Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy
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
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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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 4

Ly6Clo monocyte infiltration during anti-VEGFR2 treatment recruits neutrophils via CXCL5.

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Ly6Clo monocyte infiltration during anti-VEGFR2 treatment recruits neutr...
(A–D) Representative flow cytometry plots depicting subset-specific depletion of myeloid cells in (A) WT control, (B) Cx3cr1–/– (Ly6Clo monocyte), (C) Ccr2–/– (Ly6Chi monocyte), and (D) anti-Ly6G antibody–treated mice (Ly6G+ neutrophil). (E–G) Monocytes and neutrophils in SL4 tumors. C57BL/6 Cx3cr1–/– (E), Ccr2–/– (F), or WT (E–G) mice bearing SL4 tumors were treated with either control rat IgG (C), anti-Ly6G antibody (G), DC101 (D), or anti-Ly6G antibody plus DC101 (G+D). Each subset of myeloid cells in tumor infiltrate was analyzed on day 12 by flow cytometry. n = 8/group. Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05. The graphs depict the absolute number of cells per mg of tumor tissue (E–G). Data are representative of 3 independent experiments. (H) In vitro migration assay. Neutrophils isolated from tumors were seeded in the upper chamber, and their migration to the bottom part of the chamber was measured. The lower chamber included either tumor-isolated Ly6Clo monocytes, Ly6Chi monocytes, or their conditioned media with or without neutralizing antibodies for the chemokine/chemokine receptor as indicated. n = 9/group. Comparison between groups was made using ANOVA with Holm-Šídák post-hoc test. *P < 0.05 versus control (first bar); #P < 0.05 versus Ly6Clo monocytes (second bar). Data are represented as mean ± SEM. FOV, field of view.