Gao Yan, Duan Qingke, Wu Ning, Xu Bo
Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education Tianjin 300060, China.
Center for Intelligent Oncology, Chongqing University Cancer Hospital, Chongqing University School of Medicine Chongqing 400030, China.
Am J Cancer Res. 2021 Feb 1;11(2):513-529. eCollection 2021.
Our understanding on transcriptional regulation of tumour cells responding to ionizing radiation (IR) has mostly come from bulk sequencing. However, due to the heterogeneity of tumour, how each individual cell responds to IR differently is unclear. We report here a heterogeneous cellular response to IR by single cell transcriptome sequencing. We utilized the barcoded Smart-seq2 single cell transcriptome sequencing technology in breast cancer cell line MDA-MB-231 both without and with IR treatment. To further understand how ATM, a major hub protein required for an optimal DNA damage response, affected the heterogeneous IR response, we also knocked down ATM gene for single cell transcriptome sequencing. Single cell t-SNE analysis showed four clusters of cells responding to IR in distinctive ways: Cluster 1 changed the least; Cluster 2 responded to IR by upregulating ribosome associated genes, while Cluster 4 upregulated both ribosome and G1/S phase associated genes; Cluster 3 was a new cluster, which appeared only in irradiated cells. In the absence of ATM kinase, cells displayed much less transcriptional changes after IR. And Cluster 4 in wild-type cells, which had the greatest change in response to IR, was not present in the ATM knock-down cells. We also selected three IR-induced genes for functional validation in both MDA-MB-231 and an additional breast cancer cell line to demonstrate their importance in radiation sensitivity. Taken together, our single cell transcriptome analysis has revealed a heterogeneous cellular response to DNA damage induced by IR and identified potential biomarkers of radiation sensitivity.
我们对肿瘤细胞对电离辐射（IR）反应的转录调控的理解大多来自批量测序。然而，由于肿瘤的异质性，每个细胞对IR的不同反应尚不清楚。我们在此报告通过单细胞转录组测序对IR的异质性细胞反应。我们在未处理和经过IR处理的乳腺癌细胞系MDA-MB-231中利用带条形码的Smart-seq2单细胞转录组测序技术。为了进一步了解ATM（一种最佳DNA损伤反应所需的主要枢纽蛋白）如何影响异质性IR反应，我们还敲低了ATM基因以进行单细胞转录组测序。单细胞t-SNE分析显示有四类细胞以不同方式对IR作出反应：第1类变化最小；第2类通过上调核糖体相关基因对IR作出反应，而第4类则上调核糖体和G1/S期相关基因；第3类是一个新类别，仅出现在受辐照的细胞中。在没有ATM激酶的情况下，细胞在IR后表现出少得多的转录变化。并且野生型细胞中对IR反应变化最大的第4类在ATM敲低的细胞中不存在。我们还选择了三个IR诱导基因在MDA-MB-231和另一个乳腺癌细胞系中进行功能验证，以证明它们在辐射敏感性中的重要性。综上所述，我们的单细胞转录组分析揭示了对IR诱导的DNA损伤的异质性细胞反应，并鉴定了辐射敏感性的潜在生物标志物。
