High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U20S cells

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-Ll expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-Ll in human osteosarcoma U20S cells. We found that the upregulation of PD-Ll expression after high LET carbon-ion irradiation was greater than that inducedby X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-Ll induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-Ll molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-Ll molecules on the cell surface after high LET carbonion irradiation compared with X-ray irradiation. Keywords: anti-PD-1/PD-Ll therapy; high linear energy transfer (LET) carbon-ion therapy; PD-L1 expression; DNA damage response