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Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells

Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells Hindawi Journal of Oncology Volume 2022, Article ID 7149686, 14 pages https://doi.org/10.1155/2022/7149686 Review Article Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells 1 2 1 1 Siwen Wu, Shubi Zhao , Dawei Cui , and Jue Xie Department of Blood Transfusion, the First Aliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China Correspondence should be addressed to Dawei Cui; daweicui@zju.edu.cn and Jue Xie; zyyyxj2011@zju.edu.cn Received 4 March 2022; Revised 12 July 2022; Accepted 2 August 2022; Published 2 September 2022 Academic Editor: Liren Qian Copyright © 2022 Siwen Wu et al. ‚is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Circulating tumor cells (CTCs) play a crucial role in tumor recurrence and metastasis, and their early detection has shown remarkable bene‰ts in clinical theranostics. However, CTCs are extremely rare, thus detecting them in the blood is very challenging. New CTC detection techniques are continuously being developed, enabling deeper analysis of CTC biology and potential clinical application. ‚is article reviews current CTC detection techniques and their clinical application. CTCs have provided, and will continue to provide, important insights into the process of metastasis, which could lead to development of new therapies for di‘erent cancers. promote self-growth and colonization of the metastatic site 1. Introduction by secreting exosomes that promote their dynamic in- Circulating tumor cells (CTCs) were ‰rst described by teraction with the tumor microenvironment [7]. ‚erefore, Ashworth in 1869 as a group of tumor cells in the peripheral CTCs provide cellular evidence for metastasis and are useful bloodstream originating from spontaneous solid tumor biomarkers for cancer progression in most cancer patients tissues (primary or metastatic) and a biomarker for cancer [8]. Several biological characteristics contribute to the diagnosis and progression [1–5]. ‚e mechanism of tumor shedding of CTCs by the primary tumor and their role in metastasis caused by circulating tumor cells is shown in metastasis. Generally, EMT promotes the formation and Figure 1. Tumor cells shed into the blood in situ cause blood- metastasis of CTCs. Metastasis is driven by cytokines, borne metastases [6]. CTCs with an epithelial-mesenchymal proteins, and transforming growth factor (TGF)-β-Smad transition (EMT) phenotype are invasive enough to pass signaling. TGF-β promotes metastasis by reducing the ex- through the extracellular matrix (ECM), dissociate from the pression of epithelial cadherin (E-cadherin). On the other marginal front, and invade the tumor vasculature. CTCs can hand, the resistance of A-kinase anchoring protein 8 evade anoikis cell death in circulation. Disseminated tumor (AKAP8) to EMT can inhibit breast cancer metastasis. ‚e cells (DTCs) exhibiting the EMT phenotype undergo in- in‰ltration of CTCs into the metastatic site is a complicated travascular stasis and develop cell protrusions to promote process. In addition to producing EMT and proteases, en- transendothelial migration (TEM) of cancer cells into the dothelial cells (ECs) secrete CXC chemokine ligand 12 metastatic site, where they may stay dormant for some time (CXCL12) to promote in‰ltration and perivascular tumor- before colonizing. ‚is allows cancer cells to evade immune associated macrophages (TAMs) to upregulate epidermal surveillance and successfully colonize distant organs. DTCs growth factor and matrix metalloproteinase-9 [9]. A hard- then acquire the mesenchymal-epithelial transition pheno- ened ECM induces the formation of invasive pseudopodia in type to proliferate and form secondary tumors. Cancer cells cancer cells, enabling them to penetrate the ECM to invade 2 Journal of Oncology Mutation Oncogene activation Proliferation Selection Normal cells Premalignent cells Metastatic tumor Mutated cells Vessels Dissemination CTC CTC Oncogene activation Proliferation Mutation Selection Normal cells Premalignent cells Primary tumor Metastatic tumor Figure 1: -e mechanism of circulating tumor cells (CTCs) driving tumor metastasis. CTCs refer to all kinds of tumor cells in the peripheral blood. Due to their spontaneous or clinic operation, most of the CTCs undergo apoptosis or are swallowed after entering the peripheral blood. A few can escape and are anchored to become metastatic. blood vessels. -e activity of cancer-associated fibroblasts in 2. Biological Characteristics of CTCs the recombinant ECM has been shown to promote drilling 2.1. Cellular Size. Due to high heterogeneity of tumor cells, and subsequent invasion of tumor cells [7]. the pore size in the CellSearch system is typically slightly CTCs are rare in healthy individuals, and even in pa- 5 7 larger than leukocytes [14]. Other researchers have suc- tients with malignancies, less than