Table_1_Irradiation enhances the malignancy-promoting behaviors of cancer-associated fibroblasts.xlsx

BackgroundCancer-associated fibroblasts (CAFs) are the important component of the tumor microenvironment (TME). Previous studies have found that some pro-malignant CAFs participate in the resistance to radiotherapy as well as the initiation and progression of tumor recurrence. However, the exact mechanism of how radiation affects CAFs remains unclear. This study aimed to explore the effect and possible mechanism of radiation-activated CAFs, and its influence on lung cancer.MethodsCAFs were isolated from surgical specimens in situ and irradiated with 8Gy x-rays. The changes in cell morphology and subcellular structure were observed. CAFs marker proteins such as FAP and α-SMA were detected by Western Blotting. Cell counting kit-8 (CCK8) assay, flow cytometry, wound healing assay, and transwell chamber assay was used to detect the activation of cell viability and migration ability. A nude mouse xenograft model was established to observe the tumorigenicity of irradiated CAFs in vivo. The genomic changes of CAFs after radiation activation were analyzed by transcriptome sequencing technology, and the possible mechanisms were analyzed.ResultsThe CAFs showed a disorderly growth pattern after X-ray irradiation. Subcellular observations suggested that metabolism-related organelles exhibited more activity. The expression level of CAFs-related signature molecules was also increased. The CAFs irradiated by 8Gy had good proliferative activity. In the (indirect) co-culture system, CAFs showed radiation protection and migration induction to lung cancer cell lines, and this influence was more obvious in radiation-activated CAFs. The radiation protection was decreased after exosome inhibitors were applied. Vivo study also showed that radiation-activated CAFs have stronger tumorigenesis. Transcriptome analysis showed that genes were enriched in several pro-cancer signaling pathways in radiation-activated CAFs.ConclusionsOur study confirmed that CAFs could be activated by ionizing radiation. Irradiation-activated CAFs could promote cancer cell proliferation, migration, radiotherapy tolerance, and tumorigenesis. These results suggested that irradiation-activated CAFs might participate in the recurrence of lung cancer after radiotherapy, and the inhibition of CAFs activation may be an important way to improve clinical radiotherapy efficacy.

背景：肿瘤相关成纤维细胞（CAFs）是肿瘤微环境（TME）中的重要组成部分。既往研究表明，部分促恶性CAFs参与了对放射治疗的抵抗，以及肿瘤复发的发展和进程。然而，辐射如何影响CAFs的确切机制尚不明确。本研究旨在探讨辐射激活的CAFs的作用及其可能的机制，以及其对肺癌的影响。
方法：从手术标本中原位分离CAFs，并使用8Gy X射线进行照射。观察细胞形态和亚细胞结构的变化。通过Western Blotting检测CAFs标记蛋白如FAP和α-SMA。使用细胞计数试剂盒-8（CCK8）试验、流式细胞术、伤口愈合试验和Transwell小室试验检测细胞活力和迁移能力的激活。建立裸鼠异种移植模型，观察照射后CAFs的体内肿瘤发生性。通过转录组测序技术分析辐射激活后CAFs的基因组变化，并分析可能的机制。
结果：X射线照射后，CAFs呈现出紊乱的生长模式。亚细胞观察表明，与代谢相关的细胞器表现出更高的活性。与CAFs相关的标志性分子的表达水平也升高。8Gy照射的CAFs具有良好的增殖活性。在（间接）共培养系统中，CAFs表现出对肺癌细胞系的辐射保护和迁移诱导，这种影响在辐射激活的CAFs中更为显著。应用外泌体抑制剂后，辐射保护作用降低。体内研究也表明，辐射激活的CAFs具有更强的肿瘤发生性。转录组分析显示，基因在辐射激活的CAFs中富集于多个促癌信号通路。
结论：本研究证实，CAFs可以被电离辐射激活。辐射激活的CAFs可以促进癌细胞增殖、迁移、放射治疗耐受性和肿瘤发生。这些结果表明，辐射激活的CAFs可能参与放射治疗后肺癌的复发，抑制CAFs的激活可能是提高临床放射治疗效果的重要途径。