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Eighteenth Annual Pezcoller Symposium: Tumor Microenvironment and Heterotypic Interactions

¹ Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts and ² Pharmacology and Therapeutics Department, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Enrico Mihich, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-3314; Fax: 716-845-3351; E-mail: enrico.mihich{at}roswellpark.org.

	Abstract

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This symposium was held in Trento, Italy, from June 27 to 29, 2006, and was co-chaired by Robert Weinberg and Enrico Mihich. The interactions between tumor cells and their microenvironment were discussed with particular emphasis on their molecular mechanisms. The roles of transforming growth factor ß signaling, urokinase, and matrix metalloproteinases in matrix remodeling; the effects of matrix-tumor interactions on cell proliferation and migration; the tumor-promoting effects of inflammation and of related host cell and cytokine functions; the signaling mechanisms affecting the biology of the stroma; and the mechanisms governing angiogenesis were discussed. (Cancer Res 2006; 66(24): 11550-3)

	Introduction

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Robert Weinberg emphasized that the mechanisms whereby carcinoma cells acquire the ability to invade and metastasize are complex and remain poorly understood. Four transcription factors (Tf), Twist, FOXC2, Slug, and Goosecoid, when ectopically expressed, are able to induce a mesenchymal phenotype in epithelial Madine-Darby canine kidney cells. This epithelial-mesenchymal transition (EMT) enables carcinoma cells to acquire phenotypes associated with high-grade malignancy, including metastatic dissemination. Expression of some of these Tfs is induced by transforming growth factor (TGF) ß. Acquisition of colonizing ability may require cancer cells to adapt to their newly found microenvironment by developing capabilities that are distinct from those programmed by these Tfs. Inhibition of Twist expression by small interfering RNA has no effect on growth of a mouse mammary carcinoma primary tumor but causes a major inhibition of metastasis. Ectopic expression of Goosecoid increases metastasis of human breast cancer cells. Slug expression is required for metastasis by experimentally transformed melanocytes. Expression of these various Tfs is associated with various types of malignancies; this seems to depend on the developmental lineage of the cells and, possibly, on their exposure to heterotypic signals released by the inflammatory stroma of primary tumors, which act together with the mutant genotype of the primary carcinoma cells to induce expression of these key regulators.

Harold Moses (Vanderbilt-Ingram Cancer Center, Nashville, Tennessee) discussed the role of TGFß signaling in the regulation epithelial of cell proliferation and provided evidence that it is tumor suppressive at both early and late stages of carcinogenesis. Tgfbr2fspKO mice have deletion of the TGFßII receptor gene in stromal fibroblasts: this resulted in increased abundance of stromal cells and, in certain organs, epithelial tumors. Activation of paracrine hepatocyte growth factor (HGF) signaling by the stromal cells was one key mechanism stimulating epithelial proliferation, leading to tumorigenesis. Tgfbr2fspKO mammary fibroblasts transplanted with mammary carcinoma cells promoted growth and invasion, with increased phosphorylation of erbB1, erbB2, RON, and c-Met receptors. The absence of TGFß signaling affected host stromal cells, whereas increased TGFß release affected both epithelial cell EMT and host cells. The role of stromal TGFß signaling in mammary tissue homeostasis and tumor progression via regulation of TGF as well as HGF signaling was shown. Thus, TGFß signaling in fibroblasts modulated the growth and oncogenic potential of adjacent epithelia in selected tissues.

Francesco Blasi (Institute San Raffaele, Milan, Italy) discussed the role of the urokinase receptor (uPAR) in cell proliferation, adhesion, and migration. In humans, the level of uPAR and tumor relapse-free survival is strictly correlated. uPAR and epidermal growth factor (EGF) receptor (EGFR) are constitutively associated. The phosphorylation of the EGFR is uPAR dependent and is blocked by uPAR binding. Using uPAR^–/– mice, the uPAR role in the mobilization of hematopoietic stem cells in skin wound healing and carcinogenesis was evaluated: carcinogenesis and wound healing were reduced; keratinocytes differentiated faster but grew more slowly, whereas fibroblasts grew faster. Thus, uPAR has a complex role in cell proliferation, migration, and carcinogenesis.

