濒死细胞经外泌体途径促进胰腺肿瘤再增殖的分子机制及干预策略

Tumor repopulation is one of the main reasons for chemoradiation resistance and therapeutic failure in clinical pancreatic cancer, yet the underlying mechanisms remain unclear. Based on our previous researches, we hypothesize that dying cells from chemoradiation would release exosomes and further deliver them to accelerate pancreatic cancer repopulation. This proposal would investigate the underlying molecular mechanisms of pancreatic cancer repopulation, and explore the feasibility of low-dose aspirin use for intervention of pancreatic cancer after chemoradiation from the multiple level of cancer cells, clinical tissues and tumor-burdened animal models by using comprehensive modern technologies of cancer biology. We would first explore the effects of exosomes from dying cells on pancreatic cancer repopulation after chemoradiation. We would further illuminate that the exosomal microRNAs (such as tumor suppressor miR-26a, et al) could arrest the cell cycle of the damaged pancreatic cancer stem cells and promote its DNA repair in the immediate early response to chemoradiation, and then cascade with PGE2 for persevering the “seed” for pancreatic cancer cell proliferation and repopulation. Furthermore, we would explore the feasibility of low-dose aspirin use for the clinical intervention of pancreatic cancer repopulation by down-regulating exosomal miR-26a and blocking PGE2 signaling pathways. This study will help to decipher the underlying molecular mechanisms of pancreatic cancer repopulation after chemoradiation, and provide novel views for clinical intervention of pancreatic cancer with chemoradiation.

肿瘤再增殖是临床上胰腺癌治疗失败的主要原因之一，但其内在机制仍不清楚。基于既往研究工作，我们认为放化疗所致濒死细胞可经外泌体途径促进胰腺癌再增殖。本课题拟综合应用现代肿瘤生物学研究技术，从细胞、组织和模型动物等层面探讨濒死细胞促进胰腺癌再增殖的级联反应机制，探索阿司匹林干预胰腺癌再增殖的可行性。课题拟首先分析放化疗所致濒死细胞释放的外泌体对胰腺癌再增殖的影响，继而阐释外泌体中miRNA（如抑癌的miR-26a等）在放化疗后的反应早期阻滞存活细胞的细胞周期进展、促进其DNA损伤修复以保存肿瘤再增殖的“火种”，并在反应后期与PGE2等细胞增殖信号形成级联反应，从而促进胰腺癌快速再增殖；其次探索阿司匹林通过下调外泌体中miR-26a含量、阻断PGE2信号通路、继而实现临床上干预放化疗后胰腺癌再增殖的可行性。本研究有助于系统性理解放化疗后肿瘤再增殖的分子机制，也为临床干预胰腺癌再增殖提供新的思路。

【目的】放射治疗是肿瘤临床治疗最重要的手段之一，在肿瘤局部治疗中显示出强大作用。胰腺癌是恶性程度最高的肿瘤之一，对放化疗等治疗反应差。肿瘤再增殖作为肿瘤放射生物学的“5R”特征之一，是造成放疗失败的重要原因。我们的既往研究提示濒死细胞在肿瘤再增殖中发挥关键作用，能促进存活的肿瘤再增殖细胞的快速增殖。然而，肿瘤再增殖细胞同样在放疗时受到了DNA损伤，细胞在有大量DNA损伤的情况下如何快速增殖，是一个尚待解决的重要科学问题。本研究旨在探究放疗后濒死细胞对受损肿瘤再增殖细胞存活和再增殖的影响及其机制，并由此发掘可能的干预措施。.【方法】 应用体内和体外的肿瘤再增殖模型观察放疗后胰腺癌细胞的再增殖情况，并探究其具体分子机制。应用RNA测序技术明确放疗前后细胞内及细胞释放的外泌体中显著改变的信号通路及分子。应用定量PCR、免疫印迹、免疫组织化学、免疫荧光、流式细胞术、克隆形成、彗星实验等技术分析细胞及分子水平的变化。.【结果】体内外实验研究发现濒死细胞对存活肿瘤细胞的增殖影响表现为先抑制后促进的动态调控过程。其后的机制研究发现濒死细胞经由其所释放的外泌体运输miR-194-5p至残存的ALDH1A1+ 的肿瘤活细胞（肿瘤再增殖细胞）中，抑制E2F3的表达，诱导细胞发生G1/S细胞周期阻滞并促进DNA损伤反应，最终促进了残存的肿瘤再增殖细胞的存活及损伤修复。此外，miR-194-5p也减轻了促癌的HMGA2在放疗时对肿瘤再增殖细胞的损害作用。此后，濒死细胞还释放出大量的PGE2以促进完成修复的肿瘤再增殖细胞进入快速增殖，完成了对整个再增殖过程的调控。体内外实验研究还发现低剂量的阿司匹林能通过减少外泌体及PGE2分泌来抑制胰腺癌细胞的再增殖。.【结论】放疗所导致的濒死细胞经由其所分泌的外泌体中的miR-194-5p增强残存的肿瘤再增殖细胞的DNA损伤反应并促进肿瘤再增殖细胞存活，为后续的快速增殖埋下“火种”。阿司匹林联合放疗可能有助于胰腺癌患者获得更好的疗效。