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IVIS Imaging System

Discovery at the Speed of Light TM

Jack Yang

Product Manager

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Non-invasive in vivo imaging

Continued monitor

Low variation

3R (Replacement, Reduction, Refinement)

Drug Discovery Today (2002) 7:125

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microPET

microCT

microSPECT

microMRI

Optical bioluminescence

Optical fluorescence

Ultrasound

GENE & DEVELOPMENT 2003 17:545

Multi-type of in vivo imaging

Aspect M2

Micro MRI

Quantum FX

Micro CT

Vevo 2100

HR Ultrasound

Vevo LAZR

Photoacustic

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Genisys4

Micro PET

Cellvizio

In vivo microscopy IVIS

Optical imaging

Maestro

Fluorescence

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Advantage of optical imaging

1.High throughput

2.No radiation

3.Easy to operation

4.Dual function

(fluorescence and bioluminescence)

5.Functional analysis

IVIS can cover multi-optical imaging

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Basic Method

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Reporter Molecules

Luciferase, Fluorescent Protein

Fluorescent dyes

Quantum dots

Label Cells Label Bacteria Label Genes

ATP and O2 required for luciferase

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Autofluorescence Autoluminescence

Luminescent or Fluorescent source

Imaging system

Imaging Principle

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An “Imaging” Window of Opportunity

Excitation Emission Fluorophore Spectra Luciferase Spectra

Demonstration of Light Transmission

Tissue is not transparent - Light absorbance depends on wavelength

Doyle et al, 2004

Nature Medicine

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Bioluminescence

Oxyluciferin

luciferin

+ATP, Mg2+, O2

Light

+

+AMP, PPi, CO2

The source of light

Renilla Luciferase

Fluorescence

GFP

Quantum dot NIR dye

ground

state

Excitation

Wavelength

Emission

Wavelength

excited

state

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Bioluminescence

Just light out

No background

Fluorescence

Light in and Light out

Autofluorescence interference

Bioluminescence and fluorescence

Quantified images

Luminance detection

Fluorescence detection

NATURE MEDICINE (2004) 10: 1257

Journal of Biomedical Optics, (2001)

絕對定量功能 Absolute Quantification reference

Raw Signal

(Counts)

Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec

Binning: small small small small medium medium

Day: 1 2 3 4 5

6

Peak

Counts

1600

800

1200

400

Calibrated Physical Units vs Raw Signal - Example

Calibrated

Signal

(Photons per

second)

Radiance:

Photons per

second

Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec

Binning: small small small small medium medium

Day: 1 2 3 4 5

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Calibrated Physical Units vs Raw Signal - Example

Autoexposure feature available for

bioluminescence and fluorescence

User definable autoexposure settings

Image Wizard - autoexposure

Bioluminescence Imaging Sensitivity

Cooled (-90C) camera with large CCD chip area for high

sensitivity light detection

Low f-number and large diameter lens

Most sensitive system available Resolves multiple bioluminescent

reporters Detects down to even a single cell in vivo

In vivo imaging of s.c. implanted T cells transduced with optimized firefly luciferase (left) and a ‘single’ 4T1 breast cancer

cell (right)

Precision filters

Gives high sensitivity and uniform light collection

Rabinovich et al, PNAS,2008

Single cell (flux: 460-528 photons/sec)

Kim et al, pLOs One et al, 2010

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16

8

256

Do

x (n

g)

128

64

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Spectral Unmixing enables detection of as little as 8 ng of Doxorubicin in vivo

IVIS enables direct correlation between Doxorubicin signal and concentration both in vitro and in vivo

Simple to use Unmixing software significantly improves signal to background ratio at low concentrations

Non-unmixed image

Unmixed image

Non-unmixed image

Unmixed image

R² = 0.9671

0.E+00

2.E-06

4.E-06

0 64 128 192 256

Flu

orescen

ce

Doxorubicin (ng)

In Vivo Quantitation of Doxorubicin Using Spectral Unmixing

Fluorescence Imaging Sensitivity

Molecular Imaging in Drug Development Your Success is Our Success

IVIS imaging performance is measured by your results: 1000 systems in laboratories worldwide 2,600 peer-reviewed publications

14 drugs in clinical trials (that we know of…we estimate there are a whole lot more)

IVIS Preclinical Imaging •All therapeutic areas

•Mode of action •Efficacy studies

•Pharmacokinetic model •Dosing and treatment

IVIS Bringing Compounds to the Clinic Research using IVIS contributed to 5 FDA approved drugs and 9 drugs currently

in clinical trials.

