Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Abstract

Whether cultured in vitro or part of a complex tissue in vivo, a cell's phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell's spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV 10 light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and singlecell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental 20 insights into the connections between tissue microenvironments and biological (dys)function.

Optophysiology: Illuminating cell physiology with optogenetics

“…Cell tagging has been used for spatiotemporally labeling cell types in organoids and isolated tumors with photocage compounds, such as near-UV light-responsive calcein nitroveratryloxycarbonyl caging dye. This photopharmacology method allows spatially resolved live cell tagging and isolation in a nondestructive manner using protected photoactivatable cell dyes ( 313 ). To tag cells dependent on their activities and study the causal relationship in the cell circuits with genetically encoded tools, a robust tool designated Fast light- and Ca 2+ -regulated expression (FLiCRE; FIGURE 7 ) has been developed to convert the transient activation signal (Ca 2+ influx) in neurons into a stably expressed transcript, such as a reporter gene ( 314 ).…”