基于特异性识别多肽和流式细胞术的肿瘤源性外泌体检测及分子分型

Cancer is the leading cause of death among all the diseases in China, and metastasis is the main attribute to cancer death. Personalized target therapy according to the molecular phenotyping of tumor has become a trend for cancer treatment. Currently the diagnosis and molecular phenotyping of tumor is based on the analysis of solid biopsies. However, it is difficult to obtain solid biopsies due to the invasive collection procedure and the risk for metastasis through the circulating system. This largely limits the application of personalized target therapy. In this regard, liquid biopsies serve as ideal substitutes for tissue biopsies for monitoring the progress and treatment response of cancer. Exosomes are small (30 – 100 nm) extracellular membrane vesicles of endocytic origin. They are secreted by almost all the cells, and are largely present in various body fluids including blood, urine, etc. They carry the nucleic acids and proteins from the cells of origin, and transfer them to the targeting cells, and therefore play key roles in cell-cell communications. Accumulating evidence has shown that exosomes play important roles in cancer progress and metastasis, and are involved in immune response to tumor. Therefore, there is increasing interest in identification, isolation, quantification and molecular phenotyping of tumor-derived exosomes from the peripheral blood for monitoring cancer progress and treatment response. However, isolation of exosomes has been technically challenging due to the contamination of other materials such as free proteins, microvesicles, and cell debris in the body fluids. Moreover, the nano-scaled size of exosomes limits the application of flow cytometry in the molecular analysis of exosomes. This project will take breast cancer as an example, and utilize immunoaffinity microbeads functionalized with peptides in combination with flow cytometry to detect tumor-derived exosomes in the serum, and perform molecular phenotyping, especially HER2 phenotyping of the captured exosomes. Multi-angle light scattering nano-flow cytometry will also be used to directly detect tumor-derived exomes in the serum without pre-purification or the assistance of immunoaffinity microbeads. These methods provide technique support for the real-time monitoring of tumor progress and treatment response, and provide important information for cancer treatment management and for understanding the mechanisms of tumor metastasis and drug resistance.

恶性肿瘤是我国致死率最高的疾病，转移是肿瘤致死的主要因素，个性化靶向治疗成为肿瘤治疗的趋势。然而组织标本获取困难，发展无损的液体活检技术对实时监测肿瘤进程和疗效评估意义重大。肿瘤源性外泌体大量存在于体液中，携带和传递来源细胞的核酸和蛋白质，成为理想液体活检对象。目前外泌体的富集和检测仍是难点，传统离心纯化耗时长、产量和纯度不高，造成外泌体水平的分子分型和肿瘤耐药分析的极大挑战性。流式细胞术被广泛用于生物微粒定量和分选，然而外泌体的纳米级尺度限制了流式细胞术在其检测和分型上的应用。本项目以乳腺癌为切入点，利用多肽功能化微珠多价富集外泌体，提高富集灵敏度，发展基于流式细胞术的外泌体检测和分子分型技术；同时利用新型纳米流式细胞术，建立不经预纯化和微珠辅助，直接快速检测复杂样品中外泌体的新方法。本项目研究将为实时动态监测乳腺癌等重要肿瘤的进程和疗效评估提供重要信息和指导。

恶性肿瘤是我国致死率最高的疾病，转移是肿瘤致死的主要因素，个性化靶向治疗成为肿瘤治疗的趋势。然而组织标本获取困难，发展无损的液体活检技术对实时监测肿瘤进程和疗效评估意义重大。肿瘤源性外泌体大量存在于体液中，携带和传递来源细胞的核酸和蛋白质，成为理想液体活检对象。目前外泌体的富集和检测仍是难点，传统离心纯化耗时长、产量和纯度不高，造成外泌体水平的分子分型和肿瘤耐药分析的极大挑战性。流式细胞术被广泛用于生物微粒定量和分选，然而外泌体的纳米级尺度限制了流式细胞术在其检测和分型上的应用。本项目利用醛基化乳胶微珠高效富集外泌体，并利用针对外泌体表面标志蛋白的荧光抗体都被捕获外泌体进行标记，结合流式细胞术对被捕获外泌体进行检测和分子分析，建立微珠辅助流式细胞术，解决了外泌体纳米级尺寸超过传统流式细胞仪检测极限的问题。并积极开发该技术的临床应用价值，利用微珠辅助流式细胞术的外泌体检测，证明上皮细胞粘附分子（EpCAM）阳性血清外泌体可有效用于乳腺癌检测，人表皮生长因子受体2（HER2）阳性血清外泌体可有效用于乳腺癌HER2分型，表皮生长因子受体（EGFR）阳性血清外泌体可有效用于胶质瘤检测和恶性程度评估，血清外泌体叶酸受体（FOLR1）和EpCAM表达水平可用于垂体瘤侵袭性评估。本项目研究为基于血清的无创肿瘤检测，预后评估，实时动态监测肿瘤进程和疗效评估提供重要方法和信息指导。