Metabolic profiling of transferred mitochondria using FLIM intensity based image segmentation (FIBIS)

Bulk metabolic assays on cell populations have shown that mitochondria transfer trigger breast cancer cells turn towards oxidative phosphorylation (OXPHOS) in favor of proliferation, migration, and cell growth. Yet, the current methods lack single cell resolution of interaction between endogenous and exogenous mitochondria to help understand the consequence metabolic alterations and other cell fate decisions. The phasor approach to fluorescence lifetime imaging microscopy (FLIM) has been widely used to measure the free to bound fraction of reduced form of NADH to quantify metabolic changes in live cells but not at the single mitochondrial scale. Here, we developed the FIBIS algorithm as a robust approach to recognize mitochondria from NADH intensity and further analyze mitochondrial metabolic states to investigate mitochondria transfer. We demonstrate that the NADH autofluorescence perfectly localizes with Mito7-mRuby labeled mitochondria and is free from artifactual emission spectral overlaps. Our data indicates that there was a 40% and a 20% increased fraction of bound NADH after mitochondria transfer in MCF7 and MB231 cells, respectively. This suggests that there is an enhancement of OXPHOS for both breast cancer cells. This was in correlation with the results from the Seahorse XF analyzer where the oxygen consumption rate was significantly higher after uptake of isolated mitochondria from breast epithelial cells. We also used Mitometer to show that there were increasing numbers of mitochondria and branching which imply that the fusion of exogenous and endogenous mitochondria are highly adaptable and increase cellular respiration. In summary, the FIBIS enables single mitochondrial metabolic profiling and aids in understanding the effects of transferred mitochondria to recipient cells. Our results also indicate that transferred mitochondria in cancer cells enhances OXPHOS, increased branching, and mitochondrial numbers, thereby decreasing cell viability and increasing sensitivity to chemotherapy agents.