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 7

In vivo nanoparticle delivery of siCX3CL1 inhibits Ly6Clo monocyte infiltration and enhances efficacy of anti-VEGFR2 therapy.

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In vivo nanoparticle delivery of siCX3CL1 inhibits Ly6Clo monocyte infil...
(A) Schematic of 7C1 nanoparticle formulated with siRNA. (B) In vitro screening of siCX3CL1 candidate duplexes. Relative Cx3cl1 expression level normalized to siLUC (luciferase) control is plotted for candidate duplexes in 0.1 nM or 10 nM. Each siRNA was transfected twice, and mRNA analysis was run in triplicate. Red box indicates the best duplex selected for large-scale synthesis and subsequent nanoparticle formulation. Black box indicates siRNA control that targets luciferase. (C–F) C57BL/6 WT mice bearing orthotopically grown syngeneic SL4 CRCs were treated with either control rat IgG (C), 7C1-Axo-siCX3CL1 (7C1), DC101 (D), or 7C1-Axo-siCX3CL1 plus DC101 (7+D). (C) Relative Cx3cl1 mRNA expression levels in endothelial cells isolated from SL4 tumors were determined by quantitative real-time PCR, normalized against Gapdh. 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. (D) Western blot analysis of CX3CL1 protein expression in SL4 tumors treated as indicated. CX3CL1 protein levels were measured on day 12 after treatment. (E) Ly6Clo monocytes in SL4 tumors treated as indicated. Ly6Clo monocytes in tumor infiltrate were analyzed on day 12 after treatment by flow cytometry. n = 8/group. The graphs depict the absolute number of cells per mg of tumor tissue. (F) Tumor volume of SL4 measured on day 12 after treatment. n = 8/group. Data are represented as mean ± SEM. Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05.