腫瘤胞外體腔內載物客製化與空間功能解析平台之建立

Abstract

胞外體 (Extracellular vesicles, EVs) 因具備高度生物相容性與跨膜運輸能力，近年來被視為具潛力的新興藥物載體。尤其是腫瘤來源的胞外體具有自體靶向性，能優先回到其母源腫瘤細胞，因此被視為極具潛力的癌症載藥平台。然而，這些腫瘤來源胞外體同時攜帶多種致癌蛋白與致癌相關核酸，使其直接應用於治療時存在安全疑慮。此外，胞外體本身具有高度異質性，且其表面與腔內成分在分佈與功能上各具不同角色，進一步增加功能解析與生物醫學應用的挑戰。為解決此問題，本研究提出CLEAR (Cargo Luminal Elimination to Attenuate Risk) 方法，旨在選擇性清除囊泡內部載物，同時維持表面蛋白與膜結構完整性，並結合實驗室先前已建立之 eSimoa (EV single-molecule array) 超高靈敏胞外體蛋白定量與SWITCHER (SWITCHable EV Releaser) 胞外體子群分離平台，形成整合性的eBFR (EV Bimodal Functional Regulator) 分析功能系統。 經比較四種物理化學方法確立最佳CLEAR條件，包括凍融法 (freeze-thaw)、超音波 (sonication)、皂苷法 (saponin) 與電穿孔 (electroporation)，並以胰臟導管腺癌(Pancreatic ductal adenocarcinoma, PDAC) 來源的PANC-1細胞及其分泌之胞外體作為主要模型進行驗證。結果顯示200 V電穿孔具備最佳效能，可達到約80%的腔內載物清除效率，並保留約九成的腫瘤來源胞外體表面蛋白。CLEAR方法不會影響腫瘤胞外體對原癌細胞的特異靶向性 (保有原本90%的細胞攝取量)。有趣的是，進一步應用邏輯閘分析，發現腫瘤胞外體被癌細胞攝取可由其表面蛋白單獨驅動，腔內載物的存在與否並不影響其進入癌細胞的能力；而細胞增殖與遷移等現象則需同時具備表面與腔內載物，適用「AND 閘」規律，確認致癌現象需要內部載物的存在。為了進一步驗證腫瘤胞外體經過CLEAR處理後的成分改變，本研究透過蛋白質體 (proteomics) 與小RNA (miRNA) 定序分析，結果顯示CLEAR EV中44項致癌蛋白與32項致癌相關的小RNA顯著下降 (log2 fold change < −1)。 為了驗證eBFR在胞外體蛋白空間功能解析之應用，本研究建立一個獨立的胞外體模型：帶有SARS-CoV-2 Spike蛋白的腫瘤胞外體 (Spike EVs)。結果顯示，Spike僅分布於胞外體表面，表現ACE2 (angiotensin-converting enzyme 2) 受體的細胞可透過特異性結合內化Spike EVs；CLEAR處理後仍能維持此表面功能。相對地，腔內致癌蛋白則主要驅動細胞增生，突顯Spike EVs表面與腔內載物在功能上的空間分工。此外，本研究亦觀察到Spike EVs在進入ACE2細胞後，其表面Spike蛋白可再經由第二代胞外體的釋放傳遞至鄰近細胞。 最後，本研究進一步驗證經過CLEAR處理後的腫瘤胞外體於抗癌藥物吉西他濱 (GEM) 遞送之應用。結果顯示CLEAR 顯著提升藥物裝載量，又以「邊清除邊載藥」為最佳策略，可提升藥物裝載量達8430倍，並使胰臟癌細胞抑制效果較未經CLEAR處理的腫瘤胞外體顯著提升約37%，大幅提升抗癌治療效果。 綜合而言，本研究建立CLEAR以降低腫瘤胞外體腔內致癌載物所造成之安全疑慮，並提升其載藥能力，同時結合eSimoa與SWITCHER，形成可解析胞外體蛋白空間性的eBFR平台，未來可望用於開發更具安全性與標靶性的胞外體藥物遞送系統，並為胞外體生物功能研究與疾病診斷之生物標記開發奠定基礎。

Extracellular vesicles (EVs) have emerged as promising drug carriers due to their biocompatibility and ability to transport biomolecules across cellular barriers. Tumor-derived EVs exhibit intrinsic autologous homing that enables them to preferentially return to their parental cancer cells, making them attractive candidates for targeted drug delivery. However, their clinical application is limited by the presence of oncogenic luminal cargos, as well as by the intrinsic heterogeneity of EVs and the distinct functional roles played by their surface and intraluminal components. To address these challenges, this study introduces CLEAR (Cargo Luminal Elimination to Attenuate Risk), a method designed to selectively remove luminal cargos while preserving surface proteins and membrane integrity. CLEAR was further combined with two previously established platforms, namely eSimoa (EV single-molecule array) for ultrasensitive EV protein quantification and SWITCHER (SWITCHable EV Releaser) for the biocompatible isolation of EV subpopulations. Together, these technologies form an integrated analytical and functional system referred to as the EV Bimodal Functional Regulator (eBFR). A comparison of four physicochemical approaches, including freeze–thaw, sonication, saponin, and electroporation, was conducted to determine the optimal CLEAR condition. Using PANC-1 cells derived from pancreatic ductal adenocarcinoma (PDAC) and their secreted EVs as the primary model, electroporation at 200 V demonstrated the best performance, achieving approximately 80% removal of intraluminal cargos while preserving about 90% of the surface proteins on tumor-derived EVs. CLEAR-treated EVs retained approximately 90% of their cellular uptake capacity and maintained their preferential homing toward parental tumor cells. Logic-gate analysis further revealed that EV uptake is driven solely by surface proteins, whereas phenotypic outputs such as proliferation and migration require both surface and luminal factors, consistent with an AND-gate mechanism. Multi-omics analyses supported these findings, showing significant decreases in 44 oncogenic proteins and 32 cancer-associated miRNAs (log₂ fold change < −1) in CLEAR EVs. To validate the applicability of eBFR for spatially resolving EV protein functions, this study established an independent EV model displaying the SARS-CoV-2 Spike protein (Spike EVs). The results showed that Spike was localized exclusively on the EV surface, and ACE2-expressing cells internalized Spike EVs through specific receptor-mediated interactions; this surface-driven functionality was preserved after CLEAR treatment. In contrast, luminal oncogenic proteins primarily drove proliferative responses, highlighting the spatial division of function between the EV surface and its intraluminal cargos. In addition, this study observed that, after entering ACE2-positive cells, Spike EVs could re-release their surface Spike protein via secondary EVs, enabling propagation of Spike to neighboring cells. Finally, this study further evaluated the application of CLEAR-processed tumor-derived EVs for delivering the anticancer drug gemcitabine (GEM). CLEAR markedly increased drug-loading capacity, with the co-loading strategy providing the greatest enhancement, achieving up to an 8,430-fold increase in drug incorporation. Moreover, GEM-loaded CLEAR EVs enhanced the inhibition of pancreatic cancer cells by approximately 37% compared with untreated tumor EVs, substantially improving overall anticancer efficacy. Overall, this study establishes CLEAR as an effective strategy for reducing the safety concerns associated with oncogenic intraluminal cargos in tumor-derived extracellular vesicles while simultaneously enhancing their drug-loading capacity. By incorporating CLEAR with eSimoa and SWITCHER, we developed the eBFR platform, which enables spatially resolved analysis of EV-associated proteins. This integrated system provides a foundation for the future development of safer and more target-specific EV-based drug delivery systems and supports advances in EV functional biology and biomarker discovery for disease diagnosis.