PECULIARITIES OF THE MALIGNANT STROMA

1. PECULIARITIES OF THE  ?? MALIGNANT ?? STROMA Mathew A Cherian Fellow in Hematology-Oncology Washington University, St.Louis

2. No financial disclosures

3. Components of the tumour stroma 1.Fibroblasts/Cancer Associated Fibroblasts/Activated fibroblasts/Myofibroblasts 2.Mesenchymal Stem Cells a.Circulating b.Tissue resident Mesenchymal Stem Cells 3.Cells of Hematopoietic origin a.Tumour associated macrophages b.Tumour infiltrating lymphocytes i)Immunostimulatory ii)Immunosuppressive 4.Endothelial cells i)Vascular ii)Lymphatic 5.Matrix

4. Transformation:?not completely a cell autonomous phenomenon Cancer cells have certain cardinal features which define them as being transformed: 1.Lack of contact inhibition 2.Anchorage independence 3.Independence from growth factors 4.Lack of responsiveness to normal growth limiting mechanisms 5.Ability to form tumours in immunodeficient hosts

5. All the above mentioned features are cell autonomous. However experiments have demonstrated that: Subcutaneous implants of tumour cells in nude mice below the minimum tumorigenic dose grow in the presence of co-inoculated fibroblasts(Cancer Res 1986;46:3290-3294. ) Cancer Res 1986;46:3290-3294. Published online July 1, 1986. Odile Picard, Yvette Rolland and Marie France Poupon Fibroblast-dependent Tumorigenicity of Cells in Nude Mice: Implication for Implantation of MetastasesCancer Res 1986;46:3290-3294. Published online July 1, 1986. Odile Picard, Yvette Rolland and Marie France Poupon Fibroblast-dependent Tumorigenicity of Cells in Nude Mice: Implication for Implantation of Metastases

6. Co-culture with stromal fibroblasts mediates resistance to tyrosine kinase inhibitors in otherwise sensitive cell lines. Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Wei Wang,1 Qi Li,1 Tadaaki Yamada,1 Kunio Matsumoto,2 Isao Matsumoto,3 Makoto Oda,3 Go Watanabe,3 Yoshiyuki Kayano,4 Yasuhiko Nishioka,5 Saburo Sone,5 and Seiji Yano1 Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.Clin Cancer Res 2009;15(21) November 1, 2009)Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Wei Wang,1 Qi Li,1 Tadaaki Yamada,1 Kunio Matsumoto,2 Isao Matsumoto,3 Makoto Oda,3 Go Watanabe,3 Yoshiyuki Kayano,4 Yasuhiko Nishioka,5 Saburo Sone,5 and Seiji Yano1 Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.Clin Cancer Res 2009;15(21) November 1, 2009)

7. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1050�1055, February 1998 Immortalized keratinocytes are transformed by the stable transfection of PDGF, though they do not express the receptor, via stromal cell activation. (Tumorigenic conversion of immortal human keratinocytes through stromal cell activation. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1050�1055, February 1998) Cell Biology Tumorigenic conversion of immortal human keratinocytes through stromal cell activation MIHAELA SKOBE* AND NORBERT E. FUSENIG� Cancer Res.�2000 Mar 1;60(5):1254-60. Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells. Barcellos-Hoff MH,�Ravani SA.Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1050�1055, February 1998 Immortalized keratinocytes are transformed by the stable transfection of PDGF, though they do not express the receptor, via stromal cell activation. (Tumorigenic conversion of immortal human keratinocytes through stromal cell activation. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1050�1055, February 1998) Cell Biology Tumorigenic conversion of immortal human keratinocytes through stromal cell activation MIHAELA SKOBE* AND NORBERT E. FUSENIG� Cancer Res.�2000 Mar 1;60(5):1254-60. Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells. Barcellos-Hoff MH,�Ravani SA.

8. Non random chromosomal changes present selectively in the stromal compartment have repeatedly been detected in tumours.

9. Non random chromosomal changes present selectively in the stromal compartment have repeatedly been detected in tumours. Activating mutations in classical protooncogenes and deletion or inactivating mutations in classical tumour suppressor genes restricted to the stromal component have been detected in multiple experiments.

10. Non random chromosomal changes present selectively in the stromal compartment have repeatedly been detected in tumours. Activating mutations in classical protooncogenes and deletion or inactivating mutations in classical tumour suppressor genes restricted to the stromal component have been detected in multiple experiments. Mutant genes engineered to be to be expressed selectively in the stroma causes transformation in neighboring epithelial cells in animal models. Carcinogen exposed bladder with denuded epithelium proved to be a superior soil for carcinogen exposed urothelial cells than normal unexposed bladder(J Urol.�1990 Mar;143(3):618-21. Stimulation of urinary bladder tumorigenesis by carcinogen-exposed stroma.Uchida K,�Samma S,�Momose H,�Kashihara N,�Rademaker A,�Oyasu R. ) Carcinogen exposed bladder with denuded epithelium proved to be a superior soil for carcinogen exposed urothelial cells than normal unexposed bladder(J Urol.�1990 Mar;143(3):618-21. Stimulation of urinary bladder tumorigenesis by carcinogen-exposed stroma.Uchida K,�Samma S,�Momose H,�Kashihara N,�Rademaker A,�Oyasu R. )

