肿瘤细胞治疗研究
英文原题：DRP1 depletion protects NK cells from hypoxia-induced dysfunction.
DRP1 depletion protects NK cells from hypoxia-induced dysfunction.
这些发现表明，DRP1 失活可在缺氧条件下支持 NK 细胞功能，而代谢工程可能增强 CAR-NK 在实体瘤中的疗效。
**目的：**细胞治疗在实体瘤中的疗效不理想，其中一个主要障碍是肿瘤微环境中的缺氧。本研究考察缺氧对自然杀伤（NK）细胞功能的影响，并评估恢复其缺氧环境下活性的方法。**方法：**将未改造或 CAR-NK 细胞置于常氧（21% O₂）或缺氧（1% O₂）条件下，再进行实验检测。通过共聚焦显微镜评估线粒体含量和形态，通过流式细胞术检测膜电位和活性氧（ROS），并利用 RNA 测序分析整体转录变化。以肿瘤细胞系及患者来源癌症类器官评估细胞毒性，后者还进行了 RNA 测序表征。采用药理学方法或 CRISPR-Cas9 介导的敲除抑制 DRP1 功能。**结果：**缺氧降低 NK 细胞线粒体含量和膜电位，同时增加线粒体 ROS，并广泛改变应激反应通路的转录。NK 细胞细胞毒活性显著受损，CD70-CAR-IL-15 工程化也无法避免这一影响。药理学抑制 DRP1 可恢复线粒体含量及细胞毒功能。为验证 DRP1 的作用，研究构建了 CRISPR-Cas9 介导的 DRP1 敲除（KO）NK 细胞，发现其在缺氧下仍能维持线粒体负荷和膜电位；DRP1 KO CAR-NK 细胞在缺氧条件下对癌细胞系仍保留细胞毒活性。具有不同转录特征的患者来源微肿瘤模型，对 DRP1 野生型和 KO CAR-NK 细胞表现出不同应答。**结论：**DRP1 失活有助于 NK 细胞在缺氧环境中维持功能；代谢工程可能提高 CAR-NK 治疗实体瘤的疗效。
OBJECTIVES: The efficacy of cellular therapies has been disappointing in solid tumors. A major barrier that contributes to the low success rate, is hypoxia within the tumor microenvironment. In this study, we investigated the influence of hypoxia on natural killer (NK) cell function and to evaluated a strategy to restore their activity in hypoxia. METHODS: Unarmed or CAR NK cells were placed in normoxia (21% O 2 ) or hypoxia (1% O 2 ) prior to experimental readouts. Mitochondrial content and morphology were assessed by confocal microscopy, membrane potential and reactive oxygen species (ROS) by flow cytometry, and global transcriptional changes by RNA sequencing. Cytotoxicity was evaluated against tumor cell lines and patient-derived cancer organoids, which were characterized by RNA sequencing. DRP1 function was inhibited pharmacologically or through CRISPR-Cas9-mediated knockout. RESULTS: Hypoxia reduced NK cell mitochondrial content and membrane potential, while increasing mitochondrial ROS and inducing broad transcriptional changes in stress response pathways. Their cytotoxic activity was drastically impaired, which could not be prevented by CD70-CAR-IL-15 engineering. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, CRISPR-Cas9-mediated DRP1 knockout (KO) NK cells preserved mitochondrial load and membrane potential under hypoxia, and DRP1 KO CAR NK cells retained cytotoxic activity under hypoxic conditions against cancer cell lines. Patient microtumor models with distinct transcriptomic profiles exhibited divergent responses to DRP1 WT and DRP1 KO CAR NK cells. CONCLUSION: These findings indicate that DRP1 inactivation supports NK cell function in hypoxia and metabolic engineering may enhance CAR-NK efficacy in solid tumors.
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