EpCAM-CAR-NK细胞对EpCAM表达相关性靶向杀伤及体内抗肿瘤效应

EpCAM Expression-Associated Targeted Cytotoxicity and in Vivo Antitumor Effects of EpCAM-CAR-NK Cells

摘要: 目的：探究靶向上皮细胞黏附分子(epithelial cell adhesion molecule, EpCAM)的嵌合抗原受体修饰自然杀伤细胞(EpCAM-CAR-NK)对结直肠癌细胞体内外杀伤效应及其与EpCAM表达水平的关系。方法：采用流式细胞术检测HT-29、HT-115及HT-29-OE-EpCAM-2细胞的EpCAM表达水平，细胞计数法预测细胞增殖能力，CCK-8法评价不同效靶比下EpCAM-CAR-NK细胞和NK-92细胞的体外杀伤作用。分别建立HT-29及HT-29-OE-EpCAM-2细胞裸鼠皮下移植瘤模型，随机分为移植瘤对照组(PBS组)、NK-92组和EpCAM-CAR-NK组，每组5只。NK-92组及EpCAM-CAR-NK组经尾静脉输注相应细胞4.5 × 10⁶个/只，隔日1次，共3次；PBS组给予等体积无菌PBS。间隔2 d测量1次移植瘤体积，实验终点称取瘤质量；采用DiR荧光标记及活体成像观察效应细胞体内分布，HE染色观察主要脏器组织形态变化。结果：HT-29-OE-EpCAM-2、HT-29及HT-115细胞EpCAM阳性表达情况明显，组间差异均有统计学意义(P < 0.001)。HT-29-OE-EpCAM-2细胞增殖能力显著高于HT-29和HT-115细胞(P < 0.001)。随效靶比升高，EpCAM-CAR-NK细胞对3种结直肠癌细胞的杀伤率总体升高，且其杀伤作用强于NK-92细胞；其中对HT-29-OE-EpCAM-2细胞杀伤作用最强，对HT-29细胞次之，对HT-115细胞相对较弱。体内实验显示，EpCAM-CAR-NK组显著抑制HT-29及HT-29-OE-EpCAM-2移植瘤生长；HT-29模型中，EpCAM-CAR-NK组终点肿瘤体积低于PBS组和NK-92组(P < 0.01, P < 0.05)；HT-29-OE-EpCAM-2模型中，EpCAM-CAR-NK组终点肿瘤体积低于PBS组及NK-92组(P < 0.001)。两种模型中，EpCAM-CAR-NK组终点瘤质量均明显降低，且在EpCAM高表达模型中的抑瘤效应更为突出。荧光成像显示，EpCAM-CAR-NK细胞在肿瘤部位出现明显信号富集且主要脏器HE染色后未见明显治疗相关组织损伤。结论：EpCAM-CAR-NK细胞可通过靶向识别EpCAM增强对结直肠癌细胞的杀伤作用，并显著抑制裸鼠移植瘤生长，其抗肿瘤效应与靶细胞EpCAM表达水平相关。EpCAM-CAR-NK细胞具有一定的肿瘤归巢能力和良好的初步安全性，为EpCAM阳性结直肠癌的靶向细胞免疫治疗提供实验依据。

Abstract: Objective: To investigate the in vitro cytotoxic activity and in vivo antitumor efficacy of epithelial cell adhesion molecule (EpCAM) targeted chimeric antigen receptor-engineered natural killer cells (EpCAM-CAR-NK cells) against colorectal cancer cells, and to examine their association with EpCAM expression levels. Methods: EpCAM expression in HT-29, HT-115, and HT-29-OE-EpCAM-2 cells was determined by flow cytometry. Cell proliferative capacity was assessed by cell counting, and the in vitro cytotoxicity of EpCAM-CAR-NK cells and NK-92 cells at different effector-to-target ratios was evaluated using the Cell Counting Kit-8 assay. Subcutaneous xenograft models were established in nude mice using HT-29 and HT-29-OE-EpCAM-2 cells, respectively. Mice were randomly assigned to the xenograft control group (PBS group), NK-92 group, and EpCAM-CAR-NK group, with 5 mice per group. Mice in the NK-92 and EpCAM-CAR-NK groups received tail-vein infusions of the corresponding cells at a dose of 4.5 × 10⁶ cells per mouse every other day for a total of 3 administrations, whereas mice in the PBS group received an equal volume of sterile PBS. Tumor volume was measured every 2 days, and tumor weight was determined at the experimental endpoint. DiR fluorescence labeling combined with in vivo imaging was used to assess the biodistribution of effector cells, and hematoxylin and eosin staining was performed to examine histopathological changes in major organs. Results: EpCAM expression differed significantly among HT-29-OE-EpCAM-2, HT-29, and HT-115 cells, with a statistically significant difference among the groups (P < 0.001). HT-29-OE-EpCAM-2 cells exhibited significantly greater proliferative capacity than HT-29 and HT-115 cells (P < 0.001). With increasing effector-to-target ratios, the cytotoxicity of EpCAM-CAR-NK cells against three types of colorectal cancer cell lines increased overall and was greater than that of NK-92 cells. The strongest cytotoxic effect was observed against HT-29-OE-EpCAM-2 cells, followed by HT-29 cells, whereas the effect against HT-115 cells was relatively weak. In vivo, EpCAM-CAR-NK treatment significantly inhibited tumor growth in both HT-29 and HT-29-OE-EpCAM-2 xenograft models. In the HT-29 model, endpoint tumor volume was significantly lower in the EpCAM-CAR-NK group than in the PBS group and the NK-92 group (P < 0.01, P < 0.05). In the HT-29-OE-EpCAM-2 model, endpoint tumor volume was significantly lower in the EpCAM-CAR-NK group than in the PBS and NK-92 groups (P < 0.001). Endpoint tumor weight was also markedly reduced in the EpCAM-CAR-NK group in both models, with a more pronounced tumor-inhibitory effect in the EpCAM-high-expressing model. Fluorescence imaging demonstrated prominent accumulation of EpCAM-CAR-NK cells at tumor sites. No apparent treatment-related histopathological injury was observed in major organs after hematoxylin and eosin staining. Conclusion: EpCAM-CAR-NK cells enhance cytotoxicity against colorectal cancer cells through targeted recognition of EpCAM and significantly inhibit xenograft tumor growth in nude mice. Their antitumor activity is associated with EpCAM expression levels in target cells. EpCAM-CAR-NK cells exhibit tumor-homing potential and a favorable preliminary safety profile, providing experimental evidence for targeted cellular immunotherapy in EpCAM-positive colorectal cancer.