Mass spectrometry imaging for in-situ evaluating the lipid metabolic heterogeneity of 5-fluorouracil in HepG2 spheroids

Spatial lipidomics analysis of three-dimensional (3D) tumor cell spheroids enables in-situ investigation of metabolic reprogramming across distinct regions cellular regions within a single spheroid. However, it is still a challenge to study the effect of 5-fluorouracil (5-FU) with 3D HepG2 spheroid on the alteration of abundance and spatial distribution of endogenous lipid species. Herein, we systematically optimized the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) methodologies for extending spatial lipidomics to in-situ screening and visualizing lipid changes in 3D HepG2 spheroids treated with 5-FU at the microregional level. The embedding protocols in carboxymethyl cellulose (CMC), gelatin, and optimal cutting temperature (OCT) compound of 3D HepG2 spheroid were evaluated for MALDI MSI analysis with 10 μm spatial resolution, respectively. Subsequently, CMC embedding of 3D HepG2 spheroid completely preserved the 3D spatial structure and enabled the division into the core necrotic area, intermediate quiescent area, and outer proliferative area from the spatial lipidic ion spectra. Furthermore, we spatially screened and identified 35, 40, and 30 distinct lipid molecules associated with 5-FU treatment in the 3D HepG2 spheroid of necrotic, quiescent, and proliferative areas, respectively. The main dysregulated metabolic pathway was the biosynthesis of glycerophospholipid metabolism, providing the reliable evidence for the mechanism of 5-FU anticancer effect on hepatocellular carcinoma (HCC). In addition, the spatial lipidomic results demonstrated that PA, PC, PG, PI, and SM contents in 5-FU treated tumor spheroids were significantly changed and limited the formation of the central necrotic area. This innovative combination of MALDI MSI with 3D HepG2 spheroids can be conducive to our better understanding of the biological processes between 5-FU triggered lipid metabolism and HCC heterogeneous structure.