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Supplemental Figure 1. from VEGF Potentiates GD3-Mediated Immunosuppression by Human Ovarian Cancer Cells

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posted on 2023-03-31, 18:15 authored by Irina V. Tiper, Sarah M. Temkin, Sarah Spiegel, Simeon E. Goldblum, Robert L. Giuntoli, Mathias Oelke, Jonathan P. Schneck, Tonya J. Webb

Ovarian cancer associated ascites contains soluble inhibitory factors. The ascites was cleared of cellular debris by centrifugation as previously described (18). (A, B) The ascites was filtered 0.2 micron filter and fractionated- f1: <5KDa; f2: 5-50KDa; f3: >50. (C) The ascites was treated with 50 μg/ml proteinase K at 37{degree sign}C for 1 hour and then incubated at 95{degree sign}C for 10min to heat inactivate the enzyme. The antigen presenting cells were treated with the clarified supernantants for 4 h at 37{degree sign}C, then washed extensively with PBS, and cocultured with NKT hybridomas for 20-24 h at 37{degree sign}C. Cytokine released (IL-2) was measured as an indication of NKT cell activation and was measured by standard sandwich ELISA.

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NIH

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Gynecologic Cancer Foundation/Ann Schreiber Ovarian Cancer Research

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ARTICLE ABSTRACT

Purpose: Natural killer T (NKT) cells are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which inhibits NKT-cell activation. Ovarian cancers also secrete high levels of VEGF. In this study, we sought to test the hypothesis that VEGF production by ovarian cancers suppresses NKT-cell–mediated antitumor responses.Experimental Design: To investigate the effects of VEGF on CD1d-mediated NKT-cell activation, a conditioned media model was established, wherein the supernatants from ovarian cancer cell lines (OV-CAR-3 and SK-OV-3) were used to treat CD1d-expressing antigen-presenting cells (APC) and cocultured with NKT hybridomas. Ovarian cancer–associated VEGF was inhibited by treatment with bevacizumab and genistein; conditioned medium was collected, and CD1d-mediated NKT-cell responses were assayed by ELISA.Results: Ovarian cancer tissue and ascites contain lymphocytic infiltrates, suggesting that immune cells traffic to tumors, but are then inhibited by immunosuppressive molecules within the tumor microenvironment. OV-CAR-3 and SK-OV-3 cell lines produce high levels of VEGF and GD3. Pretreatment of APCs with ascites or conditioned medium from OV-CAR-3 and SK-OV-3 blocked CD1d-mediated NKT-cell activation. Inhibition of VEGF resulted in a concomitant reduction in GD3 levels and restoration of NKT-cell responses.Conclusions: We found that VEGF inhibition restores NKT-cell function in an in vitro ovarian cancer model. These studies suggest that the combination of immune modulation with antiangiogenic treatment has therapeutic potential in ovarian cancer. Clin Cancer Res; 22(16); 4249–58. ©2016 AACR.

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