肿瘤细胞治疗研究
英文原题：Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells.
这种spike-in探针增强的scRNA-seq工作流程能够稳健地检测和高分辨率地表征synNotch-CAR-T细胞动态，并为在各种临床背景下监测工程化免疫细胞提供了一个广泛适用的平台。
我们之前开发了用于胶质母细胞瘤的synthetic Notch（synNotch）-嵌合抗原受体（CAR）-T细胞，以提高CAR-T疗法的安全性和有效性。在该系统中，抗EphA2/IL13R 2-双CAR仅在识别肿瘤或脑特异性“启动”抗原EGFRvIII（称为E-SYNC细胞）或brevican（B-SYNC）时表达，其中E-SYNC目前正在进行I期临床评估（NCT06186401）。然而，在体内追踪和分析这些工程化细胞仍然具有挑战性，限制了对其活性和治疗潜力的理解。为弥补这一空白，我们开发了一种单细胞RNA测序（scRNA-seq）工作流程，并针对synNotch-CAR转录本设计了定制spike-in探针，从而能够同时检测工程化细胞并进行转录组分析。在体外，使用机器学习辅助分类器整合多个探针，以98.0%的特异性检测到78.2%的E-SYNC细胞和60.0%的B-SYNC细胞。在异种移植模型中，synNotch阳性细胞在脾、肺和脑中被检测到，其中在脑中观察到最高频率以及最强健的启动和激活。单细胞转录组分析揭示了组织特异性分化程序，包括细胞毒性、增殖、代谢活性以及获得组织驻留记忆表型，这些均受到环境线索和synNotch介导的抗原识别的共同塑造。总之，这种spike-in探针增强的scRNA-seq工作流程能够稳健检测并以高分辨率表征synNotch-CAR-T细胞动态，并提供了一个可广泛适用的平台，用于在多种临床背景下监测工程化免疫细胞。一句话总结：我们的spike-in探针增强单细胞RNA测序方法能够分析synNotch-CAR-T细胞的组织依赖性激活和转录状态，为工程化细胞疗法的体内追踪和转录组分析提供了一个稳健且可扩展的平台。
UNLABELLED: We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R 2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation ( NCT06186401 ). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro , integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. ONE SENTENCE SUMMARY: Our spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.
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