Circulating tumor cells as an emerging tool in cancer therapy
Majority of the cancer-related deaths are related to metastasis during which cancer cells invade the surrounding tissues, enter (intravasation) and exit (extravasation) the peripheral circulation and seed distant organs. The Circulating Tumor Cells (CTCs) exist in peripheral blood as single cells or as oligoclonal clusters of tumor cells along with platelets and lymphocytes. Detection of CTCs allows characterizing the tumors by their genotype and in predicting the prognosis and response to therapy and explants derived from these cells can be used in drug screening. In this review, we highlight the methods used for isolation and culture of CTCs and their clinical use.
Our understanding of the molecular mechanisms involved in tumorigenesis has singificantly improved in the last decade. The genomic data sets from TCGA (The Cancer Genome Atlas) (1) and ICGC (International Cancer Genome Consortium) (2) have a significant impact on the understanding of the biology of tumor cells and thereby leading to the new era of precision medicine. However, with the sophisticated technologies to sequence cancer cells, we narrowed down to the static view of tumor development. But it is well understood that the patient on chemotherapy/ targeted therapy would develop resistance over the course of time (3, 4). Hence, the treatment regimen needs to be continuously monitored during the treatment. Therefore, the current clinical setting demands a rather dynamic view, which would enhance our knowledge on the patient-specific mutational changes for a particular treatment.
Circulating tumor cells (CTCs) are the cancer cells in blood circulation, which shed from primary sites or from metastatic deposits. CTCs exist either as a single cell or as a cluster of cells in the form of microemboli, held together by intercellular junctions. Recent studies suggest that CTCs provide real-time information on the dynamic changes of tumor progression (6-10). The molecular characterization of CTCs would potentially be an important aspect to understand the metastatic cascade in tumor development (11, 12). With the recent technological advancements, several CTCs detection and isolation methods have emerged in the last decade. However, the complexity of their biology in relation to cancer progression and metastasis is still a major challenge towards clinical utility. Only a few studies until today has been reported, where the researchers have successfully cultured CTCs in ex-vivo condition for its functional validation (13) (Table 1). In this review, we summarize various techniques and challenges in CTCs isolation, culture methods, CTCs-derived preclinical models and the role of CTCs in clinical research.
|Cancer type||No of patients included in the study||No of established CTC lines||Method for isolation of CTC||Culture conditions||Ref|
|Breast||36||6||CTC-iChip (74) Principle: Hydrodynamic cell sorting Inertial focusing and Magnetophoresis||Ultra low attachment plate; RPMI-1640 medium with EGF (20ng/ml), basic FGF (20ng/ml) and B27 (10ml) in hypoxic condition (4% O2)||73|
|Colon||71||1||RosetteSep (CD45 –ve selection)||Medium 1: DMEM/F12 medium with insulin (20 μg/mL), 1% N2 complement, EGF: 20 ng/mL, L-Glutamine (2mM), FGF2: 10 ng/mL and 2% FBS for 2 weeks in hypoxic condition (2% O2) Medium 2: RPMI1640 medium with EGF, FGF-2, Insuline-Transferine-Selenium supplement and L-Glutamine in normoxia||75|
|Prostate||17||1||Ficoll-Paque density gradient separation and RosetteSep (CD45 –ve selection)||DMEM/F12 medium with EGF (50 ng/ml), R-spondin 1 (5% v/v), Noggin (10% v/v), FGF10 (10 ng/ml), FGF2 (1 ng/ml), DHT (0.1nM for CRPC samples, 1nM for hormone-sensitive samples), Nicotinamide (10 mM), A83-01 (0.5 μM), SB202190 (10 μM), Y-27632 (10 μM), B27 additive (1X), N-Acetyl-L-cysteine (1.25 mM), glutamax (2 mM), HEPES (10 mM) and primocin (1:100 v/v)||76|
|Small cell, Lung||30||3||Ficoll-Paque density gradient separation||RPMI-1640 medium with insulin, IGF-1, transferrin, and selenite||136|
3. CTCs isolation methods and challenges
3.1. Methods for isolation of CTCs
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.
3.1.1. Isolation of CTCs by biological properties
Antibody-based enrichment techniques can be broadly classified into positive and negative selection. In positive selection, epithelial markers such as EpCAM and cytokeratin are used for CTCs isolation, while in negative selection CD45 is used to deplete leukocytes. Till date, the CellSearch® system is the only FDA approved system to enrich CTCs. It is designed to isolate CTCs of epithelial origin (EpCAM +ve, cytokeratin (CK 8, 18 and 19) +ve and CD45 –ve) and has been clinically tested in breast (14), prostate (15) and colorectal (16) cancer patients. MagSweeper is another patented device, which uses magnetic beads functionalized with EpCAM antibody and has been reported clinically in the breast (17) and prostate (18) cancer patients. EPHESIA CTCs chip (19) and CTCs-chip (20) are two microfluidic-based devices, which capture CTCs by using specific antibodies. Moreover, CTCs can also be captured in vivo directly from the blood vessels of cancer patients. GILUPI CellCollector® used EpCAM functionalized medical wire, which is inserted into the cubital vein for 30 minutes which screens 1.5-3 liters of blood (21). It has been used to isolate CTCs from lung (22-25), breast (22, 26), prostate (27-30), neuroendocrine (31), head and neck (32) cancer patients.
3.1.2. Isolation of CTCs by physical properties
126.96.36.199. Size exclusion
The difference in thesize of CTCs from normal blood cells is reported in many cancers such as breast (33, 34) and prostate (35) (Table 2). Thus, the size-based exclusion of CTCs allows label-free enrichment by filtering out the cells of a certain size. It will allow the isolation of CTCs without any antibodies; hence the isolation of CTCs is not biased based on their EMT status. The size-based selection of CTCs is proven to be effective in non-small cell lung carcinoma (36). ScreenCell® is a commercially available filtration system which isolates, quantify and analyze the CTCs by allowing blood to pass through a microporous filter (37-39).
|Cell type||Size (µm)||Cell number (per ml)||References|
|Erythrocytes||6 - 8||4 - 6.2 billion||137-140|
|Neutrophils||15 - 17||1.8 - 7.7 million|
|Basophils||10 - 14||0.25 - 1 million|
|Eosinophil||12 - 15||0.05 - 0.4 million|
|Monocyte||15 - 20||0.1 - 0.8 million|
|Lymphocyte||6 - 15||1 - 4 million|
|Platelets||2 - 3||0.1 - 0.4 billion|
|CTCs||12 - 25||1-10|
Membrane microfilter, which consists of a semipermeable membrane having a pore size of 8 µm is also suitable for CTCs isolation (40). The deposition of trapped cells in the filter can later inhibit the filtration process. This was overcome by the introduction of 3D filters (41), in which two filters are separated by a space. The upper layer has a pore size of 9 µm and the lower filter has a pore size of 8 µm. The smaller cells like RBCs and WBCs can transverse through the gap, whereas the CTCs will get trapped on the top layer. The relatively low flow rate of the 3D filters is overcome by using 2D membrane filters with slots. Higher viability and recovery rate are reported for 2D membrane slot filters. Spiral microfluidic device allows label-free size-based isolation of viable CTCs using hydrodynamic force. The spiral system could recover more than 85% of CTCs across multiple cancer cell lines (42).
