Isolation of CTCs from the blood enables better characterization of the representative cells which accounts for the cancer progression and response to treatment. The rarity of CTCs in peripheral blood demands enrichment, which increases the probability to capture these cells for further genomic and functional characterization. As there is no standard protocol for the isolation of CTCs, many strategies were developed in recent years. These techniques are either based on the physical or biological properties of CTCs (Figure 1). The isolation of CTCs by its biological properties relies on specific surface markers that are detected by antibodies. CTCs can also be enriched on the basis of physical properties through the filtration-based method; a custom made microfluidic devices; centrifugation on ficoll density gradient and also based on dielectrophoresis.

Figure 1

Current isolation techniques for CTCs.

CTCs play a vital role in metastatic cascade and hence can be exploited as a promising prognostic marker. The enumeration of CTCs has been correlated with poor prognosis; early relapse; progression-free and overall survival; recurrence; the advancement of the disease and the response to treatment in various cancer types. Identification of CTCs in the circulatory system has been associated with poor overall survival in breast and metastatic prostate cancers (54, 55). A meta-analysis of non-metastatic colorectal cancer patients has demonstrated that the presence of CTCs is associated with aggressive disease progression and poor survival (56). The enumeration of CTCs from the peripheral circulatory system has been related to the progression-free and overall survival of patients with breast (54, 57-59), non- small cell lung (60, 61), squamous cell carcinoma of head and neck (62), colorectal (63) and prostate cancers (64). The CTCs count in patients with bone-metastatic castration-resistant-prostate cancer (mCRPC) treated with docetaxelwas reported to be used as a predictor of overall survival (65). In breast cancer patients the advancement in the TNM stage is positively correlated to the number of CTCs detected (66, 67). Moreover, CTCs from lung cancer patients has been related to histological as well as TNM staging (61). In non-small cell lung cancer patients, the post-operative identification of CTCs using magnetic bead enrichment and laser scanning cytometry (MAINTRAC^®), was related to tumor relapse (68). The presence of CTCs in the peripheral circulation of esophageal cancer patients has been related to poor prognosis, disease progression, and poor overall survival, thus suggesting it as a potential tool to improve the prognosis of patients (69).

Though CTCs have been widely suggested as a non-invasive prognostic marker for many cancers, it has not been widely exploited in clinics. As CTCs counts vary with the patients, determining a standard baseline of CTCs count is difficult. Though there are a number of available methods for CTCs isolation, choosing the right method is the primary challenge (70). The difficulty in evaluating the sensitivity and reproducibility for the identification of CTCs using current methods also limits the use of CTCs as biomarkers. The cost-effectiveness of the current methods also creates a bottleneck in the clinical utility of CTCs.

Even though monitoring of CTCs can serve as a potential diagnostic, prognostic and predictive tool in various cancer types, it is not widely used in clinics. Although there are many techniques for CTCs isolation, very few methods are cost-effective and commercially feasible for clinical use. The lack of standardization of the methods also makes it difficult to choose among them. For instance, the CellSearch system has been approved by the FDA for CTCs detection in metastatic breast, prostate, and colon cancer patients, but not in metastatic lung cancer patients (70). The isolation based on biological properties relies on the epithelial markers of CTCs. Owing to intra- and inter-tumor heterogeneity, the separation based on the specific antibody is not an optimal approach. The alteration of transcription pattern during the epithelial to mesenchymal transition (EMT) also downregulates the epithelial markers, which possibly result in missing out of CTCs in invasive cancers. Robust and more sensitive assays have to be developed for CTCs isolation and further to be tested in clinics.

It is worthwhile to note that the isolation of CTCs by any enrichment strategies would inevitably lead to cell loss. For instance, enrichment of CTCs based on the epithelial markers will fail to capture those CTCs, which are partially mesenchymal and vice-versa. Consequently, few research groups have tried short-term culturing of CTCs without prior enrichment (71, 72). In spite of several advanced techniques to isolate CTCs, there is still an urgent need to develop a highly reliable method.

The low number of CTCs is the primary challenge to isolate CTCs from the peripheral blood. In spite of the recent advances in single-cell analysis, only a few techniques maintain cell viability (15, 16). Hence further functional characterization of the isolated CTCs remains a challenge. The ability to expand CTCs in ex vivo condition could yield a reasonable number of cells for further validation and characterization. In the past decade, researchers delved into various possible methods to expand CTCs in the cell culture conditions, which could enable a wide range of clinical applications including early detection, the evolution of tumor genotypes during disease progression and to predict response to a given treatment (Figure 2).

Figure 2

Current methodologies for expansion of CTCs and clinical applications of CTCs.

