肿瘤细胞治疗研究
英文原题：Ultrasonication-derived CAR-T vesicles preserve antigen-specific cytotoxic function.
Ultrasonication-derived CAR-T vesicles preserve antigen-specific cytotoxic function.
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超声衍生 CAR-AVs 在实体瘤模型中保留可检测的 CAR 展示并保留抗原相关的抗肿瘤活性。这些发现支持 CAR-AVs 作为 CAR-T 疗法有前景且可能可扩展的无细胞补充，同时强调需要进行颗粒分辨定量、剂量反应研究和体内验证。
嵌合抗原受体（CAR）T细胞疗法已彻底改变血液系统恶性肿瘤的治疗，但在实体瘤中仍基本无效，其中基质屏障和免疫抑制微环境限制了T细胞的浸润和功能。治疗性淋巴细胞的无细胞囊泡衍生物可能有助于克服这些局限，同时保留抗原特异性。
通过短暂超声处理后差速离心，从 CAR-T 细胞中制备了人工囊泡（CAR-AVs）。采用扫描电子显微镜、DiO 标记和重组 CD19-PE 的流式细胞术、实时阻抗分析以及凋亡相关基因的 qRT-PCR，在体外评估了其理化性质和功能特性。
扫描电子显微镜显示，CAR-AVs 为纳米级、膜包裹的囊泡，平均 SD 直径为 132 76 nm，T 细胞来源囊泡（T-AVs）为 149 63 nm。流式细胞术检测到 52.7 20.5% 的 CAR-AVs 表达 CAR，而 T-AVs 的背景染色为 8.3 6.1%，亲本 CAR-T 细胞中 CD19-PE 阳性率为 64.2 14.4%。在 15 g mL-1 的蛋白归一化剂量下，CAR-AVs 选择性抑制表达 CD19 的肿瘤细胞增殖，使 PC3M(CD19+) 培养物中的细胞指数降低约 26-27%，使 A431(CD19+) 培养物中的细胞指数降低 68%，而对抗原阴性对应细胞影响极小。T-AVs 仅诱导轻微的、非抗原依赖性抑制。在抗原阳性模型中，CAR-AV 处理上调了 MDM2、CDKN1A/p21、BBC3/PUMA 和 BAX，这与应激和凋亡相关信号通路的激活一致。
Artificial vesicles were generated from CAR-T cells (CAR-AVs) by brief ultrasonication followed by differential centrifugation. Their physicochemical and functional properties were evaluated in vitro using scanning electron microscopy, flow cytometry with DiO labeling and recombinant CD19-PE, real-time impedance analysis, and qRT-PCR of apoptosis-associated genes.
Scanning electron microscopy revealed nanoscale, membrane-enclosed vesicles with mean SD diameters of 132 76 nm for CAR-AVs and 149 63 nm for T cell-derived vesicles (T-AVs). Flow cytometry detected CAR expression on 52.7 20.5% of CAR-AVs, compared with 8.3 6.1% background staining in T-AVs and 64.2 14.4% CD19-PE-positive parental CAR-T cells. At a protein-normalized dose of 15 g mL-1, CAR-AVs selectively suppressed the proliferation of CD19-expressing tumor cells, reducing the cell index by approximately 26-27% in PC3M(CD19+) cultures and 68% in A431(CD19+) cultures, with minimal effects on antigen-negative counterparts. T-AVs induced only modest, antigen-independent suppression. In antigen-positive models, CAR-AV treatment upregulated MDM2, CDKN1A/p21, BBC3/PUMA , and BAX , consistent with activation of stress- and apoptosis-associated signaling pathways. DISCUSSION: Ultrasonication-derived CAR-AVs preserve detectable CAR display and retain antigen-associated antitumor activity in solid-tumor models. These findings support CAR-AVs as a promising and potentially scalable cell-free complement to CAR-T therapy, while highlighting the need for particle-resolved quantification, dose-response studies, and in vivo validation.
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