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

Cell phenotypes and functions are influenced by dynamic interactions with their microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function. Overall design: Tissue samples from lung tumor were sliced into ~1 mm sections using an acrylic tissue slicer (Braintree Scientific, Inc.). After sectioning, tissue was stained with calcein NVOC as described in “SPACECAT Staining” for at least 30 min prior to imaging and photoactivation. Tissue sections were then imaged and photoactivated as described in “SPACECAT Photoactivation of In Vitro Cell Lines,” using 12 s pulses of 405 nm light at 4X magnification. After photoactivating the lung tumor sections, samples were dissociated by mechanical and enzymatic methods using the gentleMACS Tissue Dissociation Kit (Miltenyi Biotec). Resultant cell suspensions were stained with calcein violet (ThermoFisher, 1:1,000 dilution in PBS0) as an orthogonal indicator of viability. For lung tumor sections, cells were sorted by gating on positive for both calcein violet (viability following dissociation) and calcein NVOC (presence in the region of interest). All experiments leveraged calcein NVOC stained without photoactivation negative control for determining proper gating. Tumor samples were sorted into 500 µL RPMI + 10% FBS and kept on ice for subsequent single-cell RNA-seq.