Cancer Stem Cells and the Microenvironment

Figure 10-1 The tumor microenvironment Elevated levels of cytokines/growth factors produced by tumor cells enhance the proliferation and survival of cancer stem cells, induce angiogenesis, and recruit tumor-associated macrophages, neutrophils, and mast cells, which secrete additional growth factors, forming a positive feedback loop that promotes tumor cell invasion and distant metastasis.

Cellular Components of the Tumor Microenvironment That Influence Cancer Stem Cells

During tumor progression, tumor cells recruit a diverse collection of cells that make up the microenvironment, and through iterative interactions both the tumor cell and its microenvironment co-evolve. ⁴² For example, because of increased oxidative stress in the prostate tumor microenvironment, stromal cells acquire the ability to mimic other cell types such as mesenchymal and vascular stem cells. ⁴⁸ Although early studies suggested that some of the cells in the tumor microenvironment harbored mutations, ²² more recent evidence suggests that mutations are limited to the tumorigenic cells, which through paracrine interactions modify the epigenetic program of nontumorigenic cells in the tumor microenvironment.^22,41,49,50The cells in the microenvironment in turn interact with and generate epigenetic changes in tumor cells.^41,42This reciprocal interaction is illustrated by changes in the tumor microenvironment that occur during the evolution of preinvasive ductal carcinoma in situ to invasive carcinoma of the breast, which involves sequential epigenetic changes in both the tumor as well as the stromal microenvironment.^41,42Human bone stromal cells were able to induce aggressive mouse prostate tumors that acquired an EMT phenotype associated with resistance to radiation therapy. ⁵¹

In human breast cancers, mesenchymal cells are recruited from the bone marrow ⁴³ or from the normal breast stroma. ⁴⁵ As is the case in breast cancer cells, ALDH expression is elevated in MSCs that are recruited from the bone marrow. These MSCs interact with breast CSCs through cytokine loops involving IL6 and CXCL7. ⁴³ These cytokine loops stimulate the self-renewal of breast CSCs. ⁴³ Immunohistochemical analysis has confirmed the existence of such MSC–breast CSC interactions in biopsies obtained from breast cancer patients. ⁴³ In addition, MSCs have the ability to differentiate into adipocytes as well as tumor-associated fibroblasts that also interact with and influence tumor cells. ⁵²

The activation of fibroblasts and myofibroblasts was originally described in a study of wound healing by Gabbiani and Majno, who observed morphological changes in activated myofibroblasts compared to quiescent tumor- and wound-associated fibroblasts. ⁵³ Based on the similarities between the wound healing process and cancer, both of which involve infiltration of inflammatory cells and activation of cytokine networks, it was proposed that malignant tumor cells are “wounds that don’t heal.” ⁵⁴ It appears that the persistence of a wound healing environment promotes the persistence and/or expansion of CSCs. In an experimental mouse model, acute wounding in the mammary gland by dermal incision accelerated breast tumor growth and metastasis. ⁵⁵ Although the exact mechanisms remain unknown, paracrine signals from evolving tumors induce epigenetic changes in the surrounding stromal fibroblasts. ⁵⁶ Indeed, the gene expression profile of tumor-associated fibroblasts resembles that of wound-activated fibroblasts, and this profile is associated with poor prognosis.^57,58Growth factors such as TGF-β may be involved in these epigenetic changes, leading to activation of fibroblasts. ⁵⁹ In addition, cytokines such as SDF-1 (aka CXCL-12) produced by breast carcinoma–associated fibroblasts (but not normal fibroblasts) may promote the proliferation of tumor cells, which express the SDF-1 receptor CXCR4. ⁶⁰ The level of expression of SDF-1 in serum has been associated with poor survival in breast cancer patients.^61,62Other growth factors such as hepatocyte growth factor (HGF), produced by mammary stromal cells, may also have a profound effect on developing mammary tumors, possibly inducing the stem cell compartment. ⁶³ HGF provides a co-stimulatory signal to the Wnt pathway during colon carcinogenesis. ⁶⁴ Other important growth factors produced by activated fibroblasts include the fibroblast growth factors (FGFs). Cancer stem cells can use oxidative stress to drive stromal fibroblasts to produce necessary nutrients for their survival. ⁶⁵ It has recently been shown that estrogen regulates the breast CSC population through a paracrine mechanism involving FGF9. ⁶⁶ Additional factors produced by stromal cells in the tumor microenvironment activate a number of pathways including IGF, PDGF, Wnt, NFκB, Notch, Hedgehog, and matrix metalloproteinases (MMPs) regulating tumor proliferation, invasion, and metastasis.^67–72These pathways also have been implicated in regulating CSCs in a number of human malignancies.^{37,40,73–76}