11. Possible Source of Cancer Associated Fibroblasts

12. NATURE OF CANCER ASSOCIATED FIBROBLASTS Histology: The expression of a smooth muscle actin is considered a defining characteristic. They co-express a-SMA and vimentin and phenotypically behave like mofibroblasts. Normal fibroblasts take on the characteristics of myofibroblasts when co-cultured with cancer cells. (G. Serini and G. Gabbian. Exp. Cell Res.�250�(1999), pp. 273�283). Serini and Gabbiani, 1999�G. Serini and G. Gabbiani, Mechanisms of myofibroblast activity and phenotypic modulation,�Exp. Cell Res.�250�(1999), pp. 273�283 Serini and Gabbiani, 1999�G. Serini and G. Gabbiani, Mechanisms of myofibroblast activity and phenotypic modulation,�Exp. Cell Res.�250�(1999), pp. 273�283

13. CAFs are more competent at enhancing tumour growth than normal fibroblasts in vivo and in vitro. CAFs enhance tumour angiogenesis. CAFs enhance the recruitment of endothelial progenitor cells into the tumour microenvironment. CAFs express high levels of SDF-1 The expression of SDF-1 promotes the growth of cancer cells which express the G-protein coupled chemokine receptor CXCR4, recruits endothelial progenitor cells into the tumour promoting neoangiogenesis. Tumour enhancing properties are preserved even on culture in isolation from cancer cells for more than 10 popoulation doublings. Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion Akira�Orimo1,�Piyush B.�Gupta1,�2,�Dennis C.�Sgroi4,�Fernando�Arenzana-Seisdedos7,�Thierry�Delaunay8,�Rizwan�Naeem6,�Vincent J.�Carey5,Andrea L.�Richardson3�and�Robert A.�Weinberg1,�2,� ,� Cell, Vol. 121, 335�348, May 6, 2005 Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion Akira�Orimo1,�Piyush B.�Gupta1,�2,�Dennis C.�Sgroi4,�Fernando�Arenzana-Seisdedos7,�Thierry�Delaunay8,�Rizwan�Naeem6,�Vincent J.�Carey5,Andrea L.�Richardson3�and�Robert A.�Weinberg1,�2,� ,� Cell, Vol. 121, 335�348, May 6, 2005

14. SDF-1 in Cancer-Stromal Interactions Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion Akira�Orimo1,�Piyush B.�Gupta1,�2,�Dennis C.�Sgroi4,�Fernando�Arenzana-Seisdedos7,�Thierry�Delaunay8,�Rizwan�Naeem6,�Vincent J.�Carey5,Andrea L.�Richardson3�and�Robert A.�Weinberg1,�2,� ,� Cell, Vol. 121, 335�348, May 6, 2005Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion Akira�Orimo1,�Piyush B.�Gupta1,�2,�Dennis C.�Sgroi4,�Fernando�Arenzana-Seisdedos7,�Thierry�Delaunay8,�Rizwan�Naeem6,�Vincent J.�Carey5,Andrea L.�Richardson3�and�Robert A.�Weinberg1,�2,� ,� Cell, Vol. 121, 335�348, May 6, 2005

15. CAFs express Fibroblast Secreted Protein 1(FSP1) a calcium binding protein with intracellular effects(inhibition of p53 and extracellular effects(activation of plasmin, promotion of metastasis and angiogenesis) CAFs express the matrix protein Tenascin (Proc. Nati. Acad. Sci. USA Vol. 84, pp. 4621-4625, July 1987) CAFs secrete increased collagen,fibronectin, proteoglycans,glycosaminoglycans and express elevated levels of MMPs suggesting accelerated matrix turnover. CAFs secrete fibroblast activation Protein, a serine protease(Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6: 392�401.) Cold Spring Harb Symp Quant Biol.�2005;70:383-8. The fibroblastic coconspirator in cancer progression. Egeblad M,�Littlepage LE,�Werb Z.Cold Spring Harb Symp Quant Biol.�2005;70:383-8. The fibroblastic coconspirator in cancer progression. Egeblad M,�Littlepage LE,�Werb Z.

