Investigation of the metabolic features of cancer therapy induced senescent cells with TPEF, mass spectrometry and RNA sequencing analysis

Background. Despite major advances in cancer therapies, treatment failure remains a significant issue for a subset of patients due to therapy resistance and tumour recurrence. A key contributor to these failures is therapy-induced senescence, wherein damaged cells enter a stable cell cycle arrest while remaining metabolically active, contributing to chronic inflammation and cancer relapse. These senescent cells can promote chronic inflammation and disease relapse. However, the heterogeneous nature of senescence complicates its detection and therapeutic targeting. Methods. We employed a multimodal approach to profile metabolic alterations in senescent cancer cells. Senescent cells were induced either by doxorubicin or ?-irradiation, both widely employed strategies in anti-cancer treatment, across three human cancer cell lines: MCF7, HeLa, and TPC-1. Mitochondrial dysfunction was assessed through MitoTracker and JC-1 staining. Two-photon excitation fluorescence microscopy enabled label-free imaging of mitochondrial coenzymes NAD(P)H and FAD. Lipidomic changes were analyzed using MALDI mass spectrometry imaging following lipid extraction and matrix application. Additionally, transcriptomic profiling via RNA sequencing was performed on control, senescent, and engulfing-senescent MCF7 cells. Bioinformatic analyses included differential gene expression, gene set enrichment analysis, and pathway analysis using GO, KEGG, and Reactome databases Results. Across all models, senescent cells exhibited mitochondrial dysfunction, marked by altered NAD(P)H and FAD distribution and decreased mitochondrial membrane potential. Two-photon excited fluorescence imaging confirmed a broader intracellular redistribution of mitochondrial coenzymes. MALDI analyses revealed consistent lipid remodeling, particularly involving cardiolipin precursors such as PG(18:1/18:1) and PG(18:1/22:6). Transcriptomic profiling of senescent MCF7 cells revealed that a previously observed engulfing subpopulation is marked by enhanced oxidative metabolism and increased lipid catabolism, suggestive of an engulfing phenotype. Conclusion. Our findings delineate both conserved and divergent metabolic features across senescent cell states and highlight specific metabolic vulnerabilities. These insights offer potential avenues for the development of targeted senolytic strategies. Overall design: To obtain the senescent phenotype, adherent cells were treated with 250 nM doxorubicin (Doxo, Sigma), or irradiated at 10 Gy released by ?-rays from 137Cs sources of IBL147 biological irradiator (0.65 Gy=min). Cells were observed and/or analyzed from 6 to 8 days after treatment. Control (untreated) cells, the proliferation of which was not arrested by treatment, were split when necessary to avoid apoptosis due to overgrowth until observation (typically once after seeding and before the end point), or alternatively collected prior to full confluence, typically 72 or 96 hours after seeding. To obtain a MCF7 population enriched in engulfing cells, MCF7 cells were treated with doxorubicin as described above and, after 6 days of treatment, they were collected through trypsinization and re-seeded. Adherent cells were collected 24 hours after being re-seeded.