Size-based and non-affinity based microfluidic devices for circulating tumor cell enrichment and characterization | ПРЕЦИЗИОННАЯ ОНКОЛОГИЯ

Size-based and non-affinity based microfluidic devices for circulating tumor cell enrichment and characterization


Circulating tumor cells (CTC) are tumor cell found in cancer patient’s peripheral blood, which hold significant clinical value. Enumeration of CTC can provide prognosis information for disease management, as validated by CellSearch system in several disease settings, including breast cancer, colorectal cancer, prostate cancer, and ovarian cancer [1–4]. CellSearch system, as discussed in the previous chapter, although effective, can lead to neglect of certain subpopulations of CTC [5]. And this concern is shared among other affinity based CTC capture platforms, as affinity based CTC capture relies on expression of certain antigen on tumor cells, which can vary due to the heterogeneity observed among CTCs [6]. In contrast, non-affinity based techniques isolate CTC based on their physical properties. These antigen agnostic capture platforms may address the concerns of neglect of certain CTC subpopulations and give us a more comprehensive picture of CTC.

Current existing non-affinity based CTC capture platforms can be briefly categorized into two major types: capture platforms based on mechanical properties and capture platforms based on electrical properties. In this chapter, we discuss both types of techniques and how they are applied to isolate and characterize CTCs.

CTC capture based on mechanical properties

Circulating tumor cells, as inherent from primary tumor cells, are physically distinct from normal blood components in the aspect of size, density, and elasticity. Different technologies have been developed utilizing these properties and are discussed in the following sessions.

Density-based CTC enrichment

Density-based CTC enrichment is a standardized method for blood component separation. By layering blood onto a density gradient reagent and subsequent centrifugation, blood can be separated into plasma layer, buffy coat layer containing primarily mononuclear cells, and bottom layer containing granulocytes and erythrocytes (Fig. 3.1a). This can serve as a pre-enrichment step for CTC isolation since CTC will be retained in the buffy coat layer and isolated from granulocytes and erythrocytes. Further examination of CTC can be achieved through immunofluorescence labeling. However, density gradient centrifugation, although easy to handle and cost-friendly, suffers from poor retrieval rate as well as poor purity of CTC. Thus, several modi?cations have been made to enhance this assay for CTC isolation. The SepMate™ tubes developed by Stemcell Technologies employ a funnel shaped disk to enhance retrieval of the buffy coat and prevent mixing of the buffy coat layer with the bottom layer. Recently, the OncoQuick™ assay was developed employing a porous filter in the middle of the tube thus combining size-based isolation with density based isolation to achieve a higher retrieval rate of CTC [7].

! Circulating Tumor Cells-Springer-Verlag New York (2016) 3.1

Fig. 3.1. Principles used for non-affinity based CTC enrichment. (a) Density based CTC enrichment. (b) Size-based microfiltration for CTC enrichment. (c) Size-based microfluidics for CTC enrichment. (d) Inertial focusing based microfluidics for CTC enrichment. (e) DEP based microfluidics for CTC enrichment

Microfiltration-Based CTC Enrichment

CTCs are inherently larger than other blood components. Thus, several technologies have been developed using size-based isolation as strategy. Microfiltration was one of the first concepts developed for size-based CTC isolation (Fig. 3.1b). The first microfiltration technology was developed employing a polycarbonate filter. Brie?y, 35U ?ssion fragments were used to bombard a polycarbonate membrane, followed

by warm sodium hydroxide etching to generate pores with uniform size of 8 µm. This size cutoff can efficiently capture large CTCs whereas letting small erythrocytes and leukocytes pass through [8, 9]. Post-capture, CTCs were analyzed by Immunohistochemistry staining (IHC) to distinguish CTC from leukocytes retained on filter by the criteria of Cytokeratin (CK) positive, CD45 negative and morphological features such as cytoplasm to nuclear ratio. This Isolation by Size of Epithelial Tumor Cells (ISET) technology was validated in several disease settings, including breast cancer, lung cancer, pancreatic cancer, cutaneous melanoma, and uveal melanoma [10–14]. Furthermore, downstream molecular characterization of CTCs can be performed, which provides additional information for disease management. Vona et al. demonstrated that, by Laser Capture Microdissection (LCM), Hep 3B tumor cells captured on filter can be dissected and analyzed for p53 gene. Also, the feasibility of DNA fluorescence in situ hybridization (FISH) is also demonstrated by hybridizing the cells captured the filter with probe for centromeric chromosome 1 [9]. Later, ALK-FISH was performed by Pailler et al. on CTCs captured from Non-Small-Cell Lung Cancer (NSCLC) patients to interrogate the ALKrearrangement of these patients preand post-chemotherapy [15]. Another interesting phenomenon reported by Hou et al. using ISET technology was the capture of circulating tumor microemboli-contiguous groups of tumor cells on filter [16]. Following studies in small cell lung cancer and uveal melanoma further confirmed that presence of CTM could be correlated with worse prognosis [14, 17].

