biostudies-literatureGhislanzoni SItalian Ministry of Public HealthEuropean Union's Horizon 2020 research and innovation program2678https://www.ebi.ac.uk/biostudies/studies/S-EPMC12823619biostudies-literatureUnknown16(1)Despite advances in cancer therapies, treatment failure from resistance and recurrence remains a major clinical challenge. Therapy-induced senescence (TIS), a state of stable cell cycle arrest with sustained metabolic activity, has emerged as a driver of inflammation, tumor persistence, and relapse. However, the heterogeneity of TIS complicates its detection and targeting. Here, we applied a multi-modal strategy to characterize metabolic alterations in senescent cancer cells induced by doxorubicin or γ-irradiation across three tumor cell lines: MCF7, HeLa, and TPC-1. Mitochondrial dysfunction was assessed using MitoTracker and JC-1 staining, while two-photon excitation fluorescence (TPEF) microscopy enabled label-free visualization of intracellular NAD(P)H and FAD distribution. Lipid remodScientific reportsMetabolic profiling of therapy-induced senescent cancer cells via TPEF, MALDI-MS, and RNA-sequencing.PMC12823619101016923RF 2019-12370456Belfiore ANiero MBetti STorelli SBresci ABongarzone IBertolotti AGhislanzoni SPadelli FAgnelli LMasella APolli DfalseMetabolic profiling of therapy-induced senescent cancer cells via TPEF, MALDI-MS, and RNA-sequencing.Despite advances in cancer therapies, treatment failure from resistance and recurrence remains a major clinical challenge. Therapy-induced senescence (TIS), a state of stable cell cycle arrest with sustained metabolic activity, has emerged as a driver of inflammation, tumor persistence, and relapse. However, the heterogeneity of TIS complicates its detection and targeting. Here, we applied a multi-modal strategy to characterize metabolic alterations in senescent cancer cells induced by doxorubicin or γ-irradiation across three tumor cell lines: MCF7, HeLa, and TPC-1. Mitochondrial dysfunction was assessed using MitoTracker and JC-1 staining, while two-photon excitation fluorescence (TPEF) microscopy enabled label-free visualization of intracellular NAD(P)H and FAD distribution. Lipid remod2025-01-01T00:00:00Z2025 Dec2026-06-13T05:03:00.063Z2026-06-13T03:08:40.193ZS-EPMC128236194140810810.1038/s41598-025-32573-y