16. CAFs have been shown in different experiments, indifferent contexts to secrete a variety of growth factors including: BDNF (j.oraloncology.2010.11.002) HGF (PNAS June 15, 2010 vol. 107 no.24) IGF-2 (CANCER RESEARCH 55, 2448-2454, June 1, 1995) TGF-� (Cancer Res 2007;67:4244-4253) Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells J�zsef Dud�s a,?, Mario Bitsche a, Volker Schartinger a, Christina Falkeis b, Georg Mathias Sprinzl a, Herbert Riechelmann aJ. Dud�s et al. / Oral Oncology xxx (2010) xxx�xxx | PNAS | June 15, 2010 | vol. 107 | no. 24Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion Katharine D. Grugana,b, Charles G. Millera,b, Yao Yaoa,b, Carmen Z. Michayliraa,b, Shinya Ohashia,b, Andres J. Klein-Szantoc, J. Alan Diehld, Meenhard Herlyne, May Hanf, Hiroshi Nakagawaa,b, and Anil K. Rustgia,b,g,1 [CANCER RESEARCH 55, 2448-2454, June 1, 1995] Malignant Breast Epithelium Selects for Insulin-like Growth Factor II Expression in Breast Stroma: Evidence for Paracrine Function1 Christian Singer, Audrey Rasmussen, HélèSn.eSmith, Marc E. Lippman, Henry T. Lynch, and Kevin J. Cullar Cross-talk between Paracrine-Acting Cytokine and Chemokine Pathways Promotes Malignancy in Benign Human Prostatic Epithelium Mingfang Ao,1 Omar E. Franco,2 Dean Park,3 Dayanidhi Raman,1 Karin Williams,2 and Simon W. Hayward1,2,4 Cancer Res 2007;67:4244-4253Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells J�zsef Dud�s a,?, Mario Bitsche a, Volker Schartinger a, Christina Falkeis b, Georg Mathias Sprinzl a, Herbert Riechelmann aJ. Dud�s et al. / Oral Oncology xxx (2010) xxx�xxx | PNAS | June 15, 2010 | vol. 107 | no. 24Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion Katharine D. Grugana,b, Charles G. Millera,b, Yao Yaoa,b, Carmen Z. Michayliraa,b, Shinya Ohashia,b, Andres J. Klein-Szantoc, J. Alan Diehld, Meenhard Herlyne, May Hanf, Hiroshi Nakagawaa,b, and Anil K. Rustgia,b,g,1 [CANCER RESEARCH 55, 2448-2454, June 1, 1995] Malignant Breast Epithelium Selects for Insulin-like Growth Factor II Expression in Breast Stroma: Evidence for Paracrine Function1 Christian Singer, Audrey Rasmussen, HélèSn.eSmith, Marc E. Lippman, Henry T. Lynch, and Kevin J. Cullar Cross-talk between Paracrine-Acting Cytokine and Chemokine Pathways Promotes Malignancy in Benign Human Prostatic Epithelium Mingfang Ao,1 Omar E. Franco,2 Dean Park,3 Dayanidhi Raman,1 Karin Williams,2 and Simon W. Hayward1,2,4 Cancer Res 2007;67:4244-4253

17. CAFs have been shown in different experiments to secrete a variety of chemokines including: CXCL12(SDF-1) (Cell, Vol. 121, 335�348, May 6, 2005) CCL5(Rantes) (NATURE Vol 449:4 October 2007) CCL-2 (MCP-1) (Cancer Letters 267 (2008) 271�285) CXCL-1 CXCL-2 CXCL-8 Journal of Leukocyte Biology.Volume 89, January 2011 CXCL-14 CCL-7 Mesenchymal stem cells within tumour stroma promote breast cancer metastasis Antoine E. Karnoub1, Ajeeta B. Dash2, Annie P. Vo1, Andrew Sullivan2, Mary W. Brooks1, George W. Bell1, Andrea L. Richardson3, Kornelia Polyak4, Ross Tubo2 & Robert A. Weinberg1NATURE| Vol 449|4 October 2007 The inflammatory chemokines CCL2 and CCL5 in breast cancer Gali Soria, Adit Ben-Baruch * Cancer Letters 267 (2008) 271�285 Chemokines at the crossroads of tumor-fibroblast interactions that promote malignancy Pravin Mishra,* Debabrata Banerjee,* and Adit Ben-Baruch�,1 Journal of Leukocyte Biology.Volume 89, January 2011Mesenchymal stem cells within tumour stroma promote breast cancer metastasis Antoine E. Karnoub1, Ajeeta B. Dash2, Annie P. Vo1, Andrew Sullivan2, Mary W. Brooks1, George W. Bell1, Andrea L. Richardson3, Kornelia Polyak4, Ross Tubo2 & Robert A. Weinberg1NATURE| Vol 449|4 October 2007 The inflammatory chemokines CCL2 and CCL5 in breast cancer Gali Soria, Adit Ben-Baruch * Cancer Letters 267 (2008) 271�285 Chemokines at the crossroads of tumor-fibroblast interactions that promote malignancy Pravin Mishra,* Debabrata Banerjee,* and Adit Ben-Baruch�,1 Journal of Leukocyte Biology.Volume 89, January 2011

18. CAFs have been shown in different experiments to secrete a variety of cytokines including IL-1� (j.oraloncology.2010.11.002) IL-6 (Mol Cancer Res 2009;7:1212-1223) Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells J�zsef Dud�s a,?, Mario Bitsche a, Volker Schartinger a, Christina Falkeis b, Georg Mathias Sprinzl a, Herbert Riechelmann aJ. Dud�s et al. / Oral Oncology xxx (2010) xxx�xxx Differential Influence of Normal and Cancer-Associated Fibroblasts on the Growth of Human Epithelial Cells in an In vitro Cocultivation Model of Prostate Cancer Nicole Paland,Iris Kamer,Ira Kogan-Sakin,Shalom Madar, Naomi Goldfinger,and Varda Rotter. Mol Cancer Res 2009;7(8). August 200Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells J�zsef Dud�s a,?, Mario Bitsche a, Volker Schartinger a, Christina Falkeis b, Georg Mathias Sprinzl a, Herbert Riechelmann aJ. Dud�s et al. / Oral Oncology xxx (2010) xxx�xxx Differential Influence of Normal and Cancer-Associated Fibroblasts on the Growth of Human Epithelial Cells in an In vitro Cocultivation Model of Prostate Cancer Nicole Paland,Iris Kamer,Ira Kogan-Sakin,Shalom Madar, Naomi Goldfinger,and Varda Rotter. Mol Cancer Res 2009;7(8). August 200

