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PDF file - 7604K, Supplemental Figure S1 depicts the increased sensitivity of mesenchymal MTdeltaECad cells to salinomycin as compared to epithelial MTflECad cells. Supplemental Figure S2 shows comparable numbers of mesenchymal MTdeltaECad cells and epithelial MTflECad cells injected i.v. trapped in in lungs and no difference in cell proliferation but higher apoptosis of epithelial MTflECad cells in lung outgrowing lung metastasis. Supplemental Figure S3 presents the assessment of VEGF receptor expression, blood vessel maturation and functionality, and tumor cell proliferation and apoptosis in primary tumors formed by mesenchymal MTdeltaECad cells as compared to tumors of epithelial MTflECad cells. Supplemental Figure S4 shows the upregulated expression of pro-angiogenic genes during EMT by gene ontology and direct gene expression analysis. Supplemental Figure S5 shows an efficient siRNA-mediated ablation of VEGF-A expression in mesenchymal MTdeltaECad cells and that the ablation of VEGF-A expression, treatment with the VEGF receptor inhibitor PTK787 or the addition of recombinant VEGF-A do not affect cell proliferation and mammosphere growth of MTdeltaECad cells or MTflECad cells. Supplemental Figure S6 shows that the forced expression of VEGF-A in epithelial MTflECad cells modestly increases the cells' tumorigenicity and significantly induces tumor microvessel density and vessel functionality, it reduces apoptosis but does not affect tumor hypoxia and cell proliferation. Supplemental Figure S7 shows that other VEGF family members are upregulated in their expression during an EMT in vitro and in vivo, yet that they do not affect tumorigenicity of mesenchymal cancer cells. Supplemental Figure S8 demonstrates that VEGF-A in the supernatant of cultured mesenchymal MTdeltaECad cells induces proliferation of human umbilical vein endothelial cells (HUVEC). Supplemental Table I summarizes the flow cytometry analysis of cell surface markers in NMuMG normal murine mammary gland cell line and in the Py2T and MTflECad/MTdeltaECad murine breast cancer cell lines before and after EMT and cultured on plastic or as spheroids. Supplemental Table II presents the differences in cytokine expression between epithelial MTflECad cells and mesenchymal MTdeltaECad cells as determined by cytokine protein array analysis. Supplemental Methods provide experimental details of the general experimental procedures and reagents used in the main text and of the specific methods used to generate the supplemental data.

MOV file - 9405K, Three-dimensional confocal reconstitution.

MPG file - 3174K, Three-dimensional confocal reconstitution.

An epithelial–mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A–mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation. Cancer Res; 74(5); 1566–75. ©2014 AACR.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumor cell (DTC) dormancy in this organ is limited. Here using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micrometastatic state as the rate-limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein, thereby sequestering it from the nucleus and preventing its prometastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin‐β1‐mediated interactions. Because integrin‐β1‐targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin‐β1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin‐β1, we determined that PI3K inhibition with either a tool compounds or a compound (PF‐05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib—with or without genotoxic therapy—would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple‐negative or estrogen receptor‐positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle‐treated animals) or in combination with dose‐dense doxorubicin and cyclophosphamide (vs. animals treated only with dose‐dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin‐β1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis., Dormant disseminated tumor cells (DTCs) are a potential source of late distant breast cancer recurrences. Previously, we identified integrin‐β1 as a promising target for DTC chemosensitization and metastasis prevention. Here, we explore downstream kinases, and find that despite promising initial results, targeting PI3K lacks efficacy on its own or in combination with chemotherapy in two different pre‐clinical models.

Background: InformationTumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. Results: By employing mouse models expressing high and low levels of the laminin 1 chain (LM1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LM1, VEGFA expression triggered by the complex integrin 21-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. Conclusions: Together, we have identified VEGFA, CXCR4 and 21 integrin downstream of LM1 in colon cancer as of bad prognostic value for patient survival. Significance: This information opens novel opportunities for diagnosis and treatment of colon cancer.

