activity and

replication timing, and lamina might plays an important role in regulation of this

processes. On he other hand, tight connection of late-replicationg heterochromatin to

lamina may create additional topological constrains during replication. It can be


120

overcome by temporally dissociating replicating chromatin from lamina. We studied

correlation between spatial lamina organization and replication status of preipheral

heterochromatin using several expreimental models. First, we analysed distribution of

replicating chromatin loci relative to nuclear lamina at ultrastructural level in cell lines

expressing GFP-PCNA (Leonhard et al, 2000). Asynchronous cell culture was labeled

with mouse-anti GFP Abs, which were detected with Nanogold-coupled secondary

Abs and silver enhancement. Cells with replication pattern 3 were selected under

bright-field microscope and sectioned. We found that in many cases peripherally

located labeled replication sites moved away from nuclear lamina to a distance of 0.2-

0.3 mkm. Interestingly, this detachment was observed mainly when the label

concentrated on the distal part of replicating chromatin domain facing nuclear lamina.

Next, we studied intranuclear position of large artificial heterochromatic locus that

demonstartes orderly relocalization from its peripheral position in G1 to more central

location just before and during its replication (Li et al, 1998). Surprisingly, we found

that this locus never lost its association with nuclear lamina even during replication.

Rather, movement of the locus towards the central nuclear region was accompanied by

formation of deep intranuclear invaginations of nuclear envelope. These invaginations

always contained lamins A and B. Apparently, we failed to observe complete

dissociation of this locus from lamina during replication due to its complex replicative

organization, where individual replicative clusters fire asynchronously. Nevertheless,

our studies demonstrate dynamics of chromatin-nuclear envelope interactions and high

plasticity of nuclear lamina during replication.

This work was supported in part by RFBR (Grants No. 09-04-01352-а to I.I.K, 09-04-

00363-а to I.B.A.)


121

In-tissue lipid profiling by mass spectrometry imaging Strohalm M.

1, Volny M.

1, Faltyskova H.

1, Pol J.

1, Hulkova H.

2, Novak P.

1, Havlicek

V.1

1Institute of Microbiology, v.v.i., Laboratory of Molecular Structure Characterization,

Prague, Czech Republic, 2Institute of Inherited Metabolic Disorders, Charles

University, 1st Faculty of Medicine and Teaching Hospital, Prague, Czech Republic

There are many diseases known to be related to altered metabolism of different

species. To better understand physiology and biochemistry of the disease, information

about the spatial distribution of selected metabolites is often very important. In

addition to traditional histological techniques, recent development in mass

spectrometry has enabled a new approach called mass spectrometry imaging (MSI).

Using this method, metabolites can be identified and visualized based on the exact

mass, which allows us to monitor different species separately, whenever their masses

are not equal. Here, we provide an overview of a method for lipid identification in

MSI experiments. Matrix-assisted laser desorption/ionization (MALDI) in

combination with Fourier transform ion cyclotron resonance mass spectrometry

(FTICR MS) was utilized to determine the accurate mass and spatial distribution of

lipids in various tissue samples. Several software were involved in subsequent data

analyses. An average mass spectrum was generated for each region of interest using

in-house made software and analyzed by an open source program mMass

(www.mmass.org). This program enables fast and easy way to identify different lipids,

as well as the corresponding adducts, by comparison with publicly accessible Lipid

Maps database (www.lipidmaps.org). Detailed information about particular lipid can

be accessed from generated report, providing direct link to the Lipid Maps server.

Finally, identified lipids were visualized by FlexImaging software (Bruker Daltonics,

Germany) and combined with the results obtained by traditional histological staining.


122

Spatial proteomics: A new LC-MS/MS tissue imaging workflow

providing protein identities and their distribution in tissue Schürenberg M.

1, Lübbert C.

1, Becker M.

2, Paape R.

1, Suckau D.

1

1Bruker Daltonik GmbH, EAppM, Bremen, Germany,

2Bruker Daltonik GmbH,

MALDI Imaging Applications, Bremen, Germany

Background: MALDI-Imaging of undigested proteins in tissue sections has

established itself as a powerful new approach to biomarker discovery and

histopathological research in recent years. Digestion, on the other hand, increases the

sample complexity significantly and caused direct MS/MS identification from digested

tissue to fail providing reasonable protein coverage and more than a few identifications

of high abundant proteins. Here we introduce a novel proteomics technology that

combines the spatial information with the routine identification of proteins from tissue

sections without the need of MS/MS analysis from the tissue.

