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 6

Ly6Clo monocytes drive immunosuppression during anti-VEGFR2 treatment in CRCs.

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Ly6Clo monocytes drive immunosuppression during anti-VEGFR2 treatment in...
(A) C57BL/6 WT mice bearing syngeneic orthotopic SL4 tumors were treated with either control rat IgG or DC101. Protein levels were measured on day 12 after treatment from tumor tissue lysates (Supplemental Figure 2D). (B and C) Flow cytometric analysis of CD4+ (B) and CD8+ T cells (C) in SL4 tumors as indicated. White bar, WT mice bearing SL4 tumors treated with control rat IgG; black bar, WT mice bearing SL4 tumors treated with DC101; blue bar, Cx3cr1–/– mice bearing SL4 tumors treated with DC101 without cell transfer; gray bar, DC101-treated Cx3cr1–/– mice received adoptive transfer of tumor-isolated WT Ly6Clo monocytes. The graphs depict data for the absolute number of cells per mg of tumor tissue (B and C). The lymphocyte infiltrate in the tumor was analyzed on day 12 by flow cytometry. (D and E) Flow cytometric analysis of CD8+ T cells. The graphs depict data for granzyme B+ (D) or PD-1+ (E) populations relative to total CD8+ T cells. The lymphocyte infiltrate in the tumor was analyzed on day 12 by flow cytometry. n = 8/group. Data are represented as mean ± SEM. *P < 0.05. (F and G) CFSE-based T cell proliferation assays. CellTrace-labeled splenic CD8+ (F) or CD4+ T cells (G) from syngeneic mice were activated and coincubated with either tumor-isolated Ly6Clo monocytes, Ly6Chi monocytes, or neutrophils with or without anti–IL-10 neutralizing antibody as indicated. n = 3/group. Data are represented as mean ± SEM. (B–G) Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05. Data are representative of 3 independent experiments.