4th Annual Brain Metastases Research and Emerging Therapy Conference, September 20, 2014, Marseille, France MOLECULAR AND CELLULAR MECHANISMS OF METASTATIC CANCER CELL EXTRAVASATION ACROSS THE BLOOD BRAIN BARRIER

Ruth Lyck (PhD) Theodor Kocher Institute University of Bern Bern, Switzerland

Focus: Extravasation of MCCs across the BBB

novembre 17, 2014

Extravasation across the BBB

2

Saxena and Christofori, Mol Oncology, 2013

Nguyen, Bos, Massagué, 2009

The Blood Brain Barrier

3!

The physical barrier is formed by tight junctions of the endothelial cells

Nitta et al 2003, JCB 161: 653 Engelhardt and Sorokin, 2009

In Multiple Sclerosis (MS) encephalitogenic T cells breach the BBB

>! MS is a chronic and disabling human disease. >! In the course of MS

—! autoaggressive immune cells enter the central nervous system (CNS),

—! damage the myelin sheath of the nerve cells and —! disrupt function of the nerve fibers.

! Effector cells, eg CD4+ TEM cells, migrate across the BBB

4 novembre 17, 2014

Inflamed Healthy

Extravasation across the BBB

Meyermann, Wekerle 1986

In vitro model to study molecular events of

T cell or cancer cell extravasation

across the BBB under flow conditions

5

6

Primary mouse brain microvascular endothelial cells (pMBMECs)

Cortices Homogenate Gradient

Neural elements

discard

Vascular elements Arterioles

Venules discard

Capillaries

Enzymatic digestion

Microvascular elements

Seeding

Lyck et al., J Cereb Blood Flow Metab., 2009; Coisne et al. 2005

pMBMECs after 7 days of culture without any passage

Mouse in vitro BBB model

pMBMECs model the BBB: Endothelial, tight junction and cell adhesion molecules are expressed and properly localized

7 Lyck et al., JCBFM, 2009;

PECAM-1

Occludin

ZO-1 DAPI

vWF DAPI

JAM-A

VE-Cadherin Claudin-5

pMBMECs express "! endothelial marker proteins "! TJ proteins

Protein!Localization

(Immunofluorescence)!

Occludin! junctional!ZO-1! junctional!ZO-2! junctional!Claudin-3! junctional!Claudin-5! junctional!Jam-A! junctional!VE-Cadherin! junctional!PECAM-1! junctional!vWF! Weibel pallade bodies!ICAM-2! membrane!ICAM-1! membrane (TNF! !)!VCAM-1! membrane (TNF! !)!

Tigh

t jun

ctio

n A

dh J

EC marker

EC C

AM

Steiner et al., JCBFM, 2011

pMBMECs express " endothelial marker proteins " TJ proteins

Claudin-5 junctionalJam-A junctionalVE-Cadherin junctionalPECAM-1 junctionalvWF Weibel pallade bodiesICAM-2 membraneICAM-1 membrane (TNF! !)VCAM-1 membrane (TNF! !)

Tigh

t jun

ctio

n A

dh J

EC marker

EC C

AM

Mouse in vitro BBB model

8!

PECAM-1

Occludin

ZO-1 DAPI

vWF DAPI

JAM-A

VE-Cadherin Claudin-5

pMBMECs model the BBB: Tight monolayer barrier properties are built up

Trans-Endothelial Electrical Resistance (TEER)

Permeability (3 kDa Dextran)

Steiner et al., JCBFM, 2011 novembre 17, 2014

Mouse in vitro BBB model

Inlet tubing Outlet tubing

Flow chamber

9

pMBMECs CD4+ TEM cells Cancer cells

tubingOutlet tubing

Inverted microscope

In vitro live cell imaging under flow conditions

Automated syringe pump

Flow

In vitro live cell imaging

In vitro live cell imaging under flow conditions

novembre 17, 2014

In vitro live cell imaging

10

CD4+ TEM cells dynamically interact with inflamed pMBMECs

Encephalitogenic T cells breach the BBB

11 Coisne, Lyck and Engelhardt FBCNS 2013

pMBMECs (TNF!) (7 d)

+ encephalitogenic CD4+ T cells

Accumulation: 0.15 dyn/cm2 (initial arrest)

Physiological shear: 1.5 dyn/cm2 (dynamic interaction)

Flow

Total: 19 min

CD4+ TEM cells diapedese via the transcellular and via the paracellular pathways across pMBMECs

12

Transcellular

Paracellular

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How encephalitogenic T cells breach the BBB

Abadier et al., Eur J Imm, under revision

13 novembre 17, 2014

What about metastatic cancer cells? -! do they follow a similar mechanism -! which is the role of "v-integrin

(Intetumumab = CNTO95, Wu et al. 2012)?

CD4+ TEM cells dynamically interact with inflamed pMBMECs

Encephalitogenic T cells breach the BBB

14

MDA-MB-231BR-HER2 do not arrest on pMBMECs

novembre 17, 2014

Human “brain seeking” breast cancer cell line Yoneda et al. JBMR 2001 (Riko Nishimura) Palmieri et al. Cancer Res 2007 (Patricia Steeg)

CD4+ Th1 eff cells MDA-MB-231BR-HER2

Extravasation across the BBB – what about MCCs?

MDA-MB-231BR-HER2 arrest on basement membrane proteins (Matrigel)

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Extravasation across the BBB – what about MCCs?

MDA-MB-231BR-HER2 arrest on basement membrane proteins (Matrigel)

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Extravasation across the BBB – what about MCCs?

