Mapping cellular fate: From metabolic dynamics to DNA damage responses

Cellular homeostasis is maintained through a tightly regulated interplay of metabolic activity, cell cycle progression, programmed cell death, and genomic integrity. Perturbation of these pathways is a hallmark of oncogenesis. Analysis of these processes not only elucidates mechanisms of disease but also informs on therapeutic strategies. Here we present an integrated experimental framework designed to interrogate metabolism, cell cycle regulation, apoptosis, and DNA damage responses (DDR) in both immortalized cell lines and human primary cells. To assess cellular metabolic dependencies, we employed the SCENITH approach [1], which uses puromycin incorporation as a proxy for translational activity and, by extension, ATP production. This method enables quantification of reliance on glycolysis versus oxidative phosphorylation under defined conditions. Using metabolic inhibitors; 2-deoxyglucose to block glycolysis and oligomycin to inhibit mitochondrial ATP synthase, we observed distinct metabolic profiles. CD8⁺ T cells exhibited pronounced dependence on oxidative phosphorylation, whereas HeLa cells maintained translational activity despite mitochondrial inhibition, indicating metabolic flexibility. Parallel to metabolic profiling, we implemented flow cytometric cell cycle analysis using FxCycle™ staining to determine DNA content and the proliferative state of cells. Cell lines, MOLT-4, were exposed to therapeutics with established antiproliferative activity. Cisplatin and palbociclib induced G₁-phase arrest, consistent with DNA crosslinking and CDK4/6 inhibition, respectively, while nocodazole, a microtubule disruptor, arrested cells in G₂/M. Apoptotic responses were concurrently evaluated via caspase 3 /DAPI staining, revealing cisplatin as the most potent inducer of early and late apoptosis compared to palbociclib and nocodazole. Finally, DDR was quantified by monitoring gammaH2AX and RAD51 foci formation following cisplatin treatment or exposure to irradiation. High-content imaging facilitated dose-response characterization and demonstrated that co-treatment with an ataxia-telangiectasia mutated (ATM) kinase inhibitor markedly amplified DNA damage accumulation, underscoring the therapeutic potential of DDR modulation. Collectively, these assays provide a customizable platform for dissecting cellular vulnerabilities and resilience under pharmacological stress. Such multidimensional profiling offers critical insights for precision oncology and immunotherapy, enabling the identification of context-specific targets and combinatorial strategies to overcome resistance. [1] Arguello 2020 https://pmc.ncbi.nlm.nih.gov/articles/PMC8407169/#ABS1