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.status: publishe

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.status: publishe

Clonal fitness inferred from time-series modelling of single-cell cancer genomes

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours