New perspectives in Tumor Angiogenesis

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Institute for Cancer Research and Treatment Institute for Cancer Research and TreatmentInstitute for Cancer Research and Treatment Institute for Cancer Research and Treatment

Fondazione Piemontese per la Ricerca sul Cancro-Onlus - Fondazione Piemontese per l’Oncologia - Università degli Studi di Torino

New perspectives in Tumor Angiogenesis

Federico Bussolino, M.D., Ph.D. Scientific Director

Round Table 2 Italy-Russia@Dubna December 22th, 2010

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The features and the Mission of the Institute

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The History

Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l’Oncologia - Università degli Studi di Torino 1986: The “Fondazione Piemontese per la Ricerca sul Cancro” was funded

and signed agreements with the University of Torino and the Public Health Ministry 1998: IRCC was completed and the Research activities started

2000: Clinical activities started

2011: Starting of a new building dedicated to basic and translational research activities: the Interdisciplinary Cancer Center

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Institute for Cancer Research and Treatment

Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l’Oncologia - Università degli Studi di Torino

Basic and translation research Clinical activities

Molecular and cellular

basis of metastatic process

Molecular and cllular basis of ersonalized medicine

Gastro-intestintal cancers Breast cancers

Sarcomas

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•Area

: 2500 mq

•People

: 130

•Budget 2010

: 8800 KEuro

Core facilities

:

•Mouse House

: (breeding colonies, stock and experimental mice, genetically modified mice, BSL3 activities): 12000 mice

•Genomic platform

(Illumina platform, real time PRC,

Genome Sequencer FLX system, 454 massive DNA sequencing, Fish, ISH, shRNA libraries)

•Microscopy

(confocal microscoper, FRET, FRAP,

video-lapse microscopy, analysis, BD pathway for high throughput screening)

•FACS sorter

(DAKO MoFlo High Speed sorter)

•Mouse pathology

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PhD School: Complex Systems in Medicine and Life Sciences

http://dott-scmsv.campusnet.unito.it/cgi-bin/home.pl

PhD School: Molecular Medicine

http://dott-mm.campusnet.unito.it/cgi-bin/home.pl

3 SME: Horizon Discovery

Apavadis Biotechnologies Creabilis Therapeutics

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The Mission of

Interdisciplinary Cancer Center

(ICC)

With the completion of the human genome we understood that life

is based on dynamic molecular networks rather than on a direct

connection between genotype and phenotype.

Indeed ICC is interested in is understanding the

biological networks supporting the metastatic spreading.

Quantitative experimental data obtained at different spatial scales are integrated with the ultimate goal of being able to describe the features of metastasis network and to predict its behaviour. This thrust is

thoroughly integrated with the clinical research programs already

in place at the Center to build up methods and strategies for a predictive oncology allowing earlier diagnoses and tailored therapies and

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Core: Cancer Genetics Cell Biology Animal Models Imputs and Tools from Hard sciences

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How does Cancer - Microenvironment Ping-Pong

Influence therapy?

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The features and the Mission of the Institute

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Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid

Senger et al Science 219: 282, 1983

Isolation of a tumor factor responsible for

angiogenesis Folkman et al J Exp Med. 133:275, 1971

“Despite the striking effects of several of these

antiangiogenic agents on tumor xenografts in mice none has demonstrated efficacy in phase III clinical trials” G. Semenza Annu. Rev Med. 54:17, 2003

Bevacizumab plus irinotecan, fluorouracil,

and leucovorin for metastatic colorectal cancer.

Hurvitz et al N Engl J Med. 2004 350:2335, 2004

ANGIOGENESIS MILESTONES

1971: The beginning

1983: VEGF is isolated

2003: Doubts and hopes

2004: Clinical Trial Phase III

2010: The current use _{Anti-angiogenic regimens are extensively}

used , but their efficacy is far from the pre-clinical results

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Free angioblasts develop in the mesoderm

Vasculogenesis:

Free angioblasts assemble into cords

Angioblasts differentiate into endothelium and form tubes assemble into cords

Remodeling and maturation Recruitment of mural cells

Sprouting angiogenesis Intususception

Angiogenesis

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AUTACOIDS

SHEAR STRESS

ANGIOPOIETINS

PDGF, TGF

β, Ν

otch SEMAPHORINS

Netrins, Slit

MATRIX INTERACTIONS

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PARALLEL BETWEEN GENETIC ALTERATIONS DURING

PROGRESSION OF COLONRECTAL CARCINOMA AND

POSTULATED CHANGES IN TUMOR ANGIOGENESIS PROFILE

NORMAL EPITHELIUM HYPERPLASTIC EPITHELIUM EARLY ADENOMA INTERMEDIATE ADENOMA LATE

ADENOMA CARCINOMA METASTASIS

APC 5qLO K-RAS 18 p LOH 17p LOH OTHERS

DCC p53

Haemorrhage ? VEGF-A ? VEGF-A VEGFR-2

TSP-1

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Sprouting Angiogenesis

Cooptive Angiogenesis

Vasculogenesis

(Endothelial Precursors Mobilized from BM)

