Circulating tumor cells: a review of Non-EpCAM-based approaches for cell enrichment and isolation.
CONTENT: Most current methods are based on epithelial cell adhesion molecule (EpCAM) detection, but numerous studies have demonstrated that EpCAM is not a universal marker for CTC detection because it fails to detect both carcinoma cells that undergo epithelial-mesenchymal transition (EMT) and CTCs of mesenchymal origin. Moreover, EpCAM expression has been found in patients with benign diseases. A large proportion of the current studies and reviews about CTCs describe EpCAM-based methods, but there is evidence that not all tumor cells can be detected using this marker. Here we describe the most recent EpCAM-independent methods for enriching, isolating, and characterizing CTCs on the basis of physical and biological characteristics and point out the main advantages and disadvantages of these methods.
SUMMARY: CTCs offer an opportunity to obtain key biological information required for the development of personalized medicine. However, there is no universal marker of these cells. To strengthen the clinical utility of CTCs, it is important to improve existing technologies and develop new, non-EpCAM-based systems to enrich and isolate CTCs.
Circulating tumor cells (CTCs)  are defined as cells that originate in primary tumors, recurrences, or metastases. They circulate freely in peripheral blood and have antigenic and genetic characteristics specific to the tumor of origin (1). CTCs are important because the majority of deaths from cancer are linked to the development of disseminated metastases (2). In the last few years, emerging data have challenged the traditional theory of sequential metastasis development (3) (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol62/issue4). Several studies have pointed out that CTCs can be isolated in patients at relatively early stages of tumor growth (4, 5), even before the primary tumor mass is detected by conventional methods (6). Furthermore, current high-resolution imaging technology is not sensitive enough to detect micrometastases or early tumor cell dissemination, which are the key events in tumor progression (see online Supplemental Fig. 1).
Because they can be obtained by noninvasive methods, CTCs can be used as therapeutic markers for monitoring treatment effectiveness in real time and for detecting recurrent disease. CTCs also have potential for use in evaluating drug resistance mechanisms and may have utility in estimation of the risk of metastatic relapse and progression. Unlike the characterization of primary tumors, which provides only a static view at the time of diagnosis, analyzing CTCs may improve understanding of the different steps involved in the metastatic cascade, from invasion of tumor cells into the bloodstream to the formation of clinically detectable metastases (7).
Although studying circulating tumor cells is a promising approach for better characterizing cancer, there are certain issues inherent to the nature of CTCs that should be considered. CTCs are rare events, which are present at very low concentrations in the blood (i.e., one tumor cell in a background of millions of blood cells) (8). In addition, only a restricted number of CTCs has the ability to generate metastases (9) and consequently it is necessary to characterize them precisely to be able to distinguish metastatic and nonmetastatic CTCs. Numerous methods have been developed to isolate tumor cells, most of which are based on epithelial cell adhesion molecule (EpCAM) detection. Indeed, EpCAM is a conventional marker expressed by cancer cells of epithelial origin and has been used for carcinoma cell isolation. However, as described below, EpCAM is not expressed by all CTCs and alternative approaches need to be considered. There are multiple recent useful reviews on CTC isolation methods (10-12) but none of them have exclusively focused on non-EpCAM-based methods. The aim of this review is to provide an overview of the most recent EpCAM-independent methods for enriching, isolating, and characterizing CTCs.