one CTC per 10 to 10 cessfully separated CTCs using size-based platforms that peripheral blood mononuclear cells (PBMCs) could be exploit difference in cell sizes. CTCs in prostate cancer are observed. -us, isolation and enrichment are often the first divided into three categories based on size (diameter): very steps in CTC detection in laboratories and hospitals [10]. small nuclear CTCs (<8.54μm), small nuclear CTCs -ere are two classical approaches to separating CTCs from (8.54–14.99μm), and large nuclear CTCs (>14.99μm) [3]. blood samples: physical separation that exploits unique Jiang et al. selectively enlarged the size of tumor cells physical properties of CTCs (such as density and size) and covered with polystyrene microspheres and the modified immune adhesion, which depends on the high binding af- cells were clearly distinguishable from white blood cells. -e finity of receptors on CTCs to specific antibodies or modification method had no significant effect on cell sur- aptamers [11]. Compared with immune adhesion, physical vival and proliferation. Using this method, 15 CTC subtypes isolation is a simpler method as it obviates the need for cell were detected in 18 cases of colorectal cancer at a concen- labeling. However, the immune adhesion method achieves tration of 4–72 CTCs/mL. -us, this method has great higher purity in CTC isolation. potential in the early diagnosis and individualized treatment Due to technical limitations, few studies have in- of cancer [15]. Zavridou et al. directly compared two dif- vestigated the precision of CTC detection methods. Keller ferent methods of isolating CTCs from head and neck and Pantel discussed how CTC analysis at single-cell res- squamous cell carcinoma: a size-dependent microfluidic olution provides unique insights into tumor heterogeneity system and epithelial cell adhesion molecule (EpCAM)- [12]. Martin et al. reviewed preclinical and clinical data on dependent positive selection. -e results showed that, in the cancer treatment, CTC mobilization, and other factors that same blood sample, the label-freesize-dependent CTC may promote metastasis, establishing that advanced thera- separation system had higher sensitivity than the EpCAM- peutic strategies could benefit patients with locally advanced dependent CTC enrichment system [16]. cancer [13]. However, a systematic review of the occurrence, development, and outcome of CTCs in metastatic cancer is largely lacking. In this review, we present an overview of the 2.2. Cellular Density. Density is the physical property biological characteristics of CTCs, current CTC detection exploited in traditional separation and enrichment methods techniques, and principles and methods of CTC isolation. for CTCs [17]. In Ficoll density gradient centrifugation, Finally, potential applications of CTCs in the treatment of CTCs, plasma, and monocytes remain in the upper layer, metastatic cancer are proposed. whereas erythrocytes and polymorphonuclear leukocytes Journal of Oncology 3 metastasis [32–34]. In this process, cancer cells lose polarity settle in the bottom layer. CTCs may occur in both plasma and separation fluid. -us, some liquids above the red blood and their connection with ECM, transforming into fusiform mesenchymal cells, which are easily detached from the tu- cell layer should be collected for enrichment to prevent the loss of CTCs [5]. Huang et al. developed a new density mor cell population. Several ECM-degrading proteases are gradient centrifugation method that uses biodegradable upregulated in cancer cells with EMT, increasing their in- gelatin nanoparticles wrapped on silica beads for isolation, vasiveness [32–35]. As shown in Figure 2, the occurrence of release, and downstream analysis of CTCs from colorectal EMT in CTCs could result in the loss of specific molecular and breast cancer patients. -is method has remarkable CTC markers in epithelial cells such as EpCAM and cytokeratin capture efficiency (>80%), purity (>85%), high CTC-release and overexpression of specific molecular markers in in- efficiency (94%), and viability (92.5%) [18]. -us, this ap- terstitial cells such as vimentin and cadherin. -ese cells proach provides new opportunities for personalized cancer have a strong survival advantage and high metastasis and potential for transfer in the blood circulation [17, 36–38]. diagnosis and treatment and may also be useful in de- veloping drug treatment guidelines for cancer. Based on the EMT stage, CTCs are divided into E-type, M- type, E/M-type, and N (null)-type. Several studies have shown that E/M-type CTCs have enhanced epithelial cell 2.3. Heteromorphy in CTCs. Marrinucci et al. conducted adhesion and extravasation capacity, representing more cellular morphological evaluation of circulating components aggressive subtype of cancer cells with the highest metastatic of highly metastatic breast cancer. -ey found highly capacity [39–41]. Additionally, M-type CTCs exhibit en- polymorphic CTCs in breast