Abstract: Objective To explore the spatial heterogeneity of tumor boundary tissue metabolism in hepatocellular carcinoma (HCC) patients using time-of-flight secondary ion mass spectrometry (TOF-SIMS), and to analyze the characteristics of key metabolites in spatial distribution. Methods After obtaining adjacent non-tumor tissue samples from HCC patients, the samples were snap-frozen and embedded. Serial tissue sections were prepared using frozen section technology, and spatial metabolomic analysis was performed on the tissue sections using TOF-SIMS. The mass spectrometry data were clustered and spatially mapped at the single-cell level using the SEAM algorithm. Results TOF-SIMS combined with the SEAM algorithm successfully achieved high-resolution spatial metabolomic analysis of HCC boundary tissue. The results showed significant metabolic heterogeneity between the intra-and extra-tumoral regions, as well as within the tumor parenchyma, which could be subdivided into two cell subpopulations with different metabolic characteristics. Differential metabolite analysis identified two key phosphate-related ions (m/z=62.97 and m/z=78.96). Quantitative spatial distribution studies revealed that cell populations with high phosphate metabolism (PM+) were significantly enriched in areas close to the tumor boundary. Conclusions There is significant spatial metabolic remodeling at the HCC tumor boundary, and the boundary enrichment characteristics of PM+ cells suggest that the mechanical force environment at the tumor boundary may regulate the metabolic state of cancer cells.

Key words: hepatocellular carcinoma, spatial metabolomics, TOF-SIMS, tumor boundary, spatial heterogeneity