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 August 1, 2017; First 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 1

Anti-VEGFR2 therapy facilitates early infiltration of Ly6Clo monocytes into tumors.

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Anti-VEGFR2 therapy facilitates early infiltration of Ly6Clo monocytes i...
(A and B) Tumor volume was measured using a high-frequency ultrasound imaging system for orthotopically grown syngeneic SL4 tumors in C57BL/6 mice (A) and CT26 tumors in BALB/c mice (B). Tumors were treated with either control rat IgG (control) or monoclonal anti-VEGFR2 antibody DC101 (40 mg/kg, every 3 days). n = 8/group. (C) A representative flow cytometry plot depicting the 3 different subsets of myeloid populations. 1, Ly6Clo monocyte; 2, Ly6Chi monocyte; 3, Ly6G+ neutrophil. C57BL/6 WT mice bearing SL4 tumors were treated with DC101, and immune cells in the tumor infiltrate were analyzed on day 5 by flow cytometry. Gated on CD45+Lin–F4/80–CD11c–CD11b+. As these cells are defined as F4/80–, TAMs (F4/80+) are excluded. (D and E) C57BL/6 WT mice bearing SL4 tumors were treated with either control rat IgG (C) or DC101. Each subset of myeloid cells in tumor infiltrate was analyzed on day 5 (D) and day 12 (E) by flow cytometry. Top row, Ly6Clo monocyte; center row, Ly6Chi monocyte; bottom row, Ly6G+ neutrophil. n = 8/group. (F and G) BALB/c WT mice bearing CT26 tumors were divided into 2 different treatment groups (control, DC101), and the myeloid cell subsets in the tumor infiltrate were analyzed on day 5 (F) and day 12 (G) by flow cytometry. The graphs depict the absolute number of cells per mg of tumor tissue. n = 8 /group. Data are represented as mean ± SEM. *P < 0.05 versus control, 2-tailed t tests. Data are representative of 4 (A and B) or 3 (D–G) independent experiments.