Selective single-cell transcriptomic analysis of targeted cells – Sel-scRNA-seq

Studying populations of cells at the single-cell level is essential to fully understand normal function and disease in humans, or any other multicellular organism. The ability to analyze transcriptomes by single-cell RNA sequencing (scRNA-seq) is revolutionizing cell biology. For, example, in cancer immunotherapy, it is critical for scientists and clinicians to profile infiltrating immune cells, and especially tumor infiltrating lymphocytes (TILS), at the single-cell-level, in the complex tumor microenvironment. The past decade has seen a rapid evolution in scRNA-seq (and other single-cell “omics” systems) and the appearance of successful commercial systems, notably based on droplet microfluidics, such as the 10X Genomics system. However, there is currently no integrated technology that allows selective single-cell RNA-seq of a (sometimes rare) sub-population of cells, such as TILS. Currently, rare cells must first be pre-enriched using techniques such as fluorescence-activated cell sorting (FACS) or using magnetic beads, which are poorly adapted to precious samples containing small numbers of cells, such a tumor biopies.
The Sel-scRNA-seq project aims to develop a droplet microfluidic technology for selective single-cell transcriptomic (scRNA-seq) analysis of cells with a specific phenotype from a heterogeneous mixture. Cells will be encapsulated in picolitre volume droplets, with no more than one cell per droplet, together with single hydrogel beads carrying barcoded cDNA primers, with a unique barcode on each bead. Selective sequencing of only (rare) cells with the desired phenotype will be achieved by targeting single or multiple proteins expressed on the cell surface or secreted from the cells with DNA tagged antibodies, which will trigger a bistable molecular circuit called the DNA Toolbox. In droplets containing cells with the desired phenotype, the molecular circuit will trigger selective release of barcoded cDNA primers from the hydrogel beads, ensuring that, after reverse transcription, barcoded cDNAs are only recovered from these cells. Sequencing of the barcoded cDNAs will provide the transcriptomic landscape of only the target cells. The technology will first be validated for selective scRNA-seq of T cells from peripheral blood of healthy donors. It will then be applied to analyze rare cell types in clinical samples. Specifically, we will perform selective sequencing, with no prior enrichment, of tumor infiltrating antigen-specific T cells from tumor biopsies, recovering paired TCR ? and ? chain sequences and associated single-cell transcriptomic data. Obtaining paired TCR information from primary tumor biopsies is currently very challenging, because the low cell numbers in liquid biopsies make sorting of T cells very complicated (or impossible). Defining the nature and recurrence of TCRs in tumors is a major challenge in the field of cancer immunotherapy.
We believe that this system will represent a major breakthrough in single-cell sequencing. It can be adapted to selectively sequence many different cell types and triggered using increasingly complex combinations of cell-surface and secreted markers. In addition to single-cell transcriptomics, it should also be possible, in the future, to perform selective single-cell genomic, epigenomic, proteomic and multi-omic analysis.