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Table 1 Molecular characterization of circulating tumor cells

From: Tracking cancer progression: from circulating tumor cells to metastasis

Molecular typeTechnologyOutcomeAdvantagesLimitationsKey references
GenomeFISH• CNA• Reduced experimental time
• Reduced cost
• Allows spatial information
• Limited number of genesLeversha et al. [27]; Onozato et al. [28]
Targeted DNA sequencing• Point mutations from a small to a moderate set of genes• High sensitivity
• Reduced cost
• Limited number of genesDe Luca et al. [29]
Single-cell exome sequencing• CNA
• Point mutations from exome regions
• Comprehensive profiling in exon regions• WGA required
• High dropout levels
Lohr et al. [12]; Ni et al. [30]
Single-cell whole-genome sequencing• CNA
• DNA rearrangements
• Point mutations
• Comprehensive profiling of the genome• WGA required
• False-positive errors introduced during WGA
• High allele dropout levels
• Non-uniform coverage
• Allelic imbalance introduced during WGA
Carter et al. [31]; Heitzer et al. [32]
TranscriptomeqRT-PCR• Expression level of a moderate set of genes• High sensitivity for genes expressed at low levels
• Reduced experimental time
• Reduced cost
• Limited number of transcripts
• Requires pre-amplification of targeted cDNA
Gorges et al. [33]
RNA in situ hybridization• Expression level of a set of genes• High sensitivity for genes expressed at low levels
• Allows targeted or comprehensive profiling
• Reduced experimental time
• Allows spatial information
• Reduced cost
• Limited to transcripts that are included in the probe designGasch et al. [34]; Yu et al. [35]; Ortega et al. [36]
Single-cell RNA sequencing• Whole-transcriptome expression
• CNA
• Point mutations from cDNA regions
• Comprehensive profiling
• Allows alternative splicing analysis
• Allows discovery of new annotated transcripts
• Low success rate of WTA
• Amplification bias introduced during WTA
• Low sensitivity for transcripts with low abundance
Aceto et al. [21]; Ting et al. [37]; Miyamoto et al. [38]
EpigenomeTargeted• Epigenetic marks• High sensitivity
• Reduced cost
• Reduced experimental time
• Limited number of genesPixberg et al. [39]
Whole-genome bisulfite sequencing• Epigenetic marks from the whole genome• Comprehensive profiling• WGA required
• High dropout levels
Gkountela et al. [40]
ATAC-seq• Chromatin accessibility• Comprehensive profiling• Low coverage data
• High dropout levels
Klotz et al. [41]
ProteomeImmunostaining• Protein levels of a small set of targets• Reduced cost
• Reduced experimental time
• Limited number of proteins
• Relies on antibody specificity and proper controls
Paoletti et al. [42]
Single-cell western blot• Up to eight different targets• High specificity
• Reduced cost
• Reduced experimental time
• Limited number of proteinsSinkala et al. [43]
Single-cell mass cytometry• Up to 40 different targets• Reduced cost
• Reduced experimental time
• Limited number of proteinsGerdtsson et al. [44]
Bulk mass spectroscopy• Whole proteome levels• Comprehensive profiling• Limited number of proteins
• Low sensitivity for features with low abundance
Jordan et al. [45]
Single-cell mass spectroscopy• Whole proteome levels• Comprehensive profiling• Not well established yetAbouleila et al. [19]
Single-cell multi-omicsGenome and transcriptome• CNA
• DNA rearrangements
• Point mutations from the whole genome or exome regions
• Whole-transcriptome expression
• Allows quantitative trait loci analysis• Yields lower quality data compared to individual protocolsSzczerba et al. [46]
  1. This table shows a selection of different approaches for the characterization of circulating tumor cells (CTCs) at the molecular level, the expected outcome of molecular characterization, the advantages and limitations of these approaches, and representative references. ATAC-seq assay for transposase-accessible chromatin sequencing, CNA copy-number alteration, FISH fluorescence in situ hybridization, RT-PCR quantitative reverse transcription PCR, WGA whole-genome amplification, WTA whole-transcriptome amplification