EpCAM Is Not a Universal Biomarker for Isolating CTCs
Extensive effort and resources have been invested into developing methods for detecting CTCs in peripheral blood. In the last decade, several methods have emerged for detecting and characterizing CTCs. However, these methods and consequently the biological characterization of CTCs are still technically challenging. The first step in the detection of CTCs was the discovery that EpCAMs were expressed at variable degrees on epithelial-derived carcinomas and related cancers but were absent in peripheral blood cells (13). This finding resulted in the investigation and development of different methods for enriching and isolating CTCs on the basis of the EpCAM marker (14, 15) and led to the first and only automated EpCAM-based system (CellSearch[R]) currently approved for clinical use by the US Food and Drug Administration for the detection of CTCs. CellSearch is thus considered the gold standard for CTC detection methods (16, 17). However, recent evidence has challenged the suitability of this method; EpCAM-positive circulating epithelial cells have been reported in patients with benign colon diseases (18) and are a potential source of false-positive findings. In addition, carcinoma cells can undergo epithelial-mesenchymal transition (EMT), which results in decreased expression of epithelial markers, such as EpCAM and cytokeratin (19), and the appearance of mesenchymal markers. The loss of epithelial markers may therefore result in false-negative findings. In this context, the EpCAM marker is not suitable for isolating CTCs from carcinomas that have undergone EMT or those cancers with primary mesenchymal origin. Consequently EpCAM cannot be considered a universal marker for CTC detection. This highlights the need to develop non-EpCAM-based technologies for isolating and detecting CTCs.
Enrichment of Circulating Tumor Cells: Conventional Methods
The major challenge for isolating and characterizing CTCs is their low concentration compared to the other cell types in the peripheral blood. Enrichment approaches take into consideration several parameters: capture efficiency/recovery rate, purity, cell viability, processing speed, blood sample capacity, sample preprocessing requirements, cost of consumables and equipment, repeatability, and reliability. The optimal enrichment solution may require a compromise between these performance parameters and the intended downstream application. Current enrichment approaches include a wide range of technologies based on the different properties of CTCs that distinguish them from surrounding normal hematopoietic cells, including biological properties (cell surface protein expression, viability, invasive capacity) and physical properties (size, density, electric charges, deformability) (Fig. 1).
METHODS BASED ON PHYSICAL PROPERTIES
Cytological analyses have revealed that CTCs exhibit a greater nucleus-to-cytoplasm ratio, are larger in size, and have different nuclear morphology than normal cells (20). These cytological alterations result in the differences of their mechanical properties, providing CTCs with several capabilities. The cytoskeletal stiffness of CTCs is dynamically modified. This flexibility may facilitate their invasion to distal sites from the primary tumor and may confer their resistance to damage from fluid shear stress within the blood vessels during the metastatic process (21). These modifications in the stiffness alter the conservation of the membrane structure, which in turn affects their surface charge and electrical properties (21). Various approaches have been used to exploit the differences in physical properties between tumor cells and blood cells as a means of enriching and separating CTCs from blood samples (Fig. 1).
Density gradient centrifugation is a conventional approach for separating blood components on the basis of differences in their sedimentation coefficients. As whole blood is deposited in the liquid gradient and subjected to centrifugation, cells will distribute along the gradient depending on their density (Fig. 1, Table 1). Erythrocytes or polymorphonuclear leukocytes migrate to the bottom, whereas mononuclear leukocytes and CTCs remain at the top as a buffy coat (22). Percoll, Ficoll-Hypaque[R] (GE Healthcare Life Sciences), and OncoQuick[R] (Greiner Bio-One) are the most commonly used density gradient media in preclinical and clinical research. Ficoll-HyPaque, formed by the copolymerization of sucrose and epichlorohydrin, is mainly used in biology laboratories to recover peripheral blood mononuclear cells. Despite its long history of use in lab oratories, there are some pitfalls associated to this technique, such as the possible loss of tumor cells that migrate either to the plasma fraction or to the bottom of the gradient due to the formation of aggregates (22). It has been suggested that this cell loss may be due to the cytotoxicity of the density medium (23). An alternative to Ficoll is Percoll density (GE Healthcare Life Sciences) gradient medium made of a colloidal silica particle suspension. The main advantages over Ficoll include reduced toxicity and a wider density gradient range (23). There are certain discrepancies in the literature regarding the use of Percoll because some studies have demonstrated a high purity rate (24) while others have shown low isolation efficiencies compared to Ficoll (25). A third density system named OncoQuick is composed of a 50-mL tube with a porous barrier inserted above a separation medium. Cells are separated and pass through the barrier depending on their different buoyancy densities during centrifugation. CTCs, together with lymphocytes, will remain above the porous barrier, making them easily accessible for subsequent collection. OncoQuick has a mildly higher reported recovery rate compared to that of Ficoll density gradient (87% and 84%, respectively) (26). Moreover, the mononuclear cell depletion obtained using the OncoQuick system is significantly higher than that of Ficoll; this facilitates processing of higher sample volumes, which is beneficial for CTC characterization (26, 27). However, during the isolation process, CTCs migrate into the plasma fraction and are frequently lost (28). Overall, the major advantages of all the density centrifugation methods are that they are inexpensive and reliable (Table 1). However, the disadvantages include the loss of large CTCs and CTC aggregates that fall to the bottom (29), as well as the fact that leukocytes cannot easily be eliminated, resulting in very low purity. It is therefore necessary to combine centrifugation with another enrichment method.
