**1. Introduction of circulating tumor cells (CTCs)**

#### **1.1. Brief history of CTC researches**

Circulating tumor cells (CTCs) are cells shedding from primary tumor(s) into the adjacent vasculature and are floating around in the circulation throughout the human body. The cells, as seeds for the subsequent initiation of distant site metastases, are responsible for the cancer‐ related deaths [1]. For the first time, CTCs were described via the observation in the blood of cancer patient by Dr. Thomas Ramsden Ashworth, who postulated that "cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of origin of multiple tumors existing in the same person" in 1869 [2]. In 1906, Goldmann reported that visible venous invasion by cancer in approximately 20% of 500 necropsies and

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microscopic invasion of vasculature in nearly 10% [3]. In very early 1900s, several reports of observation of free cancer cells by morphology have been discussed in patients with melano‐ ma, gastric cancer [4–6], and lung cancer [7]. However, long being in the technical limitation on isolation of these rare cells in circulation, the realization of CTCs isolated from living cancer patients and analysis of their clinical impacts began since 1930s [8–10]. One of the first systemic surveys in 40 living cancer patients was done by Pool and Dunlop in 1934 [11]. In this period, morphology and cytochemical characteristics remained the most important method to identify "abnormal" or "atypical" cells. Tumor cell embolization was observed [12] and widely accepted to be one of the major mechanisms for dissemination of cancer [13, 14]. Since 1986 with the development of polymerase chain reaction (PCR), investigators began to utilize these nucleic acid‐based detection methods to help identify CTCs, including circulating tumor mutated DNA and mRNAs [15–23]. In early 2000s, semi‐automated devices appeared and facilitate the advances of CTC testing in clinical study enrolling healthy subjects and pa‐ tients with various types of cancer [24–26], given the fact that numerous previous methods were relatively operator‐dependent and often lacked of validated sensitivity, specificity, coefficient of variation and reproducibility [22, 27–31]. In 2004, CellSearch™ (Veridex, Janssen Diagnostics, USA) got approval from United States (US) Food and Drug Administration (FDA) for testing in patients with breast, colorectal, and prostate cancer [32, 33]. Since 2012, a rapid exploration of number of CTC isolation devices having nearly fully automated design emerged [34–40].

Recently, CTC studies are focusing on devices harboring high sensitivity, high specificity, reduced sample requirement, label‐free isolation, and the ability to catch living CTCs for in‐ vitro culture. Owing to the less invasive nature than conventional cancer tissue biopsy, CTCs, as serum circulating tumor DNA (ctDNA) and microRNA (miRNA), are termed to be "liquid biopsies."

### **1.2. Natures of CTCs**

It has been a long time after CTCs were noticed and efficiently captured by many methods; however, little is known about the behavior of CTCs [41]. Investigators have observed some phenomenon about what CTCs look like, how CTCs shed, migrate, live, defense human immune system, and initiate distant metastases.

First, CTCs were believed to be larger than normal blood cells, which contributed the devel‐ opment size‐based isolation strategy. Marrinucci et al. [42] supported that the fact of CTCs being larger than white blood cells and having high nuclear to cytoplasmic ratios with voluminous cytoplasm. In addition, the morphology of CTCs is highly similar to that of cells from original biopsied cancer tissues. Numerous devices were developed on the basis of this characteristics and collect cells with larger size (often >15 μm), including dielectrophoresis (DEP) [43, 44], optically induced dielectrophorestic (ODEP) [45] force‐dependent devices, and filter‐based systems [46–61]. However, other investigators found that the size of real CTCs could be greatly differed from cell lines [62], and might even vary interindividually and intraindividually [63, 64]. The size criteria of CTC definition remain in debate.

