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 asSTING1(log2FC = 0.81) andSELE(log2FC = 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.
背景：放射治疗（IR）不仅靶向癌细胞，还作用于肿瘤微环境，包括肿瘤血管。除了诱导肿瘤内皮细胞（TEC）凋亡外，IR还可导致TEC活化，可能增加免疫细胞浸润。然而，IR诱导内皮细胞（EC）活化的内在变化机制尚不明确。本研究在结直肠癌小鼠模型中，探讨了小鼠和人内皮细胞系在体外对IR的剂量和时间依赖性分子与功能反应，以及体内TEC的反应。 方法：使用单次2-10 Gy剂量照射HUVEC、EA.hy926、Hulec5a以及小鼠bEND.3、2H11和SVEC4-10内皮细胞系。通过Cytation 1细胞成像多模式阅读器，每24小时对所有细胞核（Hoechst 33342）和死细胞（碘化丙啶）进行染色，持续5天，评估IR后EC的增殖和存活情况。对IR后24小时和72小时接受2 Gy和5 Gy照射的HUVEC进行RNA测序分析，重点关注与EC活化相关的生物学过程。为验证RNA测序结果，对与EC活化相关的蛋白进行了免疫荧光染色，包括干扰素反应cGAMP相互作用刺激因子1（STING）、核因子κB（NF-κβ）和血管细胞粘附分子1（VCAM-1）。为验证体外结果，利用公开的RNA测序数据分析了体内TEC的反应，这些数据来源于接受单次15 Gy照射的MC38结肠癌中分离的TEC。最后，对小鼠CT26结肠癌肿瘤在IR后24和48小时，采用临床相关的分次照射方案（5×5 Gy），进行STING和NF-κβ的免疫荧光染色。 结果：2、4、6、8和10 Gy的照射剂量导致EC增殖呈剂量依赖性下降，死亡增加。RNA测序分析显示，5 Gy照射后72小时对HUVEC转录组的影响最为显著，共有1014个基因（661个下调，353个上调）表达出现显著差异。5 Gy照射导致HUVEC活化，免疫系统和细胞外基质基因上调，例如STING1（log2FC = 0.81）和SELE（log2FC = 1.09）；同时细胞周期标志物下调。此外，IR导致免疫反应和细胞外基质（ECM）相关信号通路上调，包括NF-κβ信号通路和ECM-受体相互作用，这在体内受照射小鼠TEC的转录组中也观察到。在蛋白水平上得到证实，受照射的HUVEC和体内受照射的TEC中，与EC活化相关的蛋白STING、NF-κβ和VCAM-1表达升高。 结论：IR诱导EC和TEC发生改变，以剂量和时间依赖性的方式支持其活化，这可能有助于抗肿瘤免疫反应，从而可能增加免疫细胞向肿瘤的浸润，因此提高放射治疗的整体疗效，尤其是与免疫检查点抑制剂联合使用时。
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