Summary Despite the approval of several anti-angiogenic therapies, clinical results remain unsatisfactory, and transient benefits are followed by rapid tumor recurrence. Here, we demonstrate potent anti-angiogenic efficacy of the multi-kinase inhibitors nintedanib and sunitinib in a mouse model of breast cancer. However, after an initial regression, tumors resume growth in the absence of active tumor angiogenesis. Gene expression profiling of tumor cells reveals metabolic reprogramming toward anaerobic glycolysis. Indeed, combinatorial treatment with a glycolysis inhibitor (3PO) efficiently inhibits tumor growth. Moreover, tumors establish metabolic symbiosis, illustrated by the differential expression of MCT1 and MCT4, monocarboxylate transporters active in lactate exchange in glycolytic tumors. Accordingly, genetic ablation of MCT4 expression overcomes adaptive resistance against anti-angiogenic therapy. Hence, targeting metabolic symbiosis may be an attractive avenue to avoid resistance development to anti-angiogenic therapy in patients., Graphical Abstract, Highlights • Tumors can escape anti-angiogenic therapy with multi-kinase inhibitors • A glycolytic shift underlies resistance against multi-kinase inhibitors • Metabolic symbiosis between hypoxic and oxygenated cells inspires therapy resistance • Inhibition of glycolysis or lactate export collapses metabolic symbiosis, Pisarsky et al. examine the role of metabolic symbiosis as a mechanism underlying evasive resistance to anti-angiogenic therapy by the multi-kinase inhibitors nintedanib and sunitinib. Inhibition of glycolysis or genetic ablation of the lactate exporter MCT4 in tumor cells disrupts metabolic symbiosis, overrides therapy resistance, and suppresses tumor growth.

Anti-angiogenic therapy was conceived originally as a silver bullet able to maintain tumor dormancy indefinitely. By targeting new blood vessel formation, anti-angiogenic agents were expected to suppress the growth of any type of primary or metastatic tumor, independent of their subtype or genetic landscape. However, more that 20 years after the first anti-angiogenic preclinical trial, the astonishing inhibition of metastatic outgrowth originally observed in mouse models never translated into clinics. Indeed, whereas anti-angiogenic agents (sometimes) prolong progression-free survival, they fail to impact overall survival, particularly in breast cancer. This observation revealed to be true in early- and advanced-stage breast cancer patients treated either in adjuvant or neo-adjuvant settings, suggesting that the effect of anti-angiogenic therapy on repressing growth of overt metastases - and also on preventing outgrowth of disseminated tumor cells and micrometastases - is limited. What are the reasons underlying this failure? And, more importantly, is there still room for improvement?

Anti-angiogenic therapy was conceived originally as a silver bullet able to maintain tumor dormancy indefinitely. By targeting new blood vessel formation, anti-angiogenic agents were expected to suppress the growth of any type of primary or metastatic tumor, independent of their subtype or genetic landscape. However, more that 20 years after the first anti-angiogenic preclinical trial, the astonishing inhibition of metastatic outgrowth originally observed in mouse models never translated into clinics. Indeed, whereas anti-angiogenic agents (sometimes) prolong progression-free survival, they fail to impact overall survival, particularly in breast cancer. This observation revealed to be true in early- and advanced-stage breast cancer patients treated either in adjuvant or neo-adjuvant settings, suggesting that the effect of anti-angiogenic therapy on repressing growth of overt metastases – and also on preventing outgrowth of disseminated tumor cells and micrometastases – is limited. What are the reasons underlying this failure? And, more importantly, is there still room for improvement?

Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma.By employing mouse models expressing high and low levels of the laminin α1 chain (LMα1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LMα1, VEGFA expression triggered by the complex integrin α2β1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer.Together, we have identified VEGFA, CXCR4 and α2β1 integrin downstream of LMα1 in colon cancer as of bad prognostic value for patient survival.This information opens novel opportunities for diagnosis and treatment of colon cancer.

An epithelial–mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A–mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation. Cancer Res; 74(5); 1566–75. ©2014 AACR.

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109–Jak–Stat3 axis.

Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45dimVEGFR1⁻CD31low, that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45dimVEGFR1⁻CD31low cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45dimVEGFR1⁻CD31low cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer.