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.

Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models.

Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018 International Society for Advancement of Cytometry.