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Abstract PurposeIn vitroassays are essential for studying cellular biology, but traditional monolayer cultures fail to replicate the complex three-dimensional (3D) interactions of cells in living organisms. 3D culture systems offer a more accurate reflection of the cellular microenvironment. However, 3D cultures require robust and unique methods of characterization. MethodsThe goal of this study was to create a 3D spheroid model using cancer cells and macrophages, and to demonstrate a custom image analysis program to assess structural and metabolic changes across spheroid microregions. ResultsStructural characterization shows that cells at the necrotic core show high normalized fluorescence intensities of CD206 (M2 macrophages), cellular apoptosis (cleaved caspase-3, CC3), and hypoxia (HIF-1α and HIF-2α) compared to the proliferative edge, which shows high normalized fluorescence intensities of CD80 (M1 macrophages) and cellular proliferation (Ki67). Metabolic characterization was performed using multiphoton microscopy and fluorescence lifetime imaging (FLIM). Results show that the mean NADH lifetime at the necrotic core (1.011 ± 0.086 ns) was lower than that at the proliferative edge (1.105 ± 0.077 ns). The opposite trend is shown in the A1/A2 ratio (necrotic core: 4.864 ± 0.753; proliferative edge: 4.250 ± 0.432). ConclusionOverall, the results of this study show that 3D multicellular spheroid models can provide a reliable solution for studying tumor biology, allowing for the evaluation of discrete changes across all spheroid microregions.