ISET technology, although it is effective for CTC isolation, can be limited due to the randomized generation of pores on filter. This could result in reduced filtration area and pore fusions, which could cause increased risk of clogging and loss of cells during filtration. To alleviate this concern, our group has developed a parylenebased microfilter, which is fabricated in a fashion that pores are evenly distributed in a well-controlled pattern. To achieve this, parylene C was deposited to 10 µm thickness and masked with photoresist material. Then the membrane was etched with reactive ion etching (RIE) followed by the last step, which was the stripping of photoresist using acetone. Using this novel microfilter design, Zheng et al. demonstrated on filter tumor cell capture and electrical lysis for PCR analysis [18]. Following validation reported by Lin et al. demonstrated >90% recovery using this filter design and superior sensitivity over CellSearch™ affinity based CTC capture system’s performance in the same cohort of cancer patients [19]. Another study reported by Birkhahn et al. demonstrated this novel microfilter could also be employed to enhance urine cytology performance [20].

RIE etching of parylene C technique not only provides us the advantage of controllable pore deposition but also enables us to alter the pattern and the shape of the pores of the filter. By altering these factors, our group has developed next-gen filters that can be applied for viable CTC capture. For the first generation of filter described above, the sample needs to be fixed at end concentration of 1% formalin for 10 min to achieve optimal retrieval rate of tumor cells, this is to prevent cell loss under the shear pressure of filtration. To reduce the shear pressure and achieve viable cell capture, Zheng et al. reported the fabrication of a 3D bilayer membrane filter. This filter is comprised of two layers, on the top layer, pores with 8 µm size were deposited and on the bottom layer pores with 9 µm size were deposited slightly off-setting the top layer pores. The gap distance between two layers is precisely engineered to be 6.5 µm. By this design, when cells are trapped on the top layer, the bottom layer can provide direct support in the opposite direction of the flow to reduce the pressure on trapped cells. This design is validated using model system and captured tumor cells remain viable and metabolically active at least 2 weeks post capture [21].

Another design of microfilter for viable CTC capture is the slot filter reported by Xu and Lu et al. By altering the geometry of the pores from round to slot, the ?ll factor is greatly enhanced thus reducing the shear pressure during filtration. Validation experiments confirmed tumor cells spiked into blood could be viably captured and cultured post capture. And telomerase activity can also be measured on the captured cells [22].

Another variation of the filter design for viable CTC capture is reported by Harouaka et al.—the Flexible Micro Spring Array (FMSA) design. This design utilizes a micro spring array to maximize the filtration area thus reduces the shear pressure during filtration. By using this design, spiked in tumor cells can be retrieved from blood viably and expand on filter [23]. FMSA device can also be used for on chip drug sensitivity test, and FMSA-derived cell culture can be reinject into animal models for in vivo drug sensitivity test as demonstrated by Gallant et al. in model system experiments [24].

Other microfilter platforms were also developed for CTC isolation. VyCap microsieves are fabricated using silicon nitride with evenly distributed pores of 5 µm size as reported by Coumans et al. [25]. Lim et al. also reported fabrication of microsieves using silicon-on-insulator (SOI) wafer with 10 µm sized pores [26].

Yusa et al. reported that, by fabricating a 3 dimensional phallodium filter with a pocket shape (with 30 µm sized pores on the top layer surrounding 8 µm sized pores on the bottom layer), blood sample could be processed without fixation and external pressure. The sample was processed through this 3D phallodium filter by gravity and the target cancer cells remained viable post capture [27]. Additionally, as reported by Tang et al., by replacing the cylinder shaped pores with conical shaped pores, a small differential pressure will be gained between the smaller pores facing the top and the larger pores facing the bottom of the filter, which will greatly reduce the leukocyte retention on filter and gain higher purity of CTC [28]. Also, Hosokawa, et al. reported a microcavity array (MCA) device fabricated from nickel by electro-forming to generate arrays of cavities with the average diameter of 8.4–9.1 µm [29]. And later on, a MCA system was reported using a rectangular cavity design and validated in a pilot cohort of small cell lung cancer patients[30] (Table 3.1).

