Imaging mass spectrometry (IMS) allows establishing with precision the topological distribution of hundreds of lipid species within a tissue section. In close collaboration with the University of the Basque Country, we demonstrated that the lipidome is highly cell type-specific, and capable of distinguishing between crucial biological processes as differentiation and malignization. Thus, analyzing human colon tissue, we identified a defined number of lipid species changing according to a quite simple mathematical expression (R2>0.8) and concomitant to colonocyte differentiation. Interestingly, most of these species fell all into two lipid families: phosphatidylinositols and ethanolamine plasmalogens. The lipid gradient was accompanied by a gradient in the expression of enzymes involved in the mobilization of these lipid species. To further investigate the underlying regulatory mechanisms, we sorted healthy and tumor patient-derived colonocytes according to their proliferation capacity. Next, we analyzed the lipidome and transcriptome of each subpopulation, containing approx. 15,000 cells, by IMS (n=5-8) and gene expression microarrays (n=4), respectively. Using a System Biology approach, we were able to describe the transcriptomic networks involved in the regulation of specific membrane lipid molecular species phenotype. Altogether, these results demonstrate that the spatially resolved molecular profiles will be key to describing in-depth pathological microenvironments, providing unique mechanistic information, particularly at the level of cell-to-cell interactions.