ABT-888 (Abbott): multiple diverse tumor models

RANKL Inhibitor (Amgen): denosumab and bone metastasis models

Sutent (Pfizer): subcutaneous tumor xenograft

Dasatinib (Bristol-Myers Squibb): chronic myelogenous leukemia

Tasigna (Novartis): leukemia/metastasis model

Cubicin (Cubist Pharmaceuticals): bacterial peritonitis model

CG0070 (Cell Genesys): orthotopic bladder cancer

Panzem (EntreMed Pharmaceuticals): orthotopic gliosarcoma

AEE788 (Novartis): intraperitoneal tumor model

IT-101 (Insert Therapeutics): Ewings sarcoma xenograft

PS-341 (Millennium Pharmaceuticals): intraperitoneal xenograft

Aflibercept (Sanofi-Aventis): orthotopic renal cancer

CHIR-258 (Novartis): orthotopic multiple myeloma model

NPI-0052 (Nereus Pharmaceuticals): subcutaneous tumor model

Peer reviewed references for each example in the IVIS bibliography

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Longitudinal Non-Invasive Monitoring of Disease (and Treatment)

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Spontaneous Metastases from Orthotopic Mammary Fat Pad Implant

Brachial

Lymph Node

Representative Mouse #12,

Ventral View Female Nude (Harlan)

In vivo Image

Right Brachial

Lymph Node

Ex vivo Image Histopathology

Micrometastasis Right

Brachial Lymph Node

Exp #078

Female Nude, Harlan

n=7

Images 3 min, 10 bin, FOV 15

-5000

-4000

-3000

-2000

-1000

counts

-1000

- 800

- 600

- 400

- 200

counts

Mouse #12

200x

MDA-MB-231-luc (D3H1), 2x106 cells, mfp

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Untreated 9L-Glioma (Rat)

Source: Brian Ross, Ph.D., University of Michigan Medical Center

-2 0 4 10

14 x 103

12

10

8

6

4

2

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Longitudinal Assessment of Tumor Growth and Response to Anti-Angiogenic Therapy

105 U87 glioma cells implanted 14 days prior

Control

Anti-angiogenesis inhibitors (week 4)

Szentirmai et al, 2006

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J Clinic Invest

Massauge group, 2005

Tumor Induced Damage Monitoring

Cell Model: MDA-MB-231 (breast cancer cell line)

Brain Tumor Therapy

• Intra-cranial glioma model

• U87MG-Luc Cells

• Anti-angiogenesis therapy

• Test a non-viral delivery system

(Transposon-sleep beauty, SB)

• Test anti-tumor effects of statin-AE and

sFlt-1

Mol Therapy 2005

Aflibercept (Sanofi-Aventis)

Mouse Xenograft: Orthotopic Renal Cancer Model

Transfected cells injected into subcapsular space

of left kidney.

Mice sorted according to tumor burden estimated

by luciferase expression using an IVIS system.

IVIS® imaging system used to monitor changes in

tumor burden during 30 days of treatment.

Decrease in luciferase expression measured by

IVIS correlated well with decrease in tumor weight

and with mouse survival

Aflibercept is in Phase II and III clinical trials for multiple indications including one for Renal Cancer

Verheul et al., Clin Cancer Res 2007;13(14) July 15, 2007, p. 4201–4208

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Cell Trafficking, Survival and Engraftment Patterns In Vivo

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Cell Transplantation and Trafficking Patterns

Cao et al, Stem Cells,2004

Stem Cell Foci Formation and Hematopoiesis

Transplantation of 250 Luc+ HSC into Lethally Irradiated Hosts

Image-guided cardiac cell delivery: Sprague-Dawley rats (n = 11) were imaged using high-resolution

ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the

anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After

injection, bioluminescence imaging was performed using a cooled charged-coupled camera. The signal

detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03). Optical signal

could be followed longitudinally using bioluminescence imaging.

Ultrasound Guided Delivery of

Bioluminescent Cardiomyoblasts

Rodriguez-Porcel & Gambhir et al, 2005

心成肌細胞

Image-guided cardiac cell delivery

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Stroke Kim et al, 2004

Yellew: Infarcted site

White: Injection site

Neuron Progenitor Cell Tracing

Stroke Repairing

Dynamic tracking of stem cells in an acute liver

failure model

World J Gastroenterol 2012 February 14; 18(6): 507-516

New drug development & in vivo tracking

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Neurotropic Viral Infection

• Viral Model: Sindbis Virus (Alphavirus)

