Detailed Chemical Analysis of Single Cells with Massively Multiplex Transcriptomics
Summary
The conventional high-throughput chemical screening methods, which employ broad assessments such as cell viability, fail to furnish a comprehensive understanding of the mechanisms of action, the occurrence of off-target effects, and the diverse reactions. In this study, the authors introduce "sci-Plex", a novel approach that leverages "nuclear hashing" to evaluate the global transcriptional repercussions to an array of independent perturbations at single-cell precision. As an exemplification of its feasibility, the authors apply sci-Plex to three cancer cell lines that have been subjected to 188 compounds. The analysis encompassed approximately 650,000 individual cell transcriptomes across nearly 5000 discrete samples, in a single experiment. The findings reveal the significant intercellular heterogeneity in response to individual compounds, as well as similarities in the reaction to a group of compounds, and provide insight into the differential attributes within a group. Notably, the outcomes obtained from the histone deacetylase inhibitors validate the notion that chromatin serves as a crucial repository of acetate in cancer cells.
Fig.1 Overview of sci-Plex. (Srivatsan, 2020)
Research Criteria
The authors, Srivatsan et al., have advanced the utilization of technologies for single-cell transcriptome analysis to gain a deeper understanding of biological heterogeneity by incorporating the sci-Plex method. This novel methodology integrates single-cell transcriptomics with oligo hashing, small chemical screening, and other techniques, allowing for the assessment of the effects of a substantial number of chemical compounds on three distinct cancer cell lines. The capacity to profile numerous cells is crucial for the examination of multiple chemicals, given that such screens entail a multitude of ways of perturbing individual cells. By leveraging the sci-Plex approach, the authors are able to depict the gene expression patterns of thousands of distinct experimental circumstances in a single experiment.
Fig.2 Experimental design. (Srivatsan, 2020)
Sample Type
Human cancer cell lines.
Result—Sci-Plex Enables Multiplex Chemical Transcriptomics at Single-Cell Resolution
In their study, Srivatsan et al. investigated the feasibility of utilizing nuclear hashing as a means of enabling high-content phenotypic chemical screens via single-cell transcriptional profiling. To this end, they performed a series of experiments in which the A549 cell line was exposed to a suite of chemical compounds, each at seven different doses, in triplicate. The cells were then labeled and subjected to sci-RNA-seq2, with the resulting gene expression data visualized and analyzed using various computational techniques, including Uniform Manifold Approximation and Projection (UMAP) and Louvain community detection. The study results indicated that a substantial number of genes displayed dose-dependent differential expression in response to at least one of the drugs, with gene ontology analysis revealing the involvement of drug-specific pathways. Furthermore, the researchers evaluated the suitability of the resulting "viability score" as a metric for toxicity, with the findings demonstrating concordance with established "gold standard" measurements.
Fig.3 Sci-Plex enables multiplex chemical transcriptomics at single cell resolution. (Srivatsan, 2020)
Result—Sci-Plex Scales to Thousands of Samples and Enables HTS
The authors conducted a comprehensive evaluation of the efficacy of the sci-RNA-seq method in measuring the chemical-induced transcriptional responses in three different cell lines. The visual representation of the sci-RNA-seq profiles for each cell line was an illuminating experience, revealing compound-specific transcriptional responses and patterns that were commonly observed across multiple compounds. The projection of the majority of cells in each cell line into a central mass on the Uniform Manifold Approximation and Projection (UMAP) plot was noteworthy, with smaller clusters, largely composed of cells treated with specific compounds or classes of compounds, flanking the central mass. To determine the extent to which each compound elicited similar responses across the three cell lines, the authors clustered the compounds based on the effect sizes of dose-dependent genes. The joint analysis of the three cell lines resulted in the identification of common and cell-type-specific responses to different compounds, thereby supporting previous observations of the regulation of HSP90AA1 downstream of MEK signaling and providing further insight into the utility of single-cell transcriptomes in highlighting drugs that target convergent molecular pathways.
Fig.4 Sci-Plex enables global transcriptional profiling of thousands of chemical perturbations in a single experiment. (Srivatsan, 2020)
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Reference
- Srivatsan, S.R.; et al. Massively multiplex chemical transcriptomics at single-cell resolution. Science. 2019, 367(6473): 45-51.