Methods: Highly resolved protein digests were generated by applying trypsin onto

two subsequent tissue sections by supersonic nebulization. One of the sections was

coated with DHB matrix and analyzed by MALDI imaging mass spectrometry at 50

µm spatial resolution yielding a list of 200-800 peptide signals per image.

Peptides were extracted from the other sections entire surface and submitted to

standard proteome analysis using LC-MALDI-TOF/TOF identification. The identified

peptide list is then matched to the peaklist from the image by newly developed

software tools. All matching peaks in the image can then be assigned to a protein and

the co-localization of 2 or more tryptic peptides typically confirms their protein

association.

Results: We analyzed rat brain and testis/epididymis using the new Spatial Proteomics

approach typically yielding peptide distributions at the 50-100 µm level. In both tissue

types approx. 80% of all peaks visible in the image matched to peptides identified in

LC-MS/MS. These peptides belong to ~100 different proteins. More than 20 proteins

showed co-localization of >2 IDed peptides. The developed methods and software

tools are available from the authors.

Conclusion: Simultaneous determination of protein distribution and identity was

achieved in a combined bottom-up imaging-LC-MALDI workflow for the first time


123

for ~100 proteins. In addition, the method has great potential for the MALDI imaging

analysis of FFPE tissue or membrane proteins. In this work we confirmed protein

identifications from previous top-down work on rat testis1 (e.g. b thymosins and Long

Chain fatty acid CoA ligase(19-48), at ~ 50 vs. 20 µm spatial resolution.

References:

1. Revisiting Rat Spermatogenesis with MALDI Imaging at 20-µm Resolution.

Lagarrigue M et al. MCP. 2011 Epub 2010 Dec 12.

MRI molecular imaging with targeted albumin-based nanoparticles: conceptual design strategies to create the Magic

Bullet Thurner G.C.

1, Wallnöfer E.A.

1, Rohr I.

2, Talasz H.

3, Kremser C.

1, Abdelmoez A.A.

2,4,


5, Matuszczak B.

6, Jaschke W.

1, Debbage P.

2


2Medical

University Innsbruck, Department of Anatomy, Histology & Embryology, Innsbruck,


Innsbruck, Austria, 4Assiut University, Department of Pharmaceutical Organic

Chemistry, Assiut, Egypt, 5Medical University Innsbruck, Central Animal Research

Facility, Innsbruck, Austria, 6University of Innsbruck, Institute of Pharmaceutical

Chemistry, Innsbruck, Austria

Introduction: Paul Ehrlich in 1900 [1] described the idea of a targeted vehicle

carrying a drug. His notion of a „Magic Bullet“ changed the whole concept of medical

treatment. Richard Feynmann in 1959 [2] challenged scientists to create

nanomachines. Since then Nanomedicine has promised improved drug delivery,

reduced dosages and side-effects: "personalized medicine". Why has Nanomedicine

not delivered on this promise? Two major challenges hinder realisation of this dream:

1. identification of suitable biomarkers; 2. poor availability of nanoparticles capable of

homing to biomarker molecules hiding behind intact tissue barriers. We review the

reasons for these difficulties and suggest some approaches to overcome them.

Materials and methods: Albumin-based nanoparticles bearing gadolinium were

developed and extensively characterised. The lectin LEA was attached to the particles


124

to target oligolactosamines. During MR Imaging of living rats both imaging and

quantitative approaches were applied. Coordinated with the MRI, chemical and

immunohistochemical analyses tracked components of the nanoparticles in

longitudinal time series after injection, 15 minutes to 6 weeks, obtaining large runs of

quantitative data.

Results: Our nanoparticles were ~30 nm diameter. They were pure, contained no

starting materials and had good imaging properties with relaxivities ~1 • 107 1/Ms.

They were stable in various in vitro testings though not in SDS-gel-electrophoresis. In

haemagglutination tests they agglutinated red blood cells; after intravenous injection

into living rats they gave high-resolution MR imaging of the vascular wall lasting >2

hours. The numbers of (lectin) targeting groups required for molecular imaging and of

gadolinium ions per nanoparticle necessary for high-resolution MRI were assayed in

relation to particle size and type of crosslinking.

