biostudies-literatureKarrobi KU.S. Department of DefenseNational Science Foundation3624https://www.ebi.ac.uk/biostudies/studies/S-EPMC9984376biostudies-literatureUnknown13(1)Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylatScientific reportsFluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.PMC99843761830878W81XWH-15-1-0070Zaman MFerruzzi JRoblyer DTank AKarrobi KFuzail MAKalidoss MTilbury KfalseFluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylat2023-01-01T00:00:00Z2023 Mar2025-04-26T07:39:38.027Z2025-02-18T23:35:10.214ZS-EPMC99843763686909210.1038/s41598-023-30403-7