Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy
Keehoon Jung, … , Rakesh K. Jain, Dai Fukumura
Keehoon Jung, … , Rakesh K. Jain, Dai Fukumura
Published July 10, 2017
Citation Information: J Clin Invest. 2017;127(8):3039-3051. https://doi.org/10.1172/JCI93182.
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Research Article Oncology

Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy

Abstract

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.

Authors

Keehoon Jung, Takahiro Heishi, Omar F. Khan, Piotr S. Kowalski, Joao Incio, Nuh N. Rahbari, Euiheon Chung, Jeffrey W. Clark, Christopher G. Willett, Andrew D. Luster, Seok Hyun Yun, Robert Langer, Daniel G. Anderson, Timothy P. Padera, Rakesh K. Jain, Dai Fukumura

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Figure 5

Blockade of CX3CR1-dependent infiltration of Ly6Clo monocytes improves efficacy of anti-VEGFR2 therapy.

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Blockade of CX3CR1-dependent infiltration of Ly6Clo monocytes improves e...
(A) SL4 tumors were grown in C57BL/6 WT mice or Cx3cr1–/– (Cx3cr1 KO) mice and treated with either control rat IgG (C) or DC101. Tumor weight was measured on day 12 after treatment (A–D). (B) SL4 tumors were grown in C57BL/6 WT mice or Ccr2–/– (CCR2 KO) mice and treated as indicated. (C) SL4 tumor–bearing C57BL/6 WT mice were treated with either control rat IgG (C), anti-Ly6G antibody (G), DC101 (D), or anti-Ly6G antibody plus DC101 (G+D). Data are represented as mean ± SEM. n = 8/group. Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05. Data are representative of 3 independent experiments (A–C). (D) DC101-treated Cx3cr1–/– mice received adoptive transfer of either tumor-isolated WT Ly6Clo monocytes (Ly6Clo), WT Ly6Chi monocytes (Ly6Chi), or Ly6Clo monocytes isolated from tumors of Cx3cr1–/– mice (KO Ly6Clo) twice a week from the beginning of DC101 treatment. Data are represented as mean ± SEM. n = 8/group. Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05 versus without cell transfer (black bar); #P < 0.05 versus Cx3cr1–/– control mice without cell transfer (blue bar).