cancer patients, including hanced resistance to clinically relevant chemotherapeutics. CTCs with high and low nuclear-to-cytoplasmic ratios and early and late apoptotic changes. In addition, compared with 3. Separation and Enrichment of CTCs tumor cells in other sites, the complete morphologic spec- trum of cancer cells in primary and metastatic tumors was -ere are two major approaches based on the principle of also present in peripheral blood circulation [19]. Several CTC separation and enrichment: physical property sepa- studies have found various forms of CTCs in peripheral ration and affinity-based identification [42, 43]. For the blood existing either independently or in clusters, with some physical property separation method, tumor cells are sep- CTCs even interacting with platelets to form a shell around arated from other cells based on differences in size [44, 45], them [20–23]. Aceto et al. found that CTC clusters in breast density [46], deformability, and adhesion between tumor cancer-bearing mice were shed as whole oligomeric colonies cells and normal blood cells [18, 47]. -e affinity-based rather than as simply a group of CTCs aggregating in the identification method involves identification of the spe- bloodstream [24, 25]. cific antigen on the surface of cancer cells using antibodies [44, 48], aptamer [49, 50], or E-selectin [51, 52]. Additional details are shown in Figure 3. 2.4. Proliferation and Apoptosis of CTCs. For tumor cells in circulation, only a few CTCs with high viability and potent metastatic potential survive and colonize distant organs to 3.1. Gradient Density Centrifugation. Two centrifugation- develop into metastatic foci. CTCs entering the circulatory based systems are available in the market today: Onco- system have very short survival times, typically less than Quick and AccuCyte [43, 53]. After isolation and enrich- 24 h, and vary in their indices of proliferation [26]. Driemel ment with Ficoll-Paque separation fluid, 24 CTCs were et al. found that the high expression of EpCAM was common detected in fifty-eight 1 mL blood samples from colorectal in cancer cells in the proliferation stage, while the low ex- cancer patients using real-time reverse transcription- pression of EpCAM inhibited the proliferation of CTCs [27]. polymerase chain reaction (RT-PCR) [53]. Rosenberg Studies have reported low levels of expression of pro- et al. reported that using a new OncoQuick system to isolate liferating nuclear antigen Ki-67 in CTCs, suggesting that cancer cells had a 632-fold enrichment effect compared with most CTCs may remain in a dormant state without entering less than 4-fold enrichment effect using Ficoll-Paque [53]. In the cell division cycle [28, 29]. It has also been found that, addition, 11 CTCs were detected in 37 samples of gastric several years after primary tumorigenesis, dispersed CTCs cancer patients using a combination of OncoQuick and and micrometastasis niche can remain dormant for a long RT-PCR [53]. In another study, 5 and 25 CTCs were de- time during resection of primary tumors [30]. -ese results tected in 60 cases of early breast cancer patients after im- suggest that dormant CTCs can be attached to tissues or cell munofluorescence and 63 samples of patients with advanced clusters until their activation or that of a certain factor in the breast cancer, respectively [2]. Although density-gradient isolation procedures. -e specific mechanism may be related centrifugation is uncomplicated, it lacks specificity and can to the body’s immune response. easily lead to loss of tumor cells without corresponding density. 2.5. 2e Metastatic Portent of Circulating Tumor Cells. -erefore, density gradient centrifugation is often used EMT is a biological process by which epithelial cells acquire as the first step to separate CTCs and then combined with a mesenchymal phenotype through a series of biochemical other methods to specifically bind and separate CTCs. For changes [31]. In recent years, accumulating evidence sug- example, Hu et al. used density gradient centrifugation and gests EMT phenomena in the process of cancer cell magnetic separation based on CD45 antibody to separate 4 Journal of Oncology TGF, EGF, IGF, FGF, HGF, PDGF, Hypoxia … Snail1/2 Mesenchymal traits Epithelial traits Elf5 Zeb1/2 Grhl2 Twist miR-200 FOXC2 miR-34 Six1 EMT Deceased cell-cell adhesion Cell detachment & extrusion Vessels Dissemination Figure 2: Induction of epithelial-mesenchymal transition (EMT) by various cytokines. It is generally assumed that the metastatic spread of epithelial tumors depends on EMT, a process in which cancer cells lose their polarity and cell-cell adhesion to acquire ‰broblast-like features such as migration and invasion. CTCs collection Centrifugation Filtration Ficoll centrifugation (a) (b) Adhesion Microfluids (c) (d) CTC Immunocytes Red blood cells Figure 3: Circulating tumor cell (CTC) enrichment technologies. (a) Density gradient centrifugation. (b) Micro¥uidic-based