The purpose of this study was to investigate the different immune responses between relatively radiotherapy-sensitive and -resistant tumor models in vitro and in vivo. Radiotherapy-sensitive CT26WT cell line and -resistant LL2 cell line were irradiated at different doses. Cell viability and expression levels of Sting and IFNb1 were determined in vitro. The corresponding tumor models in C57BL/6 and BALB/c mice were established. Tumor growth, expression levels of immune-related factors IFNb1, PD-1 and PD-L1, and the percentage of tumor-infiltrating lymphocytes (TILs) were measured. In radiotherapy-resistant LL2 model, activated STING pathway, along with improved host immune response was observed. However, programmed cell death proteins was also upregulated in radiotherapy-resistant LL2 model as well. These finding provide molecular basis and rationale to combine radiotherapy and immunotherapy to overcome the radiotherapy-resistant cancer in the clinical setting.
Figure1.
In vitro cell viabilities against different dosages of radiation post-72h between CT26WT and LL2. CT26WT and LL2 were inoculated into 24-well plates and treated with 0Gy, 4Gy, 8Gy, 20Gy, 30Gy, or 8Gy×3 irradiation. After the cells were attached, the plate was placed in an incubator for 72 h (1×103/well). Cell viability is performed by CCK8 and normalized as the percentage of cell viability of 0Gy group (taken as100%). Data are expressed as mean±SEM.
Figure2.
Expression of the STING pathway in LL2 and CT26WT cell lines 24 h after different dose of radiation. (A, B) mRNA levels of mouse Ifnb1(mIfnb1) was measured by RT-qPCT in LL2 and CT26WT after exposed with radiation at 0Gy, 8Gy, 20Gy, 30Gy, and 8Gy×3. (C, D) Western blot image and (E, F) quantitative analysis of western blot data of mSting protein in LL2 and CT26WT cell lines 24 h after treatment with 8Gy,20Gy,30Gy,and 8Gy×3 radiation. *P<0.05,**P<0.01.
Figure3.
Tumor growth curves and the expression of the STING pathway in LL2 and CT26WT xenograft mouse model after radiotherapy. (A, B) Tumor growth curves in CT26WT and LL2 mouse model. (C, D) Ifnb1(mIfnb1) mRNA levels expressed in the LL2 and CT26WT mouse models with radiation at 0Gy and 8Gy×3. IFNb1 expression was detected by real-time fluorescent quantitative PCR. *P<0.05,**P<0.01, ****P<0.0001.
Figure 4.
Different activation of immune cells in the LL2 and CT26WT tumor models 5 days post radiation treatment measured by flow cytometry. (A-C) The percentages of dendric cell (DC), T helper cell (Th) and effector T cell in LL2 tumor model; (D-F) the percentage of DC, Th and Effector T cell in CT26WT cell. *P<0.05,**P<0.01.
Figure5.
The expression of PD1 in the infiltrated T cells and expression of PDL1 on tumor cells in LL2 and CT26WT tumor models 5 days post radiation treatment measured by flow. (A, B) PD1 expression of T cells and PDL1 expression of tumor cells in the LL2 tumor model; (C, D) PD1 expression of T cells and PDL1 expression of tumor cells in the CT26WT tumor model. *P<0.05,**P<0.01.
Figure6.
The infiltration of CD8+ cells post-radiotherapy by Immunofluorescence detection. (A, B) In the LL2 tumor model, CD8a+ cells of the control group and of the 8Gy×3 group. (C, D) In the CT26WT tumor model, CD8a+ cells of the control group and of the 8Gy×3 group.