Impacts Of Combining Pulsed Low Dose Rate Radiotherapy With Anti-PD-1 Antibody On Lung Cancer In Mice

Research background and purpose:As an emerging anti-tumor strategy,immunotherapies has been approved for the treatment of a variety of tumors.Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade.Then,pulsed low dose rate radiotherapy(PLDR)is a new radiotherapy segmentation method.Therefore,this study investigated the inhibitory effect of PLDR combined with anti-PD-1 antibody on lung cancer in mice and its impact on tumor microenvironment.Materials and Methods:First,Lewis lung cancer cell(LLC)planted in a 6-well plate,were irradiated with RT and PLDR respectively at doses of 0 Gy,6Gy(2 Gy per session,once a day for 3consecutive days)and 10Gy(2 Gy per session,once a day for 5 consecutive days).The irradiated cells were cultured for 24 hours and harvested into flow cytometry to detect the expression level of PD-L1 on the surface of LLC cells using flow cytometry.Then,by transplanting murine LLC cells into the right leg of C57BL/6 mice with immune activity,a transplanted subcutaneous tumor model was established.The tumor grows to about 100±30mm~3,divide the mice into 5 groups:control、conventional radiotherapy(RT)+Ig G、PLDR+Ig G、RT+anti-PD-1 antibody(m Ab)、PLDR+anti-PD-1 antibody(m Ab).RT was delivered as a dose of 5×2 Gy whereas PLDR involved delivering a dose of 2 Gy as 10pulses of 0.2 Gy,each 3 minutes apart and lasting for 5 days.Anti-PD-1 antibody and isotype control were administered intraperitoneally once every three days at a dose of5mg/kg,three times in total,starting with radiotherapy on the first day.The tumor、blood and tumor-draining lymph nodes(TDLNs)were harvested 7 days after treatment,and a single cell suspension was prepared for flow cytometry to analyze the changes in the immune microenvironment of the tumor tissue and the activated systemic immune response;Then,on the 11th day after the end of treatment,tumor tissue of mice was taken for H&E staining to observe the histological changes of the tumors in each treatment group;Finally,some mice observed natural death and analyzed their survival time.Results:1、In vitro,Irradiation can up-regulate the expression of PD-L1 on the surface of LLC cells,especially in PLDR;2、PLDR combined with anti-PD-1 antibody can better inhibit the growth of tumor than RT、PLDR and RT combined group.Survival analysis demonstrated a statistically significant advantage for PLDR+anti-PD-1 than other groups.The median survival time of mice in the control group was 33 days compared with 41 d with RT 、40d with PLDR and 54 d with RT+anti-PD-1m Ab,and that in the PLDR+a PD-1m Ab group was the longest,with a median survival time of 63 days.The PLDR combined group significantly prolonged the survival time of mice;3、Both RT and PLDR induced up-regulation of PD-L1 expression on tumor surface and PD-1 expression on lymphocytes;PLDR combined with anti-PD-1 antibody can effectively reduce the up-regulated PD-L1 and PD-1,block the PD-1/PD-L1 pathway,and recover the activity of CTL;4、The frequency of CD8+T cells were dramatically higher in the PLDR combined treatment group in tumor、blood;in addition increased the ratio of CD80+DCs in draining lymph nodes,and down-regulated the infiltration of MDSCs in tumor tissues and synergistically.Conclusions:In summary,the study indicates that PLDR upregulates the expression of PD-L1 on LLC cell surface and PD-1 on lymphocyte surface;PLDR combined with anti-PD-1 monoclonal antibody can increase the infiltration of CD8+T cells into tumor tissue,effectively stimulate the maturation and activation of DCs in lymph nodes,reduce the accumulation of MDSCs,thereby changing the tumor microenvironment,achieving the synergistic effect of inhibiting the growth of mouse lung cancer,and extending the survival period.