Single-cell sequencing and tumorigenesis: improved understanding of tumor evolution and metastasis

Abstract

Extensive genomic and transcriptomic heterogeneity in human cancer often negatively impacts treatment efficacy and survival, thus posing a significant ongoing challenge for modern treatment regimens. State-of-the-art DNA- and RNA-sequencing methods now provide high-resolution genomic and gene expression portraits of individual cells, facilitating the study of complex molecular heterogeneity in cancer. Important developments in single-cell sequencing (SCS) technologies over the past 5 years provide numerous advantages over traditional sequencing methods for understanding the complexity of carcinogenesis, but significant hurdles must be overcome before SCS can be clinically useful. In this review, we: (1) highlight current methodologies and recent technological advances for isolating single cells, single-cell whole-genome and whole-transcriptome amplification using minute amounts of nucleic acids, and SCS, (2) summarize research investigating molecular heterogeneity at the genomic and transcriptomic levels and how this heterogeneity affects clonal evolution and metastasis, and (3) discuss the promise for integrating SCS in the clinical care arena for improved patient care.

Keywords: Cancer; Cancer stem cells; Circulating tumor cells; Single-cell sequencing; Tumor heterogeneity; Whole-genome amplification.

Fig. 1

Applications of single-cell sequencing in cancer research. a Resolving intratumor heterogeneity; b investigating clonal evolution in primary tumors; c studying invasion in early stage cancers; d tracing metastatic dissemination; e genomic profiling of circulating tumor cells; f investigating mutation rates and mutator phenotypes; g understanding evolution of resistance to therapy; h defining cancer stem cells and cell hierarchies; and i studying cell plasticity and the epithelial-to-mesenchymal transition [86]

Fig. 2

Single-cell isolation methods. a Methods for isolating single cells from abundant cell populations include: robotic or manual micromanipulation, serial dilution, flow-sorting, microfluidic methods, and laser-capture microdissection; b methods for isolating single cells from rare cell populations include: CellSearch™, DEP-Array™, CellCelector™, MagSweeper™, and nanofilters [16]

Fig. 3

Main approaches used for whole-genome amplification of single cells. a Degenerate Oligonucleotide-primed polymerase chain reaction (DOP-PCR) uses primers with common sequences at the 5′- and 3′-ends, but six random nucleotides near the 3′-end to allow hybridization at many sites throughout the genome; b multiple displacement amplification (MDA) uses φ29 DNA polymerase and random primers in a non-PCR based amplification reaction in which newly-synthesized strands are displaced from the original DNA molecule and serve as templates for additional DNA synthesis, resulting in a hyper-branched network; c multiple annealing and looping based amplification cycles (MALBAC) uses random primers with a common sequence at the 5′-end to amplify only the original template DNA and semi-amplicons. Full amplicons have complementary ends that allow the formation of closed-loop structures that prevent further amplification [15]

Fig. 4

Main approaches used for whole-transcriptome amplification of single cells. a The Tang method performs reverse transcription of mRNA for single-cell RNA-seq using an oligo-dT primer with an anchor sequence, then a poly-A tail is added to the 3′-end of the first cDNA and the second strand is synthesized using a different oligo-dT primer with a different anchor sequence; b Smart-seq and Smart-seq2 implement a template-switching step to increase the number of full-length cDNA transcripts with an intact 5′-end; c quartz-seq limits amplification of unwanted byproducts by removing excess primer with exonuclease I before second-strand synthesis and using suppression PCR to form hairpin structures that cannot be amplified; d cell expression by linear amplification and sequencing (CEL-Seq) includes a template-switching step and uses molecular barcodes and pooling of samples from multiple single cells prior to linear amplification; e single-cell tagged reverse transcription (STRT) permits multiplex sequencing of multiple cells in the same reaction using a template-switching mechanism to simultaneously introduce a molecular barcode and an upstream primer-binding sequence during reverse transcription; f quantitative single-cell RNA-seq generates full-length transcripts using template switching and incorporating random UMI (unique molecular identifier) sequences to label individual cDNA molecules and eliminate amplification bias [8]