188.8.131.52. Density-based centrifugation
Size and density-based isolation of CTCs can be achieved by using density gradient solutions such as sucrose gradient or Ficoll. The separation of the cellular constituents will achieve upon centrifugation of the blood sample relative to the density of suspension fluid. Subjecting to centrifugation, cells of different types will pass through the density gradient at different rates and will be settled in different layers. The cells such as RBCs, neutrophils will settle at the bottom layer whereas CTCs, plasma and mononuclear cells will settle on the top layer. Low cost and requirement of minimum equipment enable this method cheaper than others. The limitation of this method is the possibility to lose the cells of interest. Low recovery of cells has reported in Ficoll-Hypaque based density gradient separation in breast cancer (43). Percoll density gradient, which is based on silica particle suspension offers more efficiency in separating mononuclear cells, neutrophils, and platelets (46). However in another study by Chang et al., contradicts the efficiency of Percoll to the Ficoll (47). Nevertheless, the pitfalls for the density gradient based enrichment are applicable for both Ficoll and Percoll.
184.108.40.206. Electrical property-based separation
The membrane potential of a cell is maintained by the combined activity of ion channels and transporters. It is shown that the electrical and dielectric properties such as membrane capacitance, membrane resistance, cytoplasmic conductivity, and permittivity are different for cancer cells and normal cells (48). The difference in these properties of the normal and cancer cells causes differential movement of cells under the non-uniform electric field. The differential movement of the particle under non-uniform electric is referred to as dielectrophoresis (DEP). DEP devices with various geometry have been used for the isolation of cancer cells from normal cells. CTCs isolation using the difference in dielectric property has been reported in oral cancer (49, 50), breast cancer (51), colon cancer (52) and prostate cancer cells (53). The major pitfall for this type of separation is the exponential decay of the electric field owing to the increase in distance from the electrode.
3.2. CTCs as a prognostic and predictive marker
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.
3.3. Current challenges
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.
4. Ex vivo CTC culture
4.1. The necessity for CTC culture
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).
4.2. Ex vivo CTC culture
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.
4.3. Short term CTCs culture method
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.
5. CTCs clusters in tumor metastasis
5.1. Circulating tumor cells and its microenvironment
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).
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.
5.2. CTC clusters formation and cancer metastasis
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.
5.3. Correlation of CTC clusters in blood with poor prognosis
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.
5.4. CTC clusters and drug susceptibility
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.
6. Establishment of CTC-derived explants (CDX) models as a tool for preclinical drug testing
Before the advent of liquid biopsy, patient-derived xenograft (PDX) models were widely in use to predict personalized therapy (127-131). However, the development of PDX models has several technical and clinical constraints (132, 133). For the first time, Hodgkinson et al. demonstrated the tumorigenicity of the enriched CTCs from SCLC patient by successfully generating CTCs derived explants (134). The CTCs isolated from both chemosensitive and chemorefractory SCLC patients gave rise to tumors in NSG mice. Moreover, these CDX models exhibited a similar response to etoposide and platinum treatment in comparison with the corresponding donor patients.
Similarly, out of the six established CTCs lines from breast cancer patients, Yu et al. succeeded to generate three CDX models, which exhibited similar histological features with the tumor samples from the corresponding patients (73). Likewise, Cayrefourcq et al., have successfully tested the tumor-initiating potential of a CTCs line established from a colon cancer patient in a CDX model (75). The tumor formed in the mice was found to be more than 10% of their body weight by ~40 days. Therefore, it would be logical to conclude that the CDX models could be in complementary with PDX models. In addition, the CDX model could be developed for those patients, who may not have undergone surgery or biopsy. Furthermore, the xenografts, formed in the CDX are derived from a small subset of cells, which inherently have invasive capabilities, thereby closely mimicking patient tumors. However, the biggest challenge is to generate a CDX model as the success rate is low and the duration to establish and to screen for suitable drug takes more time (135).
All the preclinical models can assist researchers in understanding the basic mechanisms involved in tumor progression and to come up with a suitable strategy to treat the disease. However, in the era of personalized medicine, liquid biopsy is the more advanced strategy, which can be adapted and conducive to use in the current clinical setting. Besides CTCs, the liquid biopsy also encompasses ctDNA, which provides an alternative way to explore the genomic evolution of tumors in a patient-specific manner. In the future, CTCs and ctDNA could synergistically be used for tailoring personalized medicine.
Nonetheless, the pre-eminence of isolating and expanding CTCs ex vivo and in vivo could screen various drug combinations and turn up with the best suitable drug or drug combinations, to the continuously evolving tumors during the course of treatment. However, the currently established methods to isolate CTCs are not reliable in all cases. Moreover, the intrinsic short lifetime of CTCs also hampers the possibility of amplifying the cells ex vivo from all clinical cases. An improved and efficient way for CTCs isolation and optimization of culture conditions to expand the CTCs population will be of tremendous value for both clinicians and researchers. Thus, understating the role of CTCs in tumorigenesis will be indispensable for cancer management.
PK is a recipient of the Ramanujan Fellowship awarded by the Department of Science and Technology (DST), Government of India. IAG is a recipient of Junior research fellowship and AB is a recipient of Senior research fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India. The authors declare no potential conflicts of interest.
