Bone marrow-derived myeloid cell regulation of tumor angiogenesis and metastatic progression: Roles of VEGFR1 and CXCL4

The progression of cancer from neoplastic lesion to highly metastatic disease is a multi-step process, which requires the involvement of stromal cell types. Specifically, the contribution of bone marrow-derived myeloid (BMM) cells to the tumor and metastatic microenvironments is functionally necessary for processes such as angiogenesis, tumor cell invasion, intravasation, survival, and outgrowth in distant sites. Others and we have shown that recruited cells of the myeloid lineage express VEGF receptor 1 (VEGFR1), however, the functional role of VEGFR1 expression in these cells during tumor angiogenesis and metastasis remains unclear. Here, we report VEGFR1-deficient myeloid cells inhibit B16 melanoma growth in co-injection experiments and induce defects in blood vessel development. Gene expression analysis of myelomonocytic cells with inhibited VEGFR1 expression showed the upregulation of CXCL4 (platelet factor-4). CXCL4 is a previously characterized platelet-derived protein with potent angiostatic activity. For the first time, we show the upregulation of CXCL4 by BMM cells at the primary tumor and metastatic microenvironments and report mCXCL4-/- mice exhibit enhanced primary tumor growth. B16 tumors grown in mCXCL4-/- mice possessed vessels with large lumens and considerable pericyte coverage, indicating a "normalized" phenotype. Co-injecting mCXCL4-/- BMM cells with B16 cells recapitulated the phenotype seen in tumors in mCXCL4-/- mice, indicating BMM cell-derived CXCL4 is expressed at significant enough levels to affect pathological angiogenesis. In addition, knocking down VEGFR1 in CXCL4-/- cells failed to alter tumor growth or vessel development, indicating CXCL4 to be the primary factor involved in VEGFR1 regulation of angiogenesis in the primary tumor microenvironment.;In determining the role of VEGFR1 and CXCL4 in the metastatic microenvironment, lentiviral-mediated downregulation of VEGFR1 expression in the bone marrow drastically reduced the occurrence of advanced metastatic foci and is largely dependent on CXCL4 upregulation. Interestingly, mCXCL4-/- mice had fewer spontaneously disseminated tumor cells, however, possessed drastic acceleration of experimental macrometastasis formation. Thus, CXCL4 inhibits both primary and metastatic tumor growth by inducing disorganized vasculature, which actually promotes the dissemination stages of metastasis. Our results demonstrate an important function for VEGFR1-regulated CXCL4 expression by BMM cells in regulating angiogenesis at the primary tumor and metastatic microenvironments.