Cellular metabolism regulates immune cell activation, differentiation and effector functions, but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. In this study, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, termed single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using high-dimensional antibody-based technologies. We employed mass cytometry (cytometry by time of flight, CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naive and memory CD8⁺ T cells. We applied the approach to clinical samples and identified tissue-restricted, metabolically repressed cytotoxic T cells in human colorectal carcinoma. Combining our method with multiplexed ion beam imaging by time of flight (MIBI-TOF), we uncovered the spatial organization of metabolic programs in human tissues, which indicated exclusion of metabolically repressed immune cells from the tumor–immune boundary. Overall, our approach enables robust approximation of metabolic and functional states in individual cells.

细胞代谢调节免疫细胞激活、分化和效应器功能，但目前的代谢方法缺乏单细胞分辨率和细胞表型的同步表征。在这项研究中，我们开发了一种方法来表征单细胞的代谢调节组及其表型特征。该方法被称为单细胞代谢调节组分析（scMEP），利用基于高维抗体的技术来量化调节代谢途径活性的蛋白质。我们采用质谱流式细胞术（飞行时间细胞术，CyTOF）通过重建体外激活的幼稚和记忆 CD8 ⁺ T 细胞的代谢重塑，将 scMEP 与大量代谢测定进行基准比较。我们将该方法应用于临床样本，并在人类结直肠癌中鉴定出组织限制性、代谢抑制的细胞毒性 T 细胞。将我们的方法与飞行时间多重离子束成像（MIBI-TOF）相结合，我们发现了人体组织中代谢程序的空间组织，这表明代谢抑制的免疫细胞被排除在肿瘤-免疫边界之外。总的来说，我们的方法能够对单个细胞的代谢和功能状态进行稳健的近似。