Discussion: We determined critical parameters for MR Molecular Imaging by use of

nanoparticles. These particles require a second type of targeting group to migrate

across vascular walls and access subendothelial interstitial compartments, for

Molecular Imaging and Molecular Targeting of disease sites behind intact tissue

barriers. The concept of multiple targeting is new in Nanomedicine and represents the

hurdle that limits present-day techniques [3]. Assuming that quantitative aspects of

targeting will be similar for each of the multiple targeting groups, we already know

how to design nanoparticles for targeting both drugs and contrast agents to disease

lesions hiding behind blood-tissue barriers. Multiply-targeted nanoparticles are

Ehrlich´s "magic bullets".

Acknowledgements: Austrian Nano-Initiative (Project N201-NAN); Austrian

National Bank Jubilee Program (Projects 9273, 10844, 11574)

References:

1. Ehrlich, P. (1900) Proc R Soc Lond 66: 424 - 448.

2. Feynman RP. (1959) Miniaturization. Horace D. Gilbert, Ed. © Van Nostrand

Reinhold. New York

3. Debbage P., Thurner GC. (2010) Pharmaceuticals 3: 3371 - 3416


125

EGFR, HER2 and HER3 expression and dimerization in esophageal

cancer Timme S.

1, Fichter C.D.

1, Schoepflin A.

1, Bogatyreva L.

2, Hauschke D.

2, Tang L.

3,

Klimstra D.3, Opitz O.G.

4, Werner M.

1, Lassmann S.

1

1University medical Center Freiburg, Institute of Pathology, Freiburg, Germany,

2University medical Center Freiburg, Institute of Medical Biometry and Medical

Informatics, Freiburg, Germany, 3Memorial Sloan Kettering Cancer Center New York,

Dept. of Pathology, New York, United States, 4University medical Center Freiburg,

Tumorzentrum Ludwig Heilmeyer - Comprehensive Cancer Center, Freiburg,

Germany

Text:

Introduction: Receptor tyrosine kinases (EGFR, HER2, HER3) are therapeutic targets

in epithelial tumors, whose distinct expression and/or dimerization may affect

therapeutic responses. Here, we examined EGFR, HER2 and HER3 protein expression

and dimerization in esophageal squamous cell carcinoma (ESCC) and Barrett's

adenocarcinoma (BAC).

Materials+Methods: Serial tissue sections of 110 pre-treatment biopsies of ESCCs

and BACs from three centers were examined for EGFR/HER2/HER3 expression by

immunohistochemistry (IHC) according to routine diagnostics. HER2/HER3 double

immunofluorescence (dIF) and proximity ligation assay (PLA) was performed in

selected BAC biopsies. For evaluation of dIF and PLA, three-dimensional image

stacks were recorded on a fluorescence microscope. Statistical univariate analyses

were performed by SPSS v18.

Results: In tissue specimens, HER2 (18/61, 30%; p< 0.001) and HER3 (16/61, 26%;

p< 0.001) were significantly overexpressed in BACs. EGFR expression was found in

4/61 (6.5%) BACs and in 7/49 (14%) ESCC (p=0.088). EGFR (p=0.350), HER2

(p=0.224) and HER3 (p=0.194) expression was not associated with histologic grading.

By PLA, we found that HER2 and HER3 heterodimers do appear in invasive tumors

cells of BACs expressing high levels of HER2 and HER3. In contrast, a substantial

number of ESCCs and BACs „over“express only EGFR or HER2, suggesting presence

of EGFR homodimers in ESCCs particularly.


126

Conclusions: Together, this may contribute to differentially regulated signalling

events and effects on tumour progression in ESCCs and BACs, or subgroups thereof.

In view of current receptor tyrosine kinase targeted therapies, inhibition of EGFR

and/or HER2 may represent a therapeutic option for ESCC and BACs, respectively.

HER3 appears to be the predominant interaction partner of HER2 in BACs,

presumably affecting also therapeutic responses.

Support: Deutsche Forschungsgemeinschaft DFG CRC850 project C5, and in part

Mushett Family Foundation, Chester, NJ, US

Software for 3D MALDI imaging data: Visualization and 3D spatial

segmentation Trede D.

