Analysis of Intracellular Fatty Acid Metabolism during Doxorubicin-Induced Senescence of MCF7 Cells Using Raman Imaging

Cellular senescence, a stable growth-arrested state induced by stress or chemotherapeutic agents, is accompanied by metabolic remodeling that supports the senescence-associated secretory phenotype (SASP). Among these pathways, lipid and arachidonic acid (AA) metabolism play central roles in maintaining and propagating the senescent state. Here, we used hyperspectral confocal Raman microscopy to visualize biochemical remodeling in MCF7 human breast adenocarcinoma cells undergoing doxorubicin-induced senescence. Raman spectral analysis and principal component decomposition revealed time-dependent alterations in lipid-associated vibrational modes, particularly CH₂ and CC stretching, consistent with enhanced lipid accumulation and remodeling between Days 10 and 15 after DNA damage induction. PCA of lipid-rich compartments isolated by using true component analysis also confirms progressive increases in triacylglycerol and unsaturated lipid signatures. Using deuterated arachidonic acid (AA-<i>d</i> ₈) and COX2 inhibition, we further demonstrated real-time intracellular AA metabolism by tracking (CC)D stretching peaks (2220-2254 cm^-1) in the Raman-silent window. The ratio of these deuterium bands to CH₂ stretching provided a label-free quantitative metric for COX2-dependent AA turnover in senescent cells. Together, these findings establish hyperspectral Raman imaging as a powerful, nonperturbative tool to map lipid and oxylipin metabolism during cellular senescence, offering new avenues to identify metabolic vulnerabilities in senescent tumor cells.