Lynn Matrisian (Vanderbilt University School of Medicine, Nashville, Tennessee) outlined the function of matrix metalloproteinases (MMP) in tumor-host communications. MMPs have been considered to be major contributors to tumor invasion and progression through their ability to degrade the basement membrane and extracellular matrix (ECM) components. MMPs function as regulatory molecules in addition to their role in matrix barrier destruction. This results in inactivation or activation and solubilization of factors that mediate communications between tumor and its microenvironment. Using a two-step model of skin carcinogenesis, an orthotopic lung cancer, and a mammary tumor, she showed that in contrast to previous hypothesis, the absence of certain MMPs increased tumor progression, whereas their presence decreased it. MMP3 inhibited tumor progression in the skin model; in the lung tumor, MMP12 from macrophages inhibited angiogenesis through a plasminogen-dependent activation of angiostatin. In a mammary tumor, MMP9 deletion decreased metastasis; in a prostate carcinoma, bone metastasis was decreased by MMP7 deletion. Thus, various MMPs had differing effects on certain tumor types.

Soldano Ferrone (Roswell Park Cancer Institute, Buffalo, New York) discussed human high molecular weight melanoma-associated antigen (HMW-MAA)–mediated interactions of melanoma cells with ECM and their inhibition by antibodies. HMW-MAA is present on 85% of melanomas and 70% of gliomas on breast cancer stem cells and on tumor-activated pericytes. It has a restricted distribution in normal tissue. The survival of cancer patients treated with antibody was longer than that of patients without it. Using melanoma cell lines, the role of antigen in increasing cell migration and growth in severe combined immunodeficient mice and in enabling anchorage-independent growth was shown. Experimental and clinical evidence suggested that the HMW-MAA function could be inhibited by antibody administration or induction by HMW-MAA mimics. The anti-id monoclonal antibody MK2-23 was used to implement immunotherapy in melanoma patients.

Fran Balkwill (Barts and the London Queen Mary's School of Medicine and Dentistry, London, United Kingdom) indicated the role of tumor necrosis factor (TNF)- in inflammation and the anticancer potential of inflammation inhibitors. Risk of cancer increases at sites of chronic inflammation. Deletion of chemical mediators of inflammation found in many cancers inhibits cancer development and spread. Long-term use of nonsteroidal anti-inflammatory agents reduces mortality from some cancers. In the cancer microenvironment, TNF- can be an endogenous tumor promoter, releasing signals that lead to nuclear factor-B (NF-B) and activator protein-1 Tf activation in epithelial cells. General or cell-specific deletion/inhibition of TNF- reduces the incidence of experimental cancers. TNF- is frequently detected in human biopsies produced either by epithelial ovarian and renal cancer cells or by breast cancer stromal cells. TNF- and other inflammatory cytokines are thought to be a major cause of constitutive NF-B activity in cancer cells. In TNF- knockout mice, angiogenesis is decreased. Tumor TNF- production has been associated with poor prognosis. Anti-TNF- antibody treatment in patients with renal cell carcinoma resulted in some partial responses or stable disease.

Lisa M. Coussens (University of California San Francisco, San Francisco, California) discussed the fact that innate immunity is largely protumorigenic, whereas adaptive immunity is mostly antitumorigenic. In the K14-human papillomavirus 16 (HPV16) transgenic mouse model of epithelial carcinogenesis, inflammatory mast cells and granulocytes contributed dominantly to activation of neoplasia-associated tissue remodeling, angiogenesis, epithelial hyperproliferation, and overall cancer incidence via proteolytic enzymes release. Genetic elimination of mature T and B lymphocytes limited neoplastic progression to development of benign epithelial hyperplasias without recruitment of innate immune cells into premalignant tissue. Adoptive transfer of B lymphocytes from K14-HPV16 mice into T-cell– and B-cell–deficient/HPV16 mice reinstated full malignancy; additional signals by adaptive and innate immune cells were also required.

