Metabolic 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 remodeling was evaluated by MALDI mass spectrometry imaging, and RNA sequencing was performed on control, senescent, and engulfing-senescent MCF7 cells to identify differentially expressed genes and enriched pathways. Senescent cells displayed mitochondrial dysfunction, with altered NAD(P)H/FAD distribution and decreased membrane potential. TPEF confirmed redistribution of coenzymes, reflecting redox changes. Lipidomics revealed consistent remodeling, notably involving cardiolipin precursors. Transcriptomic profiling showed engulfing-senescent MCF7 cells possess a distinct signature marked by increased lipid metabolism, endocrine signaling, and cell-cell communication. Overall, our findings reveal conserved and cell type-specific metabolic traits of TIS, highlighting metabolic vulnerabilities for senolytic intervention.