Tumor spheroids represent valuable tools for investigating avascular solid tumors under controlled experimental conditions. In this work we investigated alterations in cellular metabolism, lipid storage, and their interactions with the extracellular matrix (ECM) using various human breast adenocarcinoma cell lines and a non-malignant breast epithelial cell line as spheroids. Three imaging methodologies, were used. The Phasor FLIM, of NADH was used to analyze metabolic signatures. Hyperspectral imaging was used to image signatures from two orthogonal solvatochromic probes: ACDAN for assessing intracellular polarity and Nile Red for detecting lipid composition. SHG images were collected through multiphoton excitation and analyzed via PLICS (Phasor analysis of Local Image Correlation Spectroscopy) to assess the cellular microenvironment and collagen remodeling within breast cancer spheroids. Notable differences emerged in NADH and lipid signatures across cell lines, spheroids, and time. Invading MDA-MB231 cells exhibited elevated hydration and lipid droplets, suggesting adaptation for invasion. These methods shed light on metastatic mechanisms and potential therapies.
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