嵌合抗原受体T细胞（CAR-T）已经在血液恶性肿瘤方面展现出了惊人的疗效，但在胶质母细胞瘤（GBM）等实体肿瘤方面并未取得同样的成功。因此需要高通量的功能筛选平台来评估CAR-T对实体肿瘤细胞的毒性。我们使用实时、无标记的细胞阻抗感知技术，评估以抗二十二烷四醇四磷酸酯（GD2）为靶点的CAR-T制品在2天和7天的体外培养中对GD2+病人来源的GBM干细胞的毒力。我们比较了两种基因转移模式下的CAR-T产品：逆转录病毒转导和无病毒CRISPR编辑，并运用终点流式细胞术、细胞因子分析和代谢组学数据来构建CAR-T细胞毒力的预测模型。结果显示，与逆转录病毒转导的CAR-T细胞相比，无病毒CRISPR编辑的CAR-T细胞在细胞溶解上更快， begingroup accompanied by增加的炎性细胞因子释放、CD8+ CAR-T细胞在共培养条件下的存在以及CAR-T细胞对3D GBM球体的浸润。计算分析表明，对于GBM干细胞的短期（2天）和长期（7天）CAR-T细胞毒力的最好预测是自噬体积和细胞体积的TB和底物削减，其中与肿瘤坏死因子α浓度的增加相关的谷氨酸、乳酸和甲酸浓度的减少最具有预测性。这些研究证实了阻抗感知作为用于实体肿瘤中CAR-T细胞毒力的高通量无标记生物学分析技术。版权所有©2023年国际细胞和基因治疗学学会。由Elsevier Inc.出版。保留所有权利。

Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells.We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency.Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells.These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.