Immunomodulatory cells may also exert inhibitory and stimulatory effects on CSCs, and the ultimate balance of these effects may profoundly influence tumor growth/progression. The importance of the immune system is illustrated by recent studies elucidating the mechanisms by which macrophages recognize and destroy CSCs. Recent studies in human leukemia and lymphoma have suggested that tumor cells express the antigen CD47, which serves as a “don’t eat me” signal to tumor-associated macrophages. ⁷⁷ At the same time, these cells express calreticulin, recognized by these macrophages as an “eat me” signal. ⁷⁸ Administration of a blocking antibody to CD47 induced macrophage phagocytosis of tumor cells in vitro and in mouse models. Importantly, it was demonstrated that leukemic stem cells as well as bulk tumor cells could be targeted by this approach.^78,79Interestingly, CD47 is widely expressed in a number of solid tumor CSCs, and targeting of this molecule suppresses tumor growth and metastasis in mouse tumor models. ⁸⁰

Endothelial cells may also play an important role in the tumor microenvironment by directly interacting with tumor cells as well as by their role in blood vessel formation. Endothelial cells constitute an important component of normal hematopoietic and neuronal stem cell niches.^81,82In addition, cytokines produced by endothelial cells directly regulate cancer stem cells.^83,84More than 40 years ago, Judah Folkman proposed that angiogenesis, the process of new blood vessel formation, was required for tumor growth and metastasis. ⁸⁵ The role of tumor angiogenesis has been demonstrated in preclinical models of many cancers, including those of the breast. ⁸⁶ This has led to the development of a number of anti-angiogenic agents as cancer therapeutics. Angiogenesis is a complex process involving interactions among multiple cell types. Bone marrow–derived endothelial progenitor cells are attracted to tumors, where they differentiate into mature endothelial cells and capillaries.^87,88These newly formed blood vessels carry oxygen and nutrients to growing tumors, facilitating progression and metastasis (see Figure 10-1). Interestingly, the tumor vasculature is vastly different from the normal vasculature, as illustrated by the finding that more than 1000 genes are differentially expressed, including FGFRs, MMPs, and JAK3. ⁸³ Although preneoplastic lesions lack angiogenic capacity, transition from hyperplasia to neoplasia requires induction of angiogenesis, a process that may be regulated by NFκB.^89,90

Tumors may also generate a vasculature by a process termed vasculogenic mimicry in which CSCs transdifferentiate into vessel-forming cells that resemble endothelium. Recent reports have demonstrated that glioblastoma CSCs are multipotent and can differentiate into endothelial cells, generating their own vasculature.^91,92Although many pro-angiogenic factors have been identified, vascular endothelial growth factor (VEGF) is the primary mediator of this process, ⁹³ and as a result it has been the principal target of anti-angiogenic therapies. A humanized monoclonal antibody targeting VEGF, bevacizumab, as well as two multikinase VEGF inhibitors, sorafenib and sunitinib, are currently approved for clinical use. These anti-angiogenic agents have shown utility in a number of tumor types, including renal and colon cancers.^94,95

Bevacizumab was initially approved for use in metastatic breast cancer on the basis of reports demonstrating that it prolonged time to tumor progression. ⁹⁶ However, more recent studies (AVADO and RIBBON1) have suggested that this effect is limited and that the addition of bevacizumab to cytotoxic chemotherapy failed to increase patient survival. ⁹⁷ These results are consistent with reports in mouse models that anti-angiogenic agents may accelerate local breast cancer invasion and distant metastasis.^98,99In mice bearing human breast cancer xenografts, these agents increase the CSC population through the generation of tissue hypoxia ¹⁰⁰ and activation of HIF1α-Wnt signaling. Anti-angiogenic agents may also stimulate CSC expansion by increasing HGF production by tumor stromal cells. ¹⁰¹ These studies provide a rationale for the addition of CSC targeting agents to anti-angiogenic agents to improve clinical efficacy.

Cytokine Networks Can Promote Cancer Stem Cell Self-Renewal

The link between inflammation and cancer is an old concept that was first proposed by Virchow in 1864, when he observed that inflammatory cells frequently infiltrate the tumor stroma. ¹⁰² Considerable clinical evidence suggests links between inflammatory states and cancer development, including the well-documented association of ulcerative colitis, hepatitis C, and chronic pancreatitis with cancers of the colon, liver, and pancreas, respectively. ¹⁰² States of chronic inflammation as assessed by serum C reactive protein or β amyloid are correlated with risk of breast cancer recurrence in women after primary therapy. ¹⁰³ This chronic inflammatory state may be mediated by cytokines including IL-1β, IL-6, and IL-8. ²⁷ Genetic polymorphisms in these cytokine genes predispose affected individuals to cancer. ¹⁰⁴ These inflammatory cytokines stimulate CSC self-renewal, which then may promote tumor growth and metastasis.^44,105

