Preparation of multicellular spheroid sections embedded with sodium carboxymethyl cellulose for mass spectrometry imaging

Compared with organic analysis, mass spectrometry imaging (MSI)-based cellular localization and molecular information investigations are rapidly becoming crucial tools in tumor research, drug screening, and toxicity testing. Fresh-frozen sections are ideal for MSI because they offer much better integrity of biological information than formalin-fixed paraffin-embedded (FFPE) sections. However, the suitable sample preparation method, especially to ensure the acquisition of intact sections from cell samples, is critical and has significant effects on MSI application. Herein, we optimized the embedding approaches and developed a thin and transparent cryoplatform using 2% sodium carboxymethyl cellulose (CMC) to prepare high-quality sections of three-dimensional heterogeneous multicellular tumor spheroids (3D heterogeneous MCTSs) for morphological and MSI analyses. More specifically, four embedding approaches, including the use of 2% CMC, optimum cutting temperature embedding medium (OCT), polyvinylpyrrolidone-hydroxypropyl methylcellulose (PVP-HPMC), and CMC-sucrose, were systematically evaluated regarding sample integrity, lipid signal interference, and lipid distributions. Among these, the 2% CMC and PVP-HPMC embedding methods exhibited optimal performance characteristics for morphological and matrix-assisted laser desorption/ionization (MALDI)-MSI analyses. More importantly, these two methods accurately highlighted the heterogeneity of the MCTSs in terms of morphological staining, spatial segmentation, and spatial distribution of lipids, including the necrotic and proliferative micro-areas. This approach allowed assessments of the metabolite expressions at the inter-cellular level, showing that phosphatidylglycerol (PG) (34 : 2), PG (32 : 1), and phosphatidylcholine (PC) (34 : 1) were enriched in the proliferative areas, while phosphatidylinositol (PI) (38 : 5), phosphatidylserine (PS) (40 : 4), and PI (38 : 4) were predominantly located in the necrotic regions. In summary, our method for preparing cell samples has greatly broadened the application range of MSI detection and allows for the extraction of more biological and pathological information associated with tumor research.