The phasor approach to FLIM for Metabolic profiling and Mitometer for tracking phenotypic changes in mitochondria in cancer cells
Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA, Laboratory for Fluorescence Dynamics, Irvine, CA, USA
The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical extracellular matrix structure may be responsible for tumor cell aggressiveness and drug resistance. While it is it known that tumor cells stiffen the ECM as the tumor progression occurs, a direct relationship between ECM stiffness and altered metabolism has not been explicitly measured. Here we apply the phasor approach technique in fluorescence lifetime imaging microscopy (FLIM) as a novel method to measure metabolic alteration as a function of ECM mechanics. We imaged and compared triple-negative breast cancer (TNBC) cells to non-cancerous cells on various ECM stiffness. Our results show that TNBC exhibit a decreased fraction of bound NADH, (indicative of glycolysis,) with increasing substrate stiffness. All other cell lines showed little to no change in fraction bound NADH on the varying collagen densities. Dysregulation of mitochondrial motion may contribute to the fueling of bioenergy demands in metastatic cancer. To measure mitochondria motion and analyze their fusion and fission events, we developed a new algorithm called “mitometer” that is unbiased and allows for automated segmentation and tracking of mitochondria in live cell 2D and 3D time-lapse images. Mitometer shows that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, Mitometer shows that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with fractions of the reduced form of NADH, in its bound form, and features such as speed and displacement, compared to the negative relationships with features such as directionality and branching in both TNBC and ER/PR+ mitochondria, but not in normal breast epithelial mitochondria. Together, the automated segmentation and tracking algorithms and the innate user interface make Mitometer a broadly accessible tool.