Cold solvent washing enhances AFADESI-MSI spatial lipidomics and lipid metabolism profiling in pancreatic cancer.

In this study, we developed a cold solvent washing-enhanced AFADESI-MSI spatial lipidomics approach that improves detection sensitivity of tissue lipid metabolites while preserving molecular spatial distribution fidelity. Using brain tissue homogenate sections as benchmark samples, the solvent composition, washing temperature, and washing time were systematically optimized, with the optimal condition determined as methanol/water (8:2, v/v) treatment at -80 °C for 1 h. The optimized method yielded an average of more than 150 enhanced mass spectral peaks in mouse brain, kidney, and liver tissues, with significant signal enhancement. Molecular imaging of fine cerebellar structures (fiber tracts, ~60 μm) confirmed that lipid spatial fidelity was largely maintained. Clinical application to pancreatic cancer tissues revealed that PC and PE species were significantly upregulated in cancer tissues, with longer-chain PC species (e.g., PC 38:5, PC 40:7) showing preferential upregulation. Notably, PC 36:3 exhibited robust discriminatory power between cancer and distal margin tissues, demonstrating the broad tissue applicability of this method in revealing previously undetectable lipid spatial distributions and providing a novel analytical tool for lipid biomarker discovery in pancreatic cancer.