**2.1. Label‐free isolation strategy**

samples in nonluminal BC subtypes. These findings suggest the potential role of CD133 as a promising marker of chemoresistance in nonluminal BC patients [148]. Similar results were also reported in recent years and the authors have come across with the same conclusion that multiple drug resistance profiling (MRPs mainly, sometimes with CD133 [148], ALDH1 [149], and ERCC1 [150]) of CTCs could predict the responses to given chemotherapies [146, 148, 150–

One direct proof of CTC exhibiting drug resistance comes from a study in 2014. Pavese et al. observed that CTC and DTC cell lines, established from mice bearing human prostate cancer orthotopic implants, exhibit increased cellular invasion in vitro, increased metastasis in mice, and express increased EMT biomarkers. In addition, CTC cell lines are selectively resistant to growth inhibition by mitoxantrone‐like agents. The findings are important and suggested that CTC formation is accompanied by phenotypic progression without obligate reversion. Their increased metastatic potential, selective therapeutic resistance, and differential expression of potential therapeutic targets provide a rational basis to test further interventions [153].

Therefore, developing an in‐vitro chemosensitivity test on CTCs is not impossible though it required large‐scale clinical trials to test and validate. Yu et al. applied pharmacogenomic (PGx) modeling testing on CTCs, while PGx testing was used on cancer tissue to predict the efficacy of chemotherapeutic agents in preclinical cancer models, and reported the feasibility in 2014. In the report, clinical benefit was seen for study participants treated with chemother‐ apy regimens predicted to be effective versus chemotherapy regimens predicted to be ineffective with regard to progression‐free (10.4 months versus 3.6 months; *P* < 0.0001; HR, 0.14) and overall survival (17.2 months versus 8.3 months; *P* < 0.0249; HR, 0.29) [151]. In another study, thymidylate synthase expression in CTCs could possibly serve as a new tool to predict 5‐fluorouracil resistance in metastatic colorectal cancer patients [152]. Other than conventional imaging studies evaluating two‐dimensional tumor size every 8–12 weeks for routine tumor assessment during anticancer therapy, CTCs could possibly serve as a rapid responding biomarker to real‐time change of cancer cells, including the early response or resistance to

There are hundreds of methods/protocols reported to be able to efficiently detect or isolate CTCs. In a simple way to discuss here, we have several common strategies of CTC isolation could be worthy of development in the future. The first one is **label‐free isolation strategy**, including size‐based, physical properties‐based, morphology‐based isolation strategy; the second one is **positive selection strategy**, including positively identification of cancer‐specific markers on nucleus or cell surface, or specific DNA mutation(s), mRNA(s) overexpression; and the third one is **negative selection strategy**, consisting of depletion of red and white blood cells by any means. Finally, the fourth one is combination of two or more strategies mentioned

152].

150 Tumor Metastasis

given therapeutic drugs [154–156].

above.

**2. The strategies for CTC isolation and enumeration**

Several novel studies using size as a key criterion of CTC identification were reported [157– 160]. Early in 2004, ISET system was used for a well‐designed clinical trial evaluating 44 patients with primary liver cancer and without metastases, 30 patients with chronic active hepatitis, 39 with liver cirrhosis, and 38 healthy individuals, and all participants were followed up for a mean period of 1 year. Both the presence (*P* = 0.01) and number (*P* = 0.02) of CTCs and microemboli were significantly associated with a shorter overall survival. Beta‐catenin mutations could be found in 3 of 60 CTCs which might be suggesting their impact on the initiation of cancer cells invasion [161]. Similar positive findings by size‐based CTC isolation were reported in melanoma [162–164], gastric cancer [76, 165], prostate cancer [166, 167], lung cancer [168–170], pancreatic cancer [103], liver cancer [127], sarcoma [171], and breast cancer [172]. Separation by physical properties, i.e., gravity, density gradients, using microfluidic technology [45, 46, 56, 60, 173–186], or microfiltration [53, 172, 187, 188] were also reported to be able to capture CTCs efficiently.

By means of label‐free isolation, combined molecular analysis could be easily performed after CTC isolation owing to no chemicals exposure and less procedures done during isolation. For instance, Zheng et al. [189] reported a novel device designed based on membrane microfilter device to isolate CTCs and then send them to PCR‐based genomic analysis by performing on‐ membrane electrolysis with embedded electrodes reaching each of the individual 16,000 filtering pores. Immunocytochemistry and FISH assays following label‐free isolation were reported to be successfully performed directly on the filter system [157, 176, 190, 191]. 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].

There are several disadvantages of physical methods should be noticed. First, the isolation process based on physical properties can cause the deformation and damage of CTCs by filter pores [192]. Second, larger size cells could not always be cancer cells and the isolated popula‐ tion often mixed up with megakaryocytes, which are very common to see in the circulation of cancer patients just underwent chemotherapy. Third, small‐size CTCs would be inevitably missed by this isolation strategy.