19. Genetic Abnormalities of the Stroma

20. Cytogenetic analysis in AML and MDS revealed abnormalities which were sometimes not shared by stromal cells and hematopoietic cells, and some which were shared. (Chromosomal aberrations in bone marrow stromal cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia. Experimental Hematology 35 (2007) 221�229) Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia Olga Blaua, Wolf-Karsten Hofmanna, Claudia Dorothea Baldusa, Gundula Thielb, Verena Serbenta, Elke Schu�mannb, Eckhard Thiela, and Igor Wolfgang Blaua. Experimental Hematology 35 (2007) 221�229Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia Olga Blaua, Wolf-Karsten Hofmanna, Claudia Dorothea Baldusa, Gundula Thielb, Verena Serbenta, Elke Schu�mannb, Eckhard Thiela, and Igor Wolfgang Blaua. Experimental Hematology 35 (2007) 221�229

21. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization Eugenia Flores-Figueroaa, Rosa Maria Arana-Trejob, Guillermo Guti�errez-Esp�indolac, Adri�an P�erez-Cabrerab, Hector Mayania,*Leukemia Research 29 (2005) 215�224Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization Eugenia Flores-Figueroaa, Rosa Maria Arana-Trejob, Guillermo Guti�errez-Esp�indolac, Adri�an P�erez-Cabrerab, Hector Mayania,*Leukemia Research 29 (2005) 215�224

22. Genetic instability and loss of heterozygosity at various loci were detected in a clinical study of patients with esophageal cancer. (Early genetic instability of both epithelial and stromal cells in esophageal squamous cell carcinomas, contrasted with Barrett�s adenocarcinomas. J Gastroenterol 2006; 41:1186�1196.) J Gastroenterol 2006; 41:1186�1196 DOI 10.1007/s00535-006-1953-4 Received: July 20, 2006 / Accepted: September 12, 2006 Reprint requests to: H. Shiraishi Early genetic instability of both epithelial and stromal cells in esophageal squamous cell carcinomas, contrasted with Barrett�s adenocarcinomas Hiroaki Shiraishi1,2, Tetuo Mikami1, Tsutomu Yoshida1, Satoshi Tanabe3, Nobuyuki Kobayashi2, Masahiko Watanabe2, and Isao Okayasu1J Gastroenterol 2006; 41:1186�1196 DOI 10.1007/s00535-006-1953-4 Received: July 20, 2006 / Accepted: September 12, 2006 Reprint requests to: H. Shiraishi Early genetic instability of both epithelial and stromal cells in esophageal squamous cell carcinomas, contrasted with Barrett�s adenocarcinomas Hiroaki Shiraishi1,2, Tetuo Mikami1, Tsutomu Yoshida1, Satoshi Tanabe3, Nobuyuki Kobayashi2, Masahiko Watanabe2, and Isao Okayasu1

23. In a study of patients with breast cancer concurrent and independent LOH in the epithelial and stromal components of tumours was detected. (Concurrent and Independent Genetic Alterations in the Stromal and Epithelial Cells of Mammary Carcinoma: Implications for Tumorigenesis. CANCER RESEARCH 60, 2562�2566, May 1, 2000) [CANCER RESEARCH 60, 2562�2566, May 1, 2000] Concurrent and Independent Genetic Alterations in the Stromal and Epithelial Cells of Mammary Carcinoma: Implications for Tumorigenesis1 Farid Moinfar,2 Yan Gao Man, Laurent Arnould, Gary L. Bratthauer, Manfred Ratschek, and Fattaneh A. Tavassoli [CANCER RESEARCH 60, 2562�2566, May 1, 2000] Concurrent and Independent Genetic Alterations in the Stromal and Epithelial Cells of Mammary Carcinoma: Implications for Tumorigenesis1 Farid Moinfar,2 Yan Gao Man, Laurent Arnould, Gary L. Bratthauer, Manfred Ratschek, and Fattaneh A. Tavassoli

24. Specific mutations:p53 P53 mutations exclusively in the stromal compartment were detected in a high proportion of breast carcinomas and were found to be mutually exclusive with PTEN mutations (Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15 ) Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas

