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

头颈鳞状细胞癌 (HNSCC) 患者血浆中肿瘤源性外泌体 (TEX) 的免疫捕获和蛋白质分析

• 批准号: 535880852
• 负责人: Dr. Ioannis Michaelides
• 金额: --
• 依托单位: Klinik und Poliklinik für Hals-Nasen-Ohren-Heilkunde
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/535880852?language=en
• 关键词: Immune; capture; protein; profiling; tumor

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.

1. 开发和优化从头颈鳞状细胞癌（HNSCC）患者血浆中靶向分离肿瘤源性外泌体（TEX）和非TEX的方法。拟议的研究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选择方法的可行性。由于尚无特异性结合 HNSCC 肿瘤抗原的单克隆抗体 (mAb)，因此针对过表达肿瘤 Ag 的 mAb 用于 TEX 的免疫检测。在该项目中，根据 Whiteside 教授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 mAb 的组合先前已被证明可以识别 Ag，该 Ag 在 HNSCC 细胞中高度过表达，并且也存在于这些细胞产生的外泌体中。因此，我们假设抗 CSPG、抗 B7-H3 和抗 CD44v3 mAb 的组合（所有这些抗体均识别三种不同的 Ag），过表达来自 HNSCC 细胞和血浆来源的 HNSCC 外泌体的 Ag，可成功用于从患者血浆中分离 HNSCC 来源的外泌体。 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以寻找表征 TEX 的抗原，包括 E2 和 E5 3. 分析分离的表型和功能影响 还将对 CD8+ T 细胞和 NK 细胞进行外泌体组分分析。