Construction of self-driving anti-αFR CAR-engineered NK cells based on IFN-γ and TNF-α synergistically induced high expression of CXCL10

IF 4.8 2区医学 Q1 Biochemistry, Genetics and Molecular Biology Neoplasia Pub Date : 2024-10-03 DOI:10.1016/j.neo.2024.101065

Min He , Xiang Ao , Yu Yang , Yanmin Xu , Tao Liu , Luoquan Ao , Wei Guo , Wei Xing , Jing Xu , Cheng Qian , Jianhua Yu , Xiang Xu , Ping Yi

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Abstract

Introduction

Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells.

Objectives

To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer.

Methods

Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo.

Results

RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue.

Conclusion

This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.

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基于 IFN-γ 和 TNF-α 协同诱导 CXCL10 高表达的自驱动抗αFR CAR 工程 NK 细胞的构建。

导言卵巢癌是恶性程度最高的妇科肿瘤。先前的研究表明，针对叶酸受体α（αFR）的嵌合抗原受体（CAR）工程化 NK-92 细胞（NK-92-αFR-CAR）可以特异性地杀死αFR 阳性的卵巢癌细胞。然而，迁移障碍限制了CAR工程细胞的抗肿瘤效果：阐明 NK-92-αFR-CAR 细胞在杀伤卵巢癌细胞过程中诱导趋化因子 CXCL10 分泌的机制。推测NK-92-αFR-CAR-CXCR3A可以靶向αFR，并具有CXCL10的趋化性，可能对卵巢癌具有更强的杀伤作用：方法：研究TNF-α和IFN-γ联合刺激卵巢癌细胞强烈诱导CXCL10表达的机制。构建共同表达 CXCR3A 和 αFR-CAR 的第四代 NK-92-αFR-CAR-CXCR3A 细胞。评估 NK-92-αFR-CAR-CXCR3A 在体外和体内的杀伤和迁移效应：结果：首先，RNA 测序（RNA-seq）显示，与 NK-92-αFR-CAR 共同培养的卵巢癌细胞中趋化因子 CXCL10 的表达水平显著增加。其次，细胞因子刺激实验证实，NK-92-αFR-CAR 分泌的 IFN-γ 和 TNF-α 能协同诱导卵巢癌细胞中 CXCL10 的高表达。进一步的信号通路实验表明，IFN-γ和TNF-α增强了IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10信号轴的活化水平。细胞毒性实验表明，NK-92-αFR-CAR-CXCR3A细胞不仅能在体外高效杀死αFR阳性卵巢癌细胞，还能分泌IFN-γ和TNF-α。利用透孔试验检测到，NK-92-αFR-CAR-CXCR3A 的迁移率高于 NK-92-αFR-CAR 的迁移率。在不同的卵巢癌小鼠异种移植模型中，NK-92-αFR-CAR-CXCR3A能有效杀死肿瘤细胞，并增加对肿瘤组织的浸润：本研究证实，αFR-CAR-工程NK细胞分泌的IFN-γ和TNF-α可协同诱导卵巢癌细胞高表达CXCL10，并构建了可基于CXCL10突破迁移障碍的自驱动αFR-CAR-工程NK细胞，为卵巢癌的治疗提供了新的武器。

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来源期刊

Neoplasia 医学-肿瘤学

CiteScore

9.20

自引率

2.10%

发文量

审稿时长

26 days

期刊介绍： Neoplasia publishes the results of novel investigations in all areas of oncology research. The title Neoplasia was chosen to convey the journal’s breadth, which encompasses the traditional disciplines of cancer research as well as emerging fields and interdisciplinary investigations. Neoplasia is interested in studies describing new molecular and genetic findings relating to the neoplastic phenotype and in laboratory and clinical studies demonstrating creative applications of advances in the basic sciences to risk assessment, prognostic indications, detection, diagnosis, and treatment. In addition to regular Research Reports, Neoplasia also publishes Reviews and Meeting Reports. Neoplasia is committed to ensuring a thorough, fair, and rapid review and publication schedule to further its mission of serving both the scientific and clinical communities by disseminating important data and ideas in cancer research.