IL-6 and IL-8 have been implicated both in chronic inflammation and in tumor growth.^106,107Within the tumor microenvironment, many cell types, including mesenchymal cells, macrophages, and immune cells, secrete both IL-6 and IL-8. ¹⁰⁶ In patients with advanced breast cancer, the serum levels of both of these cytokines have been associated with poor patient outcome.^108,109In a variety of preclinical models, IL-6 has been shown to promote tumorigenicity, angiogenesis, and metastasis.^44,110–112IL-6 has been shown to be a direct regulator of breast CSC self-renewal, a process mediated by the IL-6 receptor/GP130 complex through activation of STAT3. ³⁷ Using mouse xenografts, we have recently demonstrated that bone marrow–derived MSCs are recruited to sites of growing breast cancers by gradients of IL-6. ⁴³ IL-6 is a key component of a positive feedback loop involving these MSCs and CSCs. ⁴³ Furthermore, Sethi and colleagues recently demonstrated that IL-6–mediated Jagged1/Notch signaling promotes breast cancer bone metastasis. ⁷³ These studies identify IL-6 and its receptor as attractive therapeutic targets to deplete CSCs.

Using gene expression profiling we previously demonstrated that the IL-8 receptor CXCR1 is highly expressed on breast CSCs ¹⁰⁵ . Interestingly, cytotoxic chemotherapy induced cell death in differentiated cancer cells that resulted in increased production of IL-8, which in turn stimulated breast CSCs via activating CXCR1. A small-molecule CXCR1 inhibitor, reparaxin, significantly reduced CSC in breast cancer xenografts, inhibiting tumor growth and metastasis. Based on these preclinical studies, a clinical trial using reparaxin combined with established chemotherapy has been initiated.

The production of inflammatory cytokines including IL-6 and IL-8 is regulated by the NFκB signaling pathway. ¹¹³ The NFκB pathway plays a crucial role in inflammation and carcinogenesis. The NFκB family is composed of five related transcription factors: p50, p52, RelA (p65), c-Rel, and RelB.^114,115In resting cells, NFκB proteins are predominantly located in the cytoplasm where they associate with the IκB family of proteins; activation of NFκB by diverse signals results in ubiquitin ligase–dependent degradation of IκB, which results in nuclear translocation of NFκB protein complexes. The transcription of a number of cytokines, including IL-6 and IL-8, is activated by NFκB. ¹¹³ In addition, a positive feedback loop maintains a chronic inflammatory state in tumor cells. Interestingly, this loop involves the microRNA let7, as well as Lin28, a factor involved in embryonic stem cell self-renewal. ³⁷ This feedback loop is maintained by IL-6 through its activation of Stat3, which in turn activates NFκB and its downstream targets Lin28 and let7. The specific role of IL-6 in maintaining this inflammatory loop in BCSCs has been recently demonstrated.^37,40NFκB may play an important role in normal breast physiology as well as carcinogenesis. In a HER2/neu mouse model of mammary carcinogenesis, suppression of NFκB in mammary epithelium reduced the mammary stem cell compartment, resulting in a delayed generation of HER2-neu–induced tumors, which displayed reduced angiogenesis and infiltration by macrophages. ⁹⁰ NFκB has also been implicated in the regulation of mouse mammary stem cells during pregnancy. Elevated levels of progesterone during pregnancy induce the production of RANK ligand (RANKL) by differentiated breast epithelial cells. RANKL in turn stimulates breast stem cell self-renewal via activation of NFκB in these cells.^116,117The increased incidence of aggressive breast cancers associated with pregnancy ¹¹⁸ may result from activation of these pathways in breast CSCs.^116,117

Epidemiologic studies demonstrate that obesity is associated with a significant increase in postmenopausal breast cancer,^119,120which may be related to the known link between obesity and inflammation.^121–124Patients with type 2 diabetes mellitus (T2DM) have elevated levels of circulating proinflammatory cytokines including TNF-α, IL-6, and C-reactive protein. ¹²⁵ In addition, these elevated levels of proinflammatory cytokines have been linked to NFκB activation. ¹²⁶ The diabetes drug metformin reduces levels of circulating glucose, increases insulin sensitivity, and reduces insulin resistance-associated hyperinsulinemia. Interestingly, in preclinical mouse models, metformin has been shown to selectively inhibit CSC self-renewal, reducing the proliferation of these cells in breast tumors. ¹²⁷

Summary

As organ-like structures, tumors are composed of diverse cellular compartments and networks that play important roles in tumor progression. Accumulating evidence suggests that these cellular components and cytokine networks also regulate CSCs, which in turn drive tumor growth and metastasis. One can reframe Paget’s “seed and soil” hypothesis of tumor metastasis ¹²⁸ in a modern context. The “seeds” are the cancer stem cells, and the “soil” is the rich microenvironment composed of diverse cell types that interact with tumor cells via growth factor and cytokine networks (see Figure 10-1). These networks regulate CSCs and their progeny, which propagate CSC as well as generating non–self-renewing cells that constitute the tumor bulk. Studies in preclinical models have demonstrated that targeting of regulatory pathways such as IL-6, IL-8, and NFκB can effectively reduce the CSC populations in breast cancer as well as other tumor types.

