**2.3. Other methodologies**

In addition to pure positive and negative or label‐free methods, some investigators proposed their prototypes for CTC isolation. For example, Qin et al. [50] performed CTC isolation by the size and deformability based separation from castrate resistant prostate cancer patients using resettable cell traps. Compared with CellSearch™, the method could capture more than 10 times of CTCs for subsequent analyses. Basically, it is a label‐free method and could be rapidly processed.

Synchrotron X‐ray microimaging techniques, high‐resolution images of individual flowing tumor cells, and nanotechnology were also proposed to help identification of CTCs. Positively charged gold nanoparticles (AuNPs) which were inappropriate for incorporation into human red blood cells were selectively incorporated into tumor cells to enhance the image contrast, which was reported by Jung et al. [232]. This new technology for in vivo imaging of CTCs would contribute to improve cancer diagnosis and cancer therapy prognosis. Moreover, new chemical materials using a refined carbon‐coated pure iron‐based immunomagnetic nanopar‐ ticle‐enriched assay, and nested‐RT‐PCR was also reported to successfully isolate CTCs efficiently.

Furthermore, not only for general population of CTCs, Hosseini et al. [233] postulated an integrated nano‐electromechanical chip (NELMEC) to isolate CTCs and CTCs with EMT features from white blood cells. These new technologies hold great promising on automation, which might greatly ameliorate current problems in CTC field.

#### *2.3.1. Comparison of different strategies*

In comparison between positive selection strategy and negative selection methods, the former is most commonly used CTC isolation platform and widely validated by prospective clinical trials. Several articles of meta‐analysis confirmed the clinical impacts of CTCs obtained by CellSearch™ [107, 234–237]. However, it is relatively costly and device‐dependent. Interestingly, some investigators compared the ISET and CellSearch™ systems for their performance on CTC isolation [76, 93] and one team concluded that a combination of ISET plus CellSearch™ would have better performance in CTC detection in NCSCL patients than ISET or CellSearch™ alone [93]. That intriguing conclusion supports the combination method in the following eras; however, a long processing time of combined platforms also causes cell damage or loss which is a problem the combined systems should be noticed.

In comparison between positive selection method and label‐free strategy, Konigsberg et al. [238] compared the efficiency of CTC isolation of MACS (positive selection system) with OncoQuick (label‐free system) and found EpCAM‐negative CTCs cannot be detected by EpCAM‐dependent enrichment methods. EpCAM‐independent enrichment technologies seem to be superior to detect the entire CTC population.

Similarly, Lin et al. [229–231] postulated a protocol and a device (PowerMag) to perform red blood cell lysis and immunomagnetic beads conjugation for CD45‐positve cells and identify EpCAM‐positive cells (defined as CTCs) from the blood samples. The protocol was proven to effectively isolate CTCs from patients with colorectal, head and neck cancer and thyroid cancer. Furthermore, the CTCs isolated by this negative selection method are further proven to be

In addition to pure positive and negative or label‐free methods, some investigators proposed their prototypes for CTC isolation. For example, Qin et al. [50] performed CTC isolation by the size and deformability based separation from castrate resistant prostate cancer patients using resettable cell traps. Compared with CellSearch™, the method could capture more than 10 times of CTCs for subsequent analyses. Basically, it is a label‐free method and could be rapidly

Synchrotron X‐ray microimaging techniques, high‐resolution images of individual flowing tumor cells, and nanotechnology were also proposed to help identification of CTCs. Positively charged gold nanoparticles (AuNPs) which were inappropriate for incorporation into human red blood cells were selectively incorporated into tumor cells to enhance the image contrast, which was reported by Jung et al. [232]. This new technology for in vivo imaging of CTCs would contribute to improve cancer diagnosis and cancer therapy prognosis. Moreover, new chemical materials using a refined carbon‐coated pure iron‐based immunomagnetic nanopar‐ ticle‐enriched assay, and nested‐RT‐PCR was also reported to successfully isolate CTCs

Furthermore, not only for general population of CTCs, Hosseini et al. [233] postulated an integrated nano‐electromechanical chip (NELMEC) to isolate CTCs and CTCs with EMT features from white blood cells. These new technologies hold great promising on automation,

In comparison between positive selection strategy and negative selection methods, the former is most commonly used CTC isolation platform and widely validated by prospective clinical trials. Several articles of meta‐analysis confirmed the clinical impacts of CTCs obtained by CellSearch™ [107, 234–237]. However, it is relatively costly and device‐dependent. Interestingly, some investigators compared the ISET and CellSearch™ systems for their performance on CTC isolation [76, 93] and one team concluded that a combination of ISET plus CellSearch™ would have better performance in CTC detection in NCSCL patients than ISET or CellSearch™ alone [93]. That intriguing conclusion supports the combination method in the following eras; however, a long processing time of combined platforms also causes cell damage

In comparison between positive selection method and label‐free strategy, Konigsberg et al. [238] compared the efficiency of CTC isolation of MACS (positive selection system) with

which might greatly ameliorate current problems in CTC field.

or loss which is a problem the combined systems should be noticed.

*2.3.1. Comparison of different strategies*

alive and are capable of being cultivated for at least several weeks [229].

**2.3. Other methodologies**

processed.

154 Tumor Metastasis

efficiently.



**Table 1.** Overview of analytical methodologies for the detection and molecular characterization of CTCs.

Another report addressed the differences between positive and label‐free method was reported by Qin et al. [50]. They designed a micropore filtration platform (using resettable cell traps) to perform CTC isolation by the characteristic of CTCs (size and deformability) from patients with castrate resistant prostate cancer. Compared with CellSearch™, the method could capture CTCs 10 times more than CellSearch™ can achieve. The method was also proven to be able to perform subsequent molecular analyses.

Interestingly, some investigators compared the isolation efficiency of ISET and CellSearch™ systems [76, 93] and one team concluded that a combination of ISET plus CellSearch™ would have better performance in CTC detection in NCSCL patients than ISET or CellSearch™ alone [93].

One question which is often and needed to be asked is that how to choose a best platform for upcoming studies and trials. Before answering the question, the readers/investigators should fully understand the differences, pros and cons among these methods. Then you should choose a platform wisely according to future directions of investigations (i.e., clinical trial, CTC culture, patient‐derived xerograft model from CTCs) (i.e., genetic analysis, single cell, physical properties) and the requirements of the study materials (i.e., cells with high purity, with high cell numbers, viability, expression with specific marker, etc.). **Table 1** demonstrated the brief comparison among novel platforms. In our opinion, for genetic analysis and future personal‐ ized medicine, we need a large number of CTCs captured for cultivate, whole genome or transcriptome sequencing and avoid sampling errors by hyper‐selection of few cells to represent the whole populations of cancer. Therefore, negative selection as first step is currently most suitable strategy among all the methods.
