Translational impact of the study of the immune lipidome in patients with colon cancer: from blood to tumor infiltrates

eng Colon cancer is a leading cause of cancer-related mortality worldwide, with the tumor microenvironment (TME) significantly contributing to immune evasion and resistance to immunotherapy. Within the TME, lipids play critical roles in modulating immune cell functionality and tumor-immune interactions, presenting an opportunity to improve our understanding of these processes. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a powerful tool for spatially resolving lipid profiles in tumor sections, offering the potential to study the immune compartment within the TME at unprecedented resolution. The global aim of this study was to assess the translational impact of the lipidome of immune cells on colon cancer diagnosis and prognosis. In addition, the study sought to determine whether lipid profiles obtained under different contexts could be used to provide a comprehensive characterization of the TME of colon tumor sections imaging lipids via MALDI-IMS. However, the establishment of the lipid profiles of immune cells turned out very challenging, as the sample size was scarce. Consequently, it was first necessary to develop methodological advancements for analyzing sorted immune cells by utilizing MALDI-MS as a key analytical tool. The drop-overlay method improved sample confluence and sensitivity for MALDI-MS analysis, enabling reproducible lipid profiling of 104 immune cells. This methodological advancement allowed a comprehensive lipid profiling and facilitated the study of immune cells in both circulation and tumor contexts. After optimizing the protocol, we isolated immune cell populations from the peripheral blood of healthy donors to analyze their lipid profiles. Our goal was to determine whether distinct lipid compositions could reliably identify specific immune cell types. The lipid profiling revealed striking specificity in lipid composition among different immune cell types, reflecting their diverse functional roles. Next, we examined how immune cell activation affects lipid composition through a series of ex vivo experiments, aiming to determine whether cell-specific lipid profiles reflect functional adaptations to stimulation. Ex vivo activation experiments demonstrated significant lipid remodeling in T cells and macrophages, characterized by a consistent depletion in the relative content of arachidonic acid (AA)-containing phosphatidylethanolamine (AA-PE) and phosphatidylcholine (AA-PC) species, while the levels of monounsaturated fatty acid-containing (MUFA-containing) species increased. These changes were supported by upregulated expression of lipid-metabolizing enzymes, confirming that immune activation triggers a remodeling process consistent with the lipid changes upon activation to meet functional demands. To extend these findings to a clinical context, we analyzed the lipid profiles of peripheral blood immune cells from patients with colon cancer, focusing on the same cell types studied in healthy individuals. In these patients, circulating immune cells exhibited lipidomic changes similar to those observed during ex vivo activation, consistent with a tumor-driven systemic process of immune cell activation. Finally, we isolated tumor-infiltrating lymphocytes (T cells and B cells), tumor-associated neutrophils (TAN), and tumor-associated macrophages (TAM, specifically M1- and M2-like phenotypes) using FACS and establish their lipid profiles to evaluate whether these profiles could aid in identifying immune cell signatures in colon tumor sections imaged by MALDI-IMS. Tumor-infiltrating immune cells showed enhanced lipid remodeling compared to the circulating counterparts, with changes closely mirroring ex vivo activation findings. These results revealed how immune cells adapt metabolically within the TME, reflecting the combined influence of systemic and local environmental factors. Validation of immune cell lipid profiles using MALDI-IMS demonstrated strong alignment between the lipid profiles of isolated immune cells and segmentation clusters in tumor sections. Thus, the lipid profiles of tumor-infiltrating T cells matched those of clusters within regions of acute lymphocytic infiltration, confirming the fidelity of MALDI-IMS in capturing in situ immune-tumor composition. This consistency highlights the utility of MALDI-IMS for studying spatially resolved lipidomics and elucidating immune cell metabolic state within the TME. In conclusion, this work establishes the lipidome as a defining feature of immune cell identity and functionality, with lipid composition shaping cell-specific roles in health and disease. MALDI-IMS lipidomic analysis offers a powerful framework for studying the immune compartment within the TME, advancing our understanding of tumor-immune interactions, and guiding the development of precision immunotherapeutic strategies for colon cancer.