Fluorescence and Phosphorescence Lifetime Imaging Microscopy for Spatial Mapping of Tumor Behavior

Fluorescence intensity measurements in cellular microscopy have become a valuable tool for resolving cellular structures and quantifying protein dynamics. Furthermore, the fluorescence lifetime can reveal additional information about the local molecular environment because the lifetime of a fluorophore is sensitive to changes in excited state reactions such as dynamic quenching. The spatial resolution afforded by these photoluminescence-based methods also enables the interrogation of non-homogeneous phenomenon in complex biological systems. Here, we applied lifetime imaging methods to spatially map variations in tumor behavior. Therapeutic resistance is associated with tumor heterogeneity necessitating the development of techniques that can non-invasively identify distinct functional subpopulations. Thus, we studied glioblastoma heterogeneity using the lifetime of endogenously fluorescent NADH. With this label-free method, we identified a significant difference in the lifetime signature between tumor mass cells and stem-like tumor initiating cells. Furthermore, we were able to distinguish between the two subpopulations in a mouse xenograft model as well as monitor the transition in populations driven by culture conditions. In addition to cellular heterogeneity, in vivo nutrient and oxygen gradients can also drive phenotypic variations illustrating the need for accurate recapitulation of varying local environments. Thus, we developed a method to non-invasively monitor cellular respiration in real time. Using tumor spheroids as the 3D model, we characterized the oxygen gradient using phosphorescence lifetime imaging microscopy and found lower oxygen concentration at the center of the spheroid relative to the periphery.Finally, we analyzed changes in the fluorescence lifetime of a FRET biosensor to better understand the spatial regulation of Rac1 within a spheroid model. While the important regulatory roles of Rac1 in migration and in response to hypoxia have been studied separately in 2D, spheroid models contain both migrating cells along the surface and a hypoxic core. We found higher levels of Rac1 activation at the core relative to the surface of the spheroid, revealing how Rac1 may be spatially regulated within tumors in vivo.Overall, we have used changes in the molecular environment detected by lifetime imaging techniques to spatially map functional subpopulations of cells, oxygen concentration, and protein activation to gain insight on tumor behavior