GFP

MDA-MB-231BR-HER2 arrest on basement membrane proteins (Matrigel)

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Extravasation across the BBB – what about MCCs?

Intetumumab Ctrl

Intetumumab: Anti human !v-integrin antibody stains focal adhesion like structures of adherent MDA-MB-231BR-HER2 cells

18

z =

1.2 #m

z =

0 #

m

Does intetumumab block adhesion of MDA-MB-231BR-HER2 to BM proteins?

DAPI (nuclei)

GFP (Cancer cells)

Intetumumab (!v-integrin)

Extravasation across the BBB – what about MCCs?

Intetumumab Ctrl

Intetumumab blocks binding of MDA-MB-231BR-HER2 cells to Vitronectin

19

z =

1.2 #m

z =

0 #

m

Does intetumumab block adhesion of MDA-MB-231BR-HER2 to BM proteins?

DAPI (nuclei)

GFP (Cancer cells)

Intetumumab (!v-integrin)

Ctrl DAPI

(nuclei) GFP

(Cancer cells) Intetumumab

(!v-integrin)

Extravasation across the BBB – what about MCCs?

What about metastatic

melanoma cells?

novembre 17, 2014

Extravasation across the BBB

20

Metastatic melanoma cells (B16) do arrest on pMBMECs

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Extravasation across the BBB

21

B16 melanoma cells express !4-integrin

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Extravasation across the BBB

22

Antibody! B16-F1! B16-F10!

"4-Integrin! PS/2! +! +!

"4$7-Integrin! DATK32! -! -!

Mac-1 / "M-Integrin! M1/70! -! -!

LFA-1 ($2-Integrin)! FD441.8! -! -!

B16-F1 and B16-F10 express "4-integrin (presumably VLA-4) but not "4$7-integrin or the $2-integrins Mac-1 or LFA-1

B16 melanoma arrest on pMBMECs is !4-integrin dependent

novembre 17, 2014

Extravasation across the BBB

23

B16 melanoma arrest on pMBMECs is !4-integrin dependent

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Extravasation across the BBB

24

PS/2 Ctrl

B16

cel

ls /

FOV

Conclusions

>  Metastatic human breast carcinoma cells MDA-MB-231BR-HER2

— do not arrest on pMBMECs under flow. (= metastatic mouse breast carcinoma cells 4T1)

— do efficiently arrest on ECM proteins – as accessible when the endothelial monolayer is injured.

>  Metastatic mouse melanoma cells B16 (B16F1, B16F10)

— do arrest on pMBMECs under flow and withstand physiological shear (1.5 dyn/cm2).

>  Arrest of B16 is α4-integrin dependent. novembre 17, 2014

Extravasation across the BBB

25

Acknowledgement

Lyck Lab members > Michael Abadier > Daniel Hauser > Christoph Matti > Ana Belén García Martin > Mark Liebi

Funding •  Swiss National Foundation (SNF)"•  Suisse Multiple Sclerosis Society"•  Foundation for clinical-experimental

cancer research Bern"

Extravasation across the BBB

26

Theodor Kocher Institute (TKI) > Prof. Britta Engelhardt > Workshop of the TKI

—  Adrian Bigler, Ulrich Kindler > All colleagues of the TKI

Collaborations > Prof. Curzio Rüegg, Fribourg, CH

—  Christof Wyss > Prof. Dietmar Vestweber, Münster, D

MDA-MB-231BR HER2 > Prof. Patricia Steeg

Intetumumab > Dr. Curt Kuder, Janssen

Pharmaceutical Companies of Johnson and Johnson, PA, USA

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novembre 17, 2014

Extravasation across the BBB

27

28

In vivo: Effector T cells dynamically interact with the BBB prior to diapedesis

Bartholomäus et al., Nature, 2009 novembre 17, 2014

Encephalitogenic T cells breach the BBB

CD4+ TEM cells dynamically interact with inflamed pMBMECs

29

endothelial cell

T cell

BB

B

I. Capturing/

Rolling

III. Firm adhesion

IV. Diapedesis

II. Activation

Flow

perivascular cell astrocytes basement

membrane pericyte basement membrane

Lyck and Engelhardt, JVR 2012

Encephalitogenic T cells breach the BBB

Roles of endothelial ICAM-1, ICAM-2, VCAM-1 and ALCAM for CD4+ TEM cell dynamic interaction

with the BBB

30 novembre 17, 2014 Steiner et al., JI 2010; ALCAM: manuscript in preparation

How encephalitogenic T cells breach the BBB

Pathway of diapedesis?

31

endothelial cell

BB

B

I. Capturing/

Rolling

III. Firm adhesion

IV. Diapedesis

II. Activation

Flow

VCAM-1

ICAM-1

ICAM-2

perivascular cell astrocytes basement

membrane pericyte basement membrane

!"#$%&'()*+,$")#--.-#$((-

Pathway ???

endothelial cell

I. Capturing/

Rolling

III. Firm adhesion

II. Activation

Flow

VCAM-1

ICAM-1

ICAM-2

perivascular cellastrocytesbasement

membrane pericytebasementmembrane

!"#$%&'()*+,$")#--!"#$%&'()*+,$")#--!"#$%&'()*+,$")#--!"#$%&'()*+,$")#--.-#$((-

Transcellular Paracellular

novembre 17, 2014

How encephalitogenic T cells breach the BBB

CD4+ TEM cells diapedese via the transcellular and via the paracellular pathways across pMBMECs

32

Transcellular

Paracellular

novembre 17, 2014 Abadier et al., Eur J Imm, under revision

How encephalitogenic T cells breach the BBB