Time- and Dose-Dependent Effects of Irradiation on Endothelial and Tumor Endothelial Cells: Transcriptional, Molecular, and Functional Changes Driving Activation In Vitro and In Vivo.

Background: Irradiation (IR) targets cancer cells, but also the tumor microenvironment, including the tumor's blood vessels. In addition to tumor endothelial cell (TEC) apoptosis, IR can lead to TEC activation, potentially increasing immune cell infiltration. However, the changes underlying the IR-induced activation of endothelial cells (ECs) are poorly understood. This study investigated dose- and time-dependent molecular and functional responses of murine and human EC lines to IR in vitro and TECs in vivo in murine tumor models of colorectal carcinoma. Methods: HUVEC, EA.hy926, and Hulec5a, as well as murine bEND.3, 2H11, and SVEC4-10 EC lines, were irradiated with single doses of 2-10 Gy. EC proliferation and survival after IR were assessed by staining all nuclei (Hoechst 33342) and dead cells (propidium iodide) every 24 h for 5 days using the Cytation 1 Cell Imaging Multi-Mode Reader. RNA sequencing analysis of HUVECs irradiated with 2 Gy and 5 Gy at 24 h and 72 h after IR was conducted, focusing on processes related to EC activation. To validate the RNA sequencing results, immunofluorescence staining for proteins related to EC activation, including Stimulator of Interferon Response cGAMP Interactor 1 (STING), Nuclear factor kappa B (NF-κβ), and Vascular cell adhesion molecule 1 (VCAM-1), was performed. To validate the in vitro results, the response of TEC in vivo was analyzed using publicly available RNA sequencing data of TECs isolated from MC38 colon carcinoma irradiated with a single dose of 15 Gy. Finally, murine CT26 colon carcinoma tumors were immunofluorescently stained for STING and NF-κβ 24 and 48 h after IR with a clinically relevant fractionated regimen of 5 × 5 Gy. Results: Doses of 2, 4, 6, 8, and 10 Gy led to a dose-dependent decrease in proliferation and increased death of ECs. RNA sequencing analysis showed that the effects on the transcriptome of HUVECs were most pronounced 72 h after IR with 5 Gy, with 1014 genes (661 down-regulated and 353 up-regulated) being significantly differentially expressed. Irradiation with 5 Gy resulted in HUVEC activation, with up-regulation of the immune system and extracellular matrix genes, such as STING1 (log(2)FC = 0.81) and SELE (log(2)FC = 1.09), respectively; and down-regulation of cell cycle markers. Furthermore, IR led to the up-regulation of immune response- and extracellular matrix (ECM)-associated signaling pathways, including NF-κβ signaling and ECM-receptor interaction, which was also observed in the transcriptome of irradiated murine TECs in vivo. This was confirmed at the protein level with higher expressions of the EC activation-associated proteins STING, NF-κβ, and VCAM-1 in irradiated HUVECs and irradiated TECs in vivo. Conclusions: IR induces changes in ECs and TECs, supporting their activation in dose- and time-dependent manners, potentially contributing to the anti-tumor immune response, which may potentially increase the infiltration of immune cells into the tumor and thus, improve the overall efficacy of RT, especially in combination with immune checkpoint inhibitors.