References

1. Korkaya H. , Paulson A. , Iovino F. , Wicha M.S. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion . Oncogene . 2008 ; 27 : 6120 – 6130 .

2. Ginestier C. , Hur M.H. , Charafe-Jauffret E. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome . Cell Stem Cell . 2007 ; 1 : 555 – 567 .

3. Al-Hajj M. , Wicha M.S. , Benito-Hernandez A. , Morrison S.J. , Clarke M.F. Prospective identification of tumorigenic breast cancer cells . Proc Natl Acad Sci U S A . 2003 ; 100 : 3983 – 3988 .

4. Zhang M. , Behbod F. , Atkinson R.L. et al. Identification of tumor-initiating cells in a p53-null mouse model of breast cancer . Cancer Res . 2008 ; 68 : 4674 – 4682 .

5. Duarte S. , Loubat A. , Momier D. et al. Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect . Oncol Rep . 2012 ; 28 : 1057 – 1062 .

6. O’Brien C.A. , Kreso A. , Jamieson C.H. Cancer stem cells and self-renewal . Clin Cancer Res . 2010 ; 16 : 3113 – 3120 .

7. Bonnet D. , Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell . Nat Med . 1997 ; 3 : 730 – 737 .

8. Eramo A. , Lotti F. , Sette G. et al. Identification and expansion of the tumorigenic lung cancer stem cell population . Cell Death Differ . 2008 ; 15 : 504 – 514 .

9. Ding W. , Mouzaki M. , You H. et al. CD133⁺ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis . Hepatology . 2009 ; 49 : 1277 – 1286 .

10. Singh S.K. , Clarke I.D. , Terasaki M. et al. Identification of a cancer stem cell in human brain tumors . Cancer Res . 2003 ; 63 : 5821 – 5828 .

11. Li C. , Heidt D.G. , Dalerba P. et al. Identification of pancreatic cancer stem cells . Cancer Res . 2007 ; 67 : 1030 – 1037 .

12. O’Brien C.A. , Pollett A. , Gallinger S. , Dick J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice . Nature . 2007 ; 445 : 106 – 110 .

13. Ricci-Vitiani L. , Lombardi D.G. , Pilozzi E. et al. Identification and expansion of human colon-cancer-initiating cells . Nature . 2007 ; 445 : 111 – 115 .

14. Ma S. , Chan K.W. , Hu L. et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells . Gastroenterology . 2007 ; 132 : 2542 – 2556 .

15. Uchida N. , Buck D.W. , He D. et al. Direct isolation of human central nervous system stem cells . Proc Natl Acad Sci U S A . 2000 ; 97 : 14720 – 14725 .

16. Korkaya H. , Paulson A. , Charafe-Jauffret E. et al. Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling . PLoS Biol . 2009 ; 7 e1000121 .

17. Charafe-Jauffret E. , Ginestier C. , Iovino F. et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature . Cancer Res . 2009 ; 69 : 1302 – 1313 .

18. Richardson G.D. , Robson C.N. , Lang S.H. , Neal D.E. , Maitland N.J. , Collins A.T. CD133, a novel marker for human prostatic epithelial stem cells . J Cell Sci . 2004 ; 117 : 3539 – 3545 .

19. Collins A.T. , Berry P.A. , Hyde C. , Stower M.J. , Maitland N.J. Prospective identification of tumorigenic prostate cancer stem cells . Cancer Res . 2005 ; 65 : 10946 – 10951 .

20. Kim C.F. , Jackson E.L. , Woolfenden A.E. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer . Cell . 2005 ; 121 : 823 – 835 .

21. Ucar D. , Cogle C.R. , Zucali J.R. et al. Aldehyde dehydrogenase activity as a functional marker for lung cancer . Chem Biol Interact . 2009 ; 178 : 48 – 55 .

22. Patocs A. , Zhang L. , Xu Y. et al. Breast-cancer stromal cells with TP53 mutations and nodal metastases . N Engl J Med . 2007 ; 357 : 2543 – 2551 .

23. Yang Z.F. , Ho D.W. , Ng M.N. et al. Significance of CD90+ cancer stem cells in human liver cancer . Cancer Cell . 2008 ; 13 : 153 – 166 .