Microfiltration enrichment methods process circulating cells through an array of microscale constrictions to capture target cells on the basis of their size or a combination of size and deformability. There are multiple different microfiltration devices; some are available on the market and others currently remain prototypes (Fig. 2).
Membrane microfilters are composed of a semipermeable membrane with a 2D array of micropores. A membrane with a pore size diameter of 8 [micro]m has been demonstrated to be optimal for CTC retention (30). The typical configuration used for microfiltration is dead-end filtration (Fig. 2A), in which the blood flow is perpendicular to the membrane. The main limitation of this strategy is that the layer of cells retained on the membrane can reduce the efficiency of recovery due to the buildup of filtration resistance (31) (Table 1). To overcome this issue, Zheng et al. created a 3D membrane microfilter consisting of 2 pored layers (Fig. 2B), between which CTCs are retained (32). In contrast to conventional microfiltration devices, this system reduces the tension stress on the cell plasma membrane and demonstrates a high recovery rate (86% with a theoretically fast throughput of 3.75 mL/min) (32).
Another system based on a 2D membrane slot filter (Fig. 2C) was proposed by Lu et al., in which the forces exerted on the cells are reduced, reaching viability of 90% with a high recovery (33). The bead-packed filtration device consists of a chamber where uniform beads measuring 45 [micro]m in diameter and nonuniform beads (with diameters ranging from 15 to 100 [micro]m) are packed and act as the filtration element (Fig. 2D) that retains CTCs and allows red and white blood cells to pass through (33). Studies performed by Lin et al. demonstrated a low recovery rate (between 21% and 40%) in contrast to filtration performed using membrane systems (34).
There are systems available that make it possible to enrich and isolate CTCs in a single step. For example, ScreenCell[R] technology (ScreenCell), is an innovative single-use and low-cost device. It is based on a filter that isolates and sorts tumor cells by size. There are 3 different types of device, depending on the downstream analysis: ScreenCell Cyto (molecular techniques that require fixed cells), ScreenCell CC (cell culture), and ScreenCell MB (RNA or DNA analysis) (35, 36). The main advantages of this system are its low cost, small format, and ease of use. Another platform in development is the parylene-C slot microfilter that measures telomerase activity from captured, viable CTCs. It has a 90% recovery rate (36). The 90% of cells recovered are viable and yield 200-fold sample enrichment (36). In contrast to ScreenCell, parylene-C detects only viable CTCs and can be reused.
Filtration allows for rapid CTC enrichment from large volumes of blood in minutes, with minimal processing. Recovery rates are around 90%, but further processing is required for certain downstream applications, because the final purity is typically around 10% or less. The main disadvantages associated with filtration are the heterogeneity in CTC size, cluster formation, the possibility of membrane clogging, and difficulties in the detachment of cells retained in the filter, as well as the background signal on the filters after immunostaining for CTC detection.