1, Schiffler S.

1, Becker M.

2, Wirtz S.

3, Strehlow J.

3, Kobarg J.H.

4, Aichler

M.5, Heldmann S.

6, Walch A.

5, Thiele H.

2, Maass P.

4, Alexandrov T.

4

1Steinbeis Innovation Center SCiLS (Scientific Computing in Life Sciences), Bremen,

Germany, 2Bruker Daltonik GmbH, Bremen, Germany,

3Fraunhofer MEVIS, Institute

for Medical Image Computing, Bremen, Germany, 4University of Bremen, Zentrum für

Technomathematik, Bremen, Germany, 5Helmholtz Center Munich, Institute of

Pathology, Munich, Germany, 6Fraunhofer MEVIS, Institute for Medical Image

Computing, Lübeck, Germany

In the last decade, matrix-assisted laser desorption/ionization (MALDI) imaging mass

spectrometry, also called MALDI-imaging, has proven its potential for proteomics

analysis of 2D samples and was successfully applied for histological analysis of thin

tissue slices. MALDI-imaging is used as a general analytic tool revealing the

functional proteomic structure of tissue slices, and as a discovery tool in detecting new

cancer biomarkers discriminating an annotated tumor region. Recently, a technological

breakthrough has been reported when a spatially 3D model corresponding to one m/z-

value was reconstructed from consecutive slices each measured with MALDI-imaging.

Certainly, 3D MALDI-imaging has immense potential for representing not only one

slice through the organ but showing the complete 3D model which can be virtually

sliced in any direction. However, the progress stopped after a proof-of-principle

demonstration, since 3D MALDI-imaging requires not only much measurement time


127

but a bunch of efficient and user-friendly computational algorithms specifically

developed for 3D MALDI-imaging.

Here, we present for the first time our recently developed software SCiLS Data

Explorer for import, construction, visualization, and analysis of 3D MALDI-imaging

data. It takes several 2D MALDI-imaging datasets as an input, each dataset

corresponding to a consecutive slice. Then, it creates a 3D cloud of measurement

points in the 3D Cartesian space with coordinates (x,y,z), with a spectrum assigned to

each point. We demonstrate how this huge dataset (approximately 500,000 points with

each spectrum of 10,000 m/z bins are considered) can be visualized given a specific

m/z-value, representing 3D distribution of a compound with this m/z-value. Moreover,

we illustrate how mass spectra pre-processing (baseline correction, normalization,

image denoising) improves the visualization.

Spatial segmentation of 2D MALDI-imaging data has been introduced several years

ago and is currently a well-accepted computational method in the MALDI-imaging

field. It splits a sample into regions of similar chemical composition, clustering the

spectra, and representing the clustering results with a spatial segmentation map. One

color in this map represents a region with similar mass spectra. A spatial segmentation

map can be explored and interpreted from the mass spectrometry viewpoint providing

a unique approach to fast and sensitive analysis of complex MALDI-imaging data.

In this poster, we present for the first time the 3D spatial segmentation of a real-life 3D

MALDI-imaging data. The spatial segmentation tool is of high importance for any

analysis of 3D data, because a manual annotation in 3D space can hardly be done. Our

software makes 3D histological analysis possible by automatic selection of 3D regions

of interest which can be then manually assigned to histological tissue classes.

The project is funded by the Bremen Economic Development foundation (WFB).


128

Zonal distribution of CD105+/CD166+ cells in growing rat

humerus proximal epiphyseal cartilage Ustunel I.

1, Ozbey O.

1, Acar N.

1

1Akdeniz University, School of Medicine, Histology and Embryology, Antalya, Turkey

Objectives: The coexpression of cell surface receptors, CD105 and CD166, are

characteristic of mesenchymal stem cells in cartilage. However, there is limited data

regarding their immunolocalization in the cartilage of growing rat epiphyseal cartilage.

The purpose of this study was to determine the presence of CD105+/CD166+ cells in

the proximal epiphyseal cartilage of developing rat humerus and specify their zonal

distribution with age.

Materials and methods: The tissues of rat humerus were taken on embryonic day 15

(E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and

we detected the immunolocalization of CD105+/CD166+ cells.