Alberto Mantovani (Instituto Clinico Humanitas, Milan, Italy) discussed the role of smoldering and polarized inflammation in tumor progression. Tumor-associated macrophages (TAM) modulate inflammation and adaptive immunity; promote cell proliferation by producing growth factors; scavenge debris by expressing scavenger receptors; and promote angiogenesis, tissue remodeling, and repair. Recent studies address the relationship between genetic events causing cancer and activation of protumorigenic inflammatory reactions. Monocyte differentiation, macrophage polarization and increased proliferation, and TAM functions are involved in the development of protumorigenic chronic inflammatory events. High levels of the CXCR4 chemokine receptor are found in metastatic pancreatic cancer cells. Thus, certain chemokines and changes in cells producing them are essential factors determining protumor inflammatory reaction and should be considered prime targets for intervention.

Jacques Pouyssegur (Institute of Signaling, Developmental Biology and Cancer Research, Nice, France) outlined the function of the hypoxia-inducible factor (HIF)-1, which is central to modulating tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. HIF-1 is activated by a prolyl hydroxylase domain (PHD) 2 and inhibited by the asparaginyl hydroxylase inhibiting HIF-1 (FIH-1) enzyme. Silencing in normoxia, of either PHD2 or FIH-1, partially induced hypoxic genes, whereas combined PHD2/FIH-1 silencing generated a full hypoxic gene response. In human cell lines, the induction of the hypoxic genes CAIX, PHD3, PGKI, BNIP3 as well as 30 other genes responded differently toward HIF-1 expression. In the tumor microenvironment, HIF-1 could induce either cell survival or cell death. High expression of HIF corresponds to bad prognosis with resistance to irradiation and chemotherapy plus selection of metastatic variants. Thus, HIF is an important target for drug development.

Hartmut Beug (Institute of Molecular Pathology, Vienna, Austria) discussed the relationship between EMT and cancer progression. Complete EMT is a correlate of local invasion and metastasis. EMT may require cooperation of endogenous TGFß signaling with oncogenic Ras. The cooperation of autocrine platelet-derived growth factor receptor (PDGFR) signaling with oncogenic Ras hyperactivates phosphatidylinositol 3-kinase and is required for survival during EMT. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Overexpression of a dominant-negative PDGFR or application of Gleevec interfered with experimental metastasis in mice. In mouse mammary tumor virus-neu transgenic mice, TGFß enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. A novel, secreted, interleukin (IL)-like EMT inducer (ILEI) was identified. Stable overexpression of ILEI in normal and Ras-transformed mammary epithelial cells (EpRas) caused EMT, tumor growth, and metastasis. RNA interference–mediated knockdown of ILEI in EpRas cells before and after EMT prevented and reverted TGFß-dependent EMT, respectively, and also abrogated metastasis. ILEI cytoplasmic overexpression was associated with induction of EMT in human colon cancer and predicted metastasis and poor clinical prognosis in human breast cancer patients. Thus, ILEI is a promising target for therapeutic intervention.

Jeffrey W. Pollard (Institute of Signaling, Developmental Biology and Cancer Research, Nice, France) outlined the role of macrophages in promoting tumor progression and metastasis. The six traits indicating macrophages effects on cancer are increased invasion, extravasation, seeding, inflammation, angiogenesis, and matrix modeling. In transgenic mouse mammary tumors, reduction of TAM by genetic ablation of colony stimulating factor-1 (CSF-1) resulted in delayed tumor progression and reduction of metastasis. Restoration of macrophages by mammary gland–specific expression of CSF-1 reversed this effect, and overexpression of CSF-1 enhanced tumor progression and doubled the rate of metastasis in wild-type mice. Macrophages are recruited into the tumor-associated stroma and the resulting TAMs are educated within the tumor microenvironment to become tumor promoting. Interactions between macrophages and tumor cells are required for tumor cell migration, invasion, and intravasation. Tumor-produced CSF-1 signals to macrophages to synthesize EGF, which in turn stimulates the EGFR-bearing tumor cells to migrate. A high density of TAM enhances tumor cell invasion and intravasation and increases the number of vessels through which the tumor cells can escape. Thus, inhibition of specific macrophage functions should have clinical benefit.

