Exploration of Circulating Tumour Cell (CTC) Biology: A Paradigm Shift in Liquid Biopsy

Abstract

Circulating tumour cells (CTCs), are disseminated tumour cells found in the blood in solid tumour malignancies. Identification of CTCs act as emerging tools in the field of the Liquid Biopsy. Majority of the studies focused on detection and enumeration of CTCs due to technological challenges those results from the rarity of CTCs in the blood. Enumeration of CTCs has already proven their value as prognostic as well as predictive biomarkers for disease prognosis. However, recent advances in technology permitted to study the molecular and functional features of CTCs and these features have the potential to change the diagnostic, prognostic and predictive landscape in oncology. In this review, we summarize the paradigm shift in the field of liquid biopsy-based cancer diagnostics using CTC isolation and detection. We have discussed recent advances in the technologies for molecular characterization of CTCs which have aided a shift from CTC enumeration to an in-depth analysis of the CTC genome, transcriptomes, proteins, epigenomes along with various functional features. Finally, as a prognosticating strategy, the potentials of CTCs as a tool of liquid biopsy to predict micrometastasis, monitor prognosis and how to use them as an additional tool for cancer staging has been discussed.

Keywords: Cancer staging; Circulating Tumour Cells (CTCs); Diagnosis; Genomics; Liquid biopsy; Molecular characteristics of CTCs; Prognosis; Proteomics; Transcriptomics.

Fig. 1

CTCs as a tool for liquid biopsy. CTCs isolated from the blood of patient having solid tumour can be explored as a promising tool for liquid biopsy in real time. CTC enrichment followed by their detection and ultimately characterizing their molecular features could lead to a paradigm shift in the field of liquid biopsy

Fig. 2

Enrichment of CTCs. Approaches for CTC enrichment either exploit the difference between CTCs and blood cells in their the biological (marker dependent) or physical properties(marker independent). Marker-dependent methods include positive (using antibodies against tumour-associated or tissue-specific antigens) and negative selection (using antibodies against blood cell-specific antigens). Marker independent approach utilizes the differences in physical properties like size, charge etc

Fig. 3

Detection of CTCs. After successful enrichment, CTCs can be detected by using various strategies. Exploiting protein expression pattern of CTCs by using immunocytochemistry/FACS. Molecular biology techniques either include detection of genetic mutations/translocations/methylation pattern using PCR assay or detection of specific mRNAs using RT-PCR. Function properties like specific proteins secreted can also be exploited for CTC detection

Fig. 4

Molecular Characterization of CTCs. Various molecular features of CTCs can be characterized by using various recent technologies. CTC genomics can be studied by detecting various genomic aberrations using assays like FISH, WGA followed by aCGH/NGS and WES. CTC gene expression, as well as CTC-specific isoforms, can be demonstrated using technologies like multiplex RT-PCR, RNA-sequencing and RNA–in situ hybridization. The proteome of CTCs can be explored using recent advanced techniques such as multiplex immunoassays, on-chip western blotting, imaging mass cytometry and microfluidic qPCR. CTC-specific epigenomes can be studied using techniques like multiplexed-scAEBS and methylomics. Functional properties of CTCs such as their potential to form overt metastases could be explored by developing CDX models. In vitro, CTC cultures might be used for drug screening, personalized treatment and can provide insights into new pathways specific to metastasis-initiator CTCs and thus new therapeutic targets. FISH: Fluorescence in situ hybridizations; WGA: Whole genome amplification CGH; Array competitive genome hybridization; NGS: Next generation sequencing; WES: Whole exome sequencing; scAEBS: single-cell- agarose-embedded bisulfite sequencing; CDX- CTC-derived explants