The recent significant advances in the development of single-cell analytical tools have enabled increasingly deep cellular analyses at the genomic, transcriptomic and proteomic levels.
Genomic and transcriptomic methods utilize next-generation sequencing technologies, which complement protocols for improved quantification and cost-effective analyses of statistically significant numbers of single cells.
Single-cell proteomics methods range from cytometry tools to microchip platforms. All these methods rely on antibodies, but different platforms yield different levels of quantification.
Single-cell analyses reveal biology that is masked when cell populations or tissues are analysed. Illustrative examples include tracing the lineage of diseased cells back to the healthy tissue of origin, or a deep analysis of how targeted inhibitors can alter the structure of signalling pathways.
Single-cell analysis tools are already playing important parts in drug discovery, particularly in the rapidly emerging field of cancer immunotherapy.
The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed.
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The authors acknowledge the following funding agencies and grants for support of some of the work described in this Review: The National Cancer Institute (5R01CA170689 to J.R.H. as the principal investigator (PI) and A.R. as the co-PI, and 5U54 CA119347 to J.R.H. as the PI); Stand up to Cancer Foundation (to A.R. and J.R.H.); the Cancer Research Institute (to A.R. and J.R.H.); The Ben and Catherine Ivy Foundation (to P.S.M. and J.R.H.); and the Jean Perkins Foundation (to J.R.H. as the PI).
J.R.H. and A.R. are on the Scientific Advisory Board of Isoplexis, which is seeking to commercialize certain aspects of the single-cell barcode chip technology.
- Whole-genome amplification
A method, first reported using PCR by Arnheim's group, for nonselectively amplifying all DNA sequences present in a given sample, including a single cell.
- Multiple displacement amplification
A non-PCR based, room temperature DNA amplification technique reported by Lasken's group that is commonly used for whole-genome amplification.
- Multiple annealing and looping-based amplification cycles
(MALBAC). A PCR-type approach reported by Xie's group in which the enzymatic amplification of cDNAs proceeds via a linear process.
- Exome sequencing
Genome sequencing that is limited to only the small fraction (1%) of the genome that is protein encoding.
(RNA-seq). Also called whole transcriptome shotgun sequencing, RNA-seq is a method for analysing the transcriptome of a sample using next-generation sequencing tools.
- Molecular barcoding
An approach through which a DNA sequence or some other molecular identifier is used as an identifier of a specific cell or a specific transcript generated by that cell.
A microchip-based single-cell transcriptomics method reported by Fodor's group at Cellular Research in 2015.
A nanodrop-based single-cell transcriptomics method reported by Klein and others in 2015.
- Unique molecular index
(UMI). A molecular barcode used to identify a specific transcript from a specific cell.
A nanodrop-based single-cell transcriptomics method reported by Macosko and others in 2015.
Microfluidics methods in which individual assays are carried out in isolated nanolitre-size droplets of water, separated from one another by oil.
- Mass cytometry
A single cell proteomics method based on traditional flow cytometry methods but uses mass labels and mass spectrometry for protein analysis.
A microfluidics single-cell proteomics method.
- Single-cell barcode chips
(SCBCs). A single-cell proteomics method
- Single-cell western blottings
(scWesterns). A microchip- based method for carrying out western blotting assays on single cells.
Small peptide fragments that contain a genetic mutation. These fragments may be recognized by T cells during an antitumour immune response.
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Heath, J., Ribas, A. & Mischel, P. Single-cell analysis tools for drug discovery and development. Nat Rev Drug Discov 15, 204–216 (2016). https://doi.org/10.1038/nrd.2015.16
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