separation technology. (c) Di‘erent ‰ltration systems depending on the size of blood cells used for separation and enrichment of CTC. (d) Adhesion is dependent on the a¦nity and speci‰c binding of antibodies or aptamers (e.g., immune adhesion) to the CTC receptor. Magnetic beads Journal of Oncology 5 hepatocellular carcinoma with conventional methods is CTCs [46]. Different from the traditional negative enrich- ment, Hu et al. applied the subtraction enrichment and significantly limited, leading to false-negative detection of such CTCs. Wang et al. reported for the first time the ex- immunostaining fluorescence in situ hybridization (SE- iFISH) strategy to detect CTCs, which effectively removed istence of small-sized CTCs (<5μm WBC) with cytogenetic red blood cells by centrifugation rather than using hypotonic abnormalities in aneuploid chromosome 8, which is pre- injury [54]. dominantly detected in hepatocellular carcinoma (HCC) patients [69]. 3.2. Method for Separation and Capture Based on Cell Size. -e method takes advantage of the larger size of CTCs 3.3.ImmunomagneticBeads. Almost all cells in the blood are compared with erythrocytes [55]. Isolation by size of epi- diamagnetic or weakly magnetic [70]. -erefore, tumor cells thelial tumor cells (ISET) and ScreenCell systems have been are usually labeled with antibody-conjugated magnetic used for clinical trials in melanoma, breast, lung, and beads or nanoparticles. -ese antibodies bind primarily to pancreatic cancers [56, 57]. For the first time, Zheng et al. tumor-cellsurface-specific antigens, including some in- used parylene-C to make circular and oval microporous tracellular antigens [71]. -e number of CTCs counted using filters, achieving a CTC capture efficiency of 90% [58]. A cell search has been used for prognosis of some cancers after model for gene analysis and analysis of cells after chip metastasis [72–74]. Wu et al. developed a magnetic cell electrolysis developed by Birkhahn et al. [59] was sub- centrifugation platform (MCCP) combining the separation sequently applied to the detection of exfoliated cells from mechanism of magnetically labeled cells with the size-based urinary bladder cancer. Hosokawa et al. integrated nickel method and obtained target cells with 97% purity, high microporous sieves made from micro-electroforming into throughput of 2μL/s, and a sample enrichment factor of a microfluidic chip. -e team also applied the improved 66 times [75]. Overall, the performance of the immuno- nickel microporous sieves to the detection of CTCs in the magnetic particle separation method mainly includes the blood of patients with small-cell lung cancer [60]. following factors: (1) the expression level and specificity of To improve the capture efficiency of CTCs, Coumans the target antigen and the binding ability of the corre- et al. studied factors affecting the trapping of filter cells [61]. sponding antibody and (2) the efficiency of immuno- In microfluidic chips, the precise fabrication of shapes and magnetic particle labeling. Immunomagnetic particles used microstructures in microchannels makes it possible to for cell separation have high recovery and purity and even separate and enrich tumor cells on a size-by-size basis [62]. detect CTCs in one step [76–78]. Erythrocytes have stronger deformability and smaller vol- ume, thus can easily cross various microstructures [63]. Nicinski ´ et al. proposed a new tool that uses microfluidic 3.4. Chip Technology. In the 1990s, Manz et al. proposed devices, photovoltaic (PV)-based SERS activity platform, a microfluidic chip technology [79]. Commonly used CTC and shell separation nanoparticles (shins) for simultaneous antibodies include human EpCAM and leukocyte common separation and unlabeled analysis of circulating tumor cells antigen CD45 [80]. Affinity sorting includes two types of in blood samples. -e results demonstrated the potential of capture methods. -e first type is the positive sorting SERS-based tools for isolating tumor cells from whole blood method, which directly targets and specifically captures samples in a simple and minimally invasive way in a scaled- target cells. -e second type is the negative sorting method, up detection and molecular identification pipeline [64]. which involves the capture nontarget cells, which are then Ohnaga et al. used a microchannel to capture circulating discarded. A schematic diagram of the working principle is tumor cells in esophageal and breast cancers [65]. Zeinali shown in Figure 4. et al. used a sensitive microfluidic CTC capture device to analyze circulating epithelium and EMT-like CTCs in 3.4.1. Positive Sorting Methods. -e traditional affinity pancreatic cancer [66]. To capture CTCs larger than the maximum pore size sorting method involves direct binding of the antibody to the microfluidic chip channel [81]. Sequist et al. developed the regardless of cell surface expression, blood is filtered through pores (usually 8μm in diameter). However, the