25. Mutant p53 has been detected in ALL stromal cells (Experimental HematologyVolume 31, Issue 8, August 2003, Pages 693-701 ). Inherited breast cancer was found to be associated with somatic mutations of p53;stromal, but not epithelial mutations were significantly associated with nodal metastases (N Engl J Med.�2007 Dec 20;357(25): 2543-51 ) Hematological malignancies Mutant�p53�in�bone marrow�stromal cells�increases�VEGF�expression and supportsleukemia�cell growth abbcbcbbAru�Narendran, Hooman�Ganjavi, Natalie�Morson, Alison�Connor, Jason W.�Barlow, Edward�Keystone, David�Malkin ,� �and Melvin H.Freedman N Engl J Med.�2007 Dec 20;357(25):2543-51. Breast-cancer stromal cells with TP53 mutations and nodal metastases. Patocs A,�Zhang L,�Xu Y,�Weber F,�Caldes T,�Mutter GL,�Platzer P,�Eng C. Genomic Medicine Institute, Cleveland Clinic Foundation, Cleveland 44195, USAHematological malignancies Mutant�p53�in�bone marrow�stromal cells�increases�VEGF�expression and supportsleukemia�cell growth abbcbcbbAru�Narendran, Hooman�Ganjavi, Natalie�Morson, Alison�Connor, Jason W.�Barlow, Edward�Keystone, David�Malkin ,� �and Melvin H.Freedman N Engl J Med.�2007 Dec 20;357(25):2543-51. Breast-cancer stromal cells with TP53 mutations and nodal metastases. Patocs A,�Zhang L,�Xu Y,�Weber F,�Caldes T,�Mutter GL,�Platzer P,�Eng C. Genomic Medicine Institute, Cleveland Clinic Foundation, Cleveland 44195, USA

26. PTEN PTEN mutations exclusively in the stromal compartment were detected in a high proportion of breast carcinomas and were found to be mutually exclusive with p53 mutations (Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15 ) Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas Nat Genet.�2002 Nov;32(3):355-7. Epub 2002 Oct 15. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas

27. In mouse models specific mutations targeted selectively to the stroma have been shown to cause transformation in the epithelial compartment -PTEN,Cyclin D1,APC and TGF beta receptor2 KO. (Cancer Res. 2011 Feb 15;71(4):1203-7. Epub 2011 Feb 8, Cancer Res.�2007 Sep 1;67(17):8188-97 Cancer Res.�2011 Mar 1;71(5):1584-96 Science.�2004 Feb 6;303(5659):848-51) Cancer Res.�2011 Feb 15;71(4):1203-7. Epub 2011 Feb 8. Pten in the breast tumor microenvironment: modeling tumor-stroma coevolution. Wallace JA,�Li F,�Leone G,�Ostrowski MC. Cancer Res.�2007 Sep 1;67(17):8188-97. Tissue-specific consequences of cyclin D1 overexpression in prostate cancer progression. He Y,�Franco OE,�Jiang M,�Williams K,�Love HD,�Coleman IM,�Nelson PS,�Hayward SW. Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA Cancer Res.�2011 Mar 1;71(5):1584-96. Stromal Deletion of the APC Tumor Suppressor in Mice Triggers Development of Endometrial Cancer. Tanwar PS,�Zhang L,�Roberts DJ,�Teixeira JM. Science.�2004 Feb 6;303(5659):848-51. TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Bhowmick NA,�Chytil A,�Plieth D,�Gorska AE,�Dumont N,�Shappell S,�Washington MK,�Neilson EG,�Moses HL. Cancer Res.�2011 Feb 15;71(4):1203-7. Epub 2011 Feb 8. Pten in the breast tumor microenvironment: modeling tumor-stroma coevolution. Wallace JA,�Li F,�Leone G,�Ostrowski MC. Cancer Res.�2007 Sep 1;67(17):8188-97. Tissue-specific consequences of cyclin D1 overexpression in prostate cancer progression. He Y,�Franco OE,�Jiang M,�Williams K,�Love HD,�Coleman IM,�Nelson PS,�Hayward SW. Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA Cancer Res.�2011 Mar 1;71(5):1584-96. Stromal Deletion of the APC Tumor Suppressor in Mice Triggers Development of Endometrial Cancer. Tanwar PS,�Zhang L,�Roberts DJ,�Teixeira JM. Science.�2004 Feb 6;303(5659):848-51. TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Bhowmick NA,�Chytil A,�Plieth D,�Gorska AE,�Dumont N,�Shappell S,�Washington MK,�Neilson EG,�Moses HL.

28. Controversies over Stromal Mutations 1.Stromal Cells are separated/segregated from epithelial cells by various means. Laser capture micro-dissection ( by an experienced operator ) is the gold standard. This is performed on formalin fixed paraffin embedded tissue. Typically the number of cells sampled is in the order of 104 to 105 cells.(N Engl J Med 2007;357:2543-51. A Patocs, C Eng).

29. Formalin fixed paraffin embedded tissue use may lead to processing induced artefacts Small initial sample size may lead to small artefacts being amplified during PCR leading to errors(detection of mutations which are not truly present). The negative control should therefore be extremely stringent consisting of multiple samples of microdissected normal tissue from the same specimen processed in the same way consisting of the same small number of cells that the experimental sample contains. Few studies adhere to this standard.

