Lab Med Breast Cancer: From Basics to Beyond. Basic Cancer Biology and Model Systems ZENA WERB. 4:30-6 pm November 1, 2010

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1 Lab Med Breast Cancer: From Basics to Beyond Basic Cancer Biology and Model Systems ZENA WERB 4:30-6 pm November 1, 2010

3 Advantages of mice as breast cancer models hlp://mammary.nih.gov/atlas/histology/ Wellings001/Srwtxt/index.htm

4 Advantages of mice as breast cancer models Mice are small, tumors can occur quickly Similarity of mouse and human gene8cs Can study precancerous state Inbred giving homogeneity for cancer studies Suscep8ble to chemical and environmental agents, diet Tumors can be imaged Can be gene8cally modified with oncogenes, suppressor genes, chromosomes, congenic strains Can be used for allograas and xenograas Experimental therapeu8cs

5 Disadvantages Hard to study late stages of cancer including metastasis because of short life span and animal welfare concerns Hormonal and physiological differences with human while tumors of a given oncogene are very similar, there is greater heterogeneity in breast cancer in human

6 Mouse models of breast cancer many oncogenes including myc, wnt, Her2/ Neu many suppressor genes including Brca1 Metastasis- lung, bone (brain more difficult) XenograAs (human) and allograas( cell, 8ssue) Preven8on research; e.g. chemical carcinogens +/- xenoestrogens aging

7 GeneraQng Animal Models Mouse mammary tumor virus (Wnt) Using the MMTV promoter to deliver wnt, myc, etc. Knockouts, transgenics, recombineering, etc. Inbred strains with different Chemical carcinogens +/- xenoestrogens, ionizing (allograj, and stem cell transplant, developmental) aging

8 Characterizing Animal Models can assess tumor growth, metastasis, angiogenesis, immune system,etc. many models exist and can be obtained from Mouse Models of Cancer Consor8um or inves8gators gene8c background of mice can influence breast cancer phenotyping the breast cancer- mice tend to have much shorter ER+ stages than human metastasis via blood or lympha8cs epigene8c varia8ons recurrence aaer surgery or treatment to remove primary tumor

9 Transplanta8on models AllograAs: immune competent- orthotopic, models like i.v. or i.c., s.c. Site is cri8cal can transplant tumor +/- stroma XenograAs: usually immunodeficient- Cells, 8ssues and targets, humanizing the immune system Can use mutant mice and test effects of host

10 TransplantaQon Mouse Models Must be aware of immune responses to transgenes, e.g., luciferase, Neu, SV40 T- an8gen can transplant tumor cells (mouse or human), primary tumor, breast 8ssue that can become cancer, tumor- associated fibroblasts, inflammatory cells advantage in tes8ng effects on tumor vs. host genes

11 Other species for animal models Rats Rabbits Opossums Chickens Zebrafish Drosophila Tasmanian devils

12 Tissues consist of multiple cell types that communicate with each other Cancer cells VEGF BMPs S100A4 CXCL12 Wnts Gremlin1 Endothelial cells Leukocytes Fibroblasts Proteinases Growth factors Extracellular matrix Type I collagen Fibronectin Adapted from Y. DeClerck

13 MMTV-polyoma middle T model of luminal cancer

14 The microenvironment changes during tumor progression Hyperplasia Early carcinoma Late carcinoma edge Late carcinoma H&E Collagen Myeloid cells Vasculature

15 What is the role of the inflammatory microenvironment during tumor progression and response to therapy? 15

16 Motile myeloid cells are found at the tumor border in areas with dense collagen: In vivo imaging Mikala Egeblad Hematoxylin fibrillar collagen Velocity: 4.9 m/min ± 0.08 N=544 (moving cells only) Cancer Cells (CFP) Myeloid cells (GFP) Tumor Tumor Stromal border Stromal Border MMTV-PyMT picrosirius red staining (cross polarized light) 900x real time, 2h/loop, MMTV-PyMT;ACTB-ECFP;c-fms-EGFP

17 Motile myeloid cells are found at the tumor border in areas with dense collagen: In vivo imaging Mikala Egeblad Velocity: 4.9 m/min ± 0.08 N=544 (moving cells only) 900x real time, 2h/loop, MMTV-PyMT;ACTB-ECFP;c-fms-EGFP

18 Motile myeloid cells are found at the tumor border in areas with dense collagen: In vivo imaging Mikala Egeblad Velocity: 4.9 m/min ± 0.08 N=544 (moving cells only) 900x real time, 2h/loop, MMTV-PyMT;ACTB-ECFP;c-fms-EGFP

19 Marked heterogeneity of inflammatory cells responding to tumor progression. Gr1 + myeloid cells patrol tumor vessels, extravasate and migrate in the tumor stroma. M2 macrophages take up dextran. MMTV-PyMT; ACTB-ECFP; c-fms-egfp mouse injected with70kd dextran and anti- Gr1 antibody. Live imaging:spinning disk confocal microscopy, 12 hours Egeblad, Ewald,et al. Disease Mod Mech 1: , 2008

Marked heterogeneity of inflammatory cells responding to tumor progression. Gr1 + myeloid cells patrol tumor vessels, extravasate and migrate in the tumor stroma. M2 macrophages take up dextran.

