Radiotherapy is used to treat many types of cancer, but many treated patients relapse with local tumor recurrence. Tumor-infiltrating myeloid cells (TIM), including CD11b (ITGAM)⁺F4/80 (EMR1)⁺ tumor-associated macrophages (TAM), and CD11b⁺Gr-1 (LY6G)⁺ myeloid-derived suppressor cells (MDSC), respond to cancer-related stresses and play critical roles in promoting tumor angiogenesis, tissue remodeling, and immunosuppression. In this report, we used a prostate cancer model to investigate the effects of irradiation on TAMs and MDSCs in tumor-bearing animals. Unexpectedly, when primary tumor sites were irradiated, we observed a systemic increase of MDSCs in spleen, lung, lymph nodes, and peripheral blood. Cytokine analysis showed that the macrophage colony-stimulating factor CSF1 increased by two-fold in irradiated tumors. Enhanced macrophage migration induced by conditioned media from irradiated tumor cells was completely blocked by a selective inhibitor of CSF1R. These findings were confirmed in patients with prostate cancer, where serum levels of CSF1 increased after radiotherapy. Mechanistic investigations revealed the recruitment of the DNA damage-induced kinase ABL1 into cell nuclei where it bound the CSF1 gene promoter and enhanced CSF1 gene transcription. When added to radiotherapy, a selective inhibitor of CSF1R suppressed tumor growth more effectively than irradiation alone. Our results highlight the importance of CSF1/CSF1R signaling in the recruitment of TIMs that can limit the efficacy of radiotherapy. Furthermore, they suggest that CSF1 inhibitors should be evaluated in clinical trials in combination with radiotherapy as a strategy to improve outcomes. Cancer Res; 73(9); 2782–94. ©2013 AACR.

Tumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b+F4/80+ tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b+Gr-1loLy6Chi mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti–VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.

Growing evidence suggests that tumor-associated macrophages (TAM) promote cancer progression and therapeutic resistance by enhancing angiogenesis, matrix-remodeling, and immunosuppression. In this study, prostate cancer under androgen blockade therapy (ABT) was investigated, demonstrating that TAMs contribute to prostate cancer disease recurrence through paracrine signaling processes. ABT induced the tumor cells to express macrophage colony-stimulating factor 1 (M-CSF1 or CSF1) and other cytokines that recruit and modulate macrophages, causing a significant increase in TAM infiltration. Inhibitors of CSF1 signaling through its receptor, CSF1R, were tested in combination with ABT, demonstrating that blockade of TAM influx in this setting disrupts tumor promotion and sustains a more durable therapeutic response compared with ABT alone. Cancer Res; 75(6); 950–62. ©2015 AACR.

Lymph node involvement denotes a poor outcome for patients with prostate cancer. Our group, along with others, has shown that initial tumor cell dissemination to regional lymph nodes via lymphatics also promotes systemic metastasis in mouse models. The aim of this study was to investigate the efficacy of suppressive therapies targeting either the angiogenic or lymphangiogenic axis in inhibiting regional lymph node and systemic metastasis in subcutaneous and orthotopic prostate tumor xenografts. Both androgen-dependent and more aggressive androgen-independent prostate tumors were used in our investigations. Interestingly, we observed that the threshold for dissemination is lower in the vascular-rich prostatic microenvironment compared with subcutaneously grafted tumors. Both vascular endothelial growth factor-C (VEGF-C) ligand trap (sVEGFR-3) and antibody directed against VEGFR-3 (mF4-31C1) significantly reduced tumor lymphangiogenesis and metastasis to regional lymph nodes and distal vital organs without influencing tumor growth. Conversely, angiogenic blockade by short hairpin RNA against VEGF or anti–VEGFR-2 antibody (DC101) reduced tumor blood vessel density, significantly delayed tumor growth, and reduced systemic metastasis, although it was ineffective in reducing lymphangiogenesis or nodal metastasis. Collectively, these data clarify the utility of vascular therapeutics in prostate tumor growth and metastasis, particularly in the context of the prostate microenvironment. Our findings highlight the importance of lymphangiogenic therapies in the control of regional lymph node and systemic metastasis. [Cancer Res 2008;68(19):7828–37] [Cancer Res 2008;68(19):7828–37]

Malignant ascites is a common complication in the late stages of epithelial ovarian cancer (EOC) that greatly diminishes the quality of life of patients. Malignant ascites is a known consequence of vascular dysfunction, but current approved treatments are not effective in preventing fluid accumulation. In this study, we investigated an alternative strategy of targeting macrophage functions to reverse the vascular pathology of malignant ascites using fluid from human patients and an immunocompetent murine model (ID8) of EOC that mirrors human disease by developing progressive vascular disorganization and leakiness culminating in massive ascites. We demonstrate that the macrophage content in ascites fluid from human patients and the ID8 model directly correlates with vascular permeability. To further substantiate macrophages' role in the pathogenesis of malignant ascites, we blocked macrophage function in ID8 mice using a colony-stimulating factor 1 receptor kinase inhibitor (GW2580). Administration of GW2580 in the late stages of disease resulted in reduced infiltration of protumorigenic (M2) macrophages and dramatically decreased ascites volume. Moreover, the disorganized peritoneal vasculature became normalized and sera from GW2580-treated ascites protected against endothelial permeability. Therefore, our findings suggest that macrophage-targeted treatment may be a promising strategy toward a safe and effective means to control malignant ascites of EOC. Cancer Res; 75(22); 4742–52. ©2015 AACR.

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineeredVHLgene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. TheVHLgene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL⁺) cells. Renal tumors with either VHL⁺or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL⁺cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL⁻) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.