24. Schepers A.G. , Snippert H.J. , Stange D.E. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas . Science . 2012 ; 337 : 730 – 735 .

25. Driessens G. , Beck B. , Caauwe A. , Simons B.D. , Blanpain C. Defining the mode of tumour growth by clonal analysis . Nature . 2012 ; 488 : 527 – 530 .

26. Chen J. , Li Y. , Yu T.S. et al. A restricted cell population propagates glioblastoma growth after chemotherapy . Nature . 2012 ; 488 : 522 – 526 .

27. Coussens L.M. , Werb Z. Inflammation and cancer . Nature . 2002 ; 420 : 860 – 867 .

28. Neurath M.F. , Finotto S. IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer . Cytokine Growth Factor Rev . 2011 ; 22 : 83 – 89 .

29. Mantovani A. , Pierotti M.A. Cancer and inflammation: a complex relationship . Cancer Lett . 2008 ; 267 : 180 – 181 .

30. Liu H. , Patel M.R. , Prescher J.A. et al. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models . Proc Natl Acad Sci U S A . 2010 ; 107 : 18115 – 18120 .

31. Eyler C.E. , Rich J.N. Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis . J Clin Oncol . 2008 ; 26 : 2839 – 2845 .

32. Eyler C.E. , Foo W.C. , LaFiura K.M. , McLendon R.E. , Hjelmeland A.B. , Rich J.N. Brain cancer stem cells display preferential sensitivity to Akt inhibition . Stem Cells . 2008 ; 26 : 3027 – 3036 .

33. Li X. , Lewis M.T. , Huang J. et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy . J Natl Cancer Inst . 2008 ; 100 : 672 – 679 .

34. Vera-Ramirez L. , Sanchez-Rovira P. , Ramirez-Tortosa C.L. et al. Gene-expression profiles, tumor microenvironment, and cancer stem cells in breast cancer: latest advances towards an integrated approach . Cancer Treat Rev . 2010 ; 36 : 477 – 484 .

35. Baylin S.B. , Herman J.G. DNA hypermethylation in tumorigenesis: epigenetics joins genetics . Trends Genet . 2000 ; 16 : 168 – 174 .

36. Jaenisch R. , Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals . Nat Genet . 2003 ; 33 ( suppl ) : 245 – 254 .

37. Iliopoulos D. , Hirsch H.A. , Struhl K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation . Cell . 2009 ; 139 : 693 – 706 .

38. Liu S. , Dontu G. , Mantle I.D. et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells . Cancer Res . 2006 ; 66 : 6063 – 6071 .

39. Dontu G. , Jackson K.W. , McNicholas E. , Kawamura J. , Abdallah W.M. , Wicha M.S. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells . Breast Cancer Res . 2004 ; 6 : R605 – R615 .

40. Iliopoulo D. , Jaeger S.A. , Hirsch H.A. , Bulyk M.L. , Struhl K. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer . Mol Cell . 2010 ; 39 : 493 – 506 .

41. Ma X.J. , Dahiya S. , Richardson E. , Erlander M. , Sgroi D.C. Gene expression profiling of the tumor microenvironment during breast cancer progression . Breast Cancer Res . 2009 ; 11 : R7 .

42. Polyak K. , Haviv I. , Campbell I.G. Co-evolution of tumor cells and their microenvironment . Trends Genet . 2009 ; 25 : 30 – 38 .

43. Liu S. , Ginestier C. , Ou S.J. et al. Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks . Cancer Res . 2011 ; 71 : 614 – 624 .

44. Sansone P. , Storci G. , Tavolari S. IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland . J Clin Invest . 2007 ; 117 : 3988 – 4002 .

45. Karnoub A.E. , Dash A.B. , Vo A.P. et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis . Nature . 2007 ; 449 : 557 – 563 .

46. Mantovani A. Cancer: inflaming metastasis . Nature . 2009 ; 457 : 36 – 37 .

47. Yu H. , Pardoll D. , Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3 . Nat Rev Cancer . 2009 ; 9 : 798 – 809 .

48. Josson S. , Matsuoka Y. , Chung L.W. , Zhau H.E. , Wang R. Tumor-stroma co-evolution in prostate cancer progression and metastasis . Semin Cell Dev Biol . 2010 ; 21 : 26 – 32 .

49. Trimboli A.J. , Cantemir-Stone C.Z. , Li F. et al. Pten in stromal fibroblasts suppresses mammary epithelial tumours . Nature . 2009 ; 461 : 1084 – 1091 .

50. Campbell I.G. , Qiu W. , Polyak K. , Haviv I. Breast-cancer stromal cells with TP53 mutations . N Engl J Med . 2008 ; 358 : 1634 – 1635 author reply 1636 .

