Associated anisotropy of intrinsic NAD(P)H for monitoring changes in the metabolic activities of breast cancer cells (4T1) in three-dimensional collagen matrix.

The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture. Recent studies, however, suggest that 2D cell culture promotes specific types of aberrant cell behaviors due to the growth on non-physiologically stiff surfaces and the lack of the tissue-based extracellular matrix. Here, we investigate the sensitivity of the two-photon (2P) rotational dynamics of the intrinsic reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, to changes in the metabolic state of the metastatic murine breast cancer cells (4T1) in 2D monolayer and three-dimensional (3D) collagen matrix cultures. Time-resolved 2P-associated anisotropy measurements reveal that the rotational dynamics of free and enzyme-bound NAD(P)H in 4T1 cells are correlated to changes in the metabolic state of 2D and 3D cell cultures. In addition to the type of cell culture, we also investigated the metabolic response of 4T1 cells to treatment with two metabolic inhibitors (MD1 and TPPBr). The statistical analyses of our results enabled us to identify which of the fitting parameters of the observed time-resolved associate anisotropy of cellular NAD(P)H were significantly sensitive to changes in the metabolic state of 4T1 cells. Using a black-box model, the population fractions of free and bound NAD(P)H were used to estimate the corresponding equilibrium constant and the standard Gibbs free energy changes that are associated with underlying metabolic pathways of 4T1 cells in 2D and 3D cultures. These rotational dynamics analyses are in agreement with the standard 2P-fluorescence lifetime imaging microscopy (FLIM) measurements on the same cell line, cell cultures, and metabolic inhibition. These studies represent an important step towards the development of a noninvasive, time-resolved associated anisotropy to complement 2P-FLIM in order to elucidate the underlying cellular metabolism and metabolic plasticity in more complex in vivo, tumor-like models using intrinsic NADH autofluorescence.