success of second-generation CTC chip called herringbone (HB)-chip this process depends on many factors, including blood flow [82]. Compared with first-generation CTC chips, the rate, pore size uniformity, and membrane stiffness. High second-generationHB-chip is easy to use and more efficient, flow velocity will cause CTC to “squeeze” through pores, providing comprehensive and easy access to data. Hughes causing membrane distortion. A very slow flow rate will lead et al. integrated halloysite nanotubes into this chip [83] by to excessive accumulation of white blood cells, blood co- immobilizing E-selectin and anti-EpCAM on nanotubes. In agulation, and prolonged processing time [67]. Moreover, this design, E-selectin captures rapidly moving CTCs, tumor cells undergoing epithelial-mesenchymal transition whereas anti-EpCAM specifically captures CTCs [84, 85], increasing the purity of the captured CTCs. To simplify the (EMT) were smaller than those without EMT characteristics [68]. -erefore, CTCs receiving EMT may not be detected experimental procedures, Stott et al. designed a fishbone- based affinity sorting chip for direct analysis of whole blood using these technologies. Due to the inherent heterogeneity and dynamic expression of EpCAM and the degradation of samples with a sorting speed of up to 1 mL/h. Captured cytokeratin during the transformation of epithelial cells into circulating tumor cells could also be used for other assays or mesenchymal cells, the detection of circulating tumor cells in cell culture [86]. Sheng et al. optimized the fishbone 6 Journal of Oncology Blood sample from patients Red blood cells Immunocytes CTCs Negative sorting method Positive sorting method Microfluidic chip Target cells Non-target cells Figure 4: Peripheral blood samples from patients with non-small-cell lung cancer were obtained before any treatment and immediately processed in the circulating tumor cell (CTC) herringbone (HB)-chip that captures anti-EP-CAM-coated column epithelial cell adhesion molecules (left). Negative consumption of untargeted cells by a negative consumption method, including red blood cells and immune cells. Targeted cells such as CTCs were left in the chip for further analysis (right). structure to achieve a CTC capture e¦ciency and sorting Containing Anti-CD56 to collect CTCs in peripheral blood, purity higher than 90% and 84%, respectively [87]. ‚ese was used to monitor the occurrence and disease response to micro¥uidic chip technologies have shown good CTC treatment at di‘erent time points [91]. capture capability. However, releasing CTCs from micro- Unlike traditional negative enrichment, researchers ¥uidic chips for subsequent analysis is challenging. ‚ere- utilize subtraction enrichment (SE), independent of cell size, fore, researchers have introduced magnetic materials into cluster, or surface anchor protein expression. ‚e immu- micro¥uidic chips for CTC sorting [88]. nostained proteins were proved to be free from the re- striction of antigen epitopes inside and outside cells and membrane-related tumor biomarkers. With the clinical 3.4.2. Negative Sorting Methods. EpCAM-based a¦nity application of SE-iFISH, in addition to the traditional tumor separation cannot be applied to CTCs with weakly expressed cell types, there are more and more accidental discoveries of or nonexpressed EpCAM in the process of tumor cell me- various phenotypes of CTCs [54]. Zhang et al. have shown − + tastasis, which leads to the signi‰cant decrease or even loss of that aneuploidy CD31 CTC and CD31 CTEC may be used EpCAM expression. For example, Lee designed a chip called as a pair of biomarkers for circulating cell tumors to predict “μ-MixMACS”, which greatly increased the number of CTCs patients with non-small-cell lung cancer receiving anti- detected [89]. Sajay et al. designed a two-step negative CTC angiogenesis combined therapy [40]. Based on the SE-iFISH sorting platform where the recovered cells remain bioactive strategy, Yang et al. demonstrated that patients with early and can be further analyzed for protein or nucleic acid bladder cancer had more triploid CTCs, tetraploid CTCs, content [90]. A CTC-negative enrichment scheme, which and total circulation endothelial cells (CECs). Various CTC/ utilized the RosetteSep CTC Enrichment Cocktail CEC subtypes may have di‘erent potential function to guide ™ Journal of Oncology 7 the diagnosis, prognosis prediction, and treatment decision tests and the specificity of series tests [116]. -e molecular of bladder cancer [92]. Li et al. found that the presence of spectrum of four genes, including CK20, CK19, CEA, and circulating tumor-cell-associated white blood cell (CTC- GCC, identified 87.7% of tumor metastases with a false- WBC) clusters is an independent prognostic factor for positive rate of only 2.2% [117]. advanced non-small-cell lung cancer [93]. 