肿瘤细胞治疗研究
英文原题：Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening.
我们的研究凸显了基于 CRISPR 的基因筛选在探究肿瘤-CAR T 相互作用方面的能力，并鉴定出肿瘤细胞中可成药的潜在靶点，这些靶点赋予肿瘤细胞对 CAR T 细胞杀伤的耐药性。
背景：多形性胶质母细胞瘤（GBM）是一种高度侵袭性脑肿瘤，预后差。现有治疗选择有限且常疗效不佳。CAR-T细胞治疗血液系统恶性肿瘤已取得成功，人们也日益关注其用于实体瘤（包括GBM）的潜力。然而，由于肿瘤相关耐药机制和CAR-T细胞自身缺陷，目前CAR-T治疗GBM的临床疗效仍不足，因此亟需提高其疗效。 方法：我们在GBM细胞系U87 MG细胞中开展大规模CRISPR干扰（CRISPRi）筛选，并与靶向B7-H3的CAR-T细胞共培养，以识别能够增强CAR-T介导肿瘤杀伤的遗传调节因子。随后采用基于流式细胞术的肿瘤杀伤实验和CAR-T活化实验验证筛选结果。我们还分析整体及单细胞RNA测序数据和TCGA数据库，以阐明在U87 MG细胞中敲低所选筛选靶点后CAR-T疗效增强的机制。 结果：我们证实B7-H3是GBM CAR-T治疗的可靶向抗原。通过大规模CRISPRi筛选，发现GBM细胞中的ARPC4、PI4KA、ATP6V1A、UBA1和NDUFV1等遗传调节因子可影响CAR-T介导肿瘤杀伤的效能。此外，我们发现ARPC4或NDUFV1敲低的GBM细胞中TNFSF15均上调，并揭示TNFSF15可通过调节肿瘤与CAR-T细胞的相互作用发挥免疫刺激作用，从而增强CAR-T疗效。 结论：本研究凸显CRISPR遗传筛选研究肿瘤与CAR-T细胞相互作用的价值，并识别出可能使肿瘤细胞对CAR-T杀伤产生耐药的潜在可药物干预靶点。我们还设计出可与CAR-T治疗GBM协同作用的靶向策略。这些发现为开发治疗GBM及其他实体瘤的有效新型联合免疫疗法提供了线索。
BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. METHODS: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. RESULTS: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. CONCLUSIONS: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.
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