Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer

Significance The survival benefit of antiangiogenic therapies for cancer patients has been limited, potentially due to intrinsic/acquired resistance. Deciphering and targeting resistance mechanisms are critical to improving treatment outcome, especially in cancers where antiangiogenic therapies are standard of care, such as colorectal cancer (CRC). Consistent with our clinical findings, we found up-regulation of CXCL12/CXCR4 in orthotopic CRC models and conditional Apc mutant spontaneous rectal tumors after anti-VEGFR2 treatment. CXCR4 signaling recruited immunosuppressive innate immune cells such as Ly6Clow monocytes and Ly6G+ neutrophils to the CRCs, conferring resistance to VEGFR2 blockade. Furthermore, we successfully targeted these pathways genetically and pharmacologically, including with an FDA-approved agent Plerixafor (AMD3100), which significantly enhanced treatment response. These strategies have the potential for rapid clinical translation. Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1−/− mice), Ly6Chigh monocytes (CCR2−/− mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2–induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2–induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

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