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
View: Text | PDF
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

×

Figure 3

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

Options: View larger image (or click on image) Download as PowerPoint
Blockade of VEGF/VEGFR2 signaling upregulates CX3CL1 in both human and m...
(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.