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[27]
adjacent tissues and survive the environmental stress [24-26] . Recently, Sun et al. compared EMT-features of
CTCs isolated from different vascular sites of HCC patients prior to resection, including peripheral vein,
peripheral artery, hepatic veins, infrahepatic inferior vena cava, and portal vein. Single-cell transcriptional
characterization demonstrated that CTCs were initially epithelial phenotype at release, but they switched
to EMT-activated phenotype during hematogenous transit via Smad2- and b-catenin-related signaling
pathways. They suggested such heterogeneous EMT status during the CTC transition may be the result of
shear stress in circulation. Nevertheless, while the loss of E-cadherin increased invasion, it also reduced
cancer cell proliferation and survival of CTCs in multiple models of breast cancer, indicating the complex
phenotypic plasticity of CTCs in EMT status [28,29] .

In addition, CTCs may aggregate to form CTC clusters [also referred to as CTC microemboli (CTM)]
and travel together in circulation, with or without fibroblasts, leukocytes, endothelial cells, or platelets,
which possess significantly higher invasiveness and increased survival ability compared to individual
[30]
CTCs . Notably, clusters of circulating tumor cells (CTCs) possessed an up to 50 times greater metastatic
[31]
potential compared with single CTCs . CTC clusters have been identified in many cancer types including
HCC; these clinical studies confirmed that CTC clusters are a much stronger prognostic factor for cancer
metastasis than single CTCs [27,32-34] . A recent study comprehensively profiled the DNA methylation
landscape of single CTCs and CTC clusters from breast cancer patients and mouse models, and the results
revealed that binding sites for stemness- and proliferation-associated transcription factors were specifically
[36]
[35]
hypomethylated in CTC clusters, thus promoting the stemness and metastasis of CTC clusters . Szczerba et al.
identified a specific of CTC-neutrophil cluster in circulation and further confirmed this interaction drove
cell cycle progression within the bloodstream and expanded the metastatic potential of CTCs. Targeting
this interaction may be rational in treating cancer metastasis. Additionally, the formation of CTC clusters
can induce a hypoxic environment that drives hypoxia-inducible factor 1-alpha-mediated mitophagy,
clearing damaged mitochondria, and limiting reactive oxygen species. Such a metabolic switch may support
[37]
the survival and metastatic spread of CTCs in circulation . Although the specific mechanism driving CTC
cluster formation remains unclear, it is currently considered that CTC clusters arise from oligoclonal tumor
cell groupings from the primary tumor or intra-vascular aggregation of single CTCs in circulation [31,38] . A
recent study has proposed that CTC cluster formation was a dynamic event rather than grouped migration
derived from the primary tumor by their multi-vascular sampling, in which CTM displayed an “aggregated-
apart-aggregated” pattern during the circulatory pathway. They hypothesized that single CTCs might
[27]
aggregate spontaneously in blood vessels against the unfavorable microenvironment in the bloodstream .
Characterization of the mechanism that determines the cluster formation may identify viable therapeutic
targets for inhibiting metastasis.

CTC DETECTION TECHNOLOGY
CTCs are extremely rare and surrounded by numerous blood cells in the bloodstream, thus CTCs are
[39]
generally required to be firstly enriched from blood samples . Reliable CTC enrichment technologies
are essential to the downstream comprehensive CTC analysis. Many different technologies have been
developed for CTC detection in the past years, and they can be classified into two categories based on
whether they make use of the physical or biological properties of the target cells [Table 1] . However,
[40]
most of these technologies for CTC capture and downstream analyses are designed for scientific research,
requiring multiple batch-process steps and having relatively limited throughput. The price of CTC analysis
is still on the high side (it generally costs hundreds of dollars per patient). A standardized and easy-to-use
platform that facilities integrated CTC analysis is still needed.

Immunoaffinity-based method
The immunoaffinity-based method for CTC detection relies on the tumor-specific antibodies against cell
surface markers, including EpCAM, human epidermal growth factor receptor (EGFR)2, prostate-specific