Microfluidics includes several separation methods, which makes it possible to manipulate very small volumes of biological fluids. The past decade has seen many new technologies proposed for biological cell sorting and analysis on microchips. Arrays with pillars of varying geometries have been used to fractionate cells in blood and capture tumor cells (37). Similarly, crescent-shaped trap arrays with a fixed 5-[micro]m gap width within microfluidic chambers have been used to enrich CTCs from whole blood without preprocessing (38). The Parsortix system (Angle) (Fig. 3A) is microfluidic technology that captures CTCs based on their less deformable nature and larger size compared to other blood components. With this system, it has been reported that a higher number and purity of isolated CTCs in patient samples were obtained than with the Cell Search. Moreover, the processing time of 7.5 mL of whole blood is 2 h in contrast to the 4 h reported for the Cell Search (Cell Search, Jansen Diagnostic). It is worth noting that with the second version of Parsortix 10 mL can be processed in 2.5 h. The main drawback of this technique is the difficulty of eliminating all leukocytes owing to size overlap with CTCs (39).
In addition to the previous devices described above, ClearCell[R] FX (Clearbridge Biomedics) recovers viable cells in small sample volumes and in a short period of time (e.g., 1 mL of blood in 10 min) (Fig. 3B) (40). ClearCell FX does not require preprocessing of the blood; this decreases the possibility of losing cells of interest (Table 1). This system takes advantage of the inertial and centrifugal forces causing the smaller red and white blood cells to flow along the channel's outer wall and the larger CTCs to flow along the inner wall, recovering both fractions in different channels of the system. Unfortunately, CTCs of different sizes may escape through the white/red cell channels, and certain white blood cells can be captured in the CTC fraction.
To limit CTC loss to white and red cell channels, CTC-iChip technology (D.A. Harber, Massachusetts General Hospital Cancer Center; M. Toner, Harvard Medical School, Boston, MA) was developed (Fig. 3C). CTC-iChip technology combines continuous deterministic lateral displacement for size-based separation of red blood cells/platelets from tumor cells obtained from whole blood, inertial focusing for precise positioning of cells in a microchannel, and microfluidic magnetophoresis for immunomagnetic depletion of white blood cells. This system allows the rapid recovery of any viable cancer cell types, which are accordingly available for characterization. It can process 8 mL of blood per hour. Unfortunately, in the deterministic lateral displacement step, small CTCs are lost, and undesired large cells and aggregates pass on to the next step due to particle deformability and can limit the usefulness of this device (41).
Carefully applied microfluidic approaches are capable of achieving both excellent purity of more than 80% and high recovery rates with little disturbance to the CTCs. However, these advantages come at the expense of lower throughput requiring either reduced sample volumes or prolonged periods of time to process samples (e.g., several hours to process a full tube of blood).
Dielectrophoresis (DEP) was initially described by Pohl as "the translational motion of neutral matter caused by polarization effects in a nonuniform electric field" (42). To move a particle by DEP, the particle must be polarizable once an electrical field is applied (43). This phenomenon has inspired new approaches for the separation of cells on the basis of their electrical properties. Because the DEP force is inversely proportional to the length scale (44), microscale chambers named microchips have been developed for isolating rare cell events. These microchips integrate arrays of electrodes to generate a nonuniform alternating current field characteristic of the DEP technology.
Interdigitated gold electrodes have been used to separate cancer cells from blood cells (45). Tumor cells were attracted toward the electric field generated by the electrodes by means of positive DEP, whereas other cells were flushed away. When the electric field was turned off, the cells initially retained were released and recovered with an approximate rate of 95%. Moon et al. created a system with a DEP module integrated into a size-based hydrodynamic step, used as the enrichment stage to remove excess blood cells (46). The first commercial instrument based on DEP field flow fractionation was the ApoStream[TM] system (ApoCell) (Fig. 4A). To use this methodology, an initial enrichment step is required. Re covery rate is over 70% and the viability more than 97%; however, the purity obtained is <1%, although this can be significantly improved with additional enrichment stages at the risk of reduced recovery rate (47). The DEPArray[TM] technology (Silicon Biosystems) combines the ability to manipulate individual cells using DEP technology with high-quality image-based cell selection (Fig. 4B). The most attractive characteristics of this technology are the single-cell resolution, high-fidelity recovery, cell viability and, in the most recent version, the possibility of isolating individual cells from paraffin-embedded samples (48).
