Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers in vivo

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100–200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs [bEVs]; >200 nm, microvesicles) are less well explored. Here, we identify bEVs and sEVs as distinct EV populations, and determine that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence resonance energy transfer (BRET)-based EV reporter, reveals dose-dependent EV biodistribution at non-lethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. The bEVs and sEVs exhibit unique biodistribution profiles, yet individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass spectrometry (MS)-identified tumor progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include SLC29A1, CD9 and CD44. Remarkably, tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.

细胞外囊泡（Extracellular Vesicles, EVs）是细胞在病理与生理状态下释放、介导细胞间通讯的囊泡结构。目前小细胞外囊泡（sEVs；直径<100–200 nm，即外泌体）已得到广泛研究，但中大型细胞外囊泡（大细胞外囊泡[bEVs]；直径>200 nm，即微囊泡）的特性与功能尚未得到充分探索。本研究明确区分bEVs与sEVs为两类独立的EV亚群，并发现侵袭性人类三阴性乳腺癌（TNBC）亚型会以更高的bEV与sEV释放比例释放bEVs。基于生物发光共振能量转移（BRET）的EV报告探针PalmGRET，相较于亲脂性荧光染料，可在非致死性生理EV剂量下呈现出剂量依赖性的EV生物分布特征。bEVs与sEVs展现出独特的生物分布谱，但二者均可在同源免疫健全的TNBC乳腺癌小鼠模型中分别促进体内肿瘤生长。二者共有的经质谱（MS）鉴定的肿瘤进展相关EV表面膜蛋白（tpEVSurfMEMs）包含SLC29A1、CD9及CD44。值得注意的是，耗竭tpEVSurfMEMs可减弱EV的肺嗜性，改变其生物分布，并降低其促瘤活性。本研究阐明了乳腺癌中bEVs与sEVs各自独特的体内特性与功能，提示bEVs在疾病发生发展、诊断及治疗应用中具有重要价值。