Advances in novel strategies for isolation, characterization, and analysis of CTCs and ctDNA

Abstract

Over the past decade, the detection and analysis of liquid biopsy biomarkers such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have advanced significantly. They have received recognition for their clinical usefulness in detecting cancer at an early stage, monitoring disease, and evaluating treatment response. The emergence of liquid biopsy has been a helpful development, as it offers a minimally invasive, rapid, real-time monitoring, and possible alternative to traditional tissue biopsies. In resource-limited settings, the ideal platform for liquid biopsy should not only extract more CTCs or ctDNA from a minimal sample volume but also accurately represent the molecular heterogeneity of the patient's disease. This review covers novel strategies and advancements in CTC and ctDNA-based liquid biopsy platforms, including microfluidic applications and comprehensive analysis of molecular complexity. We discuss these systems' operational principles and performance efficiencies, as well as future opportunities and challenges for their implementation in clinical settings. In addition, we emphasize the importance of integrated platforms that incorporate machine learning and artificial intelligence in accurate liquid biopsy detection systems, which can greatly improve cancer management and enable precision diagnostics.

Keywords: CTC characterization; cancer treatment; circulating tumor DNA; circulating tumor cells; ctDNA characterization; metastasis; microfluidic.

Figure 1.

Schematic depicting the various strategies for separation and detection of circulating tumor cells (CTCs) and CTC clusters based on differences in their physical and biological features. Techniques such as density gradient, filters, electrophoresis, and microwells exploit the unique biophysical properties of CTCs to isolate them from peripheral blood. Positive/negative selection exploits the unique biological expression profile of CTCs and clusters for isolation and analysis.

Figure 2.

(a) Circulating tumor cells (CTCs) and CTC clusters found in NSCLC patients. CTCs were detected by drawing 7.5 mL of peripheral blood. CTCs were enriched by microfilter isolation, and immunofluorescence staining was performed for cytokeratins (CK) 8/18 and/or 19, EpCAM, CD45, and the nucleus identified with DAPI (a–d). CTC clusters were defined as an aggregated group of ⩾2 CTCs. CTC clusters were observed in different shapes: spherical (e,f), triangular (g,h). Reproduced with permission from Manjunath et al. under open Creative Commons Attribution License.

Figure 3.

Molecular characterization of circulating tumor cells (CTCs) and CTC clusters. CTC and CTC clusters genome analysis can be evaluated by identifying different genomic abnormalities with techniques such as FISH, WGA, and NGS. By dissociating clusters into single CTCs, the same analysis can be done on them. CTC/Cluster gene expression and CTC-specific isoforms can be demonstrated by qRT-PCR, RNA-sequencing, and in situ RNA hybridization. Immunostaining assays, on-chip western blotting, microfluidic qPCR, and imaging mass cytometry can be used to investigate the proteome of CTCs. FISH, fluorescence in situ hybridizations; NGS, next-generation sequencing; WGA, whole genome amplification.

Figure 4.

Conventional technologies implemented for ctDNA analyses and their feasibility toward clinical applications. qPCR-based technologies operate with limited targets suggesting prior knowledge of the mutation; NGS-based technologies have broad gene coverage. In gray: non-tumoral cell-free DNA. In blue: circulating tumor DNA. BEAMing, beads, emulsion, amplification, magnetics; ctDNA, circulating tumor DNA; ddPCR, digital droplet PCR; MS, mass spectrometry; NGS, new-generation sequencing; qPCR, quantitative PCR; SERS, surface-enhanced Raman scattering; TLM, tumor mutation load; UMI, unique molecular identifiers; WES, whole exome sequencing; WGS: whole exome sequencing. Reproduced with permission from Moati et al. under open Creative Commons Attribution License.

Figure 5.

Novel strategies for integrated detection and quantitation of ctDNA. (a) Schematic of self-powered bidirectional partition microfluidic chip with embedded microwells. A layered microfluidic chip was designed such that the sample was loaded into the chip through the inlet and distributed into microwells in two directions. The microchannels connected to the red channel are alternatively embedded in the row of microwells linked to the channel. The embedded microwells allow decreasing of the blank area on the chip without shortening the interval of alternate microwells. The blue line around the array denotes the hydration channel. Reproduced with permission from Geng et al. under open Creative Commons Attribution License. (b) Integrated biochip equipped with central lysis and four amplification/detection chambers linked by the fluidic channels for targeted ctDNA analysis. The on-chip electrical cell lysis releases cellular targets and the nanofluidic manipulations are under an AC field to enhance isothermal solid phase amplification. Superparamagnetic iron oxide particle nanozyme-mediated redox reaction allows for electrochemical detection of surface-immobilized amplicons. Reproduced with permission from Koo et al. Copyright 2020 American Chemical Society. (c) Schematic illustration of the pump-free, high-throughput microfluidic chip-based SERS assay for ctDNA detection. The chip was developed with Cu2O octahedra and an AuNB array as the SERS-active substrate and CHA-HCR as the dual amplification strategy. Reproduced with permission from Cao et al. under open Creative Commons Attribution License.