Second, CTCs exist in almost all staged cancer and could be detected in the course of the disease [27]. In 1995, Hansen et al. analyzed the blood samples drawn from surgical fields during 61 oncologic surgeries and 93.4% samples found tumor cells [65], which suggest one of the possible routes of early dissemination of cancer cells. In 2000, Yamashita et al. found that signals of CTCs (carcinoembryonic antigen messenger RNA, CEA mRNA) from preopera‐ tively negative to postsurgically positive might suggest a specific type of surgery could contribute to the cancer cell dissemination [66]. Similar results were reported by other investigators to support the findings in various types of cancer (but breast cancer mainly) [67– 73]. Although CTCs were found in early‐stage cancer patients, the cells do not result in metastasis all the times. The clearance of human immune systems and inadequate "soil" of distant organs are one of the plausible explanations. To look on the bright side, CTCs in very early stage cancer could help early diagnosis of cancer and prevent overwhelming dissemi‐ nation and cancer death [74].

microscopic invasion of vasculature in nearly 10% [3]. In very early 1900s, several reports of observation of free cancer cells by morphology have been discussed in patients with melano‐ ma, gastric cancer [4–6], and lung cancer [7]. However, long being in the technical limitation on isolation of these rare cells in circulation, the realization of CTCs isolated from living cancer patients and analysis of their clinical impacts began since 1930s [8–10]. One of the first systemic surveys in 40 living cancer patients was done by Pool and Dunlop in 1934 [11]. In this period, morphology and cytochemical characteristics remained the most important method to identify "abnormal" or "atypical" cells. Tumor cell embolization was observed [12] and widely accepted to be one of the major mechanisms for dissemination of cancer [13, 14]. Since 1986 with the development of polymerase chain reaction (PCR), investigators began to utilize these nucleic acid‐based detection methods to help identify CTCs, including circulating tumor mutated DNA and mRNAs [15–23]. In early 2000s, semi‐automated devices appeared and facilitate the advances of CTC testing in clinical study enrolling healthy subjects and pa‐ tients with various types of cancer [24–26], given the fact that numerous previous methods were relatively operator‐dependent and often lacked of validated sensitivity, specificity, coefficient of variation and reproducibility [22, 27–31]. In 2004, CellSearch™ (Veridex, Janssen Diagnostics, USA) got approval from United States (US) Food and Drug Administration (FDA) for testing in patients with breast, colorectal, and prostate cancer [32, 33]. Since 2012, a rapid exploration of number of CTC isolation devices having nearly fully automated design emerged

Recently, CTC studies are focusing on devices harboring high sensitivity, high specificity, reduced sample requirement, label‐free isolation, and the ability to catch living CTCs for in‐ vitro culture. Owing to the less invasive nature than conventional cancer tissue biopsy, CTCs, as serum circulating tumor DNA (ctDNA) and microRNA (miRNA), are termed to be "liquid

It has been a long time after CTCs were noticed and efficiently captured by many methods; however, little is known about the behavior of CTCs [41]. Investigators have observed some phenomenon about what CTCs look like, how CTCs shed, migrate, live, defense human

First, CTCs were believed to be larger than normal blood cells, which contributed the devel‐ opment size‐based isolation strategy. Marrinucci et al. [42] supported that the fact of CTCs being larger than white blood cells and having high nuclear to cytoplasmic ratios with voluminous cytoplasm. In addition, the morphology of CTCs is highly similar to that of cells from original biopsied cancer tissues. Numerous devices were developed on the basis of this characteristics and collect cells with larger size (often >15 μm), including dielectrophoresis (DEP) [43, 44], optically induced dielectrophorestic (ODEP) [45] force‐dependent devices, and filter‐based systems [46–61]. However, other investigators found that the size of real CTCs could be greatly differed from cell lines [62], and might even vary interindividually and

intraindividually [63, 64]. The size criteria of CTC definition remain in debate.

[34–40].

146 Tumor Metastasis

biopsies."

**1.2. Natures of CTCs**

immune system, and initiate distant metastases.

Third, CTCs would form cell clusters, clumps, or circulating tumor microemboli (CTM) and were found to highly correlate to cancer progression [75–79] and resistance to systemic anticancer therapies [78, 80], which was established on the basis of many animal model and preclinical reports [81–85]. Recently, Sarioglu et al. [86] designed a Cluster‐Chip for efficient capture of CTC clusters and enable the detailed characterization of the biological properties and role of CTC clusters in metastasis [86]. Studies of cancer metastasis have emphasized the novel concept of "seed and soil" as a key determinant of metastatic propensity [87]. This model matches the importance of mutated genetic drivers within tumor cells conferring proliferative and invasive properties, with that of the microenvironment of the distant organ or "niche," which may facilitate metastasis occurrence. However, the physical characteristics of single CTCs and CTC clusters may also contribute to metastatic propensity, especially as they impact the ability of epithelial tumor cells to survive the loss of cell adherence and shear forces in the blood stream, i.e., different survival signals among the cancer cell "seeds" may be important. For instance, in a mouse endogenous pancreatic cancer model, noncanonical Wnt signaling is elevated within CTCs, where it appears to suppress anoikis [88], while in a subcutaneous tumor xenograft model, the admixture of tumor and stromal cells within microemboli may contribute to stromal‐derived survival signals [77, 89, 90].