1. M. H. Bailey, C. Tokheim, E. Porta-Pardo, S. Sengupta, D. Bertrand, A. Weerasinghe, A. Colaprico, M. C. Wendl, J. Kim, B. Reardon, P. Kwok-Shing Ng, K. J. Jeong, S. Cao, Z. Wang, J. Gao, Q. Gao, F. Wang, E. M. Liu, L. Mularoni, C. Rubio-Perez, N. Nagarajan, I. Cortes-Ciriano, D. C. Zhou, W. W. Liang, J. M. Hess, V. D. Yellapantula, D. Tamborero, A. Gonzalez-Perez, C. Suphavilai, J. Y. Ko, E. Khurana, P. J. Park, E. M. Van Allen, H. Liang, M. S. Lawrence, A. Godzik, N. Lopez-Bigas, J. Stuart, D. Wheeler, G. Getz, K. Chen, A. J. Lazar, G. B. Mills, R. Karchin and L. Ding: Comprehensive Characterization of Cancer Driver Genes and Mutations. Cell, 174(4), 1034-1035 (2018)
2. T. J. Hudson, W. Anderson, A. Artez, A. D. Barker, C. Bell, R. R. Bernabe, M. K. Bhan, F. Calvo, I. Eerola, D. S. Gerhard, A. Guttmacher, M. Guyer, F. M. Hemsley, J. L. Jennings, D. Kerr, P. Klatt, P. Kolar, J. Kusada, D. P. Lane, F. Laplace, L. Youyong, G. Nettekoven, B. Ozenberger, J. Peterson, T. S. Rao, J. Remacle, A. J. Schafer, T. Shibata, M. R. Stratton, J. G. Vockley, K. Watanabe, H. Yang, M. M. Yuen, B. M. Knoppers, M. Bobrow, A. Cambon-Thomsen, L. G. Dressler, S. O. Dyke, Y. Joly, K. Kato, K. L. Kennedy, P. Nicolas, M. J. Parker, E. Rial-Sebbag, C. M. Romeo-Casabona, K. M. Shaw, S. Wallace, G. L. Wiesner, N. Zeps, P. Lichter, A. V. Biankin, C. Chabannon, L. Chin, B. Clement, E. de Alava, F. Degos, M. L. Ferguson, P. Geary, D. N. Hayes, A. L. Johns, A. Kasprzyk, H. Nakagawa, R. Penny, M. A. Piris, R. Sarin, A. Scarpa, M. van de Vijver, P. A. Futreal, H. Aburatani, M. Bayes, D. D. Botwell, P. J. Campbell, X. Estivill, S. M. Grimmond, I. Gut, M. Hirst, C. Lopez-Otin, P. Majumder, M. Marra, J. D. McPherson, Z. Ning, X. S. Puente, Y. Ruan, H. G. Stunnenberg, H. Swerdlow, V. E. Velculescu, R. K. Wilson, H. H. Xue, L. Yang, P. T. Spellman, G. D. Bader, P. C. Boutros, P. Flicek, G. Getz, R. Guigo, G. Guo, D. Haussler, S. Heath, T. J. Hubbard, T. Jiang, S. M. Jones, Q. Li, N. Lopez-Bigas, R. Luo, L. Muthuswamy, B. F. Ouellette, J. V. Pearson, V. Quesada, B. J. Raphael, C. Sander, T. P. Speed, L. D. Stein, J. M. Stuart, J. W. Teague, Y. Totoki, T. Tsunoda, A. Valencia, D. A. Wheeler, H. Wu, S. Zhao, G. Zhou, M. Lathrop, G. Thomas, T. Yoshida, M. Axton, C. Gunter, L. J. Miller, J. Zhang, S. A. Haider, J. Wang, C. K. Yung, A. Cros, Y. Liang, S. Gnaneshan, J. Guberman, J. Hsu, D. R. Chalmers, K. W. Hasel, T. S. Kaan, W. W. Lowrance, T. Masui, L. L. Rodriguez, C. Vergely, D. D. Bowtell, N. Cloonan, A. deFazio, J. R. Eshleman, D. Etemadmoghadam, B. B. Gardiner, J. G. Kench, R. L. Sutherland, M. A. Tempero, N. J. Waddell, P. J. Wilson, S. Gallinger, M. S. Tsao, P. A. Shaw, G. M. Petersen, D. Mukhopadhyay, R. A. DePinho, S. Thayer, K. Shazand, T. Beck, M. Sam, L. Timms, V. Ballin, Y. Lu, J. Ji, X. Zhang, F. Chen, X. Hu, Q. Yang, G. Tian, L. Zhang, X. Xing, X. Li, Z. Zhu, Y. Yu, J. Yu, J. Tost, P. Brennan, I. Holcatova, D. Zaridze, A. Brazma, L. Egevard, E. Prokhortchouk, R. E. Banks, M. Uhlen, J. Viksna, F. Ponten, K. Skryabin, E. Birney, A. Borg, A. L. Borresen-Dale, C. Caldas, J. A. Foekens, S. Martin, J. S. Reis-Filho, A. L. Richardson, C. Sotiriou, G. Thoms, L. van't Veer, D. Birnbaum, H. Blanche, P. Boucher, S. Boyault, J. D. Masson-Jacquemier, I. Pauporte, X. Pivot, A. Vincent-Salomon, E. Tabone, C. Theillet, I. Treilleux, P. Bioulac-Sage, T. Decaens, D. Franco, M. Gut, D. Samuel, J. Zucman-Rossi, R. Eils, B. Brors, J. O. Korbel, A. Korshunov, P. Landgraf, H. Lehrach, S. Pfister, B. Radlwimmer, G. Reifenberger, M. D. Taylor, C. von Kalle, P. P. Majumder, P. Pederzoli, R. A. Lawlor, M. Delledonne, A. Bardelli, T. Gress, D. Klimstra, G. Zamboni, Y. Nakamura, S. Miyano, A. Fujimoto, E. Campo, S. de Sanjose, E. Montserrat, M. Gonzalez-Diaz, P. Jares, H. Himmelbauer, S. Bea, S. Aparicio, D. F. Easton, F. S. Collins, C. C. Compton, E. S. Lander, W. Burke, A. R. Green, S. R. Hamilton, O. P. Kallioniemi, T. J. Ley, E. T. Liu and B. J. Wainwright: International network of cancer genome projects. Nature, 464(7291), 993-8 (2010)
9. W. T. Yan, X. Cui, Q. Chen, Y. F. Li, Y. H. Cui, Y. Wang and J. Jiang: Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta-analysis. Sci Rep, 7, 43464 (2017)
10. Y. Li, J. Gong, Q. Zhang, Z. Lu, J. Gao, Y. Cao and L. Shen: Dynamic monitoring of circulating tumour cells to evaluate therapeutic efficacy in advanced gastric cancer. Br J Cancer, 114(2), 138-45 (2016)
12. D. Boral, M. Vishnoi, H. N. Liu, W. Yin, M. L. Sprouse, A. Scamardo, D. S. Hong, T. Z. Tan, J. P. Thiery, J. C. Chang and D. Marchetti: Molecular characterization of breast cancer CTCs associated with brain metastasis. Nat Commun, 8(1), 196 (2017)
14. D. F. Hayes, M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, M. C. Miller, J. Matera, W. J. Allard, G. V. Doyle and L. W. Terstappen: Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res, 12(14 Pt 1), 4218-24 (2006)