• 103 CFU virus inoculated at right hind foot

J Virol Cook & Griffin, 2003

Ljubimova et al., Nanomedicine 2008

Multitargeting Drug Delivery Poly (malic acid) nanoconjugates

Polycefin family of natural-derived

polymeric drug-delivery vehicles

Heirarchic conjugation of functional

groups onto the backbone of poly(malic acid),

an alipathic polyester from the microorganism

Physarum polycephalum

Conjugated with AlexaFluor 680

Antibody to transferrin receptor -

overexpressed on tumor cells

Antibody to nucleosome specific 2C5 that

reacts with various cancer cells via tumor cell

surface-bound nucleosomes

NIRF Imaging of Arthritis

Lee et al,Biomaterials,2008

NIRF-HA based gold nanoprobes detect reactive oxygen species (ROS) and

hyaluronidase (HAdase).

in vivo fluorescence imaging of subcutaneous

U87MG glioblastoma

Cancer research (2004) 64: 8009

RGD-Cy5.5

Fluorescence probe

Cubicin (Daptomycin) is an FDA approved drug for infections caused by Staphylococcus aureus

Mortin et al., Antimicrobial Agents and Chemotherapy, May 2007, p. 1787–1794

New drug development 1. Noninvasive assessment of cancer response to

therapy. Nat Med. Mar 2008

2. Preparation of peptide-conjugated quantum dots for

tumor vasculature-targeted imaging. Nature protocols.

2008

3. Magnetically retainable microparticles for drug

delivery to the joint: efficacy studies in an antigen-

induced arthritis model in mice. Arthritis Res Ther. 2009

4. Molecular imaging of phosphorylation events for drug

development. Mol Imaging Biol. May-Jun 2009

5. 2'-Hydroxycinnamaldehyde shows antitumor activity

against oral cancer in vitro and in vivo in a rat tumor

model. Anticancer Res. Feb 2010.

6. Revisiting Old Drugs as Novel Agents for

Retinoblastoma: In vitro and In vivo Antitumor Activity

of Cardenolides. Invest Ophthalmol VisSci. 2009.

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Genetic Components of Disease Expression, Pathways and Therapy

LPTA® Animal Models

Genetically modified to emit light when a tagged gene is activated

Validated for key therapeutic areas

Accelerates clinical development candidate selection

Biophotonic

Animal Models

Drug Metabolism Toxicology

Oncology Inflammation

Neurology Metabolic Disease

Brain Kidney Pancreas Liver

Rat Insulin Promoter -islet Cells

Pancreatic Islets from RIP-luc Tg Mouse Transplanted

into NOD Host

RIP-luc Streptozotocin Treatment

Decreased Luciferase Expression

10.0

8.0

6.0

4.0

2.0

Day 0 2 7 14 21

Control

STZ

Ph

oto

ns/s

(x10

3) * = P≤0.05

** = P≤0.01

Pancreas Luminometer Assay

0

2

4

6

8

10

12

RL

U/m

g P

rote

in Control

STZ

*

Luciferase Signal

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 4 9 14 19 Days

Ph

oto

ns/s

(x10

6)

Control (n=10)

STZ (n=9)

*

*

*

* ** **

** **

**

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TRANSGENICS (LPTA) Vascular Endothelial Cell Growth Factor Receptor

VEGF - secreted during angiogenesis VEGFR2 - surface receptor for VEGF

VEGF induction of VEGFR2 promoter

causes luciferase expression

VEGFR2 Pr luc

CHROMOSOME

Day 14

Transgenic VEGFR2-luc

Embryos in a Non- Tg Mother

Reduction of VEGFR2 Expression During Development

1 Week

Signal Intensity

100,000

2 Weeks

Signal Intensity

10,000

1000

2000

3000

4000

5000

4 Weeks

Signal Intensity

200

6 Weeks

Signal Intensity

<100

200

400

600

800

1000

200

400

600

800

1000 60

35

40

45

50

55

30

x10

3

Zhang et al. (Xenogen), 2004

Induction of VEGFR-2 Expression During Wound Healing

Day 0 Day 4 Day 6 Day 11 Day 18 Day 24

Zhang et al. (Xenogen), 2004

Monitoring Angiogenesis During Tumor Development

VEGFR2-luc KI mice implanted with 106 LL/2-GFP tumor cells and imaged at day18

Tumor growth and

development imaged by

GFP fluorescence

Angiogenesis imaged by

VEGFR2-luc expressing

bioluminescence

NFĸB Induction by LPS

Control LPS

Peyer’s

patches

7

4

0

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Zhang, Xenogen & Rune Blomhoff, Oslo

Effect of Anti-inflammation Agents on NF-kB Activation

Effect of Dexamethasone and EGCG on iNOS Expression

Zhang et al. (Xenogen), 2003

Lights on Hypoxia Animal Model: HIF-Luc

Hypoxia Inducible Factor (HIF)

Promote adaptation to hypoxia

Ischemic Disease

Proc. Natl. Acad. Sci. USA

Safran, Michal et al. ,2006