Laboratory of Biotechnology and Nanomedicine, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123, Trento, Italy.
Advanced Imaging Core Facility, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123, Trento, Italy.
J Biomed Sci. 2024 Oct 15;31(1):92. doi: 10.1186/s12929-024-01084-9.
Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications.
In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors.
Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies.
This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.
细胞外囊泡 (EVs) 被认为是细胞间通讯的天然载体，是细胞分泌的颗粒。通过 EV 功能化来捕获异质分子货物并靶向特定细胞群体的能力有望推动生物医学应用的发展。然而，获得的 EV 的效率、细胞暴露的受体对 EV 相互作用的贡献以及具有潜在高相对分子质量重组 mRNA 共享的功能货物释放的可预测性对于推进靶向治疗应用中的异源 EV 至关重要。
在这项工作中，我们选择了流行的 EV 标志物 CD81 作为跨膜向导，用于融合蛋白，融合蛋白的 C 端 GFP 报告器包含或不包含针对 HER2 受体的曲妥珠单抗轻链。我们进行了高内涵成像分析，以跟踪 EV-细胞相互作用，包括 HER2 表达受调控的同基因乳腺癌细胞。我们验证了携带阿霉素的重组 EV 在 EV 供体细胞处理后递送功能性货物。然后，我们使用 JIMT-1 细胞进行了体内研究，JIMT-1 细胞通常用作 HER2 耐药、曲妥珠单抗耐药模型，以检测植入肿瘤中超过 2000 个核苷酸长度的重组 mRNA。
融合蛋白参与了囊泡运输动力学，并根据其在 HEK293T 细胞中的表达水平在分泌的 EV 上积累。尽管存在 GFP，但分泌的 EV 群体仍然保留了 HER2 受体结合能力，并被用于跟踪 EV-细胞相互作用。在 HER2 阳性 (SK-BR-3) 或阴性 (MDA-MB-231) 乳腺癌细胞系之间的全局 EV 分布没有变化的时间范围内，同基因细胞中 HER2 的暴露显著影响了异源 EV 的趋向性，证明了抗 HER2 EV 的特异性，其代表了分泌的大量囊泡的约 20%。特异性相互作用与 MDA-MB-231 中过表达 HER2 的阿霉素-EV 的细胞杀伤活性的提高以及 SK-BR-3 中敲除 HER2 受体的毒性降低强烈相关，克服了游离药物的影响。有趣的是，融合蛋白对应的转录本以全长 mRNA 的形式存在于重组 EV 中，可以到达 JIMT-1 异种移植小鼠的原位乳腺肿瘤，提高了我们在组织活检中检测穿透性货物的敏感性。
这项研究强调了创建分泌异源 EV 平台的定量方面，并展示了 EV-细胞结合机制背后的单个受体-配体相互作用的局限性，这现在成为预测功能趋向性和设计新一代 EV 为基础的纳米载体的关键步骤。