15. J. S. de Bono, H. I. Scher, R. B. Montgomery, C. Parker, M. C. Miller, H. Tissing, G. V. Doyle, L. W. Terstappen, K. J. Pienta and D. Raghavan: Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res, 14(19), 6302-9 (2008)
16. S. J. Cohen, C. J. Punt, N. Iannotti, B. H. Saidman, K. D. Sabbath, N. Y. Gabrail, J. Picus, M. Morse, E. Mitchell, M. C. Miller, G. V. Doyle, H. Tissing, L. W. Terstappen and N. J. Meropol: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol, 26(19), 3213-21 (2008)
17. G. Deng, S. Krishnakumar, A. A. Powell, H. Zhang, M. N. Mindrinos, M. L. Telli, R. W. Davis and S. S. Jeffrey: Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow. BMC Cancer, 14, 456 (2014)
18. G. M. Cann, Z. G. Gulzar, S. Cooper, R. Li, S. Luo, M. Tat, S. Stuart, G. Schroth, S. Srinivas, M. Ronaghi, J. D. Brooks and A. H. Talasaz: mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer. PLoS One, 7(11), e49144 (2012)
19. A. E. Saliba, L. Saias, E. Psychari, N. Minc, D. Simon, F. C. Bidard, C. Mathiot, J. Y. Pierga, V. Fraisier, J. Salamero, V. Saada, F. Farace, P. Vielh, L. Malaquin and J. L. Viovy: Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays. Proc Natl Acad Sci U S A, 107(33), 14524-9 (2010)
21. G. Gallerani, C. Cocchi, M. Bocchini, F. Piccinini and F. Fabbri: Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH. J Vis Exp(130) (2017)
22. N. Saucedo-Zeni, S. Mewes, R. Niestroj, L. Gasiorowski, D. Murawa, P. Nowaczyk, T. Tomasi, E. Weber, G. Dworacki, N. G. Morgenthaler, H. Jansen, C. Propping, K. Sterzynska, W. Dyszkiewicz, M. Zabel, M. Kiechle, U. Reuning, M. Schmitt and K. Lucke: A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire. Int J Oncol, 41(4), 1241-50 (2012)
23. T. M. Gorges, N. Penkalla, T. Schalk, S. A. Joosse, S. Riethdorf, J. Tucholski, K. Lucke, H. Wikman, S. Jackson, N. Brychta, O. von Ahsen, C. Schumann, T. Krahn and K. PanTel: Enumeration and Molecular Characterization of Tumor Cells in Lung Cancer Patients Using a Novel In vivo Device for Capturing Circulating Tumor Cells. Clin Cancer Res, 22(9), 2197-206 (2016)
24. Y. He, J. Shi, G. Shi, X. Xu, Q. Liu, C. Liu, Z. Gao, J. Bai and B. Shan: Using the New CellCollector to Capture Circulating Tumor Cells from Blood in Different Groups of Pulmonary Disease: A Cohort Study. Sci Rep, 7(1), 9542 (2017)
26. J. B. Li, C. Z. Geng, M. Yan, Y. S. Wang, Q. C. Ouyang, Y. M. Yin, L. N. Wu, J. He and Z. F. Jiang: (Circulating tumor cells in patients with breast tumors were detected by a novel device: a multicenter, clinical trial in China). Zhonghua Yi Xue Za Zhi, 97(24), 1857-1861 (2017)
27. G. Theil, K. Fischer, E. Weber, R. Medek, R. Hoda, K. Lucke and P. Fornara: The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study. PLoS One, 11(8), e0158354 (2016)
28. A. Kuske, T. M. Gorges, P. Tennstedt, A. K. Tiebel, R. Pompe, F. Preisser, S. Prues, M. Mazel, A. Markou, E. Lianidou, S. Peine, C. Alix-Panabieres, S. Riethdorf, B. Beyer, T. Schlomm and K. PanTel: Improved detection of circulating tumor cells in non-metastatic high-risk prostate cancer patients. Sci Rep, 6, 39736 (2016)
29. A. Markou, M. Lazaridou, P. Paraskevopoulos, S. Chen, M. Swierczewska, J. Budna, A. Kuske, T. M. Gorges, S. A. Joosse, T. Kroneis, M. Zabel, P. Sedlmayr, C. Alix-Panabieres, K. Pantel and E. S. Lianidou: Multiplex Gene Expression Profiling of In vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients. Clin Chem, 64(2), 297-306 (2018)
30. A. El-Heliebi, C. Hille, N. Laxman, J. Svedlund, C. Haudum, E. Ercan, T. Kroneis, S. Chen, M. Smolle, C. Rossmann, T. Krzywkowski, A. Ahlford, E. Darai, G. von Amsberg, W. Alsdorf, F. Konig, M. Lohr, I. de Kruijff, S. Riethdorf, T. M. Gorges, K. Pantel, T. Bauernhofer, M. Nilsson and P. Sedlmayr: In situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells. Clin Chem, 64(3), 536-546 (2018)
31. D. Mandair, C. Vesely, L. Ensell, H. Lowe, V. Spanswick, J. A. Hartley, M. E. Caplin and T. Meyer: A comparison of CellCollector with CellSearch in patients with neuroendocrine tumours. Endocr Relat Cancer, 23(10), L29-32 (2016)
32. H. Zhang, S. Gong, Y. Liu, L. Liang, S. He, Q. Zhang, M. Si and Z. Yu: Enumeration and molecular characterization of circulating tumor cell using an in vivo capture system in squamous cell carcinoma of head and neck. Chin J Cancer Res, 29(3), 196-203 (2017)
34. P. A. Kenny, G. Y. Lee, C. A. Myers, R. M. Neve, J. R. Semeiks, P. T. Spellman, K. Lorenz, E. H. Lee, M. H. Barcellos-Hoff, O. W. Petersen, J. W. Gray and M. J. Bissell: The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol, 1(1), 84-96 (2007)
35. S. Park, R. R. Ang, S. P. Duffy, J. Bazov, K. N. Chi, P. C. Black and H. Ma: Morphological differences between circulating tumor cells from prostate cancer patients and cultured prostate cancer cells. PLoS One, 9(1), e85264 (2014)
38. A. El-Heliebi, T. Kroneis, E. Zohrer, J. Haybaeck, K. Fischereder, K. Kampel-Kettner, R. Zigeuner, H. Pock, R. Riedl, R. Stauber, J. B. Geigl, B. Huppertz, P. Sedlmayr and C. Lackner: Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer? J Transl Med, 11, 214 (2013)
