Single-cell metabolic profiling of human cytotoxic T cells

Abstract

Cellular metabolism regulates immune cell activation, differentiation and effector functions but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. Here, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using a high-dimensional antibody-based approach. We employed mass cytometry (CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naïve 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 imaging mass spectrometry (MIBI-TOF), we uncovered the spatial organization of metabolic programs, 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.

Spatially resolved 3D metabolomic profiling in tissues

“…The latter direction is to profile RNA or protein signatures of metabolites of interest using readily available multiplexed transcriptional and proteomic mapping technologies. Along these lines, a recent demonstration included antibody-based detection of protein markers for enzymes to study T cell biology using CyTOF and multiplexed ion beam imaging (50). Multiplex RNA profiling of metabolic targets by fluorescent in situ hybridization (FISH)-based technologies such as sequential FISH (51,52) and multiplexed error-robust FISH (53,54) should provide metabolic regulators in tissues.…”

“…4) L = (30,34,55,57,58,59,60,66,67,68,69,71,81,83,85,88,91,93,95,97,98,100,102,104,105,111,146,166,182,184,185,190,194,196,198,206,210,212,224,226,238,240,246,252,254,256, and 282). 5) R = (50,200,201,202,203,204,205,207,208,209,214,216,217,220,221,…”

Simultaneous single cell measurements of intranuclear proteins and gene expression

“…We note that one drawback of inCITE-seq and related approaches is the need for high-quality antibodies. Future studies can combine inCITEseq with metabolic labeling 50, [92][93][94] , antibodies targeting phosphorylated forms of TFs, joint RNA and chromatin accessibility profiles 95 combined with spatial inference 96 Immunohistochemistry of mouse hippocampus. Mice were euthanized 2 hours after PBS or KA administration and perfused through the left ventricle with 20mL of PBS before gently removing the entire CNS.…”

Single‐cell sequencing in translational cancer research and challenges to meet clinical diagnostic needs

“…The tremendous technological development in single‐cell sequencing of the past decade has yielded a broad toolbox to study many modalities in cancer, such as mRNA, 37,38,57,59 DNA alterations, 141,144,146‐149,154,175 immune cell composition of tumors, 85‐87,90,92,95 chromatin changes 116‐118,122 and metabolic effectors 97 in dissociated single‐cells or nuclei as well as within the context of diseased tissue 156‐164,167,172 (Figures 3 and 4). Mono‐, bi‐, or even‐multimodal approaches have within short time facilitated cancer research at unprecedented depth and gained invaluable information on tumor composition and classification, clonal evolution in cancer, disease progression and treatment response.…”

“…Very recently, the development of single‐cell metabolic regulome profiling (scMEP) made it possible to study the highly dynamic functions exerted by immune cells manifested in metabolomic alterations at spatial resolution, deciphering metabolic profiles of CD8+ T cells in the tumor microenvironment 97 . The ability to analyze immune cell migration into diseased tissue, which is tightly regulated by the cells' metabolism, holds great promise to understand immune cell‐mediated processes in the tumor following treatment.…”