Correlation of radiosensitivity of lung carcinoma to CD137L endogenous expression levels.

Abstract

e14229 Background: Radiation therapy is a cornerstone in the management of cancer. Preclinical and clinical evidence indicate the therapeutic efficacy of radiation therapy can be augmented by immunomodulatory antibodies against targets such as CD137 and PD-1/PD-L1. Despite evidence of synergy, the mechanistic basis has yet to be fully explored. In this study, we investigated how the expression of CD137, CD137L, PD-1, and PD-L1 correlates with radiosensitivity and proliferation of lung tumor cell lines treated with radiation as well as anti-PD-L1 and agonist anti-CD137 antibodies. Methods: Expression of CD137, CD137L and PD-L1 was measured by RT-qPCR in eight NSCLC cell lines (HCC827, HCC2935, H358, H460, A549, H596, H1650, H520) treated with radiation in doses of 0-8 Gy. Clonogenic assays for radiosensitivity and the CyQUANT assay for cell proliferation were performed after treatment with radiation in combination with anti-PD-L1 (0.01-10µg/ml) and agonist anti-CD137 (0.01-10µg/ml) antibodies. Results: Out of the eight cell lines tested, four were found to be radioresistant – H460, A549, HCC827 and H358 had SF2 (survival at 2 Gy) = 0.76, 0.70, 0.67 and 0.62, respectively – two were moderately sensitive – H520 and HCC2935 (SF2 = 0.54 and 0.40) and two were sensitive to radiation – H596 and H1650 (SF2 = 0.38 and 0.27). Expression analysis by RT-qPCR showed high endogenous expression of CD137L in three out of four radioresistant cell lines while the fourth, H358, had low endogenous expression of CD137L but upon irradiation showed more than a 3-fold induction of CD137L expression. PD-L1 expression levels measured by RT-qPCR did not correlate with radiosensitivity. In vitro cell proliferation was found to be inhibited by 48-68% in all eight NSCLC cell lines after 4 Gy radiation, but combination with anti-PD-L1 and agonist anti-CD-137 antibodies did not contribute additional toxicity. Conclusions: Our results demonstrate that CD137L expression on NSCLC cell lines correlates with radioresistance. Further, addition of anti-PD-L1 and agonist anti-CD137 antibodies to radiation showed little synergistic effect in vitro on tumor cell proliferation. Additional studies to address the synergistic effect of radiation and immunomodulation on tumor cells co-cultured with immune cells is ongoing.