Fourth, CTCs could also be detected in cancer patients who underwent curative surgery, indicating minimal residual disease in the circulation [91–99] and suggesting the correlation to disease recurrence in the following months [92, 95, 100, 101]. van Dalum et al. [99] found that the presence of CTC in blood drawn pre and one and two years after surgery, but not postsurgery is associated with shorter Relapse-free survival (RFS) and OS for stages I–III breast cancer, which could partially answer the question of how frequent and how long oncologists should follow patients' CTCs up and the timing of CTC testing after curative surgery. This phenomenon indicates that postsurgical adjuvant therapy might be required in specific population on the basis of CTC testing which remains uncertain to date.

Fifth, the captured CTCs according to their method of isolation, sometimes are alive for in‐ vitro culture [48, 61, 102, 103] and might play a very important role to continuously obtain primary cancer cell lines in the near future. Furthermore, CTC‐derived cell lines and xenografts 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 tumor excision.

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 metastasis‐initiating cells [104].

#### **1.3. CTCs in cancer progression**

#### *1.3.1. Cancer migration, invasion, epithelial‐mesenchymal transition (EMT), mesenchymal‐epithelial transition (MET) and cancer stem cells (CSCs)*

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

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 allow them to travel to the site of metastasis formation without getting affected by conventional treatment [118, 120, 121]. The acquired molecular changes by CTCs undergoing EMT that facilitates cancer progression and resistance to conventional therapies [122, 123]. EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug, and N‐cadherin in CTCs, primary HCC tumors and adjacent nontumoral liver tissues were evaluated by Li et al. [123] and the twist and vimentin expression levels in CTCs could be promising biomarkers for evaluating metastasis and prognosis in liver cancer patients. Most importantly, CTCs would abandon their epithelial properties (EpCAM) [88, 124] and escape from CTC capture by positive selection strategy (will discuss below) and become one of the main downsides of the strategy of CTC isolation. Several investigators have noticed that phenomenon and suggested that there is an urgent need for optimizing CTCs detection methods through the inclusion of EMT markers [120, 125–129]. Deeper understanding of those processes is of fundamental impor‐ tance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice [122].

Stemness features of CTCs, sometimes termed as circulating cancer stem cells (CSCs), have also been getting noticed as EMT of CTCs in recent years. The CSC hypothesis claims that a small subset of cells within a tumor has the ability of both tumor initiation and sustaining tumor growth [130–132]. These cells with expression of stemness markers are capable of forming floating spheres in serum‐free medium, a property associated with stem cells and are able to differentiate into an aberrant cell phenotype constituting tumor heterogeneity [133]. Among all the possible molecular markers of stemness feature, CD133, CD44, ICAM‐1, and CXCR4 are common used antibodies for labeling the subpopulation from other CTCs and actually technically available [134–136]. These are not the only markers to identify CSCs and depend on cancer types. Sun et al. [137] found that stem cell‐like phenotypes (labeled with CD133 and ABCG2) in EpCAM‐positive CTCs, and a preoperative CTC of more than 2 cells/7.5 ml blood is a novel predictor for tumor recurrence in HCC patients after surgery, especially in patient subgroups with AFP levels of less than 400 ng/ml or low tumor recurrence risk. Many other studies in various types of cancer have come across with the similar conclu‐ sions, including breast, colorectal, gastric, liver, and NSCLC, etc. [120, 123, 138–145].

Therefore, the subpopulation of CTCs, CSCs, and CTC with EMT features is probably the one of the key determinants of future CTC and cancer metastasis investigations.
