Cellular metabolism and cell behavior are believed to be significantly different in two-dimensional (2D) cultures from that in vivo. Here, we investigate the environmental effects of the metabolic state of murine breast cancer cells line (4T1) in 2D monolayer and three-dimensional (3D) collagen matrix cultures using integrated two-photon (2P) micro-spectroscopy (FLIM) of intrinsic NAD(P)H autofluorescence. In addition, we examined the metabolic responses to two novel compounds, MD1 and TPPBr, that target cellular metabolism by disrupting monocarboxylate transporters (MCTs) or oxidative phosphorylation, respectively, using 2P-FLIM of intracellular NAD(P)H in 2D and 3D cultures. Integrating nonlinear microscopy and spectroscopy of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models is a promising approach towards in-depth understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior.
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