Table 3.1. Microfiltration-based CTC enrichment technology

Technology Modeled cells Clinical validation Efficiency (E) and sensitivity (S) Purity Viability References
ISET Hep3B, Hep G2, HeLa, LNCaP, MCF-7 Breast, lung, pancreatic cancer, melanoma S: Up to 1 cell/mL N/S N/S [9–17]
Parylene C microfilter LNCaP, MCF-7, SK-Br-3, J82, T24, RT4 Prostate, bladder, breast, colorectal cancer S: Up to 1 cell/mL

E: 89.0 ± 9.5 %

N/S N/S [18, 19]
3D bilayer parylene C filter MCF-7, LNCaP N/S E: 86.5 ± 5.3 % N/S Y [21]
Slot parylene C filter PC3, DU145 Prostate cancer E: ~70 % 1500-fold enrichment 90 % [22]
Flexible micro spring array MCF-7, MDA-MB 231, C8161, WM35 Breast, colorectal, lung cancer E: 92.6 % 14,000-fold enrichment >80 % [23, 24]
VyCaP microsieves SK-Br-3, MDA-MB-231, MDA-MB-468, MCF-7, PC3-9, COLO-320, SW-480, HL-60, K-562 N/S E: 58 % 1000-fold enrichment N/S [25]
Silicon microsieve filter HepG2, MCF-7, BT474 Breast, colorectal, prostate, cervical cancer E: ~80 % N/S N/S [26]
3D palladium filter N-87, COLM-5 Breast cancer E: >70 % N/S N/S [27]
Filter with conical shaped holes HT-29, U87 Lung, nasopharynx, mediastinal, cardiac, cervical, and breast cancer E: ~95 % 96 % WBC clearance 95 % [28]
Microcavity array MCF-7, NCI-H358, SW620, AGS, SNU-1, Hs578T N/S E: >80 % N/S 98 % [29]
Rectangular microcavity array NCI-H69, NCI-H82, NCI-H358 Small cell lung cancer E: >80 % 7000-fold enrichment Y [30]

Microfluidics-based CTC enrichment

Microfluidic devices are being increasingly employed in analytical sciences and diagnostics because they enable miniaturization, integration, and automation [31– 33]. One representative output of technology is the development of lab-on-a-chip (LOC) devices, also known as micro-total analysis systems, which combine labscale tasks on a single mini-scale chip [34, 35] to yield significantly interesting biological applications. Using intrinsic physical properties of cells, like size, density, shape, and deformability, or extrinsic properties of the cellular response to magnetic or electrical fields, or optical excitation, microfluidic platforms to allow manipulation of the cells can be created. Microfluidic platforms to manipulate cells can exploit affinity of cells to specific ligands such as antigens expressed on their surface. On the other hand, non-affinity based microfluidic platforms are interesting because no further steps are required to recover the cells following isolation. Hydrodynamic manipulations, such as inertial method [36], or the acoustophoresis method [37] are examples of non-affinity based manipulations. Apart from membrane filters, microfluidic systems are also popular in isolating CTC based on their biomechanical properties (Fig. 3.1c). An early example of such a microfluidic platform was reported by Mohamed et al. [38]. This massively parallel sieving device with 10 µm-wide and 20 µm-deep microchannels could trap neuroblastoma cells spiked into whole blood. A microfluidic platform has been developed by Tan et al. [39], which isolates the cancer cells of breast and colonic origin, using their larger size and stiffness characteristics. Their microdevice consists of multiple arrays of crescent-shaped isolation wells. A gap of 5 µm in each of the traps prevents any clogging issue. Pre-puri?cation is happening utilizing a filter with 20 µm gap size to remove larger clusters before blood stream flows into the isolation section. Their experiment of screening 5 mL sample size, utilizing three microdevices at the same time, and under 5 kPa constant pressure, takes 2.5 h. The efficiency of at least 80% capture for MCF-7, MDA-MB-231, and HT-29 has been reported.

Recent developments employ fluid flow dynamics to manipulate cells for isolation and separation. Fluid flow in microfluidics is considered a dominantly laminar regime due to the definition of non-dimensional Reynolds Number, which refers to the ratio of inertial force to viscosity [38]. Hydrodynamic particle manipulation methods are passive techniques and have high throughput. Inertial technique eliminates the physical barriers to trap the cells, instead, using hydrodynamic properties of the fluid flow to trap cells [38, 41]. Important design parameters in this technique are geometry of the microchannel, particle size, and flow rate. Different microchannel geometries have been explored, such as straight [42, 43], expansion– contraction [44, 45], spiral [46, 47], and serpentine [48].

