Metabolic activities of murine breast cancer cells in tumor-like model using two-photon fluorescence lifetime and anisotropy imaging of intrinsic NAD(P)H

Cellular metabolism and behavior of cancer cells in two-dimensional (2D) cell cultures are believed to be different from that in vivo three-dimensional (3D) tumor. Here, we investigate the environmental effects of the metabolic state of murine breast cancer cells line (4T1) in 2D monolayer and 3D collagen matrix cultures using time-resolved two-photon (2P) fluorescence lifetime imaging (FLIM) and time-resolved 2P-depolarization of intrinsic NAD(P)H as a natural biomarker. 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 (OXPHOS), respectively, using 2P-FLIM of intracellular NAD(P)H in 2D and 3D cultures. Since NAD(P)H is a key coenzyme in a myriad of reduction-oxidation reactions, we propose a black-box model for monitoring the overall metabolic changes in 4T1 cancer cells using this intrinsic biomarker, which is sensitive to protein binding. Physical restriction and chemical sensing are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways.