耐药内皮细胞释放的微小细胞膜囊泡的特性鉴定和肿瘤耐药的临床预测

Multidrug resistance (MDR) was observed initially to cytotoxic drugs and now also to targeted drugs such as anti-angiogenic inhibitors. Better understanding of the mechanisms and new methods reflecting and predicting the occurrence and the fluctuations of MDR are expected in clinic. We recently demonstrated that endothelial cells (ECs) of blood vessels developed acquired MDR in vitro and that this reduced the efficacy of chemotherapy in vivo. In the literature, it was showed that the MDR property could be transmitted from cells to cells through extracellular vesicles (Evs, mostly exosomes, also known as microparticles). Since ECs is known to release directly the Evs into blood circulation, we postulate that the biomarkers of the blood Evs released by drug-resistant ECs could be used to monitor MDR. .This program aims to characterize the MDR biomarkers of Evs released from drug-resistant ECs in vitro and analyze the blood Evs of the cancer patients in parallel. .Firstly, we will isolate the Evs from cultured paclitaxel-resistant endothelial cells and analyze the biomarkers contained in the Evs in vitro. We will analyze the quantities of the well-known MDR-related proteins carried by these EVs such as ABC family proteins P-gp, BCRP and MRP1, and flotillin2 and Trp5C (which play a key role in transmitting drug-resistance property between the cells). We will also compare and analyze the miRNA expression profilings of the Evs from cultured drug-resistant and non-resistant ECs and identify the potential miRNA biomarkers of MDR. .Next, we will evaluate the capacity of the Evs (collected in vitro from drug-resistant ECs) to induce MDR of tumors to the treatments by paclitaxel and cisplatin in athymic mouse tumor models. This will clarify the role of these Evs in the transmission of multi-drug resistance,.Clinically, we will analyze the isolated Evs samples from newly diagnosed and relapsed-refractory nasopharyngeal carcinoma patients. Totally 40 patients' samples and 10 volunteers' samples will be obtained to evaluate the levels of MDR biomarkers, such as P-gp, BCRP and TrpC5 in these Evs samples by multicolor flux cytometry. The endothelial Evs will be distinguished by labeling the Evs with anti-CD144 or/and anti-CD146 antibodies. ELISA method will also be established and used to quantify the Evs-carried biomarkers with the antibodies specific for MDR markers. The results are expected to clarify the best choice of tested markers for future clinic diagnosis. .We will compare and analyze the miRNA expression profilings of the Evs samples of above-mentioned patients. The technique of miRCURY RNA isolation kit-biofluids will be used to obtain the miRNA. Commercially available service will help to analyze global microRNA expression profiling, such as Exiqon's miRCURY LNA™ microRNA array system. These clinic data, together with the data of miRNA profiling obtained from the Evs of the cultured endothelial cells, are expected to identify new miRNA biomarkers of MDR. .Our project will clarify the role of the ECs-released Evs in the cell-cell transmission of MDR and in parallel, provide the rationale and the methods for quantifying MDR biomarkers in clinic. It is expected that establishing MDR index improve clinical management of chemotherapy.

已发表的研究揭示，化疗诱发肿瘤细胞和血管内皮细胞通过分泌微小细胞囊泡(EVs)引起肿瘤耐药范围的扩大。本计划旨在探索建立临床上检测EVs耐药指标的方法，从而辅助判断病人耐药的状况。本研究包括：1)体外研究耐药内皮细胞分泌的EVs，分析它们作为传递耐药信息的物质基础(包括耐药标志蛋白P-gp, ABCG2, MRP1,TrpC5等以及EVs所携带的miRNA表达谱)；2)证明这些EVs在活体裸鼠模型中诱导肿瘤耐药的产生；3)探索建立流式细胞和ELISA方法检测耐药鼻咽癌患者外周血中内皮细胞释放EVs的耐药分子标志物 P-gp、ABCG2、TrpC5，明确这些指标作为临床判断耐药的科学性和可行性；4)检测耐药鼻咽癌患者外周血中EVs的miRNA表达谱，寻找反映耐药状况新的标志物。本研究将补充阐明肿瘤耐药的发生机制，探索为临床早期和及时观察病人耐药状态提供新的量化方法，提高化疗的效果。

研究证实，肿瘤发生耐药时，不仅肿瘤组织内的肿瘤细胞，肿瘤组织内部的其他非肿瘤细胞成分如间质细胞，血管内皮细胞等同样可以产生耐药现象。肿瘤的耐药是肿瘤细胞及它周围的支撑细胞对药物耐药的综合反映。当其中的内皮细胞产生耐药时，耐药内皮细胞会释放具有耐药特性的微小囊泡。我们的前期研究已在电镜下观察到耐药内皮细胞释放微小囊泡的现象。相对于肿瘤细胞分泌的微小囊泡，内皮细胞分泌的微小囊泡更易直接释放到血液循环，理论上能够更大地提高检测阳性率。我们的研究通过生化分析耐药内皮细胞释放的具有耐药特性的微小囊泡的相关生物特性以及通过检测微小囊泡中miRNAs表达谱来寻找与耐药相关的分子标志物。研究从肿瘤微环境中内皮细胞释放耐药微小囊泡这个新视点补充阐明肿瘤耐药的机制。 .本研究项目完成了预期的研究目标。课题围绕耐药内皮细胞释放的微小细胞膜囊泡为核心，通过构建耐阿霉素的耐药内皮细胞系分离分析外泌体，通过QPCR及WB等实验检测耐药蛋白的表达，证实外泌体的耐药性；通过QPCR及WB、Transwell、CCK-8等技术，阐明了耐药内皮细胞释放的外泌体促进了肿瘤细胞的耐药及增殖、侵袭等；同时采用基因沉默，基因过表达等研究发现耐药细胞来源的外泌体中miR-106a-5p表达增高，miR-106a-5p通过外泌体转移加重了鼻咽癌细胞对顺铂的耐药，最后分别通过体内及体外试验证实miR-106a-5p靶向ARNT2调节Akt磷酸化促进鼻咽癌细胞的增殖、迁移和侵袭。通过课题证实耐药细胞来源的外泌体可将耐药相关信息传递给肿瘤细胞，从而促进了肿瘤细胞的恶性生物学行为及对细胞毒药物的耐药。从外泌体可以在肿瘤微环境中传递相关信息这个新视点补充阐明肿瘤耐药的机制，为早期判断机体耐药状态提供新的思路，同时分析外泌体中中miRNAs的表达,为进一步寻找新的生物标志物打下了基础。