51. Josson S. , Sharp S. , Sung S.Y. et al. Tumor-stromal interactions influence radiation sensitivity in epithelial- versus mesenchymal-like prostate cancer cells . J Oncol . 2010 doi: 10.1155/2010/232831 .

52. Pittenger M.F. , Mackay A.M. , Beck S.C. et al. Multilineage potential of adult human mesenchymal stem cells . Science . 1999 ; 284 : 143 – 147 .

53. Gabbiani G. , Majno G. Dupuytren’s contracture: fibroblast contraction? An ultrastructural study . Am J Pathol . 1972 ; 66 : 131 – 146 .

54. Dvorak H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing . N Engl J Med . 1986 ; 315 : 1650 – 1659 .

55. Stuelten C.H. , Barbul A. , Busch J.I. et al. Acute wounds accelerate tumorigenesis by a T cell-dependent mechanism . Cancer Res . 2008 ; 68 : 7278 – 7282 .

56. Hu M. , Yao J. , Cai L. et al. Distinct epigenetic changes in the stromal cells of breast cancers . Nat Genet . 2005 ; 37 : 899 – 905 .

57. Farmer P. , Bonnefoi H. , Anderle P. et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer . Nat Med . 2009 ; 15 : 68 – 74 .

58. Finak G. , Bertos N. , Pepin F. et al. Stromal gene expression predicts clinical outcome in breast cancer . Nat Med . 2008 ; 14 : 518 – 527 .

59. Bierie B. , Moses H.L. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer . Nat Rev Cancer . 2006 ; 6 : 506 – 520 .

60. Orimo A. , Gupta P.B. , Sgroi D.C. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion . Cell . 2005 ; 121 : 335 – 348 .

61. Burger J.A. , Kipps T.J. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment . Blood . 2006 ; 107 : 1761 – 1767 .

62. Chu Q.D. , Panu L. , Holm N.T. , Li B.D. , Johnson L.W. , Zhang S. High chemokine receptor CXCR4 level in triple negative breast cancer specimens predicts poor clinical outcome . J Surg Res . 2010 ; 159 : 689 – 695 .

63. Michieli P. , Mazzone M. , Basilico C. et al. Targeting the tumor and its microenvironment by a dual-function decoy Met receptor . Cancer Cell . 2004 ; 6 : 61 – 73 .

64. Vermeulen L. , De Sousa E.M.F. , van der Heijden M. et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment . Nat Cell Biol . 2010 ; 12 : 468 – 476 .

65. Martinez-Outschoorn U.E. , Balliet R.M. , Rivadeneira D.B. et al. Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: a new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells . Cell Cycle . 2010 ; 9 : 3256 – 3276 .

66. Fillmore C.M. , Gupta P.B. , Rudnick J.A. et al. Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling . Proc Natl Acad Sci U S A . 2010 ; 107 ( 59 ) : 21737 – 21742 14 .

67. Dufraine J. , Funahashi Y. , Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms . Oncogene . 2008 ; 27 : 5132 – 5137 .

68. Singer C.F. , Kronsteiner N. , Marton E. et al. MMP-2 and MMP-9 expression in breast cancer-derived human fibroblasts is differentially regulated by stromal-epithelial interactions . Breast Cancer Res Treat . 2002 ; 72 : 69 – 77 .

69. Pietras K. , Rubin K. , Sjoblom T. et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy . Cancer Res . 2002 ; 62 : 5476 – 5484 .

70. LeBedis C. , Chen K. , Fallavollita L. , Boutros T. , Brodt P. Peripheral lymph node stromal cells can promote growth and tumorigenicity of breast carcinoma cells through the release of IGF-I and EGF . Int J Cancer . 2002 ; 100 : 2 – 8 .

71. Hiscox S. , Parr C. , Nakamura T. , Matsumoto K. , Mansel R.E. , Jiang W.G. Inhibition of HGF/SF-induced breast cancer cell motility and invasion by the HGF/SF variant, NK4 . Breast Cancer Res Treat . 2000 ; 59 : 245 – 254 .

72. Dale T.C. , Weber-Hall S.J. , Smith K. et al. Compartment switching of WNT-2 expression in human breast tumors . Cancer Res . 1996 ; 56 : 4320 – 4323 .

73. Sethi N. , Dai X. , Winter C.G. , Kan Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells . Cancer Cell . 2011 ; 19 : 192 – 205 .

74. Du Z. , Li J. , Wang L. et al. Overexpression of DeltaNp63alpha induces a stem cell phenotype in MCF7 breast carcinoma cell line through the Notch pathway . Cancer Sci . 2010 ; 101 : 2417 – 2424 .

