Immune capture and protein profiling of tumor-derived exosomes (TEX) in plasma of patients with Head and neck squamous cell carcinoma (HNSCC)

1. 从头颈部鳞状细胞癌（HNSCC）患者血浆中靶向分离肿瘤源性外泌体（TEX）和非TEX方法的建立和优化提出的研究是基于广泛的临床前实验，证明了靶向分离作为生物标志物发现的选择方法的可行性。由于没有特异性结合HNSCC肿瘤抗原的单克隆抗体（mAb）是已知的，针对过表达的肿瘤Ag的单克隆抗体被用于TEX的免疫检测。在这个项目中，根据Whiteside教授的工作组最近收集的初步结果，将使用三种不同捕获抗体（Abs）的组合。抗cspg4、抗b7h3和CD44v3单克隆抗体的组合先前已被证明可以识别在HNSCC细胞中高度过表达的Ag，并且这些细胞产生的外泌体中也携带Ag。因此，我们假设，抗cspg、抗b7 - h3和抗cd44v3单克隆抗体（它们都能识别三种不同的ag，过表达的ag来自HNSCC细胞和来自血浆源性HNSCC外泌体）的组合可以成功地用于从患者血浆中分离HNSCC外泌体。2. 使用开发的方法从25名HPV（+）和25名HPV(-) HNSCC患者的血浆中靶向分离TEX和非TEX，用于随后的高分辨率质谱（HRMS）搜索蛋白质谱和病毒寻找抗原，包括E2和E5，表征TEX 3。分离的外泌体组分对CD8+ T细胞和NK细胞的表型和功能影响也将进行分析。

1. Development and optimization of a method for the targeted isolation of tumor-derived exosomes (TEX) and non-TEX from plasma of patients with head and neck squamous cell carcinoma (HNSCC). The proposed study is based on extensive preclinical experiments demonstrating the feasibility of targeted isolation as the method of choice for biomarker discovery. Since no monoclonal antibody (mAb) that specifically binds to HNSCC tumor antigens is known, mAb against overexpressed tumor Ag are used for immune detection of TEX. In this project, a combination of three different capture antibodies (Abs) will be used, based on preliminary results, which were recently collected in Prof. Whiteside's working group. The combination of anti-CSPG4, anti-B7H3 and CD44v3 mAbs has previously been shown to recognize Ag, which is highly overexpressed by HNSCC cells and which is also carried in exosomes produced by these cells. Therefore, we hypothesize that the combination of anti-CSPG, anti-B7-H3, and anti-CD44v3 mAbs, all of which recognize three different Ags, overexpressed those from HNSCC cells and from plasma-derived HNSCC exosomes worn, can be used successfully to isolate HNSCC-derived exosomes from patient plasma. 2. Use of the developed methodology for the targeted isolation of TEX and non-TEX from plasma of a cohort of 25 HPV(+) and 25 HPV(-) patients with HNSCC for subsequent high-resolution mass spectrometry (HRMS) to search for protein profiles as well as viral to look for antigens, including E2 and E5, that characterize TEX 3. Analysis of the phenotypic and functional influence of the separated exosome fractions on CD8+ T cells and NK cells will also be performed.