**1.4. The impact of CTCs on multidrug resistance**

might reveal new therapeutic targets and can be used for drug screening [104, 105]. The phenomenon is the main difference of CTCs from ctDNA possibly being released from dead cancer cells. In addition, living CTCs can also colonize their tumors of origin, in a process that is call "tumor self‐seeding." Kim et al. [106] successfully revealed the self‐seeding phenomenon in breast cancer, colon cancer, and melanoma tumors in mice model, which was predominantly mediated by CTCs with aggressive features, including those with bone, lung, or brain metastatic tropism. The cancer‐derived cytokines IL‐6 and IL‐8 acted as CTC attractants and the markers MMP1/collagenase‐1 and the actin cytoskeleton component fascin‐1 as mediators of CTC infiltration into mammary tumors. The important findings of tumor self‐seeding phenomenon could explain the relationships between tumor size, anaplasia, vascularity and prognosis, and local recurrence seeded by disseminated cells following ostensibly complete

Sixth, CTCs could represent a merged status of a whole tumor mass, including static and active parts with expression of specific functional markers [107–110] and could serve as a multifunc‐ tional biomarker [108, 111]. Functional analyses on CTCs might provide the possibility to identify the biological characteristics of metastatic cancer cells, including the identification of

*1.3.1. Cancer migration, invasion, epithelial‐mesenchymal transition (EMT), mesenchymal‐epithelial*

As mentioned above, Hansen et al. [65] found that CTCs exist in the 93% blood samples drawn from surgical fields. That correlated to one of the two common routes of cancer migration: hematologic and lymphatic spreading. In clinical aspect, tumor migration and invasion means tumor growth or progression and can be analyzed via the time from disease‐free status to recurrence or time from baseline to enlargement of tumor size. Early in 1999, Palmieri et al. have found a significant correlation among clinical stages, tumor progression, and presence of circulating cancer‐associated antigens in stages I–III melanoma patients [112]. In other cancer types, investigators widely agreed with the observation that the higher CTCs signals indicate to higher cancer stage and recurrence rate, suggesting larger number of CTCs might promote cancer progression [113–117]. However, not only CTC count but also the specific properties of cancer cells matter. Two of them have been widely reported are epithelial‐

In many animal species, EMTs normally occur during critical phases of embryonic develop‐ ment. The formation of mesenchymal cells (nonepithelial) that are loosely embedded in an extracellular matrix from a primitive epithelium is an important feature of most metazoans [119]. During this transition, mesenchymal cells acquire a morphology that is appropriate for migration in an extracellular environment and settlement in areas that are involved in organ formation, which involves interactions between epithelial and mesenchymal cells. Mesenchy‐ mal cells can also participate in the formation of epithelial organs through mesenchymal‐ epithelial transition (MET) [119]. CTCs may also undergo phenotypic EMT changes, which

mesenchymal transitions (EMTs) or stem‐like properties of CTCs [118].

tumor excision.

148 Tumor Metastasis

metastasis‐initiating cells [104].

**1.3. CTCs in cancer progression**

*transition (MET) and cancer stem cells (CSCs)*

In 2011, Gradilone et al. reported an interesting study aiming to test the hypothesis that drug‐ resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem‐like properties [146]. As the study presented, the extraction of mRNA from CTCs for multiple drug resistance proteins (MRPs) analysis are most commonly used protocol. They also found the expression status of MRP1 and MRP2 in CTCs was found to correlate to response to anthracyclines (doxorubicin or epirubicin) [147]. In 2013, Nadal et al. found an interesting phenomenon that a relative enrichment of cytokeratin CK(+)/CD133(+) CTCs in triple negative and HER2‐amplified tumors was found. While CK(+)/CTCs decreases after chemotherapy when analyzing the whole population, CK(+)/CD133(+) CTCs were enriched in posttreatment 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– 152].

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 given therapeutic drugs [154–156].
