**2.2. Positive versus negative selection‐based CTC isolation**

#### *2.2.1. Positive selection methods*

Positive selection strategy is the most commonly used method of CTC isolation in the literature. CellSearch™ is the most evidenced and the only one device having class III approval from US FDA since 2004; therefore, hundreds of clinical trials chose to apply the device for CTC testing for validation [114, 193–204], mainly in patients with breast, colorectal, and prostate cancer. Other representative positive selection platforms are magnetic‐activated cell sorting system (MACS) and Isoflux. The main process of positive selection is to label targets cells by anti‐CK (AE1/AE3) antibody with ferric beads and immunofluorescence dye. The approximate sensitivity of detection is 10-7 (CTCs/hematologic cells). Another system, MACS used 50–100 nm‐sized ferric beads. However, lower sensitivity and lower recovery rate of CTCs were observed. The device was firstly introduced in 1998 [205] and then CTCs obtained by the system could correlated with breast cancer stages [206] and could correlate with progression‐ free survival in colorectal cancer patients [207]. These systems are all based on immunomag‐ netic beads technology for CTC isolation and have long been limited by relatively low efficiency of antibody conjugation due to tumor heterogeneity [192]. This limitation further causes the difficulty of molecular analysis [208]. Fortunately, the technique of single CTC isolation and analysis has been much more mature in recent years [166, 209, 210]. However, fewer sampling (CTCs) could greatly contribute to the bias for prediction of target population behavior (the whole tumors in the body). Another downside of positive selection strategy was the limitation of EpCAM‐dependent nature. Hyun et al. [124] demonstrated that EMT‐induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy. In addition, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label‐free micro‐ fluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and potentially miss critical subpopulations.

In addition to conventional immunomagnetic bead separation methods, density separation and flow cytometry or cell sorting systems have been postulated to be potential tools of CTC isolation and identification considering their high sensitivity and purity since 1998 [27]. This method could be seen as a combination of negative selection strategy and a positive confir‐ mation with surface markers, such as EpCAM or cytokeratins. Later in 2011, leukapheresis and fluorescence‐activated cell sorting (FACS) elutriation were also reported to be effective for large volume blood process for CTC isolation with molecular analysis [211]. Recently, many microscale on‐chip sorting systems were developed considering the high purity of isolation for CTC culture or tumor related genetic analysis. In 2014, Kim et al. have postulated an on‐ chip multi‐imaging flow cytometry system to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross‐sectional area, number of nuclei, and size of clusters as "imaging biomarkers," with simultaneous acquisition and analysis of both bright‐field and fluorescent images at 200 frames per second [212]. Moreover, laser scanning cytometry is also a novel innovation developed to help identify CTCs [213, 214]. These methods, are mainly based on flow cytometry and sorting techniques, which could possibly yield an extremely high purity of CTCs (more than 80%). However, one of the drawbacks of the cytometric systems is operator‐dependent and multiple quality and internal controls are often required when setting a criterion of CTC identification. Another downside of conventional sorting systems is cell damage and decreased viability after sorting process.

Recently, by the advances of nanotechnology, nanoplates [215], nanowires [216], for positively trapping of CTCs are becoming hot devices with theoretically higher sensitive capturing efficiency than conventional ones. These techniques are often developed by biomedical engineers who are good at medicine, biology and engineering; however, the devices seem to be still in proof‐on‐concept phase. Hopefully, these new devices would facilitate the development of easy hands‐on CTC testing and validation in clinical trials in the near future. In brief, positive selection methods hold the greatest clinical application to date.

## *2.2.2. Negative selection methods*

sensitivity of detection is 10-7 (CTCs/hematologic cells). Another system, MACS used 50–100 nm‐sized ferric beads. However, lower sensitivity and lower recovery rate of CTCs were observed. The device was firstly introduced in 1998 [205] and then CTCs obtained by the system could correlated with breast cancer stages [206] and could correlate with progression‐ free survival in colorectal cancer patients [207]. These systems are all based on immunomag‐ netic beads technology for CTC isolation and have long been limited by relatively low efficiency of antibody conjugation due to tumor heterogeneity [192]. This limitation further causes the difficulty of molecular analysis [208]. Fortunately, the technique of single CTC isolation and analysis has been much more mature in recent years [166, 209, 210]. However, fewer sampling (CTCs) could greatly contribute to the bias for prediction of target population behavior (the whole tumors in the body). Another downside of positive selection strategy was the limitation of EpCAM‐dependent nature. Hyun et al. [124] demonstrated that EMT‐induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy. In addition, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label‐free micro‐ fluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and

