ABSTRACT

The time-sequential change in immune-related gene expression of the glioblastoma cell line after irradiation was evaluated to speculate the effect of combined immunotherapy with radiotherapy. The U373 MG glioblastoma cell line was irradiated with 6 Gy single dose. Next-generation sequencing (NGS) transcriptome data was generated before irradiation (control), and at 6, 24, and 48 h post-irradiation. Immune-related pathways were analyzed at each time period. The same analyses were also performed for A549 lung cancer and U87 MG glioblastoma cell lines. Western blotting confirmed the programmed death-ligand 1 (PD-L1) expression levels over time. In the U373 MG cell line, neutrophil-mediated immunity, type I interferon signaling, antigen cross-presentation to T cell, and interferon-γ signals began to increase significantly at 24 h and were upregulated until 48 h after irradiation. The results were similar to those of the A549 and U87 MG cell lines. Without T cell infiltration, PD-L1 did not increase even with upregulated interferon-γ signaling in cancer cells. In conclusions, in the glioblastoma cell line, immune-related signals were significantly upregulated at 24 and 48 h after irradiation. Therefore, the time interval between daily radiotherapy might not be enough to expect full immune responses by combined immune checkpoint inhibitors and newly infiltrating immune cells after irradiation.

摘要

评估辐射后胶质母细胞瘤细胞系免疫相关基因表达的时间序列变化，以推测联合免疫治疗与放疗的效果。用 6 Gy 单剂量照射 U373 MG 胶质母细胞瘤细胞系。下一代测序 (NGS) 转录组数据是在辐照前（对照）和辐照后 6、24 和 48 小时生成的。在每个时间段分析免疫相关途径。对 A549 肺癌和 U87 MG 胶质母细胞瘤细胞系也进行了相同的分析。蛋白质印迹证实了程序性死亡配体 1 (PD-L1) 随时间的表达水平。在 U373 MG 细胞系中，中性粒细胞介导的免疫、I 型干扰素信号传导、抗原向 T 细胞的交叉呈递和干扰素-γ信号在 24 小时开始显着增加，并在照射后 48 小时上调。结果与 A549 和 U87 MG 细胞系的结果相似。在没有 T 细胞浸润的情况下，即使癌细胞中干扰素-γ信号传导上调，PD-L1 也不会增加。总之，在胶质母细胞瘤细胞系中，免疫相关信号在照射后 24 和 48 小时显着上调。因此，每日放疗之间的时间间隔可能不足以预期联合免疫检查点抑制剂和照射后新浸润的免疫细胞产生完全免疫反应。