39. L. Wark, T. Klonisch, J. Awe, C. LeClerc, B. Dyck, H. Quon and S. Mai: Dynamics of three-dimensional telomere profiles of circulating tumor cells in patients with high-risk prostate cancer who are undergoing androgen deprivation and radiation therapies. Urol Oncol, 35(3), 112 e1-112 e11 (2017)
40. L. Zabaglo, M. G. Ormerod, M. Parton, A. Ring, I. E. Smith and M. Dowsett: Cell filtration-laser scanning cytometry for the characterisation of circulating breast cancer cells. Cytometry A, 55(2), 102-8 (2003)
41. S. Zheng, H. K. Lin, B. Lu, A. Williams, R. Datar, R. J. Cote and Y. C. Tai: 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices, 13(1), 203-13 (2011)
42. M. E. Warkiani, B. L. Khoo, L. Wu, A. K. Tay, A. A. Bhagat, J. Han and C. T. Lim: Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics. Nat Protoc, 11(1), 134-48 (2016)
43. O. Lara, X. Tong, M. Zborowski and J. J. Chalmers: Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation. Exp Hematol, 32(10), 891-904 (2004)
44. M. Aktas, T. F. Radke, B. E. Strauer, P. Wernet and G. Kogler: Separation of adult bone marrow mononuclear cells using the automated closed separation system Sepax. Cytotherapy, 10(2), 203-11 (2008)
46. W. M. Ellis, G. M. Georgiou, D. M. Roberton and G. R. Johnson: The use of discontinuous Percoll gradients to separate populations of cells from human bone marrow and peripheral blood. J Immunol Methods, 66(1), 9-16 (1984)
47. Y. Chang, P. H. Hsieh and C. C. Chao: The efficiency of Percoll and Ficoll density gradient media in the isolation of marrow derived human mesenchymal stem cells with osteogenic potential. Chang Gung Med J, 32(3), 264-75 (2009)
48. F. F. Becker, X. B. Wang, Y. Huang, R. Pethig, J. Vykoukal and P. R. Gascoyne: Separation of human breast cancer cells from blood by differential dielectric affinity. Proc Natl Acad Sci U S A, 92(3), 860-4 (1995)
50. H. J. Mulhall, F. H. Labeed, B. Kazmi, D. E. Costea, M. P. Hughes and M. P. Lewis: Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis. Anal Bioanal Chem, 401(8), 2455-63 (2011)
51. H. S. Moon, K. Kwon, S. I. Kim, H. Han, J. Sohn, S. Lee and H. I. Jung: Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP). Lab Chip, 11(6), 1118-25 (2011)
52. F. Fabbri, S. Carloni, W. Zoli, P. Ulivi, G. Gallerani, P. Fici, E. Chiadini, A. Passardi, G. L. Frassineti, A. Ragazzini and D. Amadori: Detection and recovery of circulating colon cancer cells using a dielectrophoresis-based device: KRAS mutation status in pure CTCs. Cancer Lett, 335(1), 225-31 (2013)
53. C. Huang, H. Liu, N. H. Bander and B. J. Kirby: Enrichment of prostate cancer cells from blood cells with a hybrid dielectrophoresis and immunocapture microfluidic system. Biomed Microdevices, 15(6), 941-8 (2013)
54. M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. Terstappen and D. F. Hayes: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med, 351(8), 781-91 (2004)
55. H. I. Scher, X. Jia, J. S. de Bono, M. Fleisher, K. J. Pienta, D. Raghavan and G. Heller: Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol, 10(3), 233-9 (2009)
56. Y. J. Lu, P. Wang, J. Peng, X. Wang, Y. W. Zhu and N. Shen: Meta-analysis Reveals the Prognostic Value of Circulating Tumour Cells Detected in the Peripheral Blood in Patients with Non-Metastatic Colorectal Cancer. Sci Rep, 7(1), 905 (2017)
57. N. Xenidis, M. Ignatiadis, S. Apostolaki, M. Perraki, K. Kalbakis, S. Agelaki, E. N. Stathopoulos, G. Chlouverakis, E. Lianidou, S. Kakolyris, V. Georgoulias and D. Mavroudis: Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breast cancer. J Clin Oncol, 27(13), 2177-84 (2009)
58. E. Andreopoulou, L. Y. Yang, K. M. Rangel, J. M. Reuben, L. Hsu, S. Krishnamurthy, V. Valero, H. A. Fritsche and M. Cristofanilli: Comparison of assay methods for detection of circulating tumor cells in metastatic breast cancer: AdnaGen AdnaTest BreastCancer Select/Detect versus Veridex CellSearch system. Int J Cancer, 130(7), 1590-7 (2012)
59. Z. Ye, C. Wang, S. Wan, Z. Mu, Z. Zhang, M. M. Abu-Khalaf, F. M. Fellin, D. P. Silver, M. Neupane, R. J. Jaslow, S. Bhattacharya, T. N. Tsangaris, I. Chervoneva, A. Berger, L. Austin, J. P. Palazzo, R. E. Myers, N. Pancholy, D. Toorkey, K. Yao, M. Krall, X. Li, X. Chen, X. Fu, J. Xing, L. Hou, Q. Wei, B. Li, M. Cristofanilli and H. Yang: Association of clinical outcomes in metastatic breast cancer patients with circulating tumour cell and circulating cell-free DNA. Eur J Cancer, 106, 133-143 (2019)
60. B. Tong, Y. Xu, J. Zhao, M. Chen, W. Zhong, J. Xing and M. Wang: Prognostic role of circulating tumor cells in patients with EGFR-mutated or ALK-rearranged non-small cell lung cancer. Thorac Cancer, 9(5), 640-645 (2018)
61. X. Wang, K. Ma, Z. Yang, J. Cui, H. He, A. R. Hoffman, J. F. Hu and W. Li: Systematic Correlation Analyses of Circulating Tumor Cells with Clinical Variables and Tumor Markers in Lung Cancer Patients. J Cancer, 8(15), 3099-3104 (2017)
