Characterization of the Tumor Immune Microenvironment Following SBRT in an Immune Competent, Orthotopic Murine Model of Lung Cancer

Abstract

Stereotactic body radiation therapy (SBRT) has demonstrated efficacy comparable to surgery for early stage non-small cell lung cancer (NSCLC). Recent preclinical and clinical data indicate that immune-mediated mechanisms drive the efficacy of SBRT, however little is known about the nature of the immune response in the lung. The purpose of this study was to develop an immunocompetent orthotopic murine model of NSCLC for SBRT by combining an intrapleural injection with a small animal irradiation platform in order to study the underlying immunobiology of SBRT for lung tumors. We generated an orthotopic syngeneic murine model by delivering Lewis Lung Carcinoma (LLC) cells via intrapleural injection into the right hemithorax. Mice were monitored for tumor growth using micro-CT scans until tumors reached 5 mm in size. A small animal micro-irradiator X-RAD 225Cx (Precision X-Ray) was used to treat the solitary tumors with a single-fraction of various doses of RT (2 Gy, 8 Gy, 16 Gy). After 7 days post-irradiation tumors were harvested for immune profiling using immunohistochemistry (IHC), multicolor flow cytometry, and quantitative PCR. Tumor growth was assessed on micro-CT. Pathologic tumor parameters including cell division (Ki-67), angiogenesis (CD31) and cell death (cleaved caspase 3) were quantitatively assessed using IHC and whole slide scanning with the Aperio digital pathology system. Treatment groups were compared using the Student’s t-test with significance set at a P value < 0.05. For lung tumors treated with 8 or 16 Gy we found significantly decreased density of Ki-67+ and cleaved caspase-3+ cells (P = 0.01) consistent with radiation treatment effect. Flow cytometric analysis of single cell tumor suspensions showed that intratumoral CD3+ T cells were decreased at all RT doses compared to controls (7.77% versus 19.7%, P < 0.05). The proportion of CD3+ CD4+ helper T cells (45.6%) decreased initially in the 2 Gy (38.35%) and 8 Gy groups (31.17%) but increased significantly at 16 Gy (54.57%, P < 0.05). The percentage of CD3+CD8+ cytotoxic T cells (0.40%) increased progressively in the 8 Gy (1.54%) and 16 Gy groups (2.57%, P = 0.04). Interestingly, PD-1 expression on CD3+CD8+ cytotoxic T cells was inversely correlated to radiation dose with the 16 Gy group having the lowest expression of PD-1 (0.42%) compared to controls (18.4%, P<0.05), 2 Gy (4.07%, P<0.05) and 8 Gy (6.1%, P<0.05). n = 10 mice per treatment group. Our study demonstrates that a preclinical model mimicking the microenvironment of human NSCLC can provide informative data about the immunobiology of the response to SBRT. Our experiments reveal that intratumoral CD4+ and CD8+ T cell numbers directly correlates with RT dose and, further, that higher doses of RT inversely correlate with PD-1 expression on cytotoxic T cells. Thus, herein we describe a unique model to study the immunobiology of SBRT which can be used to better inform future clinical trials combining RT and immunotherapy for NSCLC.