Density gradient centrifugation was the earliest technique to be developed. It remains the simplest way to isolate CTCs through the use of special centrifugation liquid. However, this method yields low rates of CTC enrichment. CD45 depletion can be combined with density gradient depending centrifugation to increase the yield of CTCs. It should be noted that density gradient depending centrifugation and lysis of red blood cell may cause the loss of tumor cell.

A great many CTC detection technologies are immune-magnetic based, which can be divided into those that rely on positive selection and those that rely on negative selection. EpCAM is the most commonly used label in positive selection methodologies. CellSearch system is one of the EpCAM-based CTC detection technologies and is the only approved CTC detection apparatus by FDA (US Food and Drug Administration). By using this approach, CTC has been proven as an independent prognostic biomarker on PFS (progression-free survival) and OS (overall survival) in a variety of cancers, including lung, liver, colon, breast, and prostate [8, 10–14]. The AdnaTest is also epithelial marker-based and can enrich CTCs positively. A study on metastatic breast cancer used this method and proved that CTC presence or absence was a valid prognostic and predictive marker [15]. A major drawback of this method is that it is not perfect in detecting MET-associated CTCs, since these have either downregulated EpCAM or a complete loss of it [16].

CD45 is the most commonly used label for negative selection approaches. Depletion of leukocytes with CD45-positive markers is the favorite approach for capturing CTCs that have downregulated or an absence of EpCAM expression [17]. CD45 depletion could also be used together with other label-independent method, such as density gradient depending centrifugation to increase the yield.

The ISET system is a typical size-based CTC isolation device. In general, this approach is designed to gain CTCs at a high enrichment rate. However, a major drawback of this method is that EMT-associated CTC might be larger than leukocytes or not solid enough. As such, they may get loss by the size-based filter. What’s more, even though CTC is captured on the membrane, it might be difficult to detect and examine for further genetic analysis.

Microfluidic device platforms like the CTC-Chip are promising alternatives to selectively capture CTCs [18]. Quite a large number of CTC-Chips are currently available, including the IsoFlux for EpCAM-positive tumor selection [19], CTC-iChip (a size-based filtration and affinity-based enrichment strategy) for systematic removal of PBMCs and RBCs [20], JETTATM microfluidic chip (a size- and deformability-based capture scheme) enabling single-cell analysis [21], and the spiral biochip (a size-based and EpCAM-independent separation method) with low leukocyte contamination [22]. Generally speaking, CTC-Chips are advantageous in that they have high recovery rates; however they are inconvenient to use and come at a high cost.

To circumvent sample volume limitations, an EpCAM-coated wire named CellCollector^TM was designed by GILUPI GmbH for in vivo CTC capture [23]. This device is positioned through a cannula into the vein of a cancer patient. During the 30-min application time, it is estimated that up to 1.5 L of blood flows over the detector, thus increasing the yield of detectable CTCs. A similar device employing 3-aminopropyltriethoxysilane (γ-APS) as the coupling reagent and CBMA-grafted anti-EpCAM antibody-immobilized Nylon as cannula has also been developed. This method has also been shown to be a promising new material for in vivo CTC capture [24].

In general, CTC isolation can be achieved with different strategies. A great number of approaches for CTC enrichment are currently under development and may be on the market in the near future. However, both independent and large clinical studies will be needed to determine the robustness and clinical validity of these new methods.

Recently, a new cellular- and molecular-based method of subtraction enrichment and immunostaining-based FISH (iFISH) was successfully established. The method promised effective enrichment, phenotypic identification in situ, and CTCs’ subtype characterization. This fine-grained information is critical, since different CTCs’ subtypes might contribute different clinical significances to tumor growth, relapse, metastasis, and treatment resistance [50].

A similar study was also carried out by Shen et al. Briefly, iFISH was also used to determine CTCs’ number and characteristics in advanced gastric cancer patients. The status of HER2 expression and the aneuploidy of the chromosome 8 in CTCs got from the patients were tested. Their results showed that examination of the CTC chromosome 8 copy number provided an important method for predicting chemosensitivity and monitoring treatment efficiency [51]. A similar study has also been conducted in breast cancer [52].

CTC analysis gnomically can also provide genetic mutation related to therapeutic resistance. For example, the mutations of KRAS were known to affect the efficiency of EGFR inhibitors in colorectal and lung cancer patients. An analysis of intra-patient KRAS [53] and BRAF mutation [54] heterogeneities in individual CTCs has been reported in colon cancer. The testing methods used included HRM, ASPCR, and ddPCR. The heterogeneity of PIK3CA mutation status was also investigated through single CTC detection in metastasis breast cancer patients [55]. Evaluation of PIK3CA mutational status in CTC could be a potential clinical method for tumor and treatment monitoring [56].Yet till now, no similar mutation reports are currently available in gastric cancer.

Studies that have examined tumor-associated mRNA in CTC are rare. In a recent colon cancer study, CTC-specific mRNAs were investigated and shown to have higher expression in patients with ≥3 CTCs. mRNA expression levels of KRT19, KRT20, and AGR2 have also been reported in mCRC, but no similar results have been available in gastric cancer until recently [57].