63. B. Groot Koerkamp, N. N. Rahbari, M. W. Buchler, M. Koch and J. Weitz: Circulating tumor cells and prognosis of patients with resectable colorectal liver metastases or widespread metastatic colorectal cancer: a meta-analysis. Ann Surg Oncol, 20(7), 2156-65 (2013)
64. W. Onstenk, W. de Klaver, R. de Wit, M. Lolkema, J. Foekens and S. Sleijfer: The use of circulating tumor cells in guiding treatment decisions for patients with metastatic castration-resistant prostate cancer. Cancer Treat Rev, 46, 42-50 (2016)
65. T. Okegawa, N. Itaya, H. Hara, M. Tambo and K. Nutahara: Circulating tumor cells as a biomarker predictive of sensitivity to docetaxel chemotherapy in patients with castration-resistant prostate cancer. Anticancer Res, 34(11), 6705-10 (2014)
66. Y. Zhang, Y. Lv, Y. Niu, H. Su and A. Feng: Role of Circulating Tumor Cell (CTCs) Monitoring in Evaluating Prognosis of Triple-Negative Breast Cancer Patients in China. Med Sci Monit, 23, 3071-3079 (2017)
67. A. Stathopoulou, M. Ntoulia, M. Perraki, S. Apostolaki, D. Mavroudis, N. Malamos, V. Georgoulias and E. S. Lianidou: A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer, 119(7), 1654-9 (2006)
68. A. Rolle, R. Gunzel, U. Pachmann, B. Willen, K. Hoffken and K. Pachmann: Increase in number of circulating disseminated epithelial cells after surgery for non-small cell lung cancer monitored by MAINTRAC(R) is a predictor for relapse: A preliminary report. World J Surg Oncol, 3(1), 18 (2005)
70. M. Ilie, V. Hofman, E. Long, O. Bordone, E. Selva, K. Washetine, C. H. Marquette and P. Hofman: Current challenges for detection of circulating tumor cells and cell-free circulating nucleic acids, and their characterization in non-small cell lung carcinoma patients. What is the best blood substrate for personalized medicine? Ann Transl Med, 2(11), 107 (2014)
71. B. L. Khoo, S. C. Lee, P. Kumar, T. Z. Tan, M. E. Warkiani, S. G. Ow, S. Nandi, C. T. Lim and J. P. Thiery: Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy. Oncotarget, 6(17), 15578-93 (2015)
72. B. L. Khoo, G. Grenci, T. Jing, Y. B. Lim, S. C. Lee, J. P. Thiery, J. Han and C. T. Lim: Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment. Sci Adv, 2(7), e1600274 (2016)
73. M. Yu, A. Bardia, N. Aceto, F. Bersani, M. W. Madden, M. C. Donaldson, R. Desai, H. Zhu, V. Comaills, Z. Zheng, B. S. Wittner, P. Stojanov, E. Brachtel, D. Sgroi, R. Kapur, T. Shioda, D. T. Ting, S. Ramaswamy, G. Getz, A. J. Iafrate, C. Benes, M. Toner, S. Maheswaran and D. A. Haber: Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science, 345(6193), 216-20 (2014)
74. E. Ozkumur, A. M. Shah, J. C. Ciciliano, B. L. Emmink, D. T. Miyamoto, E. Brachtel, M. Yu, P. I. Chen, B. Morgan, J. Trautwein, A. Kimura, S. Sengupta, S. L. Stott, N. M. Karabacak, T. A. Barber, J. R. Walsh, K. Smith, P. S. Spuhler, J. P. Sullivan, R. J. Lee, D. T. Ting, X. Luo, A. T. Shaw, A. Bardia, L. V. Sequist, D. N. Louis, S. Maheswaran, R. Kapur, D. A. Haber and M. Toner: Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci Transl Med, 5(179), 179ra47 (2013)
75. L. Cayrefourcq, T. Mazard, S. Joosse, J. Solassol, J. Ramos, E. Assenat, U. Schumacher, V. Costes, T. Maudelonde, K. Pantel and C. Alix-Panabieres: Establishment and characterization of a cell line from human circulating colon cancer cells. Cancer Res, 75(5), 892-901 (2015)
76. D. Gao, I. Vela, A. Sboner, P. J. Iaquinta, W. R. Karthaus, A. Gopalan, C. Dowling, J. N. Wanjala, E. A. Undvall, V. K. Arora, J. Wongvipat, M. Kossai, S. Ramazanoglu, L. P. Barboza, W. Di, Z. Cao, Q. F. Zhang, I. Sirota, L. Ran, T. Y. MacDonald, H. Beltran, J. M. Mosquera, K. A. Touijer, P. T. Scardino, V. P. Laudone, K. R. Curtis, D. E. Rathkopf, M. J. Morris, D. C. Danila, S. F. Slovin, S. B. Solomon, J. A. Eastham, P. Chi, B. Carver, M. A. Rubin, H. I. Scher, H. Clevers, C. L. Sawyers and Y. Chen: Organoid cultures derived from patients with advanced prostate cancer. Cell, 159(1), 176-187 (2014)
79. M. Cegan, K. Kolostova, R. Matkowski, M. Broul, J. Schraml, M. Fiutowski and V. Bobek: In vitro culturing of viable circulating tumor cells of urinary bladder cancer. Int J Clin Exp Pathol, 7(10), 7164-71 (2014)
81. V. Bobek, R. Matkowski, R. Gurlich, K. Grabowski, J. Szelachowska, R. Lischke, J. Schutzner, T. Harustiak, A. Pazdro, A. Rzechonek and K. Kolostova: Cultivation of circulating tumor cells in esophageal cancer. Folia Histochem Cytobiol, 52(3), 171-7 (2014)
82. Z. Zhang, H. Shiratsuchi, J. Lin, G. Chen, R. M. Reddy, E. Azizi, S. Fouladdel, A. C. Chang, L. Lin, H. Jiang, M. Waghray, G. Luker, D. M. Simeone, M. S. Wicha, D. G. Beer, N. Ramnath and S. Nagrath: Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model. Oncotarget, 5(23), 12383-97 (2014)
86. E. Bastida, A. Ordinas, S. L. Giardina and G. A. Jamieson: Differentiation of platelet-aggregating effects of human tumor cell lines based on inhibition studies with apyrase, hirudin, and phospholipase. Cancer Res, 42(11), 4348-52 (1982)
87. S. Pinto, L. Gori, O. Gallo, S. Boccuzzi, R. Paniccia and R. Abbate: Increased thromboxane A2 production at primary tumor site in metastasizing squamous cell carcinoma of the larynx. Prostaglandins Leukot Essent Fatty Acids, 49(1), 527-30 (1993)