In addition to conventional immunomagnetic bead separation methods, density separation and flow cytometry or cell sorting systems have been postulated to be potential tools of CTC isolation and identification considering their high sensitivity and purity since 1998 [27]. This method could be seen as a combination of negative selection strategy and a positive confir‐ mation with surface markers, such as EpCAM or cytokeratins. Later in 2011, leukapheresis and fluorescence‐activated cell sorting (FACS) elutriation were also reported to be effective for large volume blood process for CTC isolation with molecular analysis [211]. Recently, many microscale on‐chip sorting systems were developed considering the high purity of isolation for CTC culture or tumor related genetic analysis. In 2014, Kim et al. have postulated an on‐ chip multi‐imaging flow cytometry system to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross‐sectional area, number of nuclei, and size of clusters as "imaging biomarkers," with simultaneous acquisition and analysis of both bright‐field and fluorescent images at 200 frames per second [212]. Moreover, laser scanning cytometry is also a novel innovation developed to help identify CTCs [213, 214]. These methods, are mainly based on flow cytometry and sorting techniques, which could possibly yield an extremely high purity of CTCs (more than 80%). However, one of the drawbacks of the cytometric systems is operator‐dependent and multiple quality and internal controls are often required when setting a criterion of CTC identification. Another downside of conventional sorting systems is cell

Recently, by the advances of nanotechnology, nanoplates [215], nanowires [216], for positively trapping of CTCs are becoming hot devices with theoretically higher sensitive capturing efficiency than conventional ones. These techniques are often developed by biomedical engineers who are good at medicine, biology and engineering; however, the devices seem to

potentially miss critical subpopulations.

152 Tumor Metastasis

damage and decreased viability after sorting process.

Negative selection methods are developed on the basis of the disadvantages of positive selection methods‐losing non‐EpCAM or CK‐expressing CTCs and relatively poor recovery rate. The principle of negative selection strategy is to remove all the cells other than CTCs as its first step. Owing to the sequence of isolation has changed, in the negative depletion processing, the cancer information was preserved as possible, which makes the phenomenon that the number of CTCs isolated by a negative method would generally (but not always) larger than those by a positive one. In addition, by the CD45 depletion procedures, CTCs without expression of epithelial markers could be isolated though further clarification of the clinical significance of these cell populations is required [217]. The nature of the isolation strategy increased the sensitivity, recovery rate of CTCs but decrease the specificity with inevitable "background noises."

In the developing history of the negative selection methods, Naume et al. [29, 218] have proposed to use CD45 (a common antigen of leukocytes) coated beads to remove white blood cells from tumor cells and red blood cells depleted by lysis buffer or density separation processing for CTC isolation. Based on the concepts, Balasubramanian et al. [219] also successfully demonstrated positive staining images for cytokeratin‐positive CTC identification after negative selection processes in 32 cancer blood samples. Among all the negative selection systems, one of the representative systems is epithelial immunospot (EPISPOT) [220–224]. By the procedures, CTCs in the blood sample are enriched by anti‐CD45 immunomagnetic beads. The isolated CTCs are then cultured in tissue culture plates precoated with antibodies which capture cathepsin D, MUC1, or CK19 protein [225]. After the incubation period, cells are washed out and the released protein spots are detected by the incubation with a fluorochrome‐ conjugated antibody and counted. Each spot corresponds to one viable CTC. The device focused on the expression of CK19‐expressing cells, which were found to be detectable in up to 65 and 70% of colorectal cancer and breast cancer patients, respectively, and correlated with status of metastasis and poor survival in breast cancer [226, 227]. However, a single sample processing in EPISPOT system requires three days for analysis, which prohibits of its clinical use considering the time‐consuming problem.

Nevertheless, the background cells in a negatively isolated sample are often mixed with numerous white blood cells and red blood cells if the process is not well‐performed, thus prohibit the following molecular analyses. For this disadvantages of conventional negative selection method, a better depletion process for red and white blood cells depletion are warranted. Another device developed by Wu et al. [228], CanPatrol™ CTC enrichment, they reported a recovery rate more than 80%. Interestingly, FISH assay could be successfully performed for ALK gene rearrangement from CTC samples; however, further validation in prospective clinical trials was still required.

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