Polarization\nof Macrophages in Tumor Microenvironment\nUsing High-Throughput Single-Cell Metabolomics

Macrophages consist of a heterogeneous population of\nfunctionally\ndistinct cells that participate in many physiological and pathological\nprocesses. They exhibit prominent plasticity by changing their different\nfunctional phenotypes represented by proinflammatory (M1) and anti-inflammatory\n(M2) in response to different environmental stimuli. Emerging evidence\nillustrates the importance of intracellular metabolic pathways in\nmacrophage polarizations and functions. In the tumor microenvironment\n(TME), macrophages tend to M2 polarization, which promotes tumor growth\nand leads to adverse physiological effects. Due to the lack of highly\nspecific antigens in M1 and M2 macrophages, significant challenges\npresent in isolating these subtypes from clinical samples or in vitro\ncoculture models of tumor-immune cells. In reverse, the single-cell\ntechnique provides the possibility to investigate the factors influencing\nmacrophage polarization in the TME. In this research, we employed\ninertial microfluidic chip-mass spectrometry (IMC-MS) to conduct single-cell\nmetabolomics analysis of macrophages polarized into the two major\nphenotypes, respectively, and 213 metabolites were identified in total.\nSubsequently, differential metabolites between macrophage phenotypes\nwere analyzed using volcano plots and binary logistic regression models.\nGlutamine was pinpointed as a key metabolite for the M1 and M2 phenotypes.\nExperimental results from both monoculture and coculture cell models\ndemonstrated that M1 polarization is more reliant on the presence\nof glutamine in the culture environment than M2 polarization. Glutamine\ndeficiency resulted in failed M1 polarization, while its absence had\na less pronounced effect on M2 polarization. Replenishing an appropriate\namount of glutamine during the intermediate stages of coculture models\nsignificantly enhanced the proportion of M1 polarization and suppressed\nthe growth of tumor cells. This research elucidated glutamine as a\nkey factor influencing macrophage polarization in the TME via single-cell\nmetabolomics based on IMC-MS, offering promising insights and targets\nfor tumor therapies.