来自非小细胞肺癌患者BALF和血浆的小ev分子货物:揭示它们在气道炎症和免疫调节中的作用。

IF 8.2 2区 生物学 Q1 CELL BIOLOGY
Magdalena Dlugolecka, Jacek Szymanski, Lukasz Zareba, Karolina Soroczynska, Zuzanna Homoncik, Malgorzata Polubiec-Kownacka, Ewa Frankiewicz, Diana Wierzbicka, Kannathasan Thetchinamoorthy, Joanna Domagala-Kulawik, Malgorzata Czystowska-Kuzmicz
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引用次数: 0

摘要

背景:肿瘤来源的细胞外小泡(ev)在调节免疫反应和塑造肿瘤微环境中起着至关重要的作用;然而,它们对非小细胞肺癌气道免疫的功能影响在很大程度上仍未被探索。本研究首次尝试在模拟人肺微环境的人体三维支气管气道模型中研究非小细胞肺癌衍生ev的免疫调节和促肿瘤作用。方法:从非小细胞肺癌患者血浆和支气管肺泡灌洗液(BALF)中分离ev,采用纳米颗粒跟踪分析(NTA)和高分辨率成像流式细胞术进行分析。对匹配血液和BALF样本的淋巴细胞组成进行了分析。为了评估ev的功能影响,我们采用了一种开创性的体外3D气道共培养模型,该模型将原代人气道上皮细胞和肺泡巨噬细胞在气液界面结合在一起。对ev处理的细胞及其分泌组进行蛋白质组学分析,以确定ev驱动的免疫调节的关键分子途径。结果:令人惊讶的是,尽管cBALF中的调节性T细胞(Tregs)局部增加,但癌性肺室(cBALF)和对侧肺室(oBALF)的ev之间没有检测到显著的分子差异,表明区域免疫抑制。血浆源性ev表现出高度多样化的患者特异性分子特征,但与临床或免疫参数没有直接相关性。在3D气道模型中,对具有高表面分子载货和低表面分子载货的EV的功能研究表明,两个EV亚组都通过MCP-1分泌促进单核细胞/巨噬细胞募集、血管生成和上皮-间质转化(EMT),并诱导免疫抑制的气道微环境,增强IL-10的产生,并将巨噬细胞转向促肿瘤的M2表型。蛋白质组学分析揭示了上皮细胞和巨噬细胞群体中不同的差异表达蛋白(DEP)谱,最终导致了蛋白原性和免疫抑制结果。值得注意的是,巨噬细胞的功能富集分析显示,ev驱动的M2极化是通过抑制EGFR活性发生的,这是一种以前未被认识的机制,将ev介导的免疫抑制与肺癌进展联系起来。结论:本研究提供了第一个功能证据,证明非小细胞肺癌衍生的ev在人体3D气道模型中驱动免疫抑制和肿瘤支持变化,密切模仿体内肺条件。EGFR抑制是巨噬细胞极化的驱动因素,这一发现强调了在抗EGFR治疗中考虑巨噬细胞特异性EGFR调控以防止意外致瘤作用的必要性。这些发现为未来探索EV货物(如mirna)作为非小细胞肺癌潜在治疗靶点的研究铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Molecular cargo of small EVs from NSCLC patient BALF and plasma: unveiling their role in airway inflammation and immune regulation in a novel human 3D bronchial model.

Background: Tumor-derived small extracellular vesicles (EVs) play a crucial role in modulating immune responses and shaping the tumor microenvironment; however, their functional impact on airway immunity in NSCLC remains largely unexplored. This study represents the first attempt to investigate the immunomodulatory and tumor-promoting effects of NSCLC-derived EVs in a human 3D bronchial airway model, which closely mimics the human lung microenvironment.

Methods: EVs were isolated from the plasma and bronchoalveolar lavage fluid (BALF) of NSCLC patients and analyzed via nanoparticle tracking analysis (NTA) and high-resolution imaging flow cytometry. The lymphocyte compositions of the matched blood and BALF samples were profiled. To assess the functional effects of EVs, we employed a pioneering in vitro 3D airway coculture model that combines primary human airway epithelial cells and alveolar macrophages at the air‒liquid interface. Proteomic analysis of EV-treated cells and their secretome was performed to identify key molecular pathways underlying EV-driven immunomodulation.

Results: Surprisingly, no significant molecular differences were detected between EVs from cancerous (cBALF) and opposite (oBALF) lung compartments, despite a localized increase in regulatory T cells (Tregs) in the cBALF, suggesting regional immunosuppression. Plasma-derived EVs exhibited highly diverse, patient-specific molecular signatures but were not directly correlated with clinical or immune parameters. Functional studies of EVs with high and low surface molecular cargo in a 3D airway model revealed that both EV subgroups promoted monocyte/macrophage recruitment, angiogenesis, and epithelial-to-mesenchymal transition (EMT) via MCP-1 secretion and induced an immunosuppressive airway microenvironment, enhancing IL-10 production and shifting macrophages toward a tumor-promoting M2 phenotype. Proteomic analysis revealed distinct differentially expressed protein (DEP) profiles across epithelial and macrophage populations, ultimately resulting in protumorigenic and immunosuppressive outcomes. Notably, functional enrichment analysis of macrophages revealed that EV-driven M2 polarization occurred through the suppression of EGFR activity, a previously underrecognized mechanism that links EV-mediated immune suppression to lung cancer progression.

Conclusions: This study provides the first functional evidence that NSCLC-derived EVs drive immune suppression and tumor-supportive changes in a human 3D airway model, closely mimicking in vivo lung conditions. The identification of EGFR suppression as a driver of macrophage polarization underscores the need to consider macrophage-specific EGFR regulation in anti-EGFR therapies to prevent unintended protumorigenic effects. These findings pave the way for future studies exploring EV cargo, such as miRNAs, as potential therapeutic targets in NSCLC.

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来源期刊
CiteScore
11.00
自引率
0.00%
发文量
180
期刊介绍: Cell Communication and Signaling (CCS) is a peer-reviewed, open-access scientific journal that focuses on cellular signaling pathways in both normal and pathological conditions. It publishes original research, reviews, and commentaries, welcoming studies that utilize molecular, morphological, biochemical, structural, and cell biology approaches. CCS also encourages interdisciplinary work and innovative models, including in silico, in vitro, and in vivo approaches, to facilitate investigations of cell signaling pathways, networks, and behavior. Starting from January 2019, CCS is proud to announce its affiliation with the International Cell Death Society. The journal now encourages submissions covering all aspects of cell death, including apoptotic and non-apoptotic mechanisms, cell death in model systems, autophagy, clearance of dying cells, and the immunological and pathological consequences of dying cells in the tissue microenvironment.
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