Ly 6 Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR 2 cancer therapy

3 0 3 9 jci.org Volume 127 Number 8 August 2017 Introduction Angiogenesis is a hallmark of cancer (1). The VEGF signaling pathway is a key component of pathological angiogenesis in most cancers (2–5). To date, 10 anti-VEGF drugs have been approved by the FDA to treat various solid tumors, starting with metastatic colorectal cancer (CRC) in 2004 (1). However, the survival benefit from these drugs is modest, as tumors develop resistance to these agents (3, 4, 6–13). The mechanisms of resistance remain far from understood. The immune microenvironment has drawn great interest for its strong influence on tumor growth (7, 8, 10, 12, 14–22). Emerging data show that the immune system also plays a critical role in refractoriness to antiangiogenic therapy (13, 23, 24). Among various types of leukocytes, a growing body of evidence suggests that immunosuppressive innate immune cells contribute to this resistance in addition to cancer cell immune evasion (6, 25–27). However, these myeloid cells are a collection of diverse subsets of CD11b+ monocytic and granulocytic cells (27–30), which have often been studied together rather than as clearly defined subpopulations. Furthermore, Ly6Clo monocytes, also known as nonclassical monocytes, have not yet been clearly characterized or extensively investigated in the context of anti-VEGF cancer therapy. Particularly, it is not known whether Ly6Clo monocytes are capable of promoting immunosuppression in any context. Moreover, mechanistic studies on the role of chemokines/ chemokine receptors in each specific subpopulation of innate immune cells in cancers have not been conducted, even though the importance of chemokines in leukocyte trafficking has long been widely accepted (31, 32). The lack of suitable methods for in vivo longitudinal cellular-level monitoring of leukocytes in CRCs of small animal models has limited previous efforts to elucidate the highly dynamic immune microenvironment. Thus, the role and kinetics of specific subsets of innate immune cells in conferring resistance to anti-VEGF therapy are not known. Here, we uncover a function of Ly6Clo monocytes — immunosuppression — and reveal that they instigate a cascade of immunomodulatory events in response to anti-VEGF therapy. We found that anti-VEGF therapy upregulates CX3CL1 expression, which facilitates CX3CR1-dependent infiltration of Ly6Clo monocytes. Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6Clo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1+Ly6Clo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6Clo monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clo monocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6Clo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6Clo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies. Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy