[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.目的： 探讨诱导性多能干细胞（iPS）来源的抗间皮素（MSLN）-嵌合抗原受体自然杀伤（CAR-NK）细胞（即抗MSLN-iCAR-NK细胞）对卵巢上皮性癌（卵巢癌）细胞的杀伤作用。 方法： 收集2020年9月至2021年9月就诊于河南省人民医院行手术治疗的20例卵巢癌患者的癌组织，并收集20例同期因其他良性疾病行手术切除的正常卵巢组织。（1）采用免疫组化和免疫荧光法检测卵巢癌组织中MSLN蛋白的表达。（2）对新鲜卵巢癌组织进行原代卵巢癌细胞的提取和培养。构建抗MSLN-CAR-CD244重组慢病毒载体，并与iPS混匀进行扩增培养，获得抗MSLN-iCAR细胞，使用细胞因子诱导分化法分化为抗MSLN-iCAR-NK细胞。细胞实验分为3组，抗MSLN-iCAR-NK细胞组、自然杀伤（NK）细胞组和对照组。（3）采用流式细胞仪、活细胞染色实验检测3组卵巢癌细胞的凋亡情况。（4）采用酶联免疫吸附试验（ELISA）检测3组卵巢癌细胞中γ干扰素（IFN-γ）、肿瘤坏死因子α（TNF-α）、颗粒酶B（GZMB）、穿孔素1（PRF1）、白细胞介素（IL）6、IL-10的表达水平。 结果： （1）免疫组化法检测显示，卵巢癌组织中MSLN蛋白的阳性表达率为65%（13/20），正常卵巢组织为30%（6/20），两者比较，差异有统计学意义（χ2=4.912，P=0.027）。免疫荧光法检测显示，卵巢癌组织中MSLN蛋白的阳性表达率为70%（14/20），正常卵巢组织为30%（6/20），两者比较，差异有统计学意义（χ2=6.400，P=0.011）。（2）流式细胞仪检测显示，抗MSLN-iCAR-NK细胞组卵巢癌细胞的凋亡率为（29.27±0.85）%，NK细胞组、对照组分别为（8.44±0.34）%、（6.83±0.26）%，3组间两两比较，差异均有统计学意义（P均<0.01）。活细胞染色实验结果显示，抗MSLN-iCAR-NK细胞组卵巢癌细胞的死亡细胞数/活细胞数为（36.3±8.3）%，NK细胞组和对照组分别为（5.4±1.4）%、（2.0±1.3）%，3组间两两比较，差异均有统计学意义（P均<0.001）。（3）ELISA法检测显示，抗MSLN-iCAR-NK细胞组卵巢癌细胞中IFN-γ、TNF-α、GZMB、PRF1、IL-6、IL-10的表达水平均显著高于NK细胞组和对照组（P均<0.05）。 结论： 抗MSLN-iCAR-NK细胞对卵巢癌细胞具有较强的杀伤能力，有望成为卵巢癌细胞免疫治疗的新方法。.