30. IS THE STROMA ITSELF TRANSFORMED? PROBABLY,NO.

31. IS THE STROMA ITSELF TRANSFORMED? PROBABLY,NO. CAFs have not been shown to demonstrate anchorage independent growth in culture, are not tumourigenic by themselves on transplant into immunocompromised host mice and often senesce in culture after a finite number of population doublings(Cell, Vol. 121, 335�348, May 6, 2005). Cell, Vol. 121, 335�348, May 6, 2005, Copyright �2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.02.034 Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 SecretionAkira Orimo, 1 Piyush B. Gupta, 1,2 Dennis C. Sgroi, 4 Introduction Fernando Arenzana-Seisdedos, 7 Thierry Delaunay, 8 Rizwan Naeem, 6 Vincent J. Carey, Andrea L. Richardson, and Robert A. Weinberg 1,2, Cell, Vol. 121, 335�348, May 6, 2005, Copyright �2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.02.034 Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 SecretionAkira Orimo, 1 Piyush B. Gupta, 1,2 Dennis C. Sgroi, 4 Introduction Fernando Arenzana-Seisdedos, 7 Thierry Delaunay, 8 Rizwan Naeem, 6 Vincent J. Carey, Andrea L. Richardson, and Robert A. Weinberg 1,2,

32. However: Not all epithelial carcinomas successfully give rise to cell lines in culture Not all epithelial carcinomas from which epithelial cell lines are generated, prove to be immortal in culture a feature which can be rescued by transduction with hTERT. (Reconstitution of Human Telomerase Reverse Transcriptase Expression Rescues Colorectal Carcinoma Cells from In vitro Senescence: Evidence against Immortality as a Constitutive Trait of Tumor Cells. Cancer Res 2005; 65: (6). March 15, 2005) Reconstitution of Human Telomerase Reverse Transcriptase Expression Rescues Colorectal Carcinoma Cells from In vitro Senescence: Evidence against Immortality as a Constitutive Trait of Tumor Cells Piero Dalerba,1 Cristiana Guiducci,2 Pietro Luigi Poliani,3 Ingrid Cifola,1 Mariella Parenza,2 Milo Frattini,4 Gianfrancesco Gallino,5 Ileana Carnevali,4 Ilvia Di Giulio,1 Salvatore Andreola,3 Claudia Lombardo,6 Licia Rivoltini,1 Tama�s Schweighoffer,7 Filiberto Belli,5 Mario P. Colombo,2 Giorgio Parmiani,1 and Chiara Castelli1.Cancer Res 2005; 65: (6). March 15, 2005Reconstitution of Human Telomerase Reverse Transcriptase Expression Rescues Colorectal Carcinoma Cells from In vitro Senescence: Evidence against Immortality as a Constitutive Trait of Tumor Cells Piero Dalerba,1 Cristiana Guiducci,2 Pietro Luigi Poliani,3 Ingrid Cifola,1 Mariella Parenza,2 Milo Frattini,4 Gianfrancesco Gallino,5 Ileana Carnevali,4 Ilvia Di Giulio,1 Salvatore Andreola,3 Claudia Lombardo,6 Licia Rivoltini,1 Tama�s Schweighoffer,7 Filiberto Belli,5 Mario P. Colombo,2 Giorgio Parmiani,1 and Chiara Castelli1.Cancer Res 2005; 65: (6). March 15, 2005

