Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag

Abstract

The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8+ T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

Optical Cell Tagging for Spatially Resolved Single‐Cell RNA Sequencing

“…Staining with a streptavidin‐fluorophore conjugate enabled sorting and isolation of the cells for scRNA‐seq. Recently, the Shalek group developed a photocaged calcein, which could be trapped inside the cells and fluorescently activated by laser irradiation of specific cells [10] . Kasteren, Schumacher, et al.…”

“…Kasteren, Schumacher, et al. reported a photocaged tag–nanobody conjugate to label cell‐surface receptors, which upon photodecaging can differentiate the uncaged and caged cells with two different colors [11] . Most of these methods require pre‐installation of the photosensitive probes to cells in a nonselective manner, followed by photoactivation in a spatially resolved manner.…”