低氧驱动的M2极化巨噬细胞通过细胞外囊泡促进胶质母细胞瘤的上皮-间质转化

基础：M2样肿瘤相关巨噬细胞（TAMs）促进胶质母细胞瘤的恶性进展。然而，导致这一现象的机制尚不清楚。方法：采用RT-PCR、西方印迹和流式细胞术评估巨噬细胞的极化状态。利用RT-PCR、西方印迹或免疫组化检测circ_0003137、PTBP1、PLOD3及上皮-间质转化（EMT）标志物的表达。Transwell迁移和侵袭实验评估肿瘤细胞的迁移和侵袭能力。通过RNA测序、生物信息学分析及Pearson相关分析探讨PTBP1与circ_003137/PLOD3的关系。体内实验用于评估sh-circ_0003137加载的纳米平台的作用。结果：低氧促进巨噬细胞向M2样TAMs的极化，依赖HIF1α。随后，M2样TAMs通过运输富含circ_0003137的细胞外囊泡（EVs）向胶质母细胞瘤细胞输送，增加了胶质母细胞瘤细胞中circ_0003137的表达。circ_0003137的过表达在体外和体内均促进了胶质母细胞瘤的EMT。机制方面，circ_0003137与多嘧啶结合蛋白1（PTBP1）直接结合，增强了前胶原赖氨酰甲基化酶3（PLOD3）的稳定性，促进了胶质母细胞瘤细胞的EMT。此外，建立了一种载有shRNA的脂质体纳米平台，用于包封sh-circ_0003137。荧光显微镜追踪和细胞共培养实验表明，包封sh-circ_0003137的纳米平台稳定，且能穿透血脑屏障（BBB），最终到达中枢神经系统（CNS）。在脑内原位肿瘤模型中，尾静脉注射载有sh-circ_0003137的纳米平台显著抑制了胶质母细胞瘤的进展，并改善了裸鼠的生存率。结论：低氧可以驱动巨噬细胞极化为M2样TAMs。极化的M2样TAMs通过EVs向胶质母细胞瘤细胞运输circ_0003137，后者通过靶向PTBP1/PLOD3轴促进肿瘤的EMT。因此，靶向circ_0003137可能成为抗胶质母细胞瘤的创新治疗策略。

Rationale: M2-like tumor-associated macrophages (TAMs) promote the malignant progression of glioblastomas. However, the mechanisms responsible for this phenomenon remain unclear. Methods: RT-PCR, Western blot and flow cytometry were used to evaluate the polarization status of macrophages. RT-PCR, western blot or/and immunohistochemistry was used to determine the expression of circ_0003137, PTBP1, PLOD3 and epithelial-mesenchymal transition (EMT) markers. Transwell assay was used to assess migration and invasion ability of tumor cells. RNA sequencing, bioinformatic analysis and Pearson correlation coefficient was performed to explore the relation between PTBP1 and circ_003137/PLOD3. In vivo experiment was used to determine the role of sh-circ_0003137-loaded nanoplatform. Results: Hypoxia promoted the polarization of macrophages towards M2-like TAMs in an HIF1α dependent manner. Then, M2-like TAMs could transport circ_0003137 enriched extracellular vesicles (EVs) to glioblastoma cells, upregulating circ_0003137 in glioblastoma cells. The circ_0003137 overexpression promoted the EMT of glioblastoma cells in vitro and in vivo. Mechanistically, circ_0003137 physically binds to polypyrimidine tract binding protein 1 (PTBP1), enhancing the stability of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) and promoting the EMT of glioblastoma cells. Moreover, a liposome-based nanoplatform that delivers shRNAs was established and used to encapsulate sh-circ_0003137. The fluorescence microscope tracer and cell co-culture assays demonstrated that the nanoplatform encapsulated with sh-circ_0003137 was stable and could penetrate the blood-brain barrier (BBB), finally reaching the central nervous system (CNS). The intracranial in situ tumor model showed that injecting the sh-circ_0003137-loaded nanoplatform via the tail vein significantly inhibited glioblastoma progression and improved the nude mice's survival. Conclusions: Hypoxia can drive macrophage polarization towards M2-like TAMs. Polarized M2-like TAMs can transport circ_0003137 to glioblastoma cells through EVs. Then, circ_0003137 promotes the EMT of glioblastomas by targeting the PTBP1/PLOD3 axis. Hence, targeting circ_0003137 might be a novel therapeutic strategy against glioblastoma.