Center for Cancer Systems Therapeutics (CaST)
Advanced Single Cell Technology Innovation Core
Core Leader: Peter Sims
The Advanced Single Cell Technology Innovation Core (ASCeTIC) will provide state-of-the-art technology for single-cell genomic analysis, multiplexed single-cell molecular perturbation, and in situ validation, leveraging robust experimental pipelines for large-scale profiling of human and murine solid tumors. A key Center deliverable is the identification of Master Regulator (MR) proteins underlying drug response and cell-cell interactions in the tumor microenvironment (TME), by interrogating tumor-specific regulatory networks. ASCeTIC will combine scRNA-seq with multiplexed CRISPR perturbations of putative MRs for direct validation of their downstream targets. To this end, we have successfully implemented CRISPR droplet sequencing (CROP-seq) for multiplexed, pooled gene perturbation with direct read-out by scRNA-seq (1). The Core will generate pooled lentiviral gRNA constructs for multiplexed perturbation and validation of master regulators with CROP-seq.
The Core will assist CaST investigators in their efforts to deconvolve cell type-specific drug responses in cultured slices of solid tumors using scRNA-seq. A major challenge is distinguishing cell death from reprogramming as the drug-induced loss of cells could occur via either mechanism. Furthermore, programmed cell death (e.g. apoptosis) generally occurs post-transcriptionally and may not induce a specific gene expression signature. The Core will provide SCOPE-seq, a highly scalable technology (2) for directly linking of live cell imaging or cytometry with scRNA-seq. SCOPE-seq combines fluorescent indicators of apoptosis and cell viability with scRNA-seq to unambiguously identify drug responsive subpopulations. Additionally, the Core will provide a suite of imaging- and spatial analysis tools for targeted in situ validation of findings from single-cell genomics and analysis of cell-cell interactions, including multiplexed RNA-FISH using RNAScope technology and immunohistochemical (IHC) analysis at the protein level. Our goals include:
Providing end-to-end experimental pipelines for single-cell RNA-seq (scRNA-seq), joint analysis of gene expression and chromatin state (scRNA/ATAC-seq), and joint analysis of cellular imaging and gene expression (SCOPE-seq). ASCeTIC will provide robust protocols and platforms for sample preparation, quality control, single-cell capture, single-cell genomic library construction, and sequencing to Center investigators using commercial technology from 10x Genomics and Illumina. We will also offer SCOPE-seq, a new microfluidic technology for linking live cell imaging or cytometry with scRNA-seq at scale.
Producing pooled lentiviral libraries for joint CRISPR perturbation and scRNA-seq readout. The Core will generate gRNA libraries for CROP-seq analysis of the molecular effect of candidate MR silencing (3) and will provide commensurate single-cell analyses.
Providing access to imaging-based validation tools and multiplexed spatial proteomics technologies. For targeted in situ validation of key findings from single-cell genomic analysis, ASCeTIC will provide RNA- and protein-level analysis using RNAScope and IHC, respectively. We will also offer both cyclic immunofluorescence for spatially resolved, highly multiplexed proteomic analysis.