88. M. Zucchella, L. Dezza, L. Pacchiarini, F. Meloni, F. Tacconi, E. Bonomi, G. Grignani and A. Notario: Human tumor cells cultured "in vitro" activate platelet function by producing ADP or thrombin. Haematologica, 74(6), 541-5 (1989)
93. T. Placke, M. Orgel, M. Schaller, G. Jung, H. G. Rammensee, H. G. Kopp and H. R. Salih: Platelet-derived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells. Cancer Res, 72(2), 440-8 (2012)
94. R. K. Assoian, A. Komoriya, C. A. Meyers, D. M. Miller and M. B. Sporn: Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem, 258(11), 7155-60 (1983)
95. M. Labelle, S. Begum and R. O. Hynes: Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell, 20(5), 576-90 (2011)
96. A. N. Fischer, E. Fuchs, M. Mikula, H. Huber, H. Beug and W. Mikulits: PDGF essentially links TGF-beta signaling to nuclear beta-catenin accumulation in hepatocellular carcinoma progression. Oncogene, 26(23), 3395-405 (2007)
97. C. Lahsnig, M. Mikula, M. Petz, G. Zulehner, D. Schneller, F. van Zijl, H. Huber, A. Csiszar, H. Beug and W. Mikulits: ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression. Oncogene, 28(5), 638-50 (2009)
100. I. Gruber, N. Landenberger, A. Staebler, M. Hahn, D. Wallwiener and T. Fehm: Relationship between circulating tumor cells and peripheral T-cells in patients with primary breast cancer. Anticancer Res, 33(5), 2233-8 (2013)
101. M. Giuliano, A. Shaikh, H. C. Lo, G. Arpino, S. De Placido, X. H. Zhang, M. Cristofanilli, R. Schiff and M. V. Trivedi: Perspective on Circulating Tumor Cell Clusters: Why It Takes a Village to Metastasize. Cancer Res, 78(4), 845-852 (2018)
102. N. Aceto, A. Bardia, D. T. Miyamoto, M. C. Donaldson, B. S. Wittner, J. A. Spencer, M. Yu, A. Pely, A. Engstrom, H. Zhu, B. W. Brannigan, R. Kapur, S. L. Stott, T. Shioda, S. Ramaswamy, D. T. Ting, C. P. Lin, M. Toner, D. A. Haber and S. Maheswaran: Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell, 158(5), 1110-1122 (2014)
103. S. H. Au, B. D. Storey, J. C. Moore, Q. Tang, Y. L. Chen, S. Javaid, A. F. Sarioglu, R. Sullivan, M. W. Madden, R. O'Keefe, D. A. Haber, S. Maheswaran, D. M. Langenau, S. L. Stott and M. Toner: Clusters of circulating tumor cells traverse capillary-sized vessels. Proc Natl Acad Sci U S A, 113(18), 4947-52 (2016)
108. X. Jiang, K. H. K. Wong, A. H. Khankhel, M. Zeinali, E. Reategui, M. J. Phillips, X. Luo, N. Aceto, F. Fachin, A. N. Hoang, W. Kim, A. E. Jensen, L. V. Sequist, S. Maheswaran, D. A. Haber, S. L. Stott and M. Toner: Microfluidic isolation of platelet-covered circulating tumor cells. Lab Chip, 17(20), 3498-3503 (2017)
109. S. L. Stott, C. H. Hsu, D. I. Tsukrov, M. Yu, D. T. Miyamoto, B. A. Waltman, S. M. Rothenberg, A. M. Shah, M. E. Smas, G. K. Korir, F. P. Floyd, Jr., A. J. Gilman, J. B. Lord, D. Winokur, S. Springer, D. Irimia, S. Nagrath, L. V. Sequist, R. J. Lee, K. J. Isselbacher, S. Maheswaran, D. A. Haber and M. Toner: Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A, 107(43), 18392-7 (2010)
110. A. F. Sarioglu, N. Aceto, N. Kojic, M. C. Donaldson, M. Zeinali, B. Hamza, A. Engstrom, H. Zhu, T. K. Sundaresan, D. T. Miyamoto, X. Luo, A. Bardia, B. S. Wittner, S. Ramaswamy, T. Shioda, D. T. Ting, S. L. Stott, R. Kapur, S. Maheswaran, D. A. Haber and M. Toner: A microfluidic device for label-free, physical capture of circulating tumor cell clusters. Nat Methods, 12(7), 685-91 (2015)
111. D. G. Duda, A. M. Duyverman, M. Kohno, M. Snuderl, E. J. Steller, D. Fukumura and R. K. Jain: Malignant cells facilitate lung metastasis by bringing their own soil. Proc Natl Acad Sci U S A, 107(50), 21677-82 (2010)
112. X. J. Min, H. Li, S. C. Hou, W. He, J. Liu, B. Hu and J. Wang: Dysfunction of volume-sensitive chloride channels contributes to cisplatin resistance in human lung adenocarcinoma cells. Exp Biol Med (Maywood), 236(4), 483-91 (2011)
113. Y. F. Sun, W. Guo, Y. Xu, Y. H. Shi, Z. J. Gong, Y. Ji, M. Du, X. Zhang, B. Hu, A. Huang, G. G. Chen, P. B. S. Lai, Y. Cao, S. J. Qiu, J. Zhou, X. R. Yang and J. Fan: Circulating Tumor Cells from Different Vascular Sites Exhibit Spatial Heterogeneity in Epithelial and Mesenchymal Composition and Distinct Clinical Significance in Hepatocellular Carcinoma. Clin Cancer Res, 24(3), 547-559 (2018)
114. M. Yu, A. Bardia, B. S. Wittner, S. L. Stott, M. E. Smas, D. T. Ting, S. J. Isakoff, J. C. Ciciliano, M. N. Wells, A. M. Shah, K. F. Concannon, M. C. Donaldson, L. V. Sequist, E. Brachtel, D. Sgroi, J. Baselga, S. Ramaswamy, M. Toner, D. A. Haber and S. Maheswaran: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science, 339(6119), 580-4 (2013)
115. E. H. Cho, M. Wendel, M. Luttgen, C. Yoshioka, D. Marrinucci, D. Lazar, E. Schram, J. Nieva, L. Bazhenova, A. Morgan, A. H. Ko, W. M. Korn, A. Kolatkar, K. Bethel and P. Kuhn: Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors. Phys Biol, 9(1), 016001 (2012)