Despite the many advantages presented by DEP-based enrichment methods, there are also some limitations, such as low sample volumes that are processed in a noncontinuous manner (Table 1). Furthermore, the dielectric characteristics of cells can gradually change due to ion leakage; this requires the isolation to be completed within a short period of time after the sample processing starts (49). In addition, the electric conductivity of the medium used must be low, which is not achievable for all samples studied.
METHODS BASED ON BIOLOGICAL PROPERTIES
Antibody-based CTC isolation takes advantage of highly specific affinity reactions between capture antibodies and the target antigens present on the cells of interest. CTCs can be captured directly (positive selection) or indirectly (negative selection). Various antigens have been used to detect or isolate CTCs. The most commonly used antibody is EpCAM, because it is expressed in all epithelial cells but is absent from blood cells (13, 50). However, the universality of EpCAM may be reduced when carcinoma cells have undergone the EMT process or when detecting tumor cells of mesenchymal origin. Results from our laboratory have revealed the presence of EpCAM-expressing and -nonexpressing CTCs after the injection of either EpCAM-expressing or -nonexpressing tumoral cells in mouse paratibias (see online Supplemental Fig. 2). Several organ- or tumor-specific markers, such as CEA (carcinoembryonic antigen), EGFR (epidermal growth factor receptor), PSA (prostate-specific antigen), HER-2 (human epidermal growth factor receptor 2), MUC-1 (mucin 1), EphB4 (ephrin type-B receptor 4), IGF-1R(insulin-like growth factor 1 receptor), cadherin 11, and CSV (cell-surface vimentin) have also been reported for antibody-based isolation of CTCs (see online Supplemental Table 1).
Immunoaffinity-based CTC isolation is based on antibody-conjugated magnetic nanoparticles or microbeads that often bind to a specific surface antigen (51, 52). After antigen-antibody interaction, the sample is exposed to a nonuniform magnetic field to capture labeled cells. This method can attain high recovery and purity rates, with single-step detection and isolation of CTCs (51, 53). The performance of the immunomagnetic method depends directly on both the expression and specificity of the target antigen, as well as on the binding quality of the associated antibody, the efficiency of the immunomagnetic labeling process and magnetic particles, and the separation mechanism designed to isolate labeled cells. A "cocktail" of antibodies targeting multiple antigens can also be used to partially overcome the lack of specificity of current tumor markers (51, 54). Another approach is negative isolation of CTCs by first lysing erythrocytes and then using specific markers to magnetically deplete leukocytes. CD45 is the most frequently used marker for leukocyte depletion. The RosetteSep[R] (STEMCELL Tech) is a CTC-negative selection system based on a mixture of antibodies that specifically crosslink red blood cells to each other and to white blood cells, forming cell rosettes consisting of multiple red and white blood cells. Due to the higher density of these clusters, they can effectively be separated from CTCs by a single centrifugation step. Negative selection methods are completely independent with regard to CTC phenotype, so they are not biased by a particular CTC marker. Negative selection also leaves CTCs untouched, which may result in higher viability. To achieve an acceptable degree of CTC purity, this separation method requires a very high specificity to remove all the leukocytes and needs to avoid nonspecific CTC binding. The binding between primary antibody and magnetic particles can be a direct (single-step) or indirect (2-step) method. The latter is composed of secondary antibodies that are already bound to magnetic particles and can specifically bind to an epitope on the primary antibody, potentially reaching higher labeling efficiency. This indirect approach shows a 15-fold increase in labeling efficiency compared to direct methods (55).