5. Clinical Applications of CTCs 4. Detection of CTCs CTCs are a promising biomarker for early disease diagnosis, treatment response and disease progression evaluation, re- 4.1. Immunocytochemistry. Immunochemistry is a modern currence monitoring, and therapeutic target identification technology that binds specific monoclonal antibodies with for drug development [118]. Detection of CTCs has been CTCs, followed by conjugation of a chromogenic reagent to widely used in the diagnosis of early and metastatic cancers visualize CTCs. -e most commonly used monoclonal (Table 1). antibody are anti-CK antibodies, such as epithelial-specific markers (CK) [94], interstitial cell surface markers (Snail1, E47, and Twist) [95], E-cadherin antagonist [96], stem cell 5.1. Early Diagnosis and Staging of Cancer. Traditional im- markers (CD133+, CD44+, and CD24−), aldehyde de- aging methods cannot effectively detect early tumor lesions. hydrogenase 1 (ALDH1) [97–100], special marker Survivin CTC detection approaches can detect tumor earlier than [101, 102], estrogen receptor (ER) [103], and progesterone imaging or clinical manifestations when the lesion is<1 cm, receptor (PR) [104]. EpCAM and CK may be lost during hence facilitate early diagnosis. Besides its role in early epithelial-mesenchymal transition (EMT), leading to the tumor diagnosis, CTC is also correlated with tumor grade failure of EpCAM- and CK-dependent strategies to detect and TNM stage. Santos et al. found that CTCs have great CTCs. -erefore, Li et al. used SET-iFISH technology to potential in the early diagnosis of colorectal cancer since enrich and characterize CTCs in advanced gastric cancer they can be detected in the peripheral blood of patients with (AGC) and obtained a higher positive detection rate than early-stage colorectal cancer. -erefore, the CTC test may be that obtained using EpCAM-dependent detection strategies applied to the diagnosis of colorectal cancer [123]. Clinical (CellSearch) [105]. In addition, Li et al. captured CTCs in staging of colorectal cancer is often based on anatomical AGC through SE-iFISH and found that cHER2 phenotype is alterations of the intestine; however, it is difficult to accu- useful to understanding the treatment resistance of AGC rately identify micrometastasis during the prognosis and patients [106]. Subsequently, scientists used this method to treatment of patients [124]. Detection of CTCs in the blood characterize the markers of CTC, such as EpCAM [41], does not necessarily indicate the occurrence of metastasis. PD-L1 [107], vimentin [40], and CD44 [108]. However, several studies have shown the value of detection of CTCs in the staging of colorectal cancer in clinical practice [125]. Using an advanced CanPatrol CTC enrich- 4.2. RT-PCR. Currently, RT-PCR is considered the gold ment technique and in situ hybridization to sort and classify standard in the detection of some viruses because of its high CTCs in blood samples, 90.18% of hepatocellular carcinoma sensitivity [109, 110]. RT-PCR is also widely used in tumor (HCC) patients were found to be CTC positive, even at the detection [111, 112]. However, selecting optimal RNA early stage of HCC [126]. CTCs were also detected in 2 of 12 markers can be challenging, limiting its efficacy. An ideal patients with hepatitis B virus (HBV) infection, with both RNA marker should have the following characteristics: all patients developing small HCC tumors in less than five types of tumor cells are expressed in peripheral blood months. Another study by Wang et al. implicated CTCs in leukocytes, nontumor epithelial cells are not expressed, and tumor staging [127]. Recent studies have shown that CTCs no illegitimate transcription events [113]. Using qualitative also put into good use in hematologic malignancies. Primary RT-PCR, Wang et al. found that the expression of androgen plasma cell leukemia (pPCL) is clinically distinguishable receptor variant 7 (AR-V7) in CTCs from patients with from newly diagnosed multiple myeloma (NDMM) based prostate cancer was associated with drug resistance. -e on the proportion of circulating tumor cells of 20% [128]. upregulation of AR-V7 led to the enhancement of cancer cell Zhang et al. also used a technology based on oncolytic proliferation, suggesting poor patient prognosis [114]. Wei herpes-simplex-virus-1 to detect CTCs in non-Hodgkin’s et al. recruited 78 patients with stage I –II cervical cancer A2 A1 lymphoma [129]. who had undergone radical hysterectomy by laparotomy or laparoscopy and selected 34 uterine fibroid patients and 32 5.2. Treatment Evaluation and Recurrence Monitoring. healthy subjects as the positive control group and negative control group, respectively. RT-PCR was used to amplify Treatment evaluation and recurrence monitoring of CTCs peripheral blood CK19, CK20, and SCC-Ag from total RNA. has been extensively studied. Lin et al. measured the number -e results showed that CTCs were highly expressed in both of peripheral blood CTCs before and after NK cell immu- the open surgery group and the laparoscopic radical mas- notherapy in stage