75. Rizzo P. , Osipo C. , Pannuti A. , Golde T. , Osborne B. , Miele L. Targeting Notch signaling cross-talk with estrogen receptor and ErbB-2 in breast cancer . Adv Enzyme Regul . 2009 ; 49 : 134 – 141 .

76. Medina V. , Calvo M.B. , Diaz-Prado S. , Espada J. Hedgehog signalling as a target in cancer stem cells . Clin Transl Oncol . 2009 ; 11 : 199 – 207 .

77. Chao M.P. , Alizadeh A.A. , Tang C. et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma . Cell . 2010 ; 142 : 699 – 713 .

78. Chao M.P. , Jaiswal S. , Weissman-Tsukamoto R. et al. Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47 . Sci Transl Med . 2010 ; 2 : 63 – 94 .

79. Chao M.P. , Alizadeh A.A. , Tang C. et al. Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia . Cancer Res . 2011 ; 71 : 1374 – 1384 .

80. Willingham S.B. , Volkmer J.P. , Gentles A.J. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors . Proc Natl Acad Sci U S A . 2012 ; 109 : 6662 – 6667 .

81. Calabrese C. , Poppleton H. , Kocak M. et al. A perivascular niche for brain tumor stem cells . Cancer Cell . 2007 ; 11 : 69 – 82 .

82. Sugiyama T. , Kohara H. , Noda M. , Nagasawa T. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches . Immunity . 2006 ; 25 : 977 – 988 .

83. Bhati R. , Patterson C. , Livasy C.A. et al. Molecular characterization of human breast tumor vascular cells . Am J Pathol . 2008 ; 172 : 1381 – 1390 .

84. Keith B. , Simon M.C. Hypoxia-inducible factors, stem cells, and cancer . Cell . 2007 ; 129 : 465 – 472 .

85. Folkman J. Tumor angiogenesis: therapeutic implications . N Engl J Med . 1971 ; 285 : 1182 – 1186 .

86. Huh J.I. , Calvo A. , Stafford J. et al. Inhibition of VEGF receptors significantly impairs mammary cancer growth in C3(1)/Tag transgenic mice through antiangiogenic and non-antiangiogenic mechanisms . Oncogene . 2005 ; 24 : 790 – 800 .

87. Duda D.G. , Cohen K.S. , Kozin S.V. et al. Evidence for incorporation of bone marrow-derived endothelial cells into perfused blood vessels in tumors . Blood . 2006 ; 107 : 2774 – 2776 .

88. Lyden D. , Hattori K. , Dias S. et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth . Nat Med . 2001 ; 7 : 1194 – 1201 .

89. Folkman J. , Watson K. , Ingber D. , Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia . Nature . 1989 ; 339 : 58 – 61 .

90. Liu M. , Sakamaki T. , Casimiro M.C. et al. The canonical NF-κB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion . Cancer Res . 2010 ; 70 : 10464 – 10473 .

91. Wang R. , Chadalavada K. , Wilshire J. et al. Glioblastoma stem-like cells give rise to tumour endothelium . Nature . 2010 ; 468 : 829 – 833 .

92. Ricci-Vitiani L. , Pallini R. , Biffoni M. et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells . Nature . 2010 ; 468 : 824 – 828 .

93. Holash J. , Wiegand S.J. , Yancopoulos G.D. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF . Oncogene . 1999 ; 18 : 5356 – 5362 .

94. Okines A. , Cunningham D. Current perspective: bevacizumab in colorectal cancer—a time for reappraisal? Eur J Cancer . 2009 ; 45 : 2452 – 2461 .

95. Yang J.C. , Haworth L. , Sherry R.M. et al. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer . N Engl J Med . 2003 ; 349 : 427 – 434 .

96. Miller K. , Wang M. , Gralow J. et al. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer . N Engl J Med . 2007 ; 357 : 2666 – 2676 .

97. Petrelli F. , Barni S. Bevacizumab in advanced breast cancer: an opportunity as second-line therapy? Med Oncol . 2012 ; 29 ( 1 ) : 226 – 230 .

98. Paez-Ribes M. , Allen E. , Hudock J. et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis . Cancer Cell . 2009 ; 15 : 220 – 231 .

99. Ebos J.M. , Lee C.R. , Cruz-Munoz W. , Bjarnason G.A. , Christensen J.G. , Kerbel R.S. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis . Cancer Cell . 2009 ; 15 : 232 – 239 .

100. Santos S.J.K.P. , Heath A. , Clouthier S. , Wicha M. Anti-angiogenic agents increase breast cancer stem cells via generation of tumor hypoxia . AACR Annu Meeting . 2011 : 3336 .

101. Shojaei F. , Lee J.H. , Simmons B.H. et al. HGF/c-Met acts as an alternative angiogenic pathway in sunitinib-resistant tumors . Cancer Res . 2010 ; 70 : 10090 – 10100 .

