Time-sequential change in immune-related gene expression after irradiation in glioblastoma: next-generation sequencing analysis

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-<i>γ</i> 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-<i>γ</i> 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.

本研究通过评估胶质母细胞瘤细胞系经辐照后免疫相关基因的时序表达变化，以推测放疗联合免疫治疗的效应。研究采用单次6 Gy剂量辐照U373 MG胶质母细胞瘤细胞系，分别于辐照前（对照组）及辐照后6、24、48小时采集下一代测序（next-generation sequencing, NGS）转录组数据，并于各时间节点分析免疫相关通路。上述分析同样应用于A549肺癌细胞系与U87 MG胶质母细胞瘤细胞系。通过蛋白质印迹（Western blotting）验证了程序性死亡配体1（programmed death-ligand 1, PD-L1）的时序表达水平。在U373 MG细胞系中，中性粒细胞介导的免疫应答、I型干扰素信号通路、抗原交叉呈递至T细胞以及干扰素-γ信号均于辐照后24小时开始显著升高，并持续上调至辐照后48小时。该结果与A549及U87 MG细胞系的检测结果一致。在无T细胞浸润的情况下，即便癌细胞内干扰素-γ信号通路上调，PD-L1的表达也未出现升高。综上，胶质母细胞瘤细胞系的免疫相关信号于辐照后24小时与48小时显著上调。因此，每日放疗的时间间隔或不足以让免疫检查点抑制剂与辐照后新浸润的免疫细胞发挥完整的免疫应答效应。