Results: Our results showed that CD105+/CD166+ cells were scattered in early stages

of development in humerus epiphysis. We observed that CD105+/CD166+ cells were

only in the hypertrophic zone on day E15, predominantly in the resting and

hypertrophic zones of the epiphysis cartilage on E19, whereas we observed

CD105+/CD166+ cells only in hypertrophic zone on PN10 and PN20.

Conclusion: We observed CD105+/CD166+ cells predominantly in the resting zone

and hypertrophic cells of the epiphyseal cartilage. These results may show the

presence of stem cell-like cells in the epiphyseal cartilage and they may have an

important role in vitality, cell-matrix interaction, migration, angiogenesis, growth and

differentiation of epiphyseal cartilage.

Key words: Cartilage, CD105, CD166, Differentiation


129

Is CRIP1 a target gene of HER2? Weber E.

1, Rauser S.

2, Mylonas I.

1, Walch A.

2, Brüning A.

1

1University Hospital Munich, OB-Gyn, Munich, Germany,

2Helmholtz Zentrum

München, Institute of Pathology, Neuherberg, Germany

Aims: It has recently been observed that HER2-overexpressing breast cancer tissues

display elevated levels of CRIP1 (cysteine-rich intestinal protein 1). CRIP1 is known

to modulate cytokine expression and might thus play a role in cancer progression,

activated by HER2. We have investigated the relation between CRIP1 and HER2 at

the cellular level.

Methods: Protein expression was analyzed by FACScan and immunohistochemical

analysis. Human CRIP1 was cloned from its cDNA and transferred into a mammalian

expression vector for functional analysis.

Results: Although some HER2-overexpressing breast cancer cells revealed elevated

CRIP1 levels, no association between HER2 expression and CRIP1 expression could

be observed when a larger number of breast cancer cells was tested (n = 8). Further,

neither heregulin nor lapatinib modified CRIP1 expression in breast cancer cells.

However, CRIP1 expression varied strongly among the breast cancer cells tested,

indicating a possible role for CRIP1 in cancer progression. Overexpression of

exogenous V5-tagged CRIP1 in breast cancer cells revealed Golgi localization and

secretion of CRIP1, indicating either a paracrine or autocrine function in cancer

tissues.

Conclusions: CRIP1 expression is not associated with HER2 expression, but appears

to play an individual role in breast cancer progression.


130

Class I HDACs in the developing heart and limb of chicken embryos Aichinger C.

1, Engelmaier C.

1, Murko C.

1, Schöfer C.

1, Weipoltshammer K.

1

1Medical University of Vienna, Dept. for Cell and Developmental Biology, Vienna,

Austria

Epigenetic mechanisms are important regulators of cellular and developmental

differentiation. It has been shown that histone deacetylases are relevant factors in

epigenetic regulation exerting their influence by modification of histones on the

compaction state and thus accessibility of chromatin. Class I HDACs were previously

thought to be ubiquitously expressed. However, genetic deletion of class I HDACs in

mice led to malformations in different organ systems including the heart, central

nervous system, liver and lung (Haberland et al. 2009). We mapped the class I HDAC

expression in mice and chicken embryos (Murko et al. 2010) and found a distinct

spatio-temporal expression pattern. In the chicken limb bud strong class I HDAC

expression was seen, whereas the expression in the heart was surprisingly low

concerning the fact that HDAC k.o. mice displayed severe cardiac malformations.

Therefore we decided to study the effects of class I HDAC down regulation in these

organs in the chicken model system which allows the controlled spatio-temporal

application of HDAC-inhibiting drugs by in ovo manipulation. We either implanted

beads soaked with trichostatin A (TSA) or directly injected TSA into wing buds and

into the pericardial cavity. Subsequently the embryos were reincubated and evaluated

according to the following criteria: external anatomy (whole mount staining),

morphometry (serial sections), proliferation and apoptosis (in situ assays).

After TSA application a mild retardation of limb development was observed. When

the beads were placed in the site of the presumptive zeugopodium region of limb buds

a significant reduction in tibia size at later stages could be demonstrated by

morphometric analysis.

In the heart, we observed dramatic phenotypic effects mostly showing an aberrant ratio

of atrial and ventricular muscular mass. Here the preferential abnormality was atrial

hypertrophy. In both cases, we did not observe major changes in cell proliferation rate

and rate of apoptosis after TSA treatment.


