SPoRTS: a novel, automated workflow for spatiotemporal analysis of live ovarian cancer spheroids expressing ratiometric fluorescent biosensors
Matthew J Borrelli1,2, Bart Kolendowski1, Yudith Ramos Valdés1, Gabriel E DiMattia1,3,4, Trevor G Shepherd1,2,3,5.
1The Mary and John Knight Translational Ovarian Cancer Research Unit, Verspeeten Family Cancer Centre, London, ON, Canada; 2Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 3Oncology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 4Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 5Obstetrics & Gynaecology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada
Introduction
Formation of multicellular aggregates (spheroids) enhances the survival of epithelial ovarian cancer (EOC) cells during peritoneal metastasis, but knowledge of the underlying biology is limited. Furthermore, microenvironmental heterogeneity—innate to spheroids—complicates the analysis of bulk materials (i.e., protein lysates, RNA, etc.), which lack spatial information. To address this deficit, we developed SPoRTS (spatial profiling of ratiometric trends in spheroids), an image analysis platform for spatiotemporal monitoring of fluorescent biosensor activity within live spheroids.
Methods
EOC spheroids expressing a ratiometric biosensor for mitosis were analyzed using SPoRTS, and the results were validated via comparison to the current gold standard for spatial analyses – immunostaining of spheroid sections. SPoRTS was then applied to spheroids expressing ratiometric biosensors for autophagy and mitophagy, and sections were subsequently stained for mitochondria and mitochondrial dynamics proteins.
Results
A depth-based proliferation gradient was identified using both analysis methods, confirming the capacity for automated spatial monitoring of biosensor activity in live spheroids. Autophagy and mitophagy were each found to exhibit a unique pattern of spatial regulation in EOC spheroids, ultimately leading us to discover region-dependent differences in mitochondrial organization.
Conclusions
Image analysis is a powerful approach for interrogating the biology of EOC spheroids, but applicable methods are scarce. SPoRTS, a novel image analysis tool, enables spatiotemporal monitoring of biosensor-reportable processes in live spheroids. Coupled with broader utilization of biosensor-expressing cancer cells, intracellular processes impacting spheroid biology and metastatic potential—and the influence of spheroids’ spatial heterogeneity on those processes—can now be systematically characterized in live spheroids (and, potentially, in other 3D models).
London Run for Ovarian Cancer. Natural Sciences and Engineering Research Council of Canada. Canadian Institutes of Health Research.