Peter Carmeliet (University of Leuven, Leuven, Belgium) discussed the role of placental growth factor (PlGF), a homologue of vascular endothelial growth factor (VEGF), and of fibroblast growth factor in lymphangiogenesis. A neutralizing anti-mPlGF monoclonal antibody blocked tumor growth and angiogenesis in several orthotopic and s.c. tumors without affecting normal vessels. It also inhibited lymphatic metastasis. Anti-VEGF receptor (VEGFR) 2 antibody up-regulated PlGF, and anti-PlGF treatment amplified the effect of anti-VEGFR2 antibody. Blocking PlGF effectively inhibited in vivo tumor growth by suppressing tumor-induced neovascularization. Xenopus tadpoles were characterized to study the genetic basis of lymphangiogenesis. Cooperative regulation of lymphangiogenesis was exerted by VEGF-C, VEGF-D, and Prox1, the master gene of lymphatic cell fate differentiation.

Shahin Rafii (Weill Medical College of Cornell University, New York, New York) clarified the contribution of CXCR4⁺ hematopoietic cells to neoangiogenesis. Mobilization of proangiogenic cells into the vascular niche of bone marrow is achieved, in part, through MMP9-mediated release of soluble Kit ligand. Subsequently, stromal-derived factor-1 (SDF-1) provides for the localization of these cells and their subsequent mobilization to the circulation. A defect in ischemic revascularization in MMP9^–/– mice was reversed by soluble Kit ligand or SDF-1. Inhibition of CXCR4 blocked ischemic revascularization; luminal incorporation of endothelial cells was significantly enhanced by SDF-1. Anti-CXCR4 plus anti-VEGFR1 reduced normal vascularization. Collaboration of CXCR4 and VEGF-A receptor signaling facilitated incorporation of bone marrow cells into newly formed vessels. VEGFR1⁺ hematopoietic progenitors migration to specific organs may initiate metastasis. Hemangiocyte-derived factor 1 is involved in determining the patterns of vessels. Bone marrow endothelial progenitor cells are 20% of the bone marrow cell population and their recruitment and the magnitude of their luminal incorporation in neovessels depends on the stage of the tumor. In conclusion, SDF-1 release determines mobilization of CXCR⁺ VEGFR⁺ cells and the dynamics of the vessel regeneration. Hemangiogenic progenitors mobilization is a reliable and validated surrogate biomarker to evaluate the extent of neoangiogenesis. Inhibition of CXCR4 in conjunction with VEGF-A receptors may block growth of angiogenesis-dependent tumors.

Rakesh Jain (Massachusetts General Hospital, Boston, Massachusetts) emphasized the possibility of normalizing tumor vasculature and microenvironment by targeted therapies. Preclinical and clinical evidence suggests that judicious application of direct (e.g., Avastin) and indirect (e.g., Herceptin) antiangiogenic agents can transiently "normalize" the abnormal structure and function of tumor vasculature to make it more efficient for oxygen and drug delivery. Avastin/bevacizumab monotherapy does not increase the survival of cancer patients, but in combination with certain chemotherapeutic agents, it is effective and induces normalization of blood vessels. During the growth of LS174T tumor cells in dorsal windows, three-dimensional images of tumor vasculature showed that vessels were normalized after anti-VEGF antibody treatment. Decreases in VEGF lead to decreases of interstitial pressure, which is usually elevated in tumors. Treatments that induce vascular normalization can also normalize the tumor microenvironment by alleviating hypoxia and interstitial hypertension and thereby increase the efficacy of many conventional therapies.

Douglas Hanahan (University of California San Francisco, San Francisco, California) focused on neutrophil and macrophage dynamics and their proangiogenesis roles in mouse cancer. In genetically engineered mice with pancreatic islet tumors as well as those that develop HPV16 oncogene-driven skin and cervical carcinomas, matrix-degrading enzymes, particularly MMP9, were important for angiogenic switching and the persistence of angiogenesis during tumorigenesis. A key role of MMP9 involved the release of latent/sequestered VEGF from the ECM, enabling its association with VEGFR on the endothelium. In the Rip-Tag pancreatic islet tumors, MMP9 was expressed in neutrophils and macrophages, including (GRI⁺MacI⁺) inflammatory cells. Granulocyte-CSF increased macrophages and neutrophils inside infiltrated islets but did not increase the number of infiltrated islets: neutrophils infiltration led to an angiogenic switch. The proportion of macrophages and neutrophils in tumors was different in different types of tumor; thus, macrophages predominated in breast and cervical tumors, neutrophils and mast cells in skin tumors, and neutrophils and macrophages in Rip-Tag islets tumor. Cells of the innate immune system have proangiogenic functions and promote tumor growth.

