GSE196245
Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity
Intratumor heterogeneity is a major obstacle to effective cancer treatment. Current  methods to study intratumor heterogeneity using single-cell RNA sequencing (scRNAseq)  lack information on the spatial organization of cells. While state-of-the art spatial  transcriptomics methods capture the spatial distribution, they either lack single cell  resolution or have relatively low transcript counts. Here, we introduce spatially  annotated single cell sequencing, based on the previously developed functional single  cell sequencing (FUNseq) technique, to spatially profile tumor cells with deep scRNA-seq  and single cell resolution. Using our approach, we profiled cells located at different  distances from the center of a 2D epithelial cell mass. By profiling the cell patch in  concentric bands of varying width, we showed that cells at the outermost edge of the  patch responded strongest to their local microenvironment, behaved most invasively, and  activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to lowconfluence  areas. We inferred cell-cell communication networks and demonstrated that  cells in the outermost ~10 cell wide band, which we termed the invasive edge, induced  similar phenotypic plasticity in neighboring regions. Applying FUNseq to spatially annotate  and profile tumor cells enables deep characterization of tumor subpopulations, thereby  unraveling the mechanistic basis for intratumor heterogeneity. Overall design: scRNA-seq was performed in various regions at different distances from the center of a 2D epithelial tumor model using functional single-cell sequencing. Gene expression profiles in the center and at the edge of the tumor were analyzed to quantify the level of EMT in MCF10A cells. Two separate experimental setups were used: we either profiled the tumor model in concentric rings of equal width (i.e. center, middle and outer; approx. ~1000 um wide bands) or we profiled the invasive edge of the tumor model at a higher resolution (i.e. comparing the center with the outermost edge; ~10 cell wide band). Cells in the different regions are phototagged using photoactivatable Janelia Fluor 549 and photoactivatable Janelia Fluor 646 dyes, after which cells are sorted based on fluoresence and subjected to scRNA-seq. For each setup, 2 biological replicates are present.