Multiplexed single-cell transcriptional response profiling to define cancer vulnerabilities and therapeutic mechanism of action

Abstract

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of singlecell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as posttreatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from shortterm transcriptional responses to treatment.

Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity ofClytiaMedusa Cell Types

“…2b-d), were concordant between the two separate multiplexed experiments and robust to different transcriptome annotations ( Supplementary Fig. 5, 11,13). For some of them, cell type identity could be assigned on the basis of published information on gene expression in Clytia and/or of homologous genes in other animals, while for the others we performed in situ hybridization for selected marker genes ( Fig.…”

“…However, scRNA-seq studies involving multiple samples can be costly, and may be confounded by batch effects resulting from multiple distinct library preparations 12,13 . Recent developments in scRNA-seq multiplexing technology expand the number of samples, individuals, or perturbations that can be incorporated within runs, facilitating well-controlled scRNA-seq experiments 11,[14][15][16][17][18] . These advances have created an opportunity to explore systems biology of whole organisms at single-cell resolution, merging the concepts of cell atlas surveys with multiplexed single-cell experimentation.…”

Massively parallel phenotyping of variant impact in cancer with Perturb-seq reveals a shift in the spectrum of cell states induced by somatic mutations

“…, a WT endogenous allele is required for distinguishing dominant negative from LOF effects). The pooled nature of Perturb-Seq should allow us to readily extend this work to many cell lines, including existing pools of cell lines such as PRISM (Kinker et al, 2019; McFarland et al, 2020). Second, we used exogenously expressed cDNA constructs, where viral packaging limits the interrogation of longer (>3.5kb) genes, and where some variants may be expressed at non-physiologic levels.…”

Transcriptional census of epithelial-mesenchymal plasticity in cancer

“…ScRNA-seq data from epithelial cancer cell lines in control and drug-treated culture conditions were acquired from McFarland et al (McFarland et al, 2020) and Cook & Vanderhyden (Cook and Vanderhyden, 2020). Initial data processing was performed identically to tumour samples and cell lines with fewer than 100 measured cells were removed.…”