131

Taken together we could show that class I HDACs play important roles in the

development of chicken embryos. In case of limb development our results suggest an

effect of HDACs in skeletal development possibly by regulation of the chondrocyte

differentiation pathway. In the heart our data indicate an important role of HDACs in

chicken myocyte differentiation.

References:

Haberland M, Montgomery RL, Olson EN (2009) Nat Rev Genet 10:32-42

Murko C, Lagger S, Steiner M, Seiser C, Schoefer C, Pusch O (2010) Int J Dev Biol

54:1527-1537

The first two authors contributed equally.

MALDI-Imaging of frozen prostate tissue micro arrays Wurlitzer M.

1, Koop C.K.

2, Becker M.

3, Thiele H.

4, Maaß P.

4, Minner S.

1, Stahl P.

1,

Schlüter H.5, Sauter G.1

1University Medical Center Hamburg-Eppendorf, Inst. f. Pathology, Hamburg,

Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany,

3Bruker Daltonik GmbH, Bremen, Germany,

4Universität Bremen, Zentrum für

Technomathematik, Bremen, Germany, 5University Medical Center Hamburg-

Eppendorf, Inst. f. Clinical Chemistry, Hamburg, Germany

In cancer research, the elucidation of new diagnostic or prognostic markers for the

detection of malignant cells in biopsies and tissue sections is an important aim. Mass

Spectral Imaging (MSI) yields information about the spatial distribution of a huge

number of biomolecules in a single experiment and therefore is a helpful tool for

biomarker discovery. MSI is especially interesting for the analysis of tissue

microarrays (TMAs), since a large number of patient samples can be analysed rapidly

under identical analytical conditions [1]. A single TMA section allows the analyses of

several dozen different tumors and control tissues at once. Formalin-fixed paraffin-

embedded (FFPE) TMAs are widely available, but complicate MSI analysis because of

the formalin induced protein crosslinking. In addition, biomarkers found in FFPE

tissue samples will likely prove difficult to translate into assays based on fresh


132

biobsies. Therefore, in this study we investigated the analysis of TMAs from fresh-

frozen tissue by MALDI Imaging, focusing on peptides and proteins. To perform a

heterogeneity analysis we constructed a TMA consisting of 34 samples from different

areas of one prostate cancer biopsy. In addition, seven samples from associated lymph

node metastases were included in the TMA. For MALDI Imaging analysis, the TMA

sections were coated with sinapinic acid matrix deposition using an ImagePrep device

(Bruker Daltonik GmbH). MALDI Imaging analysis of the TMAs was performed

using an Autoflex Speed MALDI-TOF system (Bruker Daltonik GmbH). The mass

spectrometric data was processed and interpreted with flexImaging and ClinProTools

software (Bruker Daltonik GmbH). 136 signals ranging from m/z 2000 to m/z 20000

were detected in the TMA. We could elucidate one specific cancer associated signal

which was present in only a few primary tumor samples but in several lymph node

metastases. In conclusion we were able to show that MALDI Imaging from frozen

TMAs is a promising approach for searching for markers of (prostate) cancer.

Reference:

[1] Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S,

Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-

throughput molecular profiling of tumor specimens, Nature Medicine 1998, 4:844-847.

General Information

133

General Information

Organizer


Congress Venues

Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe

Klinikum der Universität München Maistraße 11

80337 München

Helmholtz Zentrum München

Ingolstädter Landstr 1

85764 Neuherberg

Congress Language

The official congress language is English.

Registration Desk and Congress Office

You will find the registration desk on E0.94C / Stair 6. Please follow the signage on-

site.

Opening hours: Wednesday, 12 October, 2011: 14:00 – 18:30

Thursday, 13 October, 2011: 07:30 – 18:00

Friday, 14 October, 2011: 08:00 – 13:30

Saturday, 15 October, 2011: 08:30 – 16:00

Name Badges

Every registered participant will receive a name badge upon registration. It is

necessary to wear this to gain access to the scientific program and coffee / lunch

breaks.

Coffee / Lunch

Coffee and lunch are included in the registration fee. They will be served of the wing

ends of the congress area.

General Information

134

Mobile Phones

Participants are kindly requested to keep their mobile phones in the off position in the

meeting rooms, as well as during the poster session.