Hou et al. [49] introduced an inertia-based microfluidic platform to isolate CTCs in a spiral microchannel con?guration which is also known as Dean Flow Fractionation (DFF) method. Secondary flows, called the Dean vortexes, move the cells back and forth along the microchannel. Here, the cells are subjected to two forces, drag force and lift force, which exert differentially on cells with different sizes and shapes. The ratio of inertial lift forces and Dean drag forces (FL/FD) will define final equilibrium position for the cells, which grows exponentially with cell size [50]. Their isolation device showed 100% CTC detection efficiency using 3 mL of whole blood [49].

Hur et al. [51] developed a passive, continuous microfluidic platform utilizing “expansion–contraction” trapping reservoirs along microchannels. Stable vortices in the reservoirs trap larger cells (like CTCs), while the smaller cells ?ush along the microchannel to the outlet. The same group of researchers [52] developed a passive and label-free sorting technique exploiting both size and deformability differences in cells. Elasticity and viscosity impose differential lateral dynamic equilibrium positions on cells, thus separating different cell populations from each other. They observed that larger and more deformable tumor cells focus at the center of microchannel. They believe this method can also be a microfluidic platform to measure the deformability of cells. Bhagat et al. [53] proposed a two-stage chip design, which starts with contraction–expansion region where cells are focused by reaching an equilibrium between the counteracting wall-induced lift forces and viscous drag, and a region towards the end with pinched-flow con?guration where large tumor cells are pinched by aligning to the central axis. They successfully reported the separation of spiked tumor cells from peripheral blood with 80% efficiency at the throughput of 400 µL per minute. Augustsson et al. [54] developed a continuous separation method utilizing ultrasound wave radiation force. Acoustophoresis method is gentle, label-free and based on the intrinsic properties of cells such as density, size, and compressibility. The chip fabrication method has been demonstrated in detail by Moradi et al. [55]. This noncontact method does not affect cell viability or proliferation. A recovery of 87% and 83% of fixed cells (DU145 and PC3, respectively) with 4.2 mL/h throughput using a single microchannel has been reported [54].

Hyun et al. [56] utilized multi-ori?ce flow fractionation (MOFF) con?guration in their device design to isolate the CTCs based on their size. The device consists of an initial filter and several parallel MOFF microchannels. There are a series of alternating contraction channels and expansion chambers, which initiate inertial forces, separating larger CTC from smaller blood cells. This platform was validated for CTC capture in a pilot study of 24 breast cancer patient samples. Microfluidic platforms are thus increasingly shown to be suitable for clinical and biological applications, with advantages such as rapid, label-free, high throughput, and cost-efficient analysis. Process parallelization can be designed in microfluidic platforms to achieve simultaneous analysis for several samples, thus expediting the process of diagnostics, therapeutics and fundamental studies in cancer biology.

CTC capture using nanoroughened surfaces

Mechanical property based label free isolation of CTC can not only be achieved based on cell size/density/inertial force but also by other properties that are not as well understood. Chen et al. reported that, CTC, when compared with normal blood cells, has a different adhesion preference to nanorough surfaces. Thus, when applied to nanoroughened surfaces, tumor cells can be selectively captured by the surface. Preliminary results demonstrated that, when MCF-7 and MDA-MB-231 breast cancer cells were spiked into blood, the capture efficiency of the nanoroughened surfaces reached 93.3 ± 1.5% and 95.4 ± 2.2% respectively [57] (Table 3.2).

Capture based on electrical properties

As addressed above, Circulating Tumor Cells are inherently distinct from the normal blood components. This distinct feature of CTC is not only reflected in its biomechanical properties, such as size, inertial force, and density as described above, but also reflected in its electrical properties. One popular example of using electrical property to isolate CTC is the use of Dielectrophoresis (DEP). DEP is first studied by Herbert Pohl in 1950 [58]. He described a phenomenon—DEP, that particles can be moved using polarization forces in an inhomogeneous electric field. This phenomenon is strongly dependent on volume and shape of the particle, the electrical property of the particle as well as the gradient of the field and the medium. Thus, DEP can be applied to isolate cells based on their size and electrical properties (Fig. 3.1e). Later on, in 1995, Becker et al. reported the distinct dielectric property of tumor cell, erythrocytes and lymphocytes and how DEP can be used to isolate breast cancer cells from blood [59]. Cheng et al. also reported isolation of HeLa cells spiked into blood using a DEP chip [60]. Additionally, An et al. reported that malignant breast cancer cells (MCF-7 breast cancer cell line) could be separated from healthy breast cells (MCF-10A cell) using DEP since they have distinct dielectric property [61]. In 2005, Park et al. reported that, by fabrication of a chip with 3D asymmetric electrodes, mouse embryonic carcinoma cell P19 can be separated from erythrocytes using DEP. This is a stepping-stone towards isolation of cancer cell from whole blood using a DEP microfluidic chip [62]. Additionally, Jen et al. reported a handheld microfluidic chip that is able to concentrate HeLa cells controlled by DEP generated from circular microelectrodes [63].