VEGF/SDF-1

mediated mobilization

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Too much hypoxia.... by aggressive

anti-VEGF regimen

Ebos et al. Accelerated Metastasis after Short-Term Treatment with a

Potent Inhibitor of Tumor Angiogenesis. Cancer Cell 15:232, 2009

Paez-Ribes et al. Antiangiogenic therapy elicits malignant progression of tumors

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AUTACOIDS

SHEAR STRESS

ANGIOPOIETINS

PDGF, TGF

β, Ν

otch SEMAPHORINS

Netrins, Slit

MATRIX INTERACTIONS

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VASCULAR GUIDANCE AND TISSUE ARCHITECTURE

CAPILLARY DISTRIBUTION ACCORDING TO O₂ DIFFUSION

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Primary capillary plexus

Primary capillary plexus Mature vascular treeMature vascular tree

Angiogenic

remodeling

Extracellular matrix

(ECM)

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Andreas Vesalius (1514-1564)

B. Anderson, 2002

Green:vessels

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REPULSION

ACTRACTION AXON GUIDANCE

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Class 3 semaphorin inhibits integrin functions

and affects vascular remodeling

0 1 2 3 4 5 6 7 8 9 % o f W o w -1 p o s it iv e c e ll s

Control VEGF-A VEGF-A+ Sema3A RCAS-GFP RCAS-∆∆∆∆Npn-1 RCAS-∆∆∆∆PlxA 0 5 10 15 20 25 30 35 0 1 2 5 10 Fibronectin (µµµµg/ml) N o . o f m ig ra te d c e ll s Control Sema 3A Endothelial cell haptotaxis

Active ββββ1 integrins

Total ββββ1 integrins

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Talin PIP2 Cytoskeleton Talin Rnd Rac-GTP Rac-GDP Toyofuku,2005; Serini, 2005

SEMA 3A inhibits integrin function by switching-off R-Ras

GAP domain

GEF

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Normal stage (onc

+

) Hyperplastic/ Dysplastic stage Angiogenic stage Tumor stage

Multistage tumorigenesis in RIP-Tag transgenic mice

N/E2 CIN-1/2 CIN-3 SCC

S S E E E E

T

S E

_T

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-10 -8 -6 -4 -2 0 2 4 6 8 10 H A T 3A 3B 3C 3D 3E 3F Sema R el a ti v e q u a n ti fi ca ti o n (c o m p a re d t o N )

Sema3 expression during stages of

tumor progression in RipTag2 and in

in

K14-HPV16/E2 mice

Sema3Aand sema3F are strongly up-regulated in the early stages of tumor angiogenesis and completely

switched-off in invasive tumor

-6 -4 -2 0 2 4 6 8 CIN1-2 CIn3 SCC R el a ti v e q u a n ti fi ca ti o n (c o m p a re d t o N /E 2 ) 3A 3B 3C 3D 3E 3F Sema Maione et al, 2009

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N A T Sema3A Meca32 Sema3A Meca32 DAPI 630x 400x T N A T N A T N A 400x A

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wtAAV rAAV –lacZ or GFP Cap

ITR

polyA p5 p19 p46 Rep lacZ or GFP

CMV Promoter polyA

ITR

Adeno-associated virus (AVV) 8 as a new gene delivery tool for pancreatic cells

AAV8-LacZ Control

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0 20 40 60 80 100 120 140 160 T u m o r b u rd en ( m m 3 ) 12 wk 16 wk 16 wk * p<0,01 LacZ Sema3A T₀ n=12

Effect of AAV8-Sema3A treatment on tumor growth and vascular

density in a regression trial (12-16 weeks) in tumor-bearing RipTag2

400x Control AAV8-Sema3A 200X 200X CD31 CD31 CD31 200x AAV8-Sema3A Exocrine pancreas

AAV8-LacZ AAV8-Sema3A Sugen

0 10 20 30 40 50 60 70 % r e d u c ti o n v a s c u la r d e n s it y Maione et al, 2008

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The landmarks of vascular normalization

Improvement of vessel shapes

Vessel pruning

Interstitial pressure reduction

Improvment of oxygen delivery

Pericytes recruitment

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A re a d en si ty ( % ) Meca32 NG2

Control tumors AAV8-Sema3A

treated tumors 0 10 20 30 40 50 60 70 80 * p<0.01 ** p<0.001 400x 400x 400x Meca32 + ΝG2 AAV8-Sema3A Control

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Sema3A replacement improves Oxygen delivery

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T Ac Ac T Ac T T T Ac T Ac AAV8-sema3A Control RipTag tumors

H&E

Collagen I

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AAV8-sema3A treatment blocks tumor invasion

0 10 20 30 40 50 60 70 80 90 _IT IC1 IC2

Control Sunitinib

AAV8-Sema3A % t o ta l t u m o r p e r a n im a l

IT= encapsulated islet tumor

IC1= microinvasive tumor

IC2= fully invasive tumor

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Treatment with Sunitinib, but not with AAV8-Sema3A

induces liver metastases in Rip-Tag2

0 10 20 30 40 50

Control Sunitinib

AAV8-Sema3A In ci d e n ce o f li v e r m e ta st a si s

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Exogenous Sema3A