众所周知，小细胞外囊泡（sEV）从癌细胞中释放出来，并通过与受体细胞的相互作用促进癌症进展。我们之前曾报道，表达αVβ3整合素（一种在侵袭性神经内分泌前列腺癌（NEPrCa）中上调的蛋白质）的sEV有助于受体细胞中的神经内分泌分化（NED）。在这里，我们研究了 αVβ3 表达对 sEV 蛋白含量、密度和功能的影响。本研究中使用的 sEV 通过碘克沙醇密度梯度进行分离，并通过纳米颗粒跟踪分析、免疫印迹和单囊泡分析进行表征。我们对含有 αVβ3 的 sEV 的蛋白质组学谱显示，与对照 sEV 相比，参与细胞凋亡和坏死的典型效应子下调，肿瘤细胞存活因子上调。我们还表明，sEV 中 αVβ3 的表达导致 EV 标记（Alix、CD81、CD9）明显重新定位到低密度 sEV 亚群。这种低密度重新定位与细胞外基质 (ECM) 蛋白与 sEV 的相互作用无关。该 sEV 子集包含 αVβ3 和 αVβ3 下游效应子 NgR2，NgR2 是 NEPrCa 的新型标记物。我们发现，与不表达 αVβ3 的 sEV 相比，含有 αVβ3 的 sEV 负载有更高量的 NgR2。从机制上讲，我们证明含有 NgR2 的 sEV 不会影响 sEV 标记物谱，但当体内瘤内注射时，它们会促进肿瘤生长并诱导 NED。我们发现表达 NgR2 的 sEV 会增加受体细胞中粘附斑激酶 (FAK) 的激活，FAK 是已知的癌细胞增殖促进剂。我们还表明，NgR2 模拟了含有 αVβ3 的 sEV 的作用，因为与对照细胞相比，NgR2 转染子在体内表现出生长增加。总体而言，我们的结果描述了含有 αVβ3 整合素及其效应子 NgR2 的癌细胞来源的 sEV 的货物、密度和功能发生的变化，而不影响 sEV 四跨膜蛋白谱。© 2024 作者。 《Journal of Extracellular Vesicles》由 Wiley periodicals LLC 代表国际细胞外囊泡学会出版。

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.