33. HOW DO STROMAL MUTATIONS ARISE: A Model for Positive Selection of Mutations in the Stroma:Co-evolution

34. Oncogene induced Growth Factor Secretion-inside out growth signaling Based on data from: Proc. Natl. Acad. Sci. USA Vol. 84, pp. 837-841, February 1987. Oncogene (2010) 29, 3335�3348. GENES & DEVELOPMENT 9:1953-1964 HMG Advance Access published April 27, 2006 HMG Advance Access published April 27, 2006. Neurofibromin is a Novel Regulator of Ras Induced Signals in Primary Vascular Smooth Muscle Cells Fang Li1,2 Amy M. Munchhof1,2,3, Hilary A. White1,2, Laura E. Mead 1,2, Theresa R. Krier1,2, Amy Fenoglio1,2, Shi Chen1,2, Xiaohua Wu1,2, Shanbao Cai1,2, Feng-Chun Yang,1,2 David A. Ingram*1,2,3 Proc. Natl. Acad. Sci. USA Vol. 84, pp. 837-841, February 1987 Medical Sciences Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17f8-estradiol or v-Ha-ras oncogene ROBERT B. DICKSON, ATTAN KASID, KAREN K. HUFF, SUSAN E. BATES, CORNELIUS KNABBE, DIANE BRONZERT, EDWARD P. GELMANN, AND MARC E. LIPPMAN Oncogenic mutations regulate tumor microenvironment through induction of growth factors and angiogenic mediators SE Wang1, Y Yu2, TL Criswell3, LM DeBusk3,4, PC Lin3,4, R Zent3,5, DH Johnson5, X Ren2 and CL Arteaga3,5,6 Oncogene (2010) 29, 3335�3348 at doi:10.1101/gad.9.16.1953 Genes Dev. 1995 9: 1953-1964 S A McCarthy, M L Samuels, C A Pritchard, et al. oncogenes. factor/diphtheria toxin receptor expression by Raf and Ras Rapid induction of heparin-binding epidermal growthHMG Advance Access published April 27, 2006. Neurofibromin is a Novel Regulator of Ras Induced Signals in Primary Vascular Smooth Muscle Cells Fang Li1,2 Amy M. Munchhof1,2,3, Hilary A. White1,2, Laura E. Mead 1,2, Theresa R. Krier1,2, Amy Fenoglio1,2, Shi Chen1,2, Xiaohua Wu1,2, Shanbao Cai1,2, Feng-Chun Yang,1,2 David A. Ingram*1,2,3 Proc. Natl. Acad. Sci. USA Vol. 84, pp. 837-841, February 1987 Medical Sciences Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17f8-estradiol or v-Ha-ras oncogene ROBERT B. DICKSON, ATTAN KASID, KAREN K. HUFF, SUSAN E. BATES, CORNELIUS KNABBE, DIANE BRONZERT, EDWARD P. GELMANN, AND MARC E. LIPPMAN Oncogenic mutations regulate tumor microenvironment through induction of growth factors and angiogenic mediators SE Wang1, Y Yu2, TL Criswell3, LM DeBusk3,4, PC Lin3,4, R Zent3,5, DH Johnson5, X Ren2 and CL Arteaga3,5,6 Oncogene (2010) 29, 3335�3348 at doi:10.1101/gad.9.16.1953 Genes Dev. 1995 9: 1953-1964 S A McCarthy, M L Samuels, C A Pritchard, et al. oncogenes. factor/diphtheria toxin receptor expression by Raf and Ras Rapid induction of heparin-binding epidermal growth

35. Oncogene induced Cytokine Secretion Carcinogenesis vol.30 no.11 pp.1841�1847, 2009 doi:10.1093/carcin/bgp198 Advance Access publication October 5, 2009 ELR1 CXC chemokines and oncogenic Ras-mediated tumorigenesis Kevin M.O�Hayer1, Donita C.Brady1 and Christopher M.Counter,1,2 doi:10.1101/gad.1549407 Genes Dev. 2007 21: 1714-1719 Brooke Ancrile, Kian-Huat Lim and Christopher M. Counter tumorigenesis Oncogenic Ras-induced secretion of IL6 is required for Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis Anke Sparmann1,2 and Dafna Bar-Sagi2,*. CANCER CELL : NOVEMBER 2004 � VOL. 6 Carcinogenesis vol.30 no.11 pp.1841�1847, 2009 doi:10.1093/carcin/bgp198 Advance Access publication October 5, 2009 ELR1 CXC chemokines and oncogenic Ras-mediated tumorigenesis Kevin M.O�Hayer1, Donita C.Brady1 and Christopher M.Counter,1,2 doi:10.1101/gad.1549407 Genes Dev. 2007 21: 1714-1719 Brooke Ancrile, Kian-Huat Lim and Christopher M. Counter tumorigenesis Oncogenic Ras-induced secretion of IL6 is required for Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis Anke Sparmann1,2 and Dafna Bar-Sagi2,*. CANCER CELL : NOVEMBER 2004 � VOL. 6

36. Cytokine induced Cytokine Secretion Am J Physiol Heart Circ Physiol 297:H1117-H1127, 2009. First published 31 July 2009; Ball and Karen E. Porter Neil A. Turner, Anupam Das, Philip Warburton, David J. O'Regan, Stephen G. expression in human cardiac myofibroblasts Interleukin-1a stimulates proinflammatory cytokine J Immunol 2002;168;861-868 Tasaki, Yoshio Araki, Yoshihide Fujiyama and Tadao Bamba Mitsue Shimada, Akira Andoh, Kazunori Hata, Kazuhito Mediators Myofibroblasts in Response to Inflammatory J Immunol 2002;168;861-868Am J Physiol Heart Circ Physiol 297:H1117-H1127, 2009. First published 31 July 2009; Ball and Karen E. Porter Neil A. Turner, Anupam Das, Philip Warburton, David J. O'Regan, Stephen G. expression in human cardiac myofibroblasts Interleukin-1a stimulates proinflammatory cytokine J Immunol 2002;168;861-868 Tasaki, Yoshio Araki, Yoshihide Fujiyama and Tadao Bamba Mitsue Shimada, Akira Andoh, Kazunori Hata, Kazuhito Mediators Myofibroblasts in Response to Inflammatory J Immunol 2002;168;861-868

37. Cytokine Induced Growth Factor Secretion Fibroblast growth factor 7, secreted by breast fibroblasts, is an interleukin-1-induced paracrine growth factor for human breast cells C Palmieri, D Roberts-Clark, A Assadi-Sabet, R C Coope, M O�Hare1, A Sunters, A Hanby2, M J Slade, J J Gomm, E W-F Lam and R C CoombesFibroblast growth factor 7, secreted by breast fibroblasts, is an interleukin-1-induced paracrine growth factor for human breast cells C Palmieri, D Roberts-Clark, A Assadi-Sabet, R C Coope, M O�Hare1, A Sunters, A Hanby2, M J Slade, J J Gomm, E W-F Lam and R C Coombes

