4th Annual Brain Metastases Research and Emerging Therapy ... · ZO-1 DAPI vWF DAPI JAM-A...
Transcript of 4th Annual Brain Metastases Research and Emerging Therapy ... · ZO-1 DAPI vWF DAPI JAM-A...
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
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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
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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
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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
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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
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In vitro live cell imaging
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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
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Transcellular
Paracellular
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How encephalitogenic T cells breach the BBB
Abadier et al., Eur J Imm, under revision
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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
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MDA-MB-231BR-HER2 do not arrest on pMBMECs
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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
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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
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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?
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Extravasation across the BBB
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Metastatic melanoma cells (B16) do arrest on pMBMECs
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Extravasation across the BBB
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B16 melanoma cells express !4-integrin
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Extravasation across the BBB
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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
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Extravasation across the BBB
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B16 melanoma arrest on pMBMECs is !4-integrin dependent
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Extravasation across the BBB
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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
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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
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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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Extravasation across the BBB
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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
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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?
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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
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Transcellular
Paracellular
novembre 17, 2014 Abadier et al., Eur J Imm, under revision
How encephalitogenic T cells breach the BBB