Abstract 3346: Modeling breast cancer extracellular vesicle-mediated degradation of the lymphatic glycocalyx using a 3D organ-on-chip platform

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. Citation Format: Justin Lau, Issahy Cano, Esak Lee. Modeling breast cancer extracellular vesicle-mediated degradation of the lymphatic glycocalyx using a 3D organ-on-chip platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3346.