Impact of radiation therapy on vascular endothelial adhesion receptor expression

Abstract

The adjunction of PD-1/PD-L1 immunotherapy and radiation therapy (RT) has proven to be an effective approach to combat cancer. Clinical studies recently confirmed that patients show increased response to immune checkpoint inhibitors if also treated with RT. One effect of RT in this setting is the facilitation of recruitment of immune cells to tumors in which T cell infiltration is low or absent. Recruitment of immune cells is primarily dependent on endothelial activation: activated endothelial cells exhibit an increased expression of endothelial adhesion receptors that mediate recruitment from the blood. Despite evidence that RT can boost T cell recruitment to tumors an in-depth analysis of endothelial adhesion receptor expression and which fractionation doses are best suited in this context are unknown. We hypothesize that RT leads to activation of the endothelium. Using the endothelial mouse cell line bEnd5, we investigated the expression of the adhesion receptors ICAM-1, ICAM-2, VCAM-1, E-selectin, and P-selectin following RT ranging from 0.5 to 10 Gy at different times points (1.5h, 4h, and 24h) with or without prior stimulation with TNF-α by qPCR, flow cytometry, and immunofluorescence. We observed upregulation of E-selectin, P-selectin, and VCAM-1 as early as 1.5h after RT. Early upregulation of selectins was independent of dose; however, sustained upregulation of selectins and upregulation of VCAM-1 was dose-dependent. Following preceding stimulation with TNF-α led to an increased baseline expression of E-Selectin and VCAM-1. E-selectin, P-selectin, and VCAM-1 expression was further increased after RT, and to a much greater extent than without prior stimulation. Again, effects were more pronounced at early time points and showed a dose dependency for VCAM-1. In contrast, while ICAM-2 was not changed by RT or was even downregulated, the addition of RT following TNF-α treatment did increase ICAM-1 levels. This effect was not observed without prior TNF-α stimulation. In vivo experiments of KrasG12D;Tp53fl/fl NSCLC tumors revealed changes in P-selectin, E-selectin, and VCAM-1 that mimicked changes of in vitro experiments with prior TNF-α stimulation. Performing adoptive T cell transfer experiments, we found that both timing and dose of RT is important for successful recruitment of transferred cells to the tissue. Our study demonstrates that RT alters endothelial receptor display on endothelial cells in vitro and in vivo and is influenced significantly by the local cytokine milieu.