Cong, Anh2021-10-132021-10-132019-08https://hdl.handle.net/11299/224924University of Minnesota M.S. thesis. August 2019. Major: Chemistry. Advisors: Ahmed Heikal, Jon Holy. 1 computer file (PDF); ix, 87 pages.The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture towards pathophysiological investigation, drug screenings, and cancer diagnostics. There is strong evidence, however, that suggests cellular behavior and metabolism in 2D cell culture is too simplistic of a model as compared with those in vivo tumor cells. In this project, we hypothesize that cancer cell metabolism and metabolic responses to external stimuli (e.g. drug treatments) are distinctly different in threedimensional (3D), tumor-like model as compared with that of the conventional 2D monolayer culture. To test this hypothesis, we employed two-photon (2P) fluorescence lifetime imaging microscopy (2P-FLIM) and time-resolved 2P-fluorescence anisotropy of the reduced nicotinamide adenine dinucleotide (NADH) in metastatic murine breast cancer cells 4T1. In addition, we investigated the cellular metabolic response of 4T1 cells in 2D monolayer and 3D collagen matrix cultures to drug treatment using two novel metabolic drugs, namely MD1 and TPPBr. Both 2P-FLIM and complementary time-resolved anisotropy approaches reveal significant differences between metabolic activities of 4T1 cells in 2D and 3D cultures. Our results suggest that these 4T1 cells in 3D culture adapt an oxidative shift but glycolysis dominances the metabolic state of 2D cells. In addition, 4T1 cells in 3D culture appear to adapt more quickly and exhibit enhanced metabolic activities in response to drug treatment. In contrast, 4T1 cells in 2D monolayer culture exhibit a mute response and are less sensitive to drug treatments. While the tumor-like 3D collagen matrix model may not be an exact replica of in vivo tumors, these studies represent a critical step towards the development of a fundamental understanding of cellular behaviors and metabolism in the more complex in vivo models. These studies would also help advance our understanding of how the cancer cell heterogeneity and microenvironmental conditions impact metabolism and metabolic plasticity in tumor growth and metastatic progression.enbreast cancercollagen matrixfluorescencemetabolismNADHtwo-photonThesis or Dissertation