PO.CL05.02 · 临床研究
线粒体重塑维持CAR-NK干性以实现卓越的肿瘤控制
Mitochondrial remodeling sustains CAR-NK stemness for superior tumor control
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：基于NK细胞的疗法是一种有前景的现货型癌症治疗策略。其固有的安全性以及无移植物抗宿主病使NK细胞成为CAR工程化的一个有吸引力的平台，将天然细胞毒性与受体特异性相结合。然而，在实体瘤中的临床疗效受限于持久性差以及在肿瘤微环境（TME）中的快速代谢耗竭。增强代谢适应性和延缓分化的策略至关重要。受干细胞培养系统的启发，我们测试了白蛋白-聚乙烯醇-必需脂质（APEL，一种用于支持iPSC分化为胚状体和NK细胞的确定成分培养基）是否能保持CAR-NK干性并改善代谢适应性和功能。
方法：将用APEL扩增的CAR-NK细胞（CAR-NK stem）与在常规NK细胞培养基中扩增的CAR-NK细胞（CAR-NK conv）进行比较。代谢分析包括线粒体质量和膜电位分析、用于测量糖酵解和氧化磷酸化的Seahorse检测、以及评估代谢灵活性的营养竞争检测。CAR-NK细胞与多种血液学和实体肿瘤细胞系共培养，随后使用2D xCelligence和3D Incucyte进行细胞毒性评估，包括用于评估连续杀伤能力的再攻击检测。体内疗效使用多种血液学癌症（MOLM14、MM1s）和实体瘤（786-0，进行中）异种移植小鼠模型进行测试。机制研究包括通过bulk RNA-seq和ATAC-seq进行的多组学分析、CyTOF以及线粒体转移检测。
结果：我们的数据显示，CAR-NK stem在扩增期间的早期代谢重编程驱动了增大且高度活跃的线粒体的形成、增加的备用呼吸能力（SRC）以及卓越的代谢灵活性。这种代谢状态稳定了干细胞样特征，并通过染色质重塑向持久性和记忆相关程序转变而限制了终末分化。在功能上，CAR-NK stem细胞表现出改善的增殖、持续的连续杀伤、卓越的营养竞争以及在体外和体内增强的肿瘤控制。在机制上，来自CAR-NK stem细胞的线粒体转移增强了受体NK细胞的细胞毒性和持久性，凸显线粒体重塑是功能适应性的关键驱动因素。
结论：这些发现表明，制造期间有针对性的代谢重编程可生成干细胞样、高度持久的CAR-NK细胞，并为癌症免疫治疗提供了一种广泛适用、可即时转化的策略。
查看英文原文 English abstract
Background: NK cell-based therapies are a promising off-the-shelf strategy for cancer treatment. Their inherent safety and lack of graft-versus-host disease make NK cells an attractive platform for CAR engineering, combining innate cytotoxicity with receptor specificity. However, clinical efficacy in solid tumors is limited by poor persistence and rapid metabolic exhaustion in the tumor microenvironment (TME). Strategies that enhance metabolic fitness and delay differentiation are essential. Inspired by stem-cell culture systems, we tested whether Albumin Polyvinylalcohol Essential Lipids (APEL), a defined medium used to support iPSC differentiation into embryoid bodies and NK cells, could preserve CAR-NK stemness and improve metabolic fitness and function.
Methods: CAR-NK cells expanded with APEL (CAR-NK stem ) were compared with CAR-NK cells expanded in conventional NK cell media (CAR-NK conv ). Metabolic profiling included mitochondrial mass and potential profiling, Seahorse assays to measure glycolysis and oxidative phosphorylation, nutrient competition assays to assess metabolic flexibility. CAR-NK cells were cultured with various hematologic and solid tumor cell lines, followed by cytotoxicity evaluation using 2D xCelligence and 3D Incucyte including rechallenge assays to assess serial killing capacity. In vivo efficacy was tested using various xenograft mouse models of hematologic cancers (MOLM14, MM1s) and solid tumors (786-0 ongoing). Mechanistic studies included multiomics profiling by bulk RNA-seq and ATAC-seq, CyTOF, and mitochondrial-transfer assays.
Results: Our data show that early metabolic reprogramming of CAR-NK stem during expansion drove the development of enlarged and highly active mitochondria, increased spare respiratory capacity (SRC), and superior metabolic flexibility. This metabolic state stabilized stem-like features and limited terminal differentiation via chromatin remodeling toward persistence and memory-associated programs. Functionally, CAR-NK stem cells showed improved proliferation, sustained serial killing, superior nutrient competition, and enhanced tumor control in vitro and in vivo. Mechanistically, mitochondrial transfer from CAR-NK stem cells enhanced cytotoxicity and persistence in recipient NK cells, highlighting mitochondrial remodeling as a key driver of functional fitness.
Conclusion: These findings demonstrate that targeted metabolic reprogramming during manufacturing can generate stem-like, highly persistent CAR-NK cells and offer a broadly applicable, immediately translatable strategy for cancer immunotherapy.
利益披露 Disclosure
S. Tiberti, None..
M. Centomo, None.