Wang Jiemin, Zhao Zhenguo, Yang Haopeng, Wang Ruixuan, Wang Shu, Yu Jiale, Wang Yujia, Liu Ruihua, Chen Yani, Liu Yueshi, Shi Kesong, Han Pengyong, Liu Miao, Miao Jing, Li Xiaoyang, Li Xiangnan, Yu Haiquan
State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot 010020, Inner Mongolia, China.
Department of Orthopaedics, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 100021, Beijing, China.
Theranostics. 2025 Jul 25;15(16):8377-8403. doi: 10.7150/thno.115860. eCollection 2025.
Melanoma remains a highly aggressive malignancy with limited effective therapies and frequent resistance to immune checkpoint blockade (ICB). Extracellular vesicles (EVs) represent a promising platform for RNA-based therapeutics, but their clinical translation is impeded by inefficient cargo loading and insufficient tumor-specific targeting. To address these limitations, we developed an engineered EV strategy integrating efficient miRNA packaging with tumor-targeting surface modifications to enhance therapeutic outcomes in melanoma. Engineered EVs (iEV-150) were generated by co-expressing miR-150-3p and Annexin A2 (ANXA2) in HEK293T cells, followed by surface modification with tumor-targeting iRGD peptides. Mechanistic insights were obtained using RNA sequencing, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and luciferase reporter assays. Ferroptosis induction was evaluated through lipid peroxidation analysis, mitochondrial membrane potential assays, and transmission electron microscopy (TEM). Therapeutic efficacy and biodistribution were assessed using subcutaneous and metastatic melanoma mouse models. Immune modulation was examined by analyzing CD8⁺ T cell activation via flow cytometry in co-cultures of patient-derived CD8⁺ T cells and melanoma cells treated with iEV-150. miR-150-3p was elevated in melanoma-derived EVs, and ANXA2 was identified as a key RNA-binding protein that selectively facilitated its loading into EVs. iEV-150 exhibited enhanced uptake by melanoma cells and improved tumor-specific accumulation . Mechanistically, iEV-150 suppressed NF2 expression, disrupted the NF2-LATS1 interaction, activated YAP signaling, and subsequently upregulated ferroptosis-related genes ACSL4 and CHAC1, thereby inducing ferroptosis through the NF2-Hippo-YAP axis. In addition to its direct anti-tumor effects, iEV-150 promoted CD8⁺ T cell infiltration and activation within the tumor microenvironment, and significantly enhanced the therapeutic efficacy of ICB in melanoma models. iEV-150 integrates ANXA2-mediated miRNA loading, tumor-specific targeting, ferroptosis induction, and immune microenvironment reprogramming. This engineered EV strategy provides an effective RNA-based therapeutic platform to overcome ICB resistance and enhance precision treatment in melanoma.
黑色素瘤仍然是一种高度侵袭性的恶性肿瘤，有效治疗方法有限，且对免疫检查点阻断（ICB）常常产生耐药性。细胞外囊泡（EVs）是基于RNA的治疗方法的一个有前景的平台，但其临床转化受到货物装载效率低下和肿瘤特异性靶向不足的阻碍。为了解决这些局限性，我们开发了一种工程化的EV策略，将高效的miRNA包装与肿瘤靶向表面修饰相结合，以提高黑色素瘤的治疗效果。通过在HEK293T细胞中共表达miR-150-3p和膜联蛋白A2（ANXA2）来生成工程化的EV（iEV-150），随后用肿瘤靶向性iRGD肽进行表面修饰。使用RNA测序、RNA免疫沉淀（RIP）、染色质免疫沉淀（ChIP）和荧光素酶报告基因检测获得了机制性见解。通过脂质过氧化分析、线粒体膜电位检测和透射电子显微镜（TEM）评估铁死亡诱导情况。使用皮下和转移性黑色素瘤小鼠模型评估治疗效果和生物分布。通过在患者来源的CD8⁺T细胞与用iEV-150处理的黑色素瘤细胞的共培养物中通过流式细胞术分析CD8⁺T细胞活化来检查免疫调节。miR-150-3p在黑色素瘤来源的EVs中升高，并且ANXA2被鉴定为一种关键的RNA结合蛋白，其选择性地促进其装载到EVs中。iEV-150表现出黑色素瘤细胞对其摄取增强以及肿瘤特异性积累改善。从机制上讲，iEV-150抑制NF2表达，破坏NF2-LATS1相互作用，激活YAP信号传导，随后上调铁死亡相关基因ACSL4和CHAC1，从而通过NF2-Hippo-YAP轴诱导铁死亡。除了其直接的抗肿瘤作用外，iEV-150还促进了肿瘤微环境中CD8⁺T细胞的浸润和活化，并显著增强了ICB在黑色素瘤模型中的治疗效果。iEV-150整合了ANXA2介导的miRNA装载、肿瘤特异性靶向、铁死亡诱导和免疫微环境重编程。这种工程化的EV策略提供了一个有效的基于RNA的治疗平台，以克服ICB耐药性并增强黑色素瘤的精准治疗。