102. Balkwill F. , Mantovani A. Inflammation and cancer: back to Virchow? Lancet . 2001 ; 357 : 539 – 545 .

103. Pierce B.L. , Ballard-Barbash R. , Bernstein L. et al. Elevated biomarkers of inflammation are associated with reduced survival among breast cancer patients . J Clin Oncol . 2009 ; 27 : 3437 – 3444 .

104. Michaud D.S. , Daugherty S.E. , Berndt S.I. et al. Genetic polymorphisms of interleukin-1B (IL-1B), IL-6, IL-8, and IL-10 and risk of prostate cancer . Cancer Res . 2006 ; 66 : 4525 – 4530 .

105. Ginestier C. , Liu S. , Diebel M.E. et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts . J Clin Invest . 2010 ; 120 : 485 – 497 .

106. Waugh D.J. , Wilson C. The interleukin-8 pathway in cancer . Clin Cancer Res . 2008 ; 14 : 6735 – 6741 .

107. Scheller J. , Rose-John S. Interleukin-6 and its receptor: from bench to bedside . Med Microbiol Immunol . 2006 ; 195 : 173 – 183 .

108. Yao C. , Lin Y. , Chua M.S. et al. Interleukin-8 modulates growth and invasiveness of estrogen receptor-negative breast cancer cells . Int J Cancer . 2007 ; 121 : 1949 – 1957 .

109. Benoy I.H. , Salgado R. , Van Dam P. et al. Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival . Clin Cancer Res . 2004 ; 10 : 7157 – 7162 .

110. Gao S.P. , Mark K.G. , Leslie K. et al. Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas . J Clin Invest . 2007 ; 117 : 3846 – 3856 .

111. Conze D. , Weiss L. , Regen P.S. et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells . Cancer Res . 2001 ; 61 : 8851 – 8858 .

112. Ara T. , Declerck Y.A. Interleukin-6 in bone metastasis and cancer progression . Eur J Cancer . 2010 ; 46 : 1223 – 1231 .

113. Barnes P.J. , Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases . N Engl J Med . 1997 ; 336 : 1066 – 1071 .

114. Hoffmann A. , Natoli G. , Ghosh G. Transcriptional regulation via the NF-kappaB signaling module . Oncogene . 2006 ; 25 : 6706 – 6716 .

115. Moynagh P.N. The NF-kappaB pathway . J Cell Sci . 2005 ; 118 : 4589 – 4592 .

116. Joshi P.A. , Jackson H.W. , Beristain A.G. et al. Progesterone induces adult mammary stem cell expansion . Nature . 2010 ; 465 : 803 – 807 .

117. Asselin-Labat M.L. , Vaillant F. , Sheridan J.M. et al. Control of mammary stem cell function by steroid hormone signalling . Nature . 2010 ; 465 : 798 – 802 .

118. Peck J.D. , Hulka B.S. , Poole C. , Savitz D.A. , Baird D. , Richardson B.E. Steroid hormone levels during pregnancy and incidence of maternal breast cancer . Cancer Epidemiol Biomarkers Prev . 2002 ; 11 : 361 – 368 .

119. Guilherme A. , Virbasius J.V. , Puri V. , Czech M.P. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes . Nat Rev Mol Cell Biol . 2008 ; 9 : 367 – 377 .

120. Manabe Y. , Toda S. , Miyazaki K. , Sugihara H. Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions . J Pathol . 2003 ; 201 : 221 – 228 .

121. Bennett N.B. , Ogston C.M. , McAndrew G.M. , Ogston D. Studies on the fibrinolytic enzyme system in obesity . J Clin Pathol . 1966 ; 19 : 241 – 243 .

122. Basen-Engquist K. , Chang M. Obesity and cancer risk: recent review and evidence . Curr Oncol Rep . 2011 ; 13 : 71 – 76 .

123. Jee S.H. , Kim H.J. , Lee J. Obesity, insulin resistance and cancer risk . Yonsei Med J . 2005 ; 46 : 449 – 455 .

124. Bianchini F. , Kaaks R. , Vainio H. Overweight, obesity, and cancer risk . Lancet Oncol . 2002 ; 3 : 565 – 574 .

125. Wellen K.E. , Hotamisligil G.S. Inflammation, stress, and diabetes . J Clin Invest . 2005 ; 115 : 1111 – 1119 .

126. Cai D. , Yuan M. , Frantz D.F. et al. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB . Nat Med . 2005 ; 11 : 183 – 190 .

127. Hirsch H.A. , Iliopoulos D. , Tsichlis P.N. , Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission . Cancer Res . 2009 ; 69 : 7507 – 7511 .