PO.TB10.15 · 肿瘤生物学

小型和大型细胞外囊泡的蛋白质组学和功能分化揭示卵巢癌进展的亚型特异性驱动因素

Proteomic and functional divergence of small and large extracellular vesicles reveal subtype-specific drivers of ovarian cancer progression

编号 3347 展板 8 时间 4/20 02:00–05:00 区域 Section 26 主讲 Kalpana Deepa Priya Dorayappan, PhD

分会场 Extracellular Vesicles and Long-Range Tumor-Host Communication

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作者与单位 Authors & Affiliations

Kalpana Deepa Priya Dorayappan¹, Fries Brian², Wei Fu³, Ganesh Yadaigiri¹, Lakshmi Narasimhan Chakrapani¹, Sydney Wiggins⁴, Shyam Sundaram⁵, Thangavel Muthusamy⁶, Sudhiksha Anbu Chelian⁷, Gabriela S. Vendrell², Qi-En Wang⁸, David E. Cohn¹, Lianbo Yu⁹, David O’Malley¹, Colin Hisey⁴, Selvendiran Karuppaiyah¹

¹OB/GYN, The Ohio State University, Columbus, OH,²The Ohio State University, Columbus, OH,³Biomedical Engineering, The Ohio State University, Columbus, OH,⁴Biomedical Engineering, Northwestern University, Evanston, IL,⁵University of Southern California, Los Angeles, CA,⁶Sree Balaji Medical College and Hospital, Chennai, India,⁷Health & Rehabilitation Sciences, The Ohio State University, Columbus, OH,⁸Radiation Oncology, The Ohio State University, Columbus, OH,⁹Medical Bioinformatics, The Ohio State University, Columbus, OH

摘要 Abstract

中文摘要

目的：高级别浆液性卵巢癌（HGSOC）的进展和治疗耐药性越来越多地被归因于细胞外囊泡（EV）介导的通讯。虽然小型 EV（SEV）已被广泛研究，但大型 EV（LEV）的蛋白质组和生物学贡献仍未明确界定。本研究旨在（i）建立基于长期生物反应器的卵巢癌细胞系细胞外囊泡（SEV 和 LEV）生产，（ii）界定亚型特异性的蛋白质组学货物，以及（iii）比较 SEV 和 LEV 在肿瘤生长、转移和免疫调节中的功能作用。方法：将 OVCAR4、CaOV3 和 SKOV3 卵巢癌细胞系在 T-175 培养瓶中扩增，并逐步过渡到生物反应器中的无血清 CDM-HD 培养基。细胞系在生物反应器中维持八周以测量 EV 分泌。通过超速离心随后进行尺寸排阻色谱（SEC）分离 SEV 和 LEV。通过纳米颗粒跟踪分析（NTA）定量颗粒数量和大小，并通过 Nano Drop 测量蛋白浓度。通过 Western blotting（CD9、CD63、CD81、LAMP1）、成像流式细胞术（ISF）和透射电子显微镜（TEM）确认 EV 的身份和纯度。对每种细胞系的 SEV 和 LEV 进行蛋白质组学分析，并使用 Ingenuity Pathway Analysis 进行下游通路分析。功能实验包括：（i）EdU 增殖实验，（ii）迁移和侵袭实验，（iii）脾细胞共培养以评估免疫调节，以及（iv）小鼠异种移植模型以评估 EV 依赖性肿瘤生长和转移播散。结果：生物反应器支持 OVCAR4 和 CaOV3 细胞中稳定的 EV 生产，而 SKOV3 适应性较差。NTA 和 ISF 确认了八周内一致的 EV 产出。SEV 富集 CD9、CD63 和 CD81，而 LEV 显示出强烈的 LAMP1 表达。蛋白质组学揭示了不同的货物谱：SEV 含有与细胞骨架重塑、迁移和转移信号相关的蛋白质，而 LEV 携带参与增殖、代谢和应激反应的蛋白质。在功能上，SEV 增强了迁移、侵袭、转移负荷和免疫抑制，而 LEV 在体内促进了细胞增殖和更大的原发肿瘤。结论：源自卵巢癌细胞的 SEV 和 LEV 表现出不同的蛋白质组学特征，并对肿瘤进展产生分化的影响。SEV 主要驱动转移潜能，而 LEV 增强增殖性肿瘤生长。EV 亚型特异性表征为 HGSOC 的生物标志物发现和靶向治疗策略提供了新的机会。

查看英文原文 English abstract

Objective: High-grade serous ovarian cancer (HGSOC) progression and therapeutic resistance are increasingly attributed to extracellular vesicle (EV) mediated communication. While small EVs (SEVs) are widely studied, the proteome and biological contributions of large EVs (LEVs) remain poorly defined. This study aimed to (i) establish long-term bioreactor-based production of extracellular vesicles (SEVs and LEVs) from ovarian cancer cell lines, (ii) define subtype-specific proteomic cargo, and (iii) compare the functional roles of SEVs and LEVs in tumor growth, metastasis, and immune modulation. Methods: OVCAR4, CaOV3, and SKOV3 ovarian cancer cell lines were expanded in T-175 flasks and gradually transitioned to serum-free CDM-HD media in Bioreactors. Cell lines were maintained for eight weeks in bioreactors to measure EV secretion. SEVs and LEVs were isolated by ultracentrifugation followed by size-exclusion chromatography (SEC). Particle number and size were quantified by nanoparticle tracking analysis (NTA), and protein concentration was measured by Nano Drop. EV identity and purity were confirmed by Western blotting (CD9, CD63, CD81, LAMP1), Imaging Flow Cytometry (ISF), and transmission electron microscopy (TEM). Proteomic profiling of SEVs and LEVs was performed for each cell line, with downstream pathway analysis using Ingenuity Pathway Analysis. Functional assays included: (i) EdU proliferation assays, (ii) migration and invasion assays, (iii) splenocyte co-culture to evaluate immune modulation, and (iv) mouse xenograft models to assess EV-dependent tumor growth and metastatic dissemination. Results: Bioreactors supported stable EV production in OVCAR4 and CaOV3 cells, while SKOV3 adapted poorly. NTA and ISF confirmed consistent EV output across eight weeks. SEVs were enriched for CD9, CD63, and CD81, whereas LEVs showed strong LAMP1 expression. Proteomics revealed distinct cargo profiles: SEVs contained proteins linked to cytoskeletal remodeling, migration, and metastatic signaling, while LEVs carried proteins involved in proliferation, metabolism, and stress responses. Functionally, SEVs enhanced migration, invasion, metastatic burden, and immune suppression, whereas LEVs promoted cell proliferation and larger primary tumors in-vivo. Conclusion: SEVs and LEVs derived from ovarian cancer cells exhibit distinct proteomic signature and exert divergent effects on tumor progression. SEVs predominantly drive metastatic potential, whereas LEVs enhance proliferative tumor growth. EV subtype specific characterization provides new opportunities for biomarker discovery and targeted therapeutic strategies in HGSOC.

利益披露 Disclosure

K. Dorayappan, None.. F. Brian, None.. W. Fu, None.. G. Yadaigiri, None.. L. Chakrapani, None.. S. Wiggins, None.. S. Sundaram, None.. T. Muthusamy, None.. S. Anbu Chelian, None.. G. S. Vendrell, None.. Q. Wang, None.. D. E. Cohn, None.. L. Yu, None.. D. O’Malley, None.. C. Hisey, None.. S. Karuppaiyah, None.

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