METHODS: Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC-MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter.
RESULTS: Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells.
CONCLUSIONS: We reported the details of the molecular mechanisms of \"vascular normalization\" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.
方法:CCL28在人肺腺癌细胞系A549和鼠肺腺癌细胞系LLC中的过表达和敲除,分别,用于建立小鼠模型。进行单细胞测序以分析肿瘤微环境(TME)中不同细胞簇的比例和代谢变化。在鼠肿瘤组织和临床活检样品中进行免疫荧光和多重免疫组织化学以评估周细胞覆盖的百分比。使用磁激活细胞分选(MACS)从肺腺癌肿瘤组织中分离原代周细胞。然后用重组人CCL28蛋白处理这些周细胞,然后进行transwell迁移测定和RNA测序分析。检查了分泌组和代谢组的变化,并使用定量实时PCR验证周细胞中视黄酸代谢的变化,西方印迹,和LC-MS技术。染色质免疫沉淀,然后进行定量PCR(ChIP-qPCR),以验证RXRα对ANGPT1启动子特异性位点的转录调节能力和亲和力。
结果:我们的研究表明,在接受抗血管生成治疗后,肿瘤呈现缺血缺氧状态,导致低氧敏感转录因子CEBPB上调CCL28在低氧肺腺癌细胞中的表达。增加的CCL28可以通过在肿瘤微环境中募集和代谢重编程周细胞来促进肿瘤血管正常化。机械上,CCL28通过RXRα在周细胞中修饰维甲酸(RA)代谢并增加ANGPT1表达,从而增强内皮细胞的稳定性。
结论:我们首次报道了抗血管生成治疗后“血管正常化”的分子机制的细节。我们的工作可能为指导抗血管生成治疗与其他疗法之间联合治疗的临床安排提供了前瞻性的分子标志物。