Analysis of Intracellular Fatty Acid Metabolism in Senescent Cells using Raman Microscopy

Cellular senescence is a permanent growth arrest triggered by stressors such as DNA damage, contributing both to tumor suppression and to age-associated dysfunction through the senescence-associated secretory phenotype (SASP). Lipid remodeling is a key but poorly understood feature of this state. Here, we apply hyperspectral Raman microscopy to visualize lipid accumulation and fatty acid metabolism in senescent human breast adenocarcinoma (MCF7) cells. Senescence was induced by Doxorubicin treatment and validated by morphology, p21 expression, and senescence-associated β-galactosidase activity. Raman imaging revealed progressive enrichment of CH ₂ stretching signals at 2850 cm ⁻¹ across 10–25 days post-treatment. Principal component analysis (PCA) distinguished proliferating and senescent cells with variance explained largely by lipid vibrational modes, including CH ₂ scissoring (1445 cm ⁻¹ ), C=C stretching (1655 cm ⁻¹ ), and CH ₂ stretching (2850 cm ⁻¹ ), indicating a marked compositional transition between 15 and 18 days of senescence. To probe functional lipid metabolism, we employed deuterated arachidonic acid (AA-d ₈ ), which displays unique (C=C)-D stretching peaks at 2220 and 2254 cm ⁻¹ within the silent Raman region. In vitro assays confirmed that cyclooxygenase-2 (COX2) metabolism of AA-d ₈ leads to a time-dependent reduction of these peaks relative to lipid signals. In senescent MCF7 cells, AA-d ₈ accumulated perinuclearly and showed heterogeneous distribution. Withdrawal of the COX2 inhibitor Cay-10404 resulted in a progressive decrease of the (C=C)-D peaks normalized to total lipids, consistent with intracellular arachidonic acid metabolism. Together, these results establish hyperspectral Raman microscopy as a label-free platform to resolve senescence-associated lipid remodeling and metabolic heterogeneity. By enabling spatial and temporal detection of fatty acid metabolism, this approach provides a powerful tool for dissecting senescence biology and supports future strategies to identify, monitor, and target senescent cells in disease and aging contexts.