IV non-small-cell lung cancer (NSCLC) tectomy group, with no significant difference between the patients, providing a useful reference for monitoring any two groups [115]. Using Survivin, hTERT and hMAM change in NK cell therapeutic effect [130]. Nagrath et al. markers to detect CTC in breast cancer patients, Shen et al. detected and monitored CTCs using a CTC chip and found found that these markers improved the sensitivity of parallel that the CTC count of patients with lung and prostate cancer 8 Journal of Oncology Table 1: Clinical applications of CTCs Patient Detection Clinical trial Cancer type Marker Significance Reference number methods no. EpCAM, In breast cancer patients with first-line chemotherapy, CTC Breast cancer 549 CellSearch NCT00382018 Paoletti et al. [119] CK counts were associated with mortality. A transcriptional profile detectable in CTCs can serve as an NCT01942837, Prostate cancer 147 mCRPC VERSA EpCAM Sperger et al. [120] independent prognostic marker in mCRPC. NCT01942837 Pancreatic CTC-positive preoperatively (≥1 CTC/7.5 mL) showed 209 patients CellSearch EpCAM NCT01919151 Hugenschmidt et al. [121] cancer a detrimental outcome despite successful tumor resections. Baseline CTCs≥ 3 were detected in 19% of the patients. CTC≥ 3 Colorectal EpCAM, 153 CellSearch at baseline and 4 weeks after therapy showed shorter overall NCT01442935 Bidard et al. [122] cancer CK survival. Journal of Oncology 9 decreased significantly before and after chemotherapy and the detection of CTCs will become a powerful and in- endocrine therapy. Although the changes in the CTC count dispensable tool for the diagnosis of circulating tumor cells (tumor DNA) with its advantages of repeatability, mutation due to treatment are affected by the differences between individual patients, they can still be used as a reference for detection at the molecular level, noninvasive diagnosis and evaluating the efficacy of tumor treatment [131–133]. In broad application potential in targeted therapy, efficacy some cases, CTCs are more sensitive than imaging, thus they testing, postoperative prognosis, radiotherapy and chemo- are included in efficacy evaluations [134]. In recent years, therapy strategy guidance, as well as in differential diagnosis. several detection techniques have been developed for CTC genotyping as well as detection of crucial gene mutations, Data Availability such as ER [135], HER2 [136], and TP53 [137]. -us, these techniques can help clinicians in treatment evaluation and -e data used to support the findings of this study are in- monitoring tumor recurrence. Zhou et al. used PCR and cluded within the article. fluorescence-activatedsingle-cell sorting (FACS) to detect + + + + levels of EpCAM mRNA CTCs and CD4 CD25 Foxp3 Conflicts of Interest Treg cells in 49 HCC patients before surgery. -e data showed that CTC/Treg levels were positively correlated with -e authors declare that they have no conflicts of interest. the risk of postoperative recurrence [138]. Due to differences in tumor type and stage among cancer Authors’ Contributions patients and occult, it is difficult to detect metastatic tumor relapse within five years of primary tumor resection. Cancer JX and DC conceived the research topic. SW and SZ con- that persists despite treatment and cannot be detected by ducted literature review, drafted the manuscript, and current medical imaging modalities is defined as minimal designed the figures and tables. JX, DC, and SW revised the residual disease (MRD), which is in the occult stage of cancer manuscript. All the authors approved the submitted version. progression. Liquid biopsy methods based on detection of Siwen Wu and Shubi Zhao contributed equally to this work. small amounts of circulating tumor cells (CTCs) or trace amounts of circulating cell-free tumor DNA (ctDNA) are Acknowledgments now available for MRD detection in patients with various malignant tumors. Monitoring CTCs and ctDNA during -e work was supported by the National Natural Science postoperative follow-up assessments can detect disease re- Foundation of China (Grant Nos. 82172335, 81971994, and currence months earlier than other medical imaging 91846103), Zhejiang Provincial Key Research and Devel- methods. Further characterization of CTCs and ctDNA opment Program (Grant No. 2020C03032), and Technology could provide insights into the molecular evolution of MRD Project of Zhejiang Provincial Health Commission (Grant during tumor progression, which has important implica- No. 2022KY155). tions for treatments that delay or even prevent metastatic recurrence. -erefore, the detection of CTCs has become the References main method for the assessment of minimal residual disease (MRD) [139]. [1] V. J. Hofman, M. I. Ilie, C. Bonnetaud et al., “Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method,” American Journal Of 6. 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Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells

