Abstract Background: Glioblastoma is one of the most aggressive brain cancers, marked by rapid progression and resistance to treatment. Its cellular heterogeneity and invasive nature make it particularly hard to manage. New tools are needed to better understand its biology and improve diagnostics and therapies. NADH Fluorescence Lifetime Imaging Microscopy (NADH-FLIM) is a promising technique that reveals metabolic states by measuring NADH fluorescence lifetime: shorter lifetimes indicate glycolysis, while longer lifetimes are linked to oxidative phosphorylation (OXPHOS). This enables the identification of metabolic heterogeneity in tumor tissues. Material and Methods: We applied NADH-FLIM to formalin-fixed paraffin-embedded (FFPE) glioblastoma sections to assess NADH lifetime distribution in key tumor structures recognized by pathologists: (1) palisading cells near necrosis, (2) microvascular proliferation, and (3) neuronal and perivascular satellitosis. Additionally, we analyzed tumor cells overexpressing FGFR3 or carrying the IDH1 mutation to study their specific metabolic profiles. Results: Each tumor structure showed a distinct NADH lifetime signature, reflecting different metabolic states. For example, microvascular proliferation displayed lifetimes consistent with OXPHOS, while the surrounding tumoral astrocytic areas showed profiles more consistent with glycolysis. FLIM also identified isolated tumor cells in regions appearing histologically normal, including areas of early invasion. In FGFR3-overexpressing and IDH1-mutant cells, FLIM highlighted unique metabolic behaviors associated with these genetic changes. Conclusion: NADH-FLIM enables precise mapping of metabolic heterogeneity in glioblastoma, offering insights beyond conventional histology. It can detect metabolically distinct tumor subpopulations, aiding in diagnosis and potentially guiding personalized therapies. This technique represents a significant step forward in understanding glioblastoma biology. Citation Format: Manuel Giacomarra, Mariangela Morelli, Gianmarco Ferri, Francesca Lessi, Sara Franceschi, Michele Menicagli, Francesco Pasqualetti, Anna Luisa Di Stefano, Orazio Santo. Santonocito, Chiara Maria. Mazzanti. Beyond conventional pathology: Adding an extra layer of insight with NADH-FLIM to reveal hidden metabolic profiles in glioblastoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr B003.
Giacomarra et al. (Mon,) studied this question.