Regarding the use of the magnetic separation procedure to recover labeled cells, there are many different alternatives. In the batch separation approach, the whole labeled sample is subjected to a magnetic field at once, resulting in the migration of labeled cells to the regions of higher magnetic frequency (56). The EasySep[TM] system (STEMCELL Tech), MojoSort[TM] (Biolegend), and Dynabeads[R] (ThermoFisher) are based on this principle. Variations of these systems have been developed to increase the processed volume. Thus, continuous-flow separation can be used in which the sample is continuously fed through the separation module. This module can have an activated filter to capture and retain the labeled cells, like the commercially available MACS[R] (Miltenyi Biotec) and MagniSort[TM] (eBioscience). Alternatively, the magnetophoresis mode can be used to selectively manipulate the direction of labeled cells within the flow and collect them at designated outputs (57, 58). Reported recovery rates using these magnetic enrichment systems have shown significant variations (10%--90%) (59 60). This variation can be explained because the magnetic gradient generated by the separation structure can only attract labeled cells within a limited distance. The MagSweeper[R] system is a proposed (Fig. 5A) solution (61) that uses a robotic arm equipped with a magnetic rod that binds labeled cells. This was initially demonstrated for the recovery of EpCAM-positive cells but can be adapted for other CTC markers. Recovery rates of 60% using this device have been reported (54).
Microscale separation devices have also been developed. Isolation efficiency in an immunomagnetic microfluidic chip is mainly governed by an equilibrium between hydrodynamic and magnetic forces acting on the labeled cells (62). Hoshino et al. described an immunomagnetic capture system for CTCs based on a microchannel on top of a stack of permanent magnets (Fig. 5B). As the sample flows into the microchannel, the magnetic gradient attracts the labeled cells. Recovery rates around 86% have been attained with this system (63).
Finally, CTCs can be recovered using adhesion-based methods that exploit the ability of CTCs to bind to a surface whose biochemical and topographical properties have been modified without the need to label the cells. In static adhesion-based assays, the sample is first incubated on the capture surface. Nonadherent, supposedly nontarget cells are washed off, leaving the CTCs attached to the surface. On the basis of this approach, the cell adhesion matrix has been used to detect and isolate the most invasive CTCs from patients with metastatic and local carcinomas of different origins (64, 65). Microfluidic adhesion-based devices consist of microchannels coated with an antibody against CTCs. Their design determines both the efficiency of the cell binding and the recovery rate by influencing the flow rate (66, 67). Among these devices, the OnQChip[TM] (On-Q-ity) and the CEE[TM] chip (Biocept Laboratories) are 2 commercialized microfluidic devices that have incorporated 3D structures (microposts) to increase the effective surface, thus promoting cell adhesion (Fig. 5C). The first com bines antibody affinity and size selection for the capture of CTCs and the second is based in immunoaffinity. In this field, Hughes et al. have developed, for instance, a microfluidic system based on the binding of E-selectin, a molecule present in the endothelium on to which CTCs adhere before their extravasation (68). This approach has attained high flow rates compared to the other adhesion-based methods (4.8 mL/h) and approximately 50% capture efficiency. Interestingly, this device demonstrated higher efficiency than the CellSearch system on the basis of the number of CTCs isolated (68).
We have highlighted the non-EpCAM-based methods for CTC enrichment/isolation. The major advantage of these techniques is that they can enrich for CTCs that do not have EpCAM expression. However, many challenges associated with current methodologies must be faced, such as the need to improve purity and recovery rates, throughput, cell viability after recovery, and enrichment rates.
It would be beneficial to identify properties exclusive to CTCs, which may take the form of a single "master" marker or a combination of antibodies able to recognize all the CTCs present in the sample. Moreover, it would be desirable if those properties or markers were able to distinguish between metastatic and nonmetastatic CTCs. Unfortunately, current knowledge does not make it possible to clearly identify and classify CTCs. This information most important for clinical use, determining the prognosis of the disease, making treatment decisions, and assessing the effectiveness of the treatment applied.
