Abstract
Unlike bulk-cell analysis, single-cell approaches have the advantage of assessing cellular heterogeneity that governs key aspects of tumor biology. Yet, their applications to circulating tumor cells (CTCs) are relatively limited, due mainly to the technical challenges resulting from extreme rarity of CTCs. Nevertheless, recent advances in microfluidics and immunoaffinity enrichment technologies along with sequencing platforms have fueled studies aiming to enrich, isolate, and sequence whole genomes of CTCs with high fidelity across various malignancies. Here, we review recent single-cell CTC (scCTC) sequencing efforts, and the integrated workflows, that have successfully characterized patient-derived CTCs. We examine how these studies uncover DNA alterations occurring at multiple molecular levels ranging from point mutations to chromosomal rearrangements from a single CTC, and discuss their cellular heterogeneity and clinical consequences. Finally, we highlight emerging strategies to address key challenges currently limiting the translation of these findings to clinical practice.
Highlights
The concept of intratumoral heterogeneity (ITH), first described in 1982 by Fidler and Hart [1], has been expanded to include genetic, phenotypic and functional heterogeneity within tumors comprising diverse malignant and non-malignant subpopulations
While CellSearch® remains the choice of primary enrichment tool in single-cell CTC (scCTC) sequencing studies, such immunoaffinity-based enrichment technology relying on epithelial cell surface markers (e.g., EpCAM or CKs) have varying capture efficiency depending on the degree of EMT, stemness, and the resulting differentiation cell state
The challenge is that the yield of amplified DNA varies significantly across circulating tumor cells (CTCs), where the success rate of amplification ranges from 11% to 100% [24,61], and Whole Genome Amplification (WGA) step itself is subjected to coverage biases and errors, such as preferential allelic amplification, GC bias, dropout events, and nucleotide copy errors [60]
Summary
The concept of intratumoral heterogeneity (ITH), first described in 1982 by Fidler and Hart [1], has been expanded to include genetic, phenotypic and functional heterogeneity within tumors comprising diverse malignant and non-malignant subpopulations. The prevalence of phenotypic plasticity involving programmed death-ligand 1 (PD-L1) [10], stemness [11], drug resistance [12], and EMT [13], within CTC populations has fueled investigation of cellular heterogeneity using single-cell high-throughput enrichment and sequencing platforms over the past five years. The leukocyte contamination is inevitable in any given primarily enriched sample due in part to extremely rare CTCs occurring at a frequency of ~1 in 107 WBCs in blood from a cancer patient [15] and the relatively low cell capture efficiency of existing cell sorting technologies, which are further limited to isolating only certain CTC subpopulations (refer to the Section 2.1 for further details). It is essential to reach single-cell resolution to precisely characterize CTCs at the genomic level and further to investigate the clinical impact of cellular heterogeneity present within CTC populations
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