Mass spectrometry imaging revealed alterations of lipid metabolites in multicellular tumor spheroids in response to hydroxychloroquine

Highlights: • The localization and accumulation of HCQ in the inner region of A549 tumor spheroids were obtained by MALDI-MSI. • MALDI-MSI observed four lipid distribution patterns that were masked in LC/MS-MS analysis. • MALDI-MSI combined with LC-MS/MS provides detailed information about endogenous metabolites in response to anticancer drugs. Three-dimensional (3D) multicellular tumor spheroids (MCTS) that mimic the complex tumor microenvironment provide a good platform for in vitro study of drug and endogenous metabolites. Hydroxychloroquine (HCQ) has shown anti-tumor activity in a variety of tumor models. However, the effect of the drug on the alteration of lipid metabolism spatial composition and distribution in the MCTS model is not clear. Herein, we utilized matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in the analysis of A549 lung cancer multicellular spheroids to investigate the in situ spatial distribution of HCQ and its effect on lipid metabolism. We have successfully observed the spatial variations of HCQ in the inner region of the spheroid at different drug-treated time points. The MSI results also demonstrated that HCQ treatment altered the spatial composition of lipids in the inner and outer regions of treated spheroids. Furthermore, the lipidomic results showed that the identified phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE), phosphatidylinositols (PI), ceramides (Cer), glucosylceramides (CerG), and diglycerides (DG) were significantly up-regulated, and phosphatidylglycerol (PG) and triglycerides (TG) were remarkable down-regulated. MSI method combined with LC-MS/MS profiling of endogenous metabolites can obtain more detailed information about how spheroids respond to drug and spatial distribution information, thus fostering a better understanding of the relationship between drug-altered lipid metabolism and cancer microenvironment.