Despite the numerous methods for isolating CTCs described in the literature, some are still at the proof-of concept stage with evidence only in cultured cells. The main drawback is that cell lines do not effectively reflect CTCs in a natural biological fluid, especially in terms of heterogeneity (69). It would be interesting to develop new cell lines that exhibit the genomic and transcriptomic heterogeneity of cancer cell lines. Recently, Cayrefourcq et al. (70) and Yu et al. (71) reported the isolation of CTCs and their growth in culture for the establishment of a cell line to examine tumor heterogeneity. Another important point is the necessary sample volume required for CTC isolation. In most cases, the inability to process whole blood is due to high cell concentration or the necessity for reducing sample volume because of the device's limited capacity. A frequently proposed solution is the dilution of samples; however, dilution is not ideal because it reduces the probability of CTC capture and the prolonged enrichment time compromises cell viability. In addition, the biological characteristics of the cells can be altered by the composition of the dilution buffer.
With the use of immunologically based enrichment methods, the wide range of phenotypes presented by CTCs make it necessary to use specific cocktails for cell surface epithelial and mesenchymal markers that do not cross-react with other blood cells (72). Yokobori et al. described plastin 3 as a good alternative for avoiding the use of large cocktails of antibodies, because this marker is not downregulated in CTCs during their EMT and is not expressed in blood cells (73). Although positive selection is very specific and a high purity can be obtained, the presence of some uncharacterized CTCs in each individual blood sample should be taken into consideration. This can be avoided by negative selection, in which the blood sample is depleted of leukocytes by use of antibodies against CD45 and other leukocyte antigens (not expressed on carcinomas or other solid tumors). However, [cytokeratin.sup.+] and [CD45.sup.+] subcell populations have been described and may be related to various artifacts such as cell doublets or nonspecific antibody bindings (74) or circulating cancer-associated macrophage-like cells (75). The role of EMT in tumor cell dissemination stimulates the development of technologies based on the depletion of normal [CD45.sup.+] hematopoietic cells to limit loss of CTCs with high phenotypic plasticity. However, it should be noted that not all [CD45.sup.-] cells in the blood are tumor cells (e.g., circulating endothelial cells) (11).
In the last decade, the strong interest in CTCs has accelerated the development of numerous isolation technologies based on EpCAM-independent methods. Technologies based on physical approaches (density gradient centrifugation, microfiltration, mircofluidics, DEP) or biological properties of CTCs (e.g., membranous markers) have been demonstrated. However, further improvements in preenrichment steps will enhance methods for the capture and characterization of these cells.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: This paper was written as a part of research project which received funding from the Seventh Framework Program [(FP7/ 2007-2013) under grant agreement no. 264817--BONE-NET and thisworkwassupportedbythe Fondation de France (Engt no. 16390)]. Expert Testimony: None declared.
Patents: None declared.
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Marta Tellez Gabriel, [1,2] Lidia Rodriguez Calleja, [1,2] Antoine Chalopin, [1,2,3] Benjamin Ory, [1,2] and Dominique Heymann [1,2,3,4] ***
 INSERM, UMR 957, Equipe LIGUE Nationale Contre le Cancer 2012, Nantes, France;  Universite de Nantes, Nantes Atlantique Universities, Pathophysiology of Bone Resorption and Therapy of Primary Bone Tumours, Nantes, France;  CHU de Nantes, Nantes, France;  Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.
* Address correspondence to this author at: University of Sheffield, Department of Oncology and Metabolism, The Medical School, Beech Hill Rd., S10 2RX England. E-mail: email@example.com.
Received September 22, 2015; accepted January 4, 2016.
Previously published online at DOI: 10.1373/clinchem.2015.249706
 Nonstandard abbreviations: CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; EMT, epithelial-mesenchymal transition; DEP, dielectrophoresis.
