Metabolic-Response Assessment Of Murine Breast Cancer Cells In 2d And 3d Cultures Using Two-Photon Fluorescence Lifetime Imaging Microscopy Of Intrinsic Nad(P)H

Cellular metabolism and behaviors are believed to be significantly different in two-dimensional (2D) cultures from that in vivo. As a result, there is a rise in interests in applying noninvasive, quantitative imaging techniques on in vivo-like three-dimensional (3D) models. Here, we investigate the environmental effects on the metabolic state of murine breast cancer cells line (4T1) in 2D and collagen matrix (3D) cultures using two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) of intrinsic NAD(P)H. In addition, we utilize the same system to examine the metabolic responses of 4T1 cells to two novel compounds, MD1 and TPPBr, that target cellular metabolism by disrupting monocarboxylate transporters (MCTs) or oxidative phosphorylation (OXPHOS), respectively, in both 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in the two culture types, forming anastomosing multicellular networks and spherical acini in collagenous 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 compared to that in 2D, 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, suggesting a disruption of OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in MD1-treated 3D cultures 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 the metabolic pathways of cancer cells. Integrating 2P-FLIM 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.