Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy - PubMed (original) (raw)

. 2017 Aug 1;127(8):3039-3051.

doi: 10.1172/JCI93182. Epub 2017 Jul 10.

Takahiro Heishi 1, Omar F Khan 2, Piotr S Kowalski 2, Joao Incio 1, Nuh N Rahbari 1, Euiheon Chung 1, Jeffrey W Clark 3, Christopher G Willett 4, Andrew D Luster 5, Seok Hyun Yun 6 7, Robert Langer 2 7, Daniel G Anderson 2 7, Timothy P Padera 1, Rakesh K Jain 1, Dai Fukumura 1

Affiliations

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

Keehoon Jung et al. J Clin Invest. 2017.

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.

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Conflict of interest statement

Conflict of interest: R.K. Jain received consultant fees from Ophthotech Corp., SPARC, SynDevRx, and XTuit. R.K. Jain owns equity in Enlight, Ophthotech Corp., SynDevRx, and XTuit and serves on the Board of Directors of XTuit and the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund. No reagents or funding from these companies was used in this work.

Figures

Figure 1

Figure 1. Anti-VEGFR2 therapy facilitates early infiltration of Ly6Clo monocytes into tumors.

(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.

Figure 2

Figure 2. Ly6Clo monocytes require CX3CR1 to infiltrate into tumors during anti-VEGFR2 therapy.

(A) Abdominal imaging window on a live mouse bearing syngeneic SL4 CRC (red arrow) in the cecum (white arrow). (B and C) Images of crawling CX3CR1+ leukocytes (green) inside the postcapillary venule (red, TRITC-dextran) in a normal cecum (B) and in the tumor (C) of a Cx3cr1gfp/+ mouse. Ly6Clo monocytes are labeled with EGFP (green). Ly6Clo monocytes are also observed in the tumor (C). (D) Snapshot image taken at 8 seconds of Supplemental Video 1 showing flowing (gray), rolling (yellow), and crawling (white) CX3CR1+Ly6Clo monocytes inside the blood vessels in an SL4 tumor. (E) Snapshot image showing CX3CR1+Ly6Clo monocytes undergoing extravasation in an SL4 tumor. Red, TRITC-dextran (blood vessels). (F) Flux of flowing, rolling, and crawling CX3CR1+Ly6Clo monocytes in blood circulation in SL4 tumor–bearing Cx3cr1gfp/+ mice treated with either control rat IgG (C) or DC101 (D). (G) Flux of flowing, rolling, and crawling Ly6Clo monocytes in blood circulation in SL4 tumor–bearing C57BL/6 WT mice at 5 days after DC101 treatment. Ly6Clo monocytes were isolated from C57BL/6 WT or Cx3cr1–/– mice (KO), fluorescently labeled, and adoptively transferred into DC101-treated SL4 tumor–bearing C57BL/6 WT animals. n = 7/group. Data are represented as mean ± SEM. *P < 0.05, 2-tailed t tests. Data are representative of 3 independent experiments (F and G). Scale bars: 100 μm (B–E).

Figure 3

Figure 3. Blockade of VEGF/VEGFR2 signaling upregulates CX3CL1 in both human and mouse CRCs.

(A and B) Representative images showing CX3CL1 (fractalkine) expression in human tissue sections from patients with rectal carcinomas (total 7 pairs) before (A) and after (B) bevacizumab treatment. Scale bar: 100 μm. (C) Averaged percentage of CX3CL1+ area out of total area from tissue sections of 7 rectal cancer patients before and after bevacizumab treatment. n = 7/ group. *P < 0.05 versus before, 2-tailed t tests. (D) CX3CL1+ area percentage of total viable area from SL4 tumors treated with either control rat IgG (C) or DC101 analyzed on day 12. n = 7/group. *P < 0.05 versus control, 2-tailed t tests. (E) CX3CL1 protein levels measured from tissue lysates of tumors treated with either control rat IgG (C) or DC101 (D). n = 5/group. *P < 0.05 versus control, 2-tailed t tests. (F) Western blot analysis of CX3CL1 protein expression in endothelial cells in vitro. Serum-starved endothelial cells were treated with either recombinant VEGFA protein, DC101, or VEGFA protein plus DC101, and CX3CL1 protein levels were measured from cell lysates. The blockade of VEGF/VEGFR2 signaling stimulates upregulation of CX3CL1 in endothelial cells. Three independent experiments showed similar findings. (G) BALB/c WT mice bearing orthotopically grown syngeneic CT26 CRCs were treated with either control rat IgG (C) or DC101 (D). Relative Cx3cl1 mRNA expression levels in endothelial cells isolated from CT26 tumors were determined on day 2 after treatment by quantitative real-time PCR, normalized against Gapdh. n = 8/group. Data are represented as mean ± SEM.

Figure 4

Figure 4. Ly6Clo monocyte infiltration during anti-VEGFR2 treatment recruits neutrophils via CXCL5.

(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.

Figure 5

Figure 5. Blockade of CX3CR1-dependent infiltration of Ly6Clo monocytes improves efficacy of anti-VEGFR2 therapy.

(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).

Figure 6

Figure 6. Ly6Clo monocytes drive immunosuppression during anti-VEGFR2 treatment in CRCs.

(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.

Figure 7

Figure 7. In vivo nanoparticle delivery of siCX3CL1 inhibits Ly6Clo monocyte infiltration and enhances efficacy of anti-VEGFR2 therapy.

(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.

Figure 8

Figure 8. Proposed mechanism of antiangiogenic therapy–induced immunosuppression.

Anti-VEGFR2 therapy upregulates the expression of CX3CL1 that recruits CX3CR1+Ly6Clo monocytes (center, early phase), which subsequently attracts neutrophils via CXCL5 (right, late phase), resulting in the formation of an immunosuppressive microenvironment with a reduction of cytotoxic T lymphocytes in the tumor. The multistep process provides multiple points of intervention to prevent immune resistance and improve the effectiveness of anti-VEGF therapy. Red arrows indicate steps in the immunosuppressive cascade, which can be targeted as demonstrated in this study (blue inhibition arrow).

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