The Effect of Dose-Fractionation Mode on the Immune Response and Therapeutic Effect of Lewis Lung Cancer Mice Model Based on Tumor Microenvironment

Abstract

The therapeutic effect of different groups of Lewis lung cancer mice and the expression of immune cells in peripheral blood and tumor tissues of mice were observed after different dose-segmentation modes of treatment, so as to select the most suitable for stimulating immune effect with good prognosis and small side effects. Female homologous inbred C57BL/6 male mice at 6-8 weeks were selected and inoculated subcutaneously on the right thigh according to LLC: L929: lymphocyte = tumor cell suspension with PBMC(inactivated lymphocyte) = 5:1:1 ratio; after tumor growth to the size of 100-200mm3, different dose-segmentation modes were used for irradiation, which was divided into 5 groups, group A: no radiotherapy; Group B: 2Gy/qd, continuous radiotherapy for 30 days; Group C: 2Gy/bid, continuous radiotherapy for 13 days; Group D: 3Gy/bid, continuous radiotherapy for 10 days; Group E: 6Gy/qd, continuous radiotherapy for 8 days. On the 3rd day after radiotherapy, some mice were sacrificed, peripheral blood lymphocytes of mice were isolated with lymphocyte separation solution, and the transplanted tumor of mice was prepared into single-cell suspension. Then, the number of CD3+T cells, CD3+B cells, NK1.1 cells, CD3+CD4+T cells, CD3+CD8+T cells, CD4+Foxp3+Treg cells in peripheral blood and single-cell suspension of mice was detected by flow cytometry. HE staining was used to observe the tumor tissue structure of mice leg transplantation. The expressions of CD4, CD8, Foxp3 and CD31 in tumor tissues of each radiotherapy group were observed by immunohistochemical staining (IHC) in paraffin sections. The survival time of mice in group C (2Gy/ bidx13d) was significantly improved compared with that in group B (2Gy/qdx30d), and the twin-daily segmentation mode could improve the elevation of Treg cells after once-daily segmentation mode radiotherapy in both peripheral blood and tumor tissues. There was no significant difference in the survival time of mice in group E (6Gy/qdx8d) and group D (3Gy/bidx10d), the number of Treg cells in peripheral blood of mice in group E was significantly lower than that in group D, but there were more Treg cells in tumor tissue of mice in group E than group D. Compared with the control mice, the overall immunity of the experimental group was inhibited. The twice-daily segmentation mode in the conventional dose radiotherapy group significantly improved Treg in peripheral blood and tumor immune microenvironment and promoted neovascularization. The twice-daily segmentation mode in the high-dose radiotherapy group improved the immunosuppression of lymphocytes in peripheral blood but increased immunosuppression in tumor microenvironment.