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Spatiotemporal analysis of ratiometric biosensors in live multicellular spheroids using SPoRTS
Matthew J Borrelli1, Bart Kolendowski2, Gabriel E DiMattia3
1The Mary and John Knight Translational Ovarian Cancer Research Unit, Verspeeten Family Cancer Centre, London, ON N6A 5W9, Canada; Department of Anatomy and Cell Biology, Western University, London, ON N6A 5C1, Canada.
Abstract:
Here, we describe SPoRTS, an open-source workflow for high-throughput spatiotemporal image analysis of fluorescence-based ratiometric biosensors in living spheroids. To achieve this, we have implemented a fully automated algorithm for the acquisition of line intensity profile data, ultimately enabling semi-quantitative measurement of biosensor activity as a function of distance from the center of the spheroid. We demonstrate the functionality of SPoRTS via spatial analysis of live spheroids expressing a ratiometric biosensor based on the fluorescent, ubiquitin-based cell-cycle indicator (FUCCI) system, which identifies mitotic cells. We compare this FUCCI-based SPoRTS analysis with spatially quantified immunostaining for proliferation markers, finding that the results are strongly correlated.