Caption: Fig. 1. Methods for CTC isolation from whole blood. 1. Methods based on biological properties. Immunologic approaches are used for targeting specific markers for selective CTC enrichment and leukocyte depletion. 2. Physical properties such assize, deformability, density, and electrical properties can also be used to separate CTCs from blood cells.
Caption: Fig. 2. Microfiltration devices for CTC enrichment. (A), Dead-end filtration. (B) 3D membrane microfilter. The smaller cells can easily traverse the gap while the large cells (e.g., tumor cells) are trapped. Two types of force are exerted in the trapped cell such that force is caused by hydrodynamic pressure from the top and supporting force from the bottom membrane. (C), 2D membrane slot filter design. (D), Bead pack-based filtration. The microchannel entrance is blocked by packing large-sized beads. Different bead sizes are used to implement a blood/plasma separator at the inlet of the microchannel. When whole blood is dropped into the inlet of the microchannel, the structure allows for the capillary flow of blood through the hetero-packed beads. During this movement of blood, the red blood cells passthrough small pores while large cells such as CTCs are blocked from flowing into the channel.
Caption: Fig. 3. Microfluidic devices for isolating CTCs. (A), Parsortix (Angle). The patented microfluidic technology inside a cassette captures CTCs on the basis of their lower deformability and larger size compared to other blood components. Left diagram, plan view; right diagram, cross section showing details of the device. (B), ClearCell FX (Clearbridge Biomedics). The inertial and centrifugal forces transport the smaller red and white blood cells along the channel's outer wall and the larger CTCs along the inner wall, recovering both fractions in different channels of the system. (C), CTCi-chip technology combines continuous deterministic lateral displacement for size-based separation of blood cells, inertial focusing for precise positioning of cells in a microchannel, and microfluidic magnetophoresis for immunomagnetic depletion of white blood cells.
Caption: Fig. 4. DEP-based approaches. (A), ApoStream from ApoCell [adapted from (47)]; (B), DepArray[TM] technology from Silicon Biosystems (http://www.siliconbiosystems.com/ deparray-system). Reproduced with permission from Silicon Biosystems.
Caption: Fig. 5. Antibody-based CTC isolation approaches. (A), MagSweeper [Fig. adapted with permission from (61)]. Magnetic beads were coated with an antibody targeting surface markers and mixed into blood samples to bind cancer cells, which are captured with the magnetic rod. After several washings, the cells are extracted using a magnetic source. (B), Microchip-based immunomagnetic assay. The sample is pumped in continuously through the microchannel, causing noncaptured blood cells to exit the chip, whereas CTCs are retained due to the magnetic force. (C), Diagram representation of OnCChip[TM] (On-Q-ity) and CEE (Biocept) devices. These cell enrichment technologies exploit the placement of posts and flow rates through mathematical modeling to enhance isolation and capture of CTCs within a microfluidic channel.
Table 1. Major advantages and disadvantages of CTC enrichment methods. Method Advantages Disadvantages Density gradient Inexpensive Loss of large centrifugation Reliable CTCs and cell aggregates Low purity Additional enrichment techniques required Microfiltration Rapid processing of Low purity large volumes Membrane clogging High efficiency Different size of CTCs Difficult to detach CTCs from the filter Microfluidics Excellent purity Long, time- High capture rates consuming process Little cell disturbance Sample preprocessing requirement to reduce volume Dielectrophoresis Single cell isolation Limited volume High cell viability Low purity in High efficiency some devices Cell electrical properties can be affected during the procedure Large number of parameters must be controlled simultaneously Immunoaffinity- High recovery Lack of cancer- based methods High purity rates specific markers High cell viability Heterogeneous using negative expression of selection markers in cells Problems with the antibody affinity or specificity
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|Title Annotation:||epithelial cell adhesion molecule|
|Author:||Gabriel, Marta Tellez; Calleja, Lidia Rodriguez; Chalopin, Antoine; Ory, Benjamin; Heymann, Dominiqu|
|Date:||Apr 1, 2016|
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