Single cell mass spectrometry analysis of drug-resistant cancer cells: Metabolomics studies of synergetic effect of combinational treatment.

Irinotecan (IRI), a topoisomerase I inhibitor blocking DNA synthesis, is a widely used chemotherapy drug for metastatic colorectal cancer. Despite being an effective chemotherapy drug, its clinical effectiveness is limited by both intrinsic and acquired drug resistance. Previous studies indicate IRI induces cancer stemness in irinotecan-resistant (IRI-resistant) cells. Metformin, an oral antidiabetic drug, was recently reported for anticancer effects, likely due to its selective killing of cancer stem cells (CSCs). Given IRI-resistant cells exhibiting high cancer stemness, we hypothesize metformin can sensitize IRI-resistant cells and rescue the therapeutic effect. In this work, we utilized the Single-probe mass spectrometry technique to analyze live IRI-resistant cells under different treatment conditions. We discovered that metformin treatment was associated with the downregulation of lipids and fatty acids, potentially through the inhibition of fatty acid synthase (FASN). Importantly, certain species can be only detected from cells in their living status. The level of synergistic effect of metformin and IRI in their co-treatment of IRI-resistant cells was evaluated using Chou-Talalay combinational index. Using enzymatic activity assay, we determined that the co-treatment exhibit the highest FASN inhibition compared with the mono-treatment of IRI or metformin. To our knowledge, this is the first single-cell MS metabolomics study demonstrating metformin-IRI synergistic effect overcoming drug resistance in IRI-resistant cells.