Media Check

Speakers will find the media check in the Main Lecture Hall. Presenters are requested

to provide their talk on an USB stick in advance (at least 2 hours before their talk or

the evening before).

Awards

The Poster Award winners are determined by a scientific committee and will be

announced at the closing plenary session.

The Young Histochemist Travel Awards will be announced at the oping plenary

session on Wednesday and they have the honour to present the contents of the

submitted abstracts at the Free topics communication session on Saturday.

Instructions for Speakers

To ensure the congress runs smoothly and fairly for all participants please don’t allow

your presentation to run overtime. The chairs are instructed to strictly monitor the time

and to interrupt the presentation if the speaking time is exceeded.

The only multimedia technology accepted is Microsoft PowerPoint. The operating

system is Microsoft Windows XP. No support is provided for overhead or slide

projectors. The conference room is equipped with laptop and data projector. Using

your own laptop is not recommended.

Smoking Policy

The symposium is a non-smoking event. Participants are kindly requested to refrain

from smoking in the congress venue, including the exhibition area.

General Information

135

Please notice the obligatory additional registration for the following workshops:

Industry Workshop (Carl Zeiss NTS GmbH):

Introduction to Energy Filtering TEM" - Live practical session on the microscope

Guided Tour at Helmholtz Zentrum München

Industry Hands-On Workshop (Intas Science Imaging & Huron Technologies):

Laser Confocal Slide Scanning System

Scan your small or big Tissue section with the unique

TissueScope4000 - Laser Confocal Slide Scanning System.

Slide Dimensions: 25x75mm up to 150x200mm

Immunhistochemistry or Fluorescence probes can be scanned in that workshop.

Access to Venues

136

Access to Venues

Frauenklinik Maistraße

Access to Venues

137

Helmholtz Zentrum München

Introduction to the Helmholtz Zentrum München and Meeting Point

(Lecture room 052 / Building 57) – (green)

Guided Tour at the Helmholtz Zentrum München

- GMC: German Mouse Clinic – (orange)

- GAC: Genom Analysis Center – (pink)

- AMSD: Research Unit Medical Radiation Physics and Diagnostics – (pink)

Industry Workshop presented by Carl Zeiss NTS GmbH (Building 37) –

(red)

Access to Venues

138

Copyright Open Street Map

OpenStreetMap is open data, licensed under the Creative Commons Attribution-ShareAlike 2.0 licence (CC-BY-SA).

Frauenklinik

Ratskeller

http://creativecommons.org/licenses/by-sa/2.0/

Social Events 139

Social Events

Opening Plenary and Welcome Reception

Date Wednesday, October 12, 2011

Time Welcome Drink 17:30; Get Together 19:45

Location Frauenklinik Maistraße 11

Symposium Banquet

Date Friday, October 14, 2011

Time 19:00

Location Ratskeller, Marienplatz, located at the Rathaus (“City Hall”)

We would like to invite all participants to

attend the Symposium Banquet. Join us and

all the other delegates from around the

world on this evening for networking within

the Histochemistry Community.

A Welcome Drink at 17:30 will be offered

to delegates and registered accompanying

persons at the exhibition area.

A Get Together directly following to the

Robert Feulgen Lecture will be offered.

All participants are invited to attend the

Welcome Drink and the Get Together of

the 53rd Symposium of the Society for

Histochemistry 2011.

Exhibitors and Sponsors

140


Gold Sponsor

Comprehensive Index of Exhibitors and Sponsors

AB SCIEX Deutschland GmbH Booth 5

Aperio Booth 8

BioLabs --

Bruker Daltonik GmbH Booth 3

Caliper Life Sciences GmbH Booth 4

DCS Innovative Diagnostik- Systeme

Booth 1

GSG-Analytical --


141

Hamamatsu Photonics Deutschland

GmbH Booth 10

Intas Science Imaging Instruments --

W.Reichert-LABTEC Booth 2

Leica Microsystems Booth 5

Nikon GmbH Booth 1a

Olympus Deutschland GmbH -

Mikroskopie Booth 6

Springer Booth 7

Carl Zeiss NTS GmbH --

Map of Exhibition Area

142

Notes

Documents

53rd Symposium of the Society for Histochemistry … › fileadmin › 01...Welcome Message 2 Dear Members of the Society for Histochemistry, Dear Scientists and Colleagues, On behalf