Raghu Kalluri (Beth Israel Deaconess Medical Center, Boston, Massachusetts) analyzed the role of endothelial cells and fibroblast recruitment in angiogenesis and that of innate immunity in cancer progression and metastasis. Tumstatin is a 28-kDa inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth, which derives from the basement membrane and is present in human and mouse blood. In tumstatin^–/– mice, tumor growth is increased and this is reversed by tumstatin. MMP9^–/– mice have decreased tumstatin and increased tumor growth. In ß integrin^–/– mice, tumstatin does not inhibit tumor growth. The growth of B16 melanoma cells is increased in endostatin^–/– mice and is inhibited by endostatin. The effects of tumstatin and endostatin depend on tumor type: tumstatin depletion affects lymphomas growth but not sarcomas growth; endostatin depletion affects early sarcomas growth but not lymphomas growth.

Dario Neri (Institute of Pharmaceutical Sciences, Zurich, Switzerland) outlined tumor vascular targeting by suitable monoclonal antibodies. Targeting neovascular structures is based on identification of antigens specifically and abundantly expressed at sites of disease and accessible to i.v. injected ligands. In a colorectal cancer orthotopic model with metastasis to lung and liver, antibody L19 with IL-2 attached induced a 15% response rate, whereas IL-2 alone had no effect. A novel methodology was developed for the discovery of marker proteins in the blood. This technology is based on the terminal perfusion of tumor-bearing rodents or the ex vivo vascular perfusion of surgical specimens from cancer patients with a reactive ester derivative of biotin. Biotinylated proteins can be purified on streptavidin resin and submitted to a comparative proteomic analysis. After digestion, the resulting peptides are analyzed by mass spectrometry. The methodology has been extended to the comparative analysis of vascular structures in metastases.

Napoleone Ferrara (Genentech, Inc., San Francisco, California) indicated that VEGF-A is a permeability factor, a mitogen, and a survival factor for endothelial cells. It is a glycoprotein, which is present in different isoforms with differing binding affinities for heparin. High expression of VEGF-A mRNA has been described in many human tumors. Anti-VEGF-A monoclonal antibodies or other VEGF inhibitors block growth and neovascularization in tumor models. A humanized anti-VEGF-A monoclonal antibody (bevacizumab), which binds to all VEGF-A isoforms, is 93% human and is being tested in cancer patients. In untreated metastatic colorectal cancer patients, addition of bevacizumab to chemotherapy results in a significant increase in survival compared with chemotherapy alone. Similar results are being obtained in patients with breast and lung cancer.

	Prospects

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Much of basic cancer research conducted over the past 3 decades has been focused on the cancer cell and its cell-autonomous functions. The scientific reports presented at this Pezcoller meeting presented a starkly different view that tumors are complex tissues and that the biology of tumorigenesis can only be understood by revealing the heterotypic interactions between cancer cells and the complex array of mesenchymal cells that they recruit. Over the next decade, it seems likely that the list of heterotypic signaling channels will expand enormously, as more and more cytokines are found to be exchanged between the neoplastic cells and the mesenchymal and inflammatory cells that have been recruited to the tumor-associated stroma. As was also made clear at this meeting, the phenotypes of high-grade malignancy are developed and stimulated by signals originating in the stroma and impinging on neoplastic cell populations. In the future, this type of research will reveal why inflammation represents a critical tumor-promoting influence in human cancer pathogenesis. Moreover, we will come to appreciate that cancer is a systemic disease, long before metastatic dissemination has taken place, because primary cancer cells are able to perturb the bone marrow in ways that facilitate stromal recruitment.

	Acknowledgments

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

The list of speakers and their affiliations is available as supplementary data online.

Received 8/25/06. Revised 10/24/06. Accepted 10/24/06.

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