One of the first microfluidic devices that can continuously separate tumor cells from whole blood is reported by Alazzam et al. in 2011. This article described a method for continuous flow separation of CTC from blood. Brie?y, interdigitated activated comb-like electrodes were positioned divergent and convergent with respect to the flow and CTCs were isolated due to their distinct response to the alternating current frequencies as compared with normal blood cells [64].

Other DEP microfluidic chips use combination of DEP of other microfluidic principles. Wang et al. reported that, by combining DEP with field-flow-fractionation (FFF), cell separations could be achieved efficiently. In this design interdigitated microelectrodes were mounted into rectangular chambers. Cells with distinct electric properties were levitated to distinct heights where the DEP forces were equilibrated with sedimentation forces. And by field flow, cells at different heights were carried at different velocities and thus separated. The authors demonstrated efficient separation of several cell types including separation of MDA-435 breast cancer cells (later revealed to be melanoma cells) from normal t-lymphocytes [65].

Table 3.2. Microfluidics-based CTC enrichment technology

Technology Modeled cells Carrier medium Clinical validation Efficiency (E) and sensitivity (S) Purity Viability References
Crescent shaped structure MCF-7, MDA-MB-231, HT-29 Diluted blood N/S E: >80 % >80 % Y [39]
DFF spiral microchannel MCF-7 Whole blood N/S E: >85 % 104–106-fold enrichment >98 % [49]
Microscale vortices HeLa, MCF-7 Diluted blood N/S E: 10–23 % ~85 % Y [51]
Deformability based inertial microfluidics HeLa, MCF-7, SAOS-2 Diluted blood N/S E: ~97 % 3.2–5.4-fold enrichment ~92 % [52]
Pinched flow coupled inertial microfluidics MCF-7, MDA-MB-231 Diluted blood N/S E: ~81 % 3.25 × 105-fold over RBC ~1.2 × 104  over leukocytes (2nd process) >90 % [53]
p-MOFF device MCF-7, MDA-MB-231 Lysed blood Breast cancer E: 91.6–93.75 % Elimination of 90.8 % leukocytes N/S [56]
Nanoroughened surface MCF-7, MDA-MB-231 Lysed blood N/S E: ~94 % N/S Y [57]

DEP can also be combined with multi-ori?ce flow fractionation (MOFF) to achieve continuous separation of cancer cells from blood at higher flow rate as reported by Moon et al. In this microfluidic device, cell mixture will first enter a separation region, where MCF-7 cells will be focused in the center of the channel together with few contaminating blood cells. This enriched population will then enter a focusing region where all cells will be aligned at the sides of the channel by DEP force. At the end, the aligned population will enter a second separation region and tumor cells will be selectively isolated via DEP. By this design, 75.81% recovery rate was achieved with the removal of 99.24% RBCs and 94.23% of WBCs at a flow rate of 126 mL/min as demonstrated by a model system using MCF-7 cell spiked blood [66].

Recently, a commercial DEP based microfluidic platform ApoStream™ was launched. In this device design, similar to the concept of DEP-FFF, CTCs were drawn close to the channel walls by the DEP force and thus collected in a collection chamber and other cells were ?ushed into a waste collection chamber due to inefficient DEP dragging forces. To test the system, SKOV-3 and MDA-MB-231 cancer cells were mixed with 12 Ч 106 peripheral blood mononuclear cells (PBMC) as model system to test the platform, and respectively 75.4 ± 3.1% and 71.2 ± 1.6% recovery rate was achieved. Since the cell isolation is fixation-free, the captured tumor cell can be then maintained in culture [67] (Table 3.3).