There are numerous studies published on the isolation of CTCs from cancer patients, but only a few research groups succeeded in culturing the CTCs in ex vivo conditions. This reflects the limited success in the attempt to culture and expands CTCs. There is only a limited study, which has been successful in establishing CTCs lines for long-term cultures (summarized in Table 1). One of the pioneer's study to expand CTCs in ex vivo condition was published by Yu et al., in which they were successful in establishing CTCs lines from six ER+ve breast cancer patients (73). They have used CTCs-iChip, a microfluidic device to enrich CTCs (74). Interestingly, these CTCs lines could survive for more than 6 months in cell culture conditions. Similarly, in another study by Cayrefourcq et al., one permanent CTCs line was established from a colon cancer patient. This established CTCs line was able to survive ex vivo for more than a year and yet, exhibited the characteristics of tumor cells from the origin (75). A CTCs line established from advanced-stage prostate cancer patient exhibited to form organoids (76). Two other CTCs lines were established from SCLC patients, exhibits both spheroidal and adherent morphologies were reported to survive for more than 4 months. The above studies illustrate the plausibility of culturing and expanding CTCs ex vivo to facilitate the progress towards personalized therapy (77). However, the success rate in establishing a permanent CTCs line is very low, as the number of CTCs isolated from blood is very meager and also is biased towards the advanced stage of cancer.

Even though cancer cells have a proliferative advantage (78), the success rate of establishing a permanent CTCs line remains a big challenge. However, several studies used isolated CTCs to culture for short-term (3-14 days). It has been reported by Khoo et al., that the short-term CTCs culture could predict the patient’s response to the treatment. In this study, the nucleated cell fraction after the RBC lysis is directly cultured in hypoxic condition (1% O₂), hence there was no prior enrichment of cells. The CTCs in the majority of the samples were proliferative till day 14, but on day 21, the number of CTCs declined, suggesting it’s poor survival capability in ex vivo conditions. Interestingly it has been reported that even though in some samples, there were no CK+ve cells in the day 0 of the culture, they could see some CK+ve cells in the subsequent days, which clearly illustrates that short term CTCs culture could be a better choice for CTCs research (71). The short term CTCs culture were also reported to be used in the bladder (79), mesothelioma (80) and esophageal cancer (81), where CTCs were isolated by MetaCell® (a size based enrichment method to filter the peripheral blood cells through a polycarbonate membrane). Short term CTCs cultures were also established using a three- dimensional co-culture system with CAF (Cancer-Associated Fibroblast) and ECM proteins. Using this model Zhang et al., expanded CTCs from 14 of 19 early-stage lung cancer patients and further found that the cultured cells carried a mutation in TP53 gene similar to the matched primary tumor cells (82). As discussed above, the short-term CTCs cultures may not have the pure CTCs population. However, the number of cells amplified in a short period of time can mitigate the use of single-cell studies and also demonstrates the possibility of functional validation of cells.

The cells from the solid primary tumor shed into the bloodstream, however, only a few cells can withstand the hemodynamic forces, the effects of fluid shear and overcome the immune elimination (83). The subpopulation of CTCs, which are Metastatic-Initiating cells (MICs) should necessarily possess the intrinsic ability to congenially interact with blood cells to evade the immune mechanisms thereby colonizing at the distant site. Therefore, it will be captivating to study the microenvironment of tumor cells in circulation, which could aid as a clue to the mechanisms through which CTCs evades the immune response and provoke metastasis.

Although the mechanisms by which CTCs survive in the hostile microenvironment of blood circulation is not well understood, it has been well understood that the interactions of CTCs with platelets and platelet-derived cytokines could assist CTCs in the metastatic cascade. The intrinsic ability of tumor cells in the vasculature to form aggregates with platelets through its cell surface receptors is termed as Tumor cell-induced platelet aggregation (TCIPA) (Figure 3) (84). TXA2, thrombin, ADP and tumor-associated proteinases are reported to promote the formation of TCIPA (85-88). In TCIPA, the platelets are attached to CTCs by the formation of the GPIIb-IIIa-fibrinogen bridge (85, 89). Moreover, the CTCs surrounded by platelets serve as a shield to conceal against NK cell-mediated tumor cell lysis (90-92). Additionally, the platelets are reported to confer major histocompatibility complex (MHC) to CTCs, which subverts the immune surveillance (93). Furthermore, the platelets in TCIPA stimulate the release of α-granules, which mainly consist of platelet-derived growth factor (PDGF) and TGF-β at several-fold higher than non-tumorous cells (94), which promotes CTCs to undergo EMT (Epithelial-Mesenchymal Transition). TGF-β derived from platelets could activate NF-κB and TGFβ/Smad pathways in tumor cells, which promotes transdifferentiation of CTCs into a highly invasive mesenchymal-like phenotype (95). TGF-β is also reported to activate β-catenin and STAT3, which upregulates PDGF in hepatocellular carcinoma (96, 97).

