Single-Cell Mass Spectrometry Studies of Secondary Drug Resistance of Tumor Cells.

Patients with epidermal growth factor receptor mutant nonsmall cell lung cancer (NSCLC) often fail to treat gefitinib because of secondary drug resistance. The development of tumor drug resistance is closely related to variations in cancer cell metabolism. Single-cell metabolomics analysis can provide unique information about tumor drug resistance. Herein, we constructed a platform to study the secondary resistance of tumor cells based on single-cell metabolomics (sSRTC-scM). A gefitinib-resistant NSCLC cell line (PC9GR) was constructed by increasing the dose step by step. The metabolic profiles of parental PC9 cells and PC9GR cells with different drug resistance levels were detected by intact living-cell electrolaunching ionization mass spectrometry at the single-cell level. The data were analyzed by statistical methods such as t-SNE, variance, volcano plot, heat map, and metabolic pathway analysis. Using this platform, we found that the metabolic fingerprints of PC9GR cells can evaluate drug resistance degrees. The metabolic fingerprints continue to be altered with the increase of drug resistance. We revealed 19 metabolic markers of secondary resistance by variance analysis and clarified that the glycerophospholipid metabolic pathway of PC9GR cells changed significantly. In addition, we found that with the increase in drug resistance levels, the heterogeneity of single-cell metabolism became greater and the number of cells with weak drug resistance gradually decreased. This phenomenon can be utilized to illustrate the drug resistance degrees of PC9GR cells. This study provides diagnostic markers for evaluating the drug resistance of tumors and gives new insight into overcoming the secondary resistance of tumors.