CTC is consisted of a very heterogeneous population, thus affinity based CTC isolation has its limitations on using specific antigen expression to capture CTC. As Zhang et al. reported, a subpopulation of CTC that has the potential to cause brain metastasis in breast cancer is EpCAM negative [68], which would have been missed if using EpCAM based CTC capture, a common antigen utilized in majority of the affinity based CTC capture platforms, including CellSearch. In contrast, nonaffinity based CTC isolation has the potential to lead to a more comprehensive understanding of CTC, as supported by Barradas et al., who demonstrated that CTC can be captured from CellSearch waste using size based CTC capture [5].

To address this concern, some CTC capture platforms are employing a combinational strategy to combine affinity based capture with mechanical based capture to enhance capture efficiency, such as geometrically enhanced differential immunocapture (GEDI) device reported by Gelghorn et al. which employs a device geometry so that desired cells with larger size will get into contact with antibody coated walls more often due to streamline distortion [69] and CTC-iChip device reported by Ozkumur et al. which combines inertial focusing with antibody based magnetic negative depletion of leukocytes [70].

However, although mechanical and electrical property based isolation of CTC has gained its popularity due to the label free process to reduce bias, the understanding of the mechanical and electrical property of CTC is still largely based on cultured cell lines and more studies need to be conducted on clinical samples to help us to understand these features better. For example, a study conducted in castrationresistant prostate cancer by Coumans et al. demonstrated that, CTC isolated by CellSearch, whether their Cytokeratin area is larger than 4 Ч 4 µm2 or not, is predictive of survival [71]. This contradicts the standard size cutoff adopted in size based CTC isolation. Hence, more studies need to be conducted in order to define clinically relevant CTC better and to develop better technologies to isolate CTCs in a more efficient and more comprehensive manner with less contamination with undesired cell population.

Table 3.3. DEP-based CTC enrichment technology

Technology Modeled cells Carrier medium Clinical validation Efficiency (E) and sensitivity (S) Purity Viability References
DEP based separation MDA-MB-231 Diluted blood N/S N/S >95 % >98 % [59]
Bioelectronic chip HeLa Whole blood N/S N/S N/S N/S [60]
3D-asymmetric microelectrodes P19 1 × 107/mL RBC N/S N/S 81.5 ± 7.6 % Y [62]
MOFF-DEP MCF-7 Mixture of RBC and WBC N/S E: 75.81 % 162.4-fold enrichment N/S [66]
ApoStream™ SKOV-3,


12 × 106  PBMC N/S E: ~70–75 % 99.33 ± 0.56 %

PBMC reduction

97.6 % [67]

As mentioned above, Herbert Pohl first described the unique DEP signature of tumor cell in 1950, and after years of improvement, the first commercial system based on this principle was finally developed in 2012. Similarly, other technologies are also being applied on CTCs to acquire a unique signature of CTC as compared with other cells. Some of these studies aim to develop a higher throughput/more efficient isolation technology, some aim to interrogate CTC to provide more information for disease management.

One example of this is interrogation of CTC by atomic force microscopy (AFM). Chen et al. used AFM to measure isolated CTC from prostate cancer patients, and demonstrated that CTCs exhibited mechanical phenotype resembling highly metastatic cancer cells in culture [72]. Additionally, Electrical impedance spectroscopy (EIS) was also applied to analyze cancer cells. Han et al. reported that, by using EIS, the membrane capacitances and resistance of cells can be measured, and the readout is distinct in MCF-10A, MCF-7 and MDA-MB-231 cells [73]. These technologies, due to their low throughput, are still limited at the level of single cell characterization. Future development is required for these findings to be translated into high-throughput, streamlined CTC isolation/characterization platforms.

Another promising area in CTC research is the functional characterization of CTC ex vivo. As demonstrated by Yu et al., ex vivo culture of CTC established from breast cancer patients can be used for drug sensitivity test and could potentially benefit the concept of personalized therapy [74]. This type of assays required CTCs to be captured viably with minimal manipulation, and label free, non-affinity based technologies can potentially benefit this field since several technologies including next-gen microfilters, microfluidic based isolations, and DEP based isolations are compatible with viable CTC capture and the elimination of labeling step can potentially benefit CTC culture due to the less manipulative process and shorter time to perform the capture process.

To conclude, CTC can be isolated from blood due to their distinct mechanical and electrical properties as compared with normal blood components. This will enable label free CTC capture without introducing bias by relying on the expression of certain antigen(s) on CTC. However, future studies will be needed to understand the physical properties of the clinically relevant CTC better. Additionally, future developments will not only focus on high-throughput, high purity CTC isolation/ characterization technologies but also focus on viable CTC capture to enable functional characterization of CTC.


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