38. Induced Autocrine Signaling through the Epidermal Growth Factor Receptor Contributes to the Response of Mammary Epithelial Cells to Tumor Necrosis Factor * Received for publication, October 2, 2003, and in revised form, February 19, 2004 Published, JBC Papers in Press, February 20, 2004, DOI 10.1074/jbc.M310874200 Wan-Nan U. Chen, Ronald L. Woodbury, Loel E. Kathmann, Lee K. Opresko, Richard C. Zangar, H. Steven Wiley, and Brian D. Thrall� From the Cell Biology Group, Biological Sciences Division, THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 18, Issue of April 30, pp. 18488�18496, 2004Induced Autocrine Signaling through the Epidermal Growth Factor Receptor Contributes to the Response of Mammary Epithelial Cells to Tumor Necrosis Factor * Received for publication, October 2, 2003, and in revised form, February 19, 2004 Published, JBC Papers in Press, February 20, 2004, DOI 10.1074/jbc.M310874200 Wan-Nan U. Chen, Ronald L. Woodbury, Loel E. Kathmann, Lee K. Opresko, Richard C. Zangar, H. Steven Wiley, and Brian D. Thrall� From the Cell Biology Group, Biological Sciences Division, THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 18, Issue of April 30, pp. 18488�18496, 2004

39. Secretome of tumour suppressor gene inactivation Proteomic identification of the wt-p53-regulated tumor cell secretome FW Khwaja1,2, P Svoboda3, M Reed3, J Pohl3 , B Pyrzynska1,2 and EG Van Meir1,2. Oncogene (2006) 25, 7650�7661Proteomic identification of the wt-p53-regulated tumor cell secretome FW Khwaja1,2, P Svoboda3, M Reed3, J Pohl3 , B Pyrzynska1,2 and EG Van Meir1,2. Oncogene (2006) 25, 7650�7661

40. Proteomic identification of the wt-p53-regulated tumor cell secretome FW Khwaja1,2, P Svoboda3, M Reed3, J Pohl3 , B Pyrzynska1,2 and EG Van Meir1,2. Oncogene (2006) 25, 7650�7661 Proteomic identification of the wt-p53-regulated tumor cell secretome FW Khwaja1,2, P Svoboda3, M Reed3, J Pohl3 , B Pyrzynska1,2 and EG Van Meir1,2. Oncogene (2006) 25, 7650�7661

41. Cancer Cell.�2005 Jun;7(6):499-500. Coevolution of cancer and stromal cellular responses. Littlepage LE,�Egeblad M,�Werb Z.Molecular mediators of stromal-epithelial interactions in tumorigenesis The cells in the tumor tissue communicate during tumor progression through the secretion of growth factors, chemokines, and cytokines.A:�Examples of stimulators of tumorigenesis secreted by one cell type and acting on another.B:�TGF-� is unique in its ability to both promote and inhibit tumorigenesis, depending on the cell type it is acting on. CAFs, carcinoma-associated fibroblasts; VEGF, vascular endothelial growth factor; SDF-1a, stromal derived factor 1a; FSP-1, fibroblast-specific protein-1; HGF, hepatocyte growth factor; MSP, macrophage-stimulating protein; EGF, epidermal growth factor; TGF-a, transforming growth factor-a; TGF-�, transforming growth factor-�; FGF, fibroblast growth factor; IL-6, interleukin 6; LIF, leukemia inhibitory factor; NGF, nerve growth factor (Bhowmick et al., 2004b, and references therein).� Cancer Cell.�2005 Jun;7(6):499-500. Coevolution of cancer and stromal cellular responses. Littlepage LE,�Egeblad M,�Werb Z.Molecular mediators of stromal-epithelial interactions in tumorigenesis The cells in the tumor tissue communicate during tumor progression through the secretion of growth factors, chemokines, and cytokines.A:�Examples of stimulators of tumorigenesis secreted by one cell type and acting on another.B:�TGF-� is unique in its ability to both promote and inhibit tumorigenesis, depending on the cell type it is acting on. CAFs, carcinoma-associated fibroblasts; VEGF, vascular endothelial growth factor; SDF-1a, stromal derived factor 1a; FSP-1, fibroblast-specific protein-1; HGF, hepatocyte growth factor; MSP, macrophage-stimulating protein; EGF, epidermal growth factor; TGF-a, transforming growth factor-a; TGF-�, transforming growth factor-�; FGF, fibroblast growth factor; IL-6, interleukin 6; LIF, leukemia inhibitory factor; NGF, nerve growth factor (Bhowmick et al., 2004b, and references therein).�

43. EVOLUTION OF CAFs

44. Therapeutic implications Therapies targeted at stromal interactions: Anti-PDGF mAb(already in trials) TKIs with PDGF-R TKI activity Antiinflammatory agents, eg anti TNFa Mab, IL-1R antagonists and Anti IL-6 Mab (all already FDA approved in rheumatoid arthritis) Anti chemokine therapies(Plerixafor)

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