Figure 3

CTCs and their microenvironment in the vasculature.

It has been reported that CTCs could differentiate monocytes to macrophages which expresses markers for TAM (Tumor-associated macrophage) (98, 99). Hamilton et al., have a cocultured CTCs line with PBMCs. The macrophages present in the culture revealed the overexpression of complement factor D (CFD)/ adipsin and vitamin D-BP (VDBP) and increased secretion of OPN (Osteopontin), lipocalin-2 (LCN2), CHI3L1, uPAR, MIP-1, and GDF-15/MIC-1. These results conclude that the CTCs through TAMs can evade the immune system, thereby gaining survival advantage in the treacherous journey through the vasculature (Figure 3). Interestingly the presence of CTCs in breast cancer patients was found to have significantly increased expression of CD95 (FAS) on the cell surface peripheral T-helper cells (100). The interaction of CD95L from the CTCs on interaction with cell surface CD95 of T-helper cells could assist the apoptosis of the helper T cells. This mechanism could also aid the immune evasion of CTCs.

CTCs have been reported in the blood either single cell or in the form of clusters. The cells in the bloodstream have several fates: it could go in apoptosis or anoikis or could be eliminated by the immune system. However, CTCs clusters, have an advantage to escape (101) and extravasate successfully to reach the distant organs and involve in micro- or macro-metastasis. A study by Aceto et al., experimentally demonstrated that the metastatic initiating potential of CTCs clusters is 23 to 50 fold more than single CTCs (102). Also, RNA sequencing at the single-cell level resulted in the upregulation of plakoglobin expression in the CTCs clusters. Moreover, knockdown of plakoglobin in mouse models resulted in decreased CTCs cluster formation and reduced the level of lung metastasis. Also, it has been validated that the level of plakoglobin resulted in poor prognosis in the breast cancer patient. Hence plakoglobin dependent cellular junctions play a vital role in the formation of CTCs clusters and thus contributing to the metastasis.

However, it is also inquisitive to dwell into how CTCs clusters, a multicellular aggregate travel through the narrow capillary vessels to colonize the distant organs. It is astounding to notice that the CTCs clusters in a microfluidic device, readily formed a reversibly single-file chain-like structure, which could make the CTCs clusters to travel through narrow passages in the capillary by reducing the hydrodynamic resistance. This was also demonstrated in vivo in the capillaries of zebrafish, where the CTCs clusters exhibited similar reorganization as observed in the microfluidic device (103). Hence, it could be fair enough to conclude that the survival advantage coupled with the ability to deform into capillary vessels bestow CTCs clusters to extravasate and reach to the distant organs more efficiently than single CTCs.

Although majority of the CTCs isolated from the blood exist as single cells, however CTCs were also reported to be present as an oligoclonal clusters of 2 or more tumor cells along with the aggregation of platelets, lymphocytes in the vasculature of cancer patients, along with endothelial cells and tumor-derived fibroblast (11, 95, 101, 104-111). The clusters of CTCs which possess three or more distinct nuclei are termed as circulating tumor microemboli (CTM) (112, 113). These clusters are formed as an aggregation of oligoclonal tumor cells in the primary site and together enters into the bloodstream (102). CTCs clusters in breast (114, 115), melanoma (116), renal (117), colorectal (118), pancreas (115), prostate (109, 115, 119) and lung cancers (109, 115, 120) have been reported. Further, studies have been shown a direct association of the presence of CTC clusters with the poor prognosis in the lung (121-123) and breast (102, 124, 125) cancer patients.

CTCs clusters are shown to be more resistant to chemotherapeutic drugs than single CTCs (126). Drug susceptibility testing on CTCs clusters would be an advantageous strategy to screen the appropriate drug from various drug combinations in vivo, which could then be used for personalized therapy. In the study by Khoo et al., demonstrated that drug response was monitored by the serial sampling of CTCs clusters using an integrated microfluidic device, where CTCs were cultured without prior enrichment (72). Moreover, the IC₅₀ value of one of the cultures obtained from the post-treatment time point was higher than the corresponding pre-treatment sample, suggesting the possible onset of resistance to the treatment. In another study from the same group reflected the persistence of CTCs clusters in the culture not only reflects the therapeutic resistance but also inversely correlates with the overall survival of breast cancer patients (71). In another study by Yu et al., demonstrated that drug susceptibility testing on established CTCs cell lines and predicted the sensitivity and resistance of various chemotherapeutic drug and drug combinations (73). Also, they could identify a few drug targets, which were mutated in CTCs but not in the primary tumor. However, there is an urgent need to establish a more robust and sensitive assay, which could be translated into the clinics.