Single-cell RNA sequencing profiles drug activity within spatially engineered 3D cultures

Spatial transcriptomics techniques have been widely used to map the spatial organisation of multiple cell types within tissue sections. Here, we used transient transfection to label cells with DNA barcodes that are compatible with multiplexed single-cell RNA sequencing in a method called single-cell transfection-enabled cell hashing (scTECH-seq). Cells were then formed into 3D spheroids in a layer-by-layer process, with the cells in each layer labelled with a unique sample barcode. Using this approach, we collected spatially resolved transcriptomics information from 3D spheroid cultures at the single-cell level. Abstract Spatial transcriptomic techniques provide a wealth of information useful in guiding drug development, while three-dimensional (3D) cell cultures have demonstrated power in accelerating drug approvals. However, techniques for robust spatial analysis of 3D cultures are limited. Here, we present a transfection-based method for constructing cellular spheroids through a layer-by-layer approach, in which DNA barcodes encode the spatial positioning of cells. Our technique facilitates multiplex single-cell RNA sequencing, providing spatial maps of gene expression and drug response, while correlative imaging reveals the locations of barcoded cell populations and quantifies local tissue elasticity. We show that model HeLa 3D spheroids display heterogeneous responses to drugs, which may arise through diffusion gradients of the drug, or from differences in metabolism, nutrient supply, and cellular stressors. The ability to create spatially encoded cellular assemblies may help to reveal spatial variation in gene expression within 3D culture models. Contents This dataset contains processed (demultiplexed) sequencing data as .RData files, the R code used to analyse the data, and raw flow cytometry and microscopy images used in the figures. Supporting information, raw sequencing data, and processed data as depicted in the figures are available elsewhere. Contacting the authors Author contact details are provided in the publication.