20 Marked heterogeneity of inflammatory cells responding to tumor progression. Gr1 + myeloid cells patrol tumor vessels, extravasate and migrate in the tumor stroma. M2 macrophages take up dextran. MMTV-PyMT; ACTB-ECFP; c-fms-egfp mouse injected with70kd dextran and anti- Gr1 antibody. Live imaging:spinning disk confocal microscopy, 12 hours Egeblad, Ewald,et al. Disease Mod Mech 1: , 2008

21 Inflammation, MMPs, VEGF, vascular permeablity and angiogenesis are all upregulated and are causally linked in tumor progression: does this affect response to chemotherapy?

22 Extravascular dextran leakage depends on the tumor microenvironment O min 2 min 24 s 16 min 201 min Hyperplasia Early Carcinoma Late Carcinoma 10 kd, 2 MD or both dextrans above threshold in MMTV-PyMT; ACTB-eCFP. Three fields imaged in parallel in the same mouse (representative of 5 exp.) Egeblad et al., DMM 2008; Mikala Egeblad, Hanne Askautrud, unpublished

23 What is responsible for vascular permeability? Candidate: TGF-

24 Vascular leakage in carcinoma is regulated by TGF- signaling: treat mice with TGFRI inhibitor 10 min 20 min 40 min 60 min Control (DMSO) 100 m TGFR I (ALK5) Inhibitor Sounni, Dehne, van Kempen, Egeblad et al., DMM kd, 70 kd and Qdots dextrans injected i.v. into MMTV-PyMT mice. Extravascular dextran is indicated (Qdots are intravascular). Mice were pre-treated for 6 days. N=5

25 Summary The tissue microenvironment is rich in cells of the innate and acquired immune system: A key function of these cells is to control vascular permeability, which in turn regulates tumor susceptibility to chemotherapy

26 A transplant approach to study tumor progression TEB Hyperplasia Transplant hyperplasia 5-18 wks Sacrifice OR 8 wks Sac into cleared fat Remove Tumor pad Kouros-Mehr et al., Cancer Cell 13: (2008)

27 Kinetics of growth of hyperplasia transplants Time to palpaable tumor=63 days Hosein Kouros-Mehr Cancer Cell 13: (2008) MMTV-PyMT; β-actin-egfp

28 Tumor dissemination starts at 8 weeks post-transplant (early carcinoma) Lung Spleen Kidney Brain Liver Kouros-Mehr, Cancer Cell 13: (2008) Vascular (Tomato Lectin) staining

29 GATA3 expression is lost during cancer progression coinciding with tumor cell dissemination GFP DAPI GATA3 Kouros-Mehr et al., Cancer Cell 13: (2008) Adenoma Early Carcinoma Late Carcinoma 5 weeks 8 weeks 18 weeks

Transplant into mammary fat pad ZF1 KRR ZF2 GATA-3 GATA-3

30 Does GATA-3 prevent metastasis? Late carcinoma GATA-3 IRES GFP Infect with Retrovirus Transplant into mammary fat pad ZF1 KRR ZF2 GATA-3 GATA PMIG-W LTR Kouros-Mehr et al., Cancer Cell 13: (2008) ψ IRES GFP LTR AMP

31 Tumors overexpressing vector alone form undifferentiated adenocarcinomas GFP GFP Kouros-Mehr et al., Cancer Cell 13: (2008) GATA-3

32 Tumors overexpressing GATA-3 show signs of tumor Duct differentiation Duct GFP GFP GATA-3 Kouros-Mehr et al., Cancer Cell 13: (2008)

33 GATA-3 infected tumors show reduced capacity for dissemination and metastasis Dissemination Frequency Vector GATA-3 Kouros-Mehr et al., Cancer Cell 13: (2008)

34 GATA-3 is a master regulator for tumor dissemination in mammary cancer GATA-3 Early Late Adenoma Carcinoma Carcinoma GATA-3 5 weeks 8 weeks 12 weeks 15 weeks 18 weeks issemination Kouros-Mehr et al., Cancer Cell 13: (2008) Loss of GATA-3 expression

35 Summary GATA3 marks luminal epithelial differentiation. Its loss during tumor progression marks malignant conversion and metastasis. Gata3 regulates the tumor microenvironment by suppressing VEGF expression Restoring GATA3 reverts the tumor phenotype to premalignant including the microenvironment and blocks metastasis to lung and bone

36 Hannah Askautrud U Oslo Audrey Brenot Jon Chou Mikala Egeblad CSHL Andrew Ewald JHU Hosein Kouros-Mehr Genentech Marja Lohela Vicki Plaks Sylvain Provot-> INSERM Paris Bryan Welm U Utah UCSF Funding: National Cancer Institute NIEHS

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