PO.TB10.15 · 肿瘤生物学
使用 3D 器官芯片平台模拟乳腺癌细胞外囊泡介导的淋巴管糖萼降解
Modeling breast cancer extracellular vesicle-mediated degradation of the lymphatic glycocalyx using a 3D organ-on-chip platform
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摘要 Abstract
中文摘要
三阴性乳腺癌（TNBC）主要依赖淋巴管进行早期转移扩散，而肿瘤分泌的因子——包括细胞外囊泡（EV）——可调理淋巴微环境以促进播散。淋巴内皮糖萼（eGCX）是一层富含糖的屏障，调节通透性和细胞黏附，但其对肿瘤驱动重塑的易感性仍知之甚少。
使用 LEC 和 TNBC 的 transwell 共培养模型，我们发现 TNBC 调理的环境显著破坏了淋巴 eGCX。MDA-MB-231 条件培养基使麦胚凝集素（WGA，总糖萼）染色降低了 45.41 ± 9.37%，使硫酸乙酰肝素（HS，最丰富的糖萼成分之一）降低了 38.87 ± 8.84%，使总体 eGCX 厚度降低了 49.37 ± 3.93%。SUM-149 条件培养基同样使糖萼厚度降低了 36.80 ± 5.11%。TNBC 暴露还诱导了淋巴内皮细胞（LEC）的形态变化，与屏障功能受损和活跃的糖萼重塑相一致。
为了鉴定这种破坏的具体驱动因素，采用膜亲和法从 TNBC 和非致瘤对照细胞中分离出 EV，并通过纳米颗粒跟踪分析进行表征。暴露于 TNBC EV 的 LEC 表现出 WGA 和 HS 染色减少，以及 VE-cadherin 连接改变。蛋白酶谱分析揭示 TNBC EV 中富集 ADAM9、组织蛋白酶 X/Z/P 和 MMP-8，提示 EV 相关蛋白酶介导了 eGCX 降解。
为了建立生理相关的基线，将人 LEC 培养在填充胶原蛋白的 PDMS 微流控芯片中，处于管腔剪切力（约 4 dynes/cm²）下。该 3D 系统产生的 eGCX 明显厚于 2D 单层（3D 相对于 2D 的 WGA 染色强度：1.311 ± 0.096；p ≤ 0.01），验证了其用于评估肿瘤和 EV 诱导的糖萼重塑的适用性。这一 3D 淋巴器官芯片模型提供了一个生理相关的平台，用于研究 eGCX 动态和 EV 介导的血管重塑。
总之，这些发现表明 TNBC 来源的 EV 携带能够降解淋巴 eGCX 的蛋白水解货物，将肿瘤分泌组信号与早期淋巴转移联系起来。我们现正使用该器官芯片系统研究单个 EV 相关蛋白酶对糖萼重塑的影响，以及靶向治疗药物在 TNBC 调理条件下保护 eGCX 完整性的潜力，目标是防止形成有利于转移的淋巴微环境。
查看英文原文 English abstract
Triple-negative breast cancer (TNBC) relies primarily on lymphatic vessels for early metastatic spread, and tumor-secreted factors-including extracellular vesicles (EVs)-can condition the lymphatic microenvironment to facilitate dissemination. The lymphatic endothelial glycocalyx (eGCX) is a sugar-rich barrier that regulates permeability and cell adhesion, yet its susceptibility to tumor-driven remodeling remains poorly understood.
Using a transwell coculture model of LECs and TNBCs, we found that TNBC-conditioned environments markedly disrupt the lymphatic eGCX. MDA-MB-231 conditioned media reduced wheat germ agglutinin (WGA, total glycocalyx) staining by 45.41 ± 9.37 percent, heparan sulfate (HS), one of the most abundant glycocalyx components, by 38.87 ± 8.84 percent, and overall eGCX thickness by 49.37 ± 3.93 percent. SUM-149 conditioned media similarly reduced glycocalyx thickness by 36.80 ± 5.11 percent. TNBC exposure also induced morphological changes in lymphatic endothelial cells (LECs) consistent with impaired barrier function and active glycocalyx remodeling.
To identify specific drivers of this disruption, EVs from TNBC and non-tumorigenic control cells were isolated using a membrane-affinity method and characterized by nanoparticle tracking analysis. LECs exposed to TNBC EVs exhibited reduced WGA and HS staining, and altered VE-cadherin junctions. Protease profiling revealed enrichment of ADAM9, Cathepsin X/Z/P, and MMP-8 in TNBC EVs, suggesting that EV-associated proteases mediate eGCX degradation.
To establish a physiologically relevant baseline, human LECs were cultured in collagen-filled PDMS microfluidic chips under luminal shear (~4 dynes/cm²). This 3D system produced a significantly thicker eGCX than 2D monolayers (WGA staining intensity in 3D relative to 2D: 1.311 ± 0.096; p ≤ 0.01), validating its use for assessing tumor- and EV-induced glycocalyx remodeling. This 3D lymphatic organ-on-chip model provides a physiologically relevant platform to study eGCX dynamics and EV-mediated vascular remodeling.
Together, these findings demonstrate that TNBC-derived EVs carry proteolytic cargo capable of degrading the lymphatic eGCX, linking tumor secretome signaling to early lymphatic metastasis. We are now using this organ-on-chip system to investigate the effects of individual EV-associated proteases on glycocalyx remodeling, as well as the potential of targeted therapeutics to preserve eGCX integrity under TNBC conditioning, with the goal of preventing the creation of a metastatic-permissive lymphatic microenvironment.
利益披露 Disclosure
J. Lau,
Merck Employment.
SonoThera Employment.
I. Cano, None..
E. Lee, None.