116. X. Luo, D. Mitra, R. J. Sullivan, B. S. Wittner, A. M. Kimura, S. Pan, M. P. Hoang, B. W. Brannigan, D. P. Lawrence, K. T. Flaherty, L. V. Sequist, M. McMahon, M. W. Bosenberg, S. L. Stott, D. T. Ting, S. Ramaswamy, M. Toner, D. E. Fisher, S. Maheswaran and D. A. Haber: Isolation and molecular characterization of circulating melanoma cells. Cell Rep, 7(3), 645-53 (2014)
117. G. Kats-Ugurlu, I. Roodink, M. de Weijert, D. Tiemessen, C. Maass, K. Verrijp, J. van der Laak, R. de Waal, P. Mulders, E. Oosterwijk and W. Leenders: Circulating tumour tissue fragments in patients with pulmonary metastasis of clear cell renal cell carcinoma. J Pathol, 219(3), 287-93 (2009)
119. B. Brandt, R. Junker, C. Griwatz, S. Heidl, O. Brinkmann, A. Semjonow, G. Assmann and K. S. Zanker: Isolation of prostate-derived single cells and cell clusters from human peripheral blood. Cancer Res, 56(20), 4556-61 (1996)
120. M. G. Krebs, J. M. Hou, R. Sloane, L. Lancashire, L. Priest, D. Nonaka, T. H. Ward, A. Backen, G. Clack, A. Hughes, M. Ranson, F. H. Blackhall and C. Dive: Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches. J Thorac Oncol, 7(2), 306-15 (2012)
121. J. M. Hou, M. G. Krebs, L. Lancashire, R. Sloane, A. Backen, R. K. Swain, L. J. Priest, A. Greystoke, C. Zhou, K. Morris, T. Ward, F. H. Blackhall and C. Dive: Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol, 30(5), 525-32 (2012)
122. V. Murlidhar, R. M. Reddy, S. Fouladdel, L. Zhao, M. K. Ishikawa, S. Grabauskiene, Z. Zhang, J. Lin, A. C. Chang, P. Carrott, W. R. Lynch, M. B. Orringer, C. Kumar-Sinha, N. Palanisamy, D. G. Beer, M. S. Wicha, N. Ramnath, E. Azizi and S. Nagrath: Poor Prognosis Indicated by Venous Circulating Tumor Cell Clusters in Early-Stage Lung Cancers. Cancer Res, 77(18), 5194-5206 (2017)
123. M. T. d. M. Diaz, E. A. Abdallah, M. S. Tariki, A. C. Braun, A. L. A. Dettino, U. R. Nicolau, V. d. S. Alves and L. T. D. Chinen: Circulating tumor cells as marker of poor prognosis in metastatic lung cancer: a pilot study. Applied Cancer Research, 38(1), 8 (2018)
124. C. Paoletti, Y. Li, M. C. Muniz, K. M. Kidwell, K. Aung, D. G. Thomas, M. E. Brown, V. G. Abramson, W. J. Irvin, Jr., N. U. Lin, M. C. Liu, R. Nanda, J. R. Nangia, A. M. Storniolo, T. A. Traina, C. Vaklavas, C. H. Van Poznak, A. C. Wolff, A. Forero-Torres and D. F. Hayes: Significance of Circulating Tumor Cells in Metastatic Triple-Negative Breast Cancer Patients within a Randomized, Phase II Trial: TBCRC 019. Clin Cancer Res, 21(12), 2771-9 (2015)
125. S. Jansson, P. O. Bendahl, A. M. Larsson, K. E. Aaltonen and L. Ryden: Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort. BMC Cancer, 16, 433 (2016)
127. A. Calles, B. Rubio-Viqueira and M. Hidalgo: Primary human non-small cell lung and pancreatic tumorgraft models--utility and applications in drug discovery and tumor biology. Curr Protoc Pharmacol, Chapter 14, Unit 14 26 (2013)
128. M. Hidalgo, F. Amant, A. V. Biankin, E. Budinska, A. T. Byrne, C. Caldas, R. B. Clarke, S. de Jong, J. Jonkers, G. M. Maelandsmo, S. Roman-Roman, J. Seoane, L. Trusolino and A. Villanueva: Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov, 4(9), 998-1013 (2014)
129. M. Ilie, M. Nunes, L. Blot, V. Hofman, E. Long-Mira, C. Butori, E. Selva, A. Merino-Trigo, N. Venissac, J. Mouroux, P. Vrignaud and P. Hofman: Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps. Cancer Med, 4(2), 201-11 (2015)
130. W. C. Anderson, M. B. Boyd, J. Aguilar, B. Pickell, A. Laysang, M. A. Pysz, S. Bheddah, J. Ramoth, B. C. Slingerland, S. J. Dylla and E. R. Rubio: Initiation and characterization of small cell lung cancer patient-derived xenografts from ultrasound-guided transbronchial needle aspirates. PLoS One, 10(5), e0125255 (2015)
131. T. L. Leong, K. D. Marini, F. J. Rossello, S. N. Jayasekara, P. A. Russell, Z. Prodanovic, B. Kumar, V. Ganju, M. Alamgeer, L. B. Irving, D. P. Steinfort, C. D. Peacock, J. E. Cain, A. Szczepny and D. N. Watkins: Genomic characterisation of small cell lung cancer patient-derived xenografts generated from endobronchial ultrasound-guided transbronchial needle aspiration specimens. PLoS One, 9(9), e106862 (2014)
134. C. L. Hodgkinson, C. J. Morrow, Y. Li, R. L. Metcalf, D. G. Rothwell, F. Trapani, R. Polanski, D. J. Burt, K. L. Simpson, K. Morris, S. D. Pepper, D. Nonaka, A. Greystoke, P. Kelly, B. Bola, M. G. Krebs, J. Antonello, M. Ayub, S. Faulkner, L. Priest, L. Carter, C. Tate, C. J. Miller, F. Blackhall, G. Brady and C. Dive: Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med, 20(8), 897-903 (2014)
139. H. M. Shapiro, E. R. Schildkraut, R. Curbelo, C. W. Laird, B. Turner and T. Hirschfeld: Combined blood cell counting and classification with fluorochrome stains and flow instrumentation. J Histochem Cytochem, 24(1), 396-401 (1976)
140. M. Hosokawa, M. Asami, S. Nakamura, T. Yoshino, N. Tsujimura, M. Takahashi, S. Nakasono, T. Tanaka and T. Matsunaga: Leukocyte counting from a small amount of whole blood using a size-controlled microcavity array. Biotechnol Bioeng, 109(8), 2017-24 (2012)