Journal of Oncology , Volume 2022 – Sep 2, 2022

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Hindawi Journal of Oncology Volume 2022, Article ID 7149686, 14 pages https://doi.org/10.1155/2022/7149686 Review Article Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells 1 2 1 1 Siwen Wu, Shubi Zhao , Dawei Cui , and Jue Xie Department of Blood Transfusion, the First Aliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China Correspondence should be addressed to Dawei Cui; daweicui@zju.edu.cn and Jue Xie; zyyyxj2011@zju.edu.cn Received 4 March 2022; Revised 12 July 2022; Accepted 2 August 2022; Published 2 September 2022 Academic Editor: Liren Qian Copyright © 2022 Siwen Wu et al. ‚is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Circulating tumor cells (CTCs) play a crucial role in tumor recurrence and metastasis, and their early detection has shown remarkable bene‰ts in clinical theranostics. However, CTCs are extremely rare, thus detecting them in the blood is very challenging. New CTC detection techniques are continuously being developed, enabling deeper analysis of CTC biology and potential clinical application. ‚is article reviews current CTC detection techniques and their clinical application. CTCs have provided, and will continue to provide, important insights into the process of metastasis, which could lead to development of new therapies for di‘erent cancers. promote self-growth and colonization of the metastatic site 1. Introduction by secreting exosomes that promote their dynamic in- Circulating tumor cells (CTCs) were ‰rst described by teraction with the tumor microenvironment [7]. ‚erefore, Ashworth in 1869 as a group of tumor cells in the peripheral CTCs provide cellular evidence for metastasis and are useful bloodstream originating from spontaneous solid tumor biomarkers for cancer progression in most cancer patients tissues (primary or metastatic) and a biomarker for cancer [8]. Several biological characteristics contribute to the diagnosis and progression [1–5]. ‚e mechanism of tumor shedding of CTCs by the primary tumor and their role in metastasis caused by circulating tumor cells is shown in metastasis. Generally, EMT promotes the formation and Figure 1. Tumor cells shed into the blood in situ cause blood- metastasis of CTCs. Metastasis is driven by cytokines, borne metastases [6]. CTCs with an epithelial-mesenchymal proteins, and transforming growth factor (TGF)-β-Smad transition (EMT) phenotype are invasive enough to pass signaling. TGF-β promotes metastasis by reducing the ex- through the extracellular matrix (ECM), dissociate from the pression of epithelial cadherin (E-cadherin). On the other marginal front, and invade the tumor vasculature. CTCs can hand, the resistance of A-kinase anchoring protein 8 evade anoikis cell death in circulation. Disseminated tumor (AKAP8) to EMT can inhibit breast cancer metastasis. ‚e cells (DTCs) exhibiting the EMT phenotype undergo in- in‰ltration of CTCs into the metastatic site is a complicated travascular stasis and develop cell protrusions to promote process. In addition to producing EMT and proteases, en- transendothelial migration (TEM) of cancer cells into the dothelial cells (ECs) secrete CXC chemokine ligand 12 metastatic site, where they may stay dormant for some time (CXCL12) to promote in‰ltration and perivascular tumor- before colonizing. ‚is allows cancer cells to evade immune associated macrophages (TAMs) to upregulate epidermal surveillance and successfully colonize distant organs. DTCs growth factor and matrix metalloproteinase-9 [9]. A hard- then acquire the mesenchymal-epithelial transition pheno- ened ECM induces the formation of invasive pseudopodia in type to proliferate and form secondary tumors. Cancer cells cancer cells, enabling them to penetrate the ECM to invade 2 Journal of Oncology Mutation Oncogene activation Proliferation Selection Normal cells Premalignent cells Metastatic tumor Mutated cells Vessels Dissemination CTC CTC Oncogene activation Proliferation Mutation Selection Normal cells Premalignent cells Primary tumor Metastatic tumor Figure 1: -e mechanism of circulating tumor cells (CTCs) driving tumor metastasis. CTCs refer to all kinds of tumor cells in the peripheral blood. Due to their spontaneous or clinic operation, most of the CTCs undergo apoptosis or are swallowed after entering the peripheral blood. A few can escape and are anchored to become metastatic. blood vessels. -e activity of cancer-associated fibroblasts in 2. Biological Characteristics of CTCs the recombinant ECM has been shown to promote drilling 2.1. Cellular Size. Due to high heterogeneity of tumor cells, and subsequent invasion of tumor cells [7]. the pore size in the CellSearch system is typically slightly CTCs are rare in healthy individuals, and even in pa- 5 7 larger than leukocytes [14]. Other researchers have suc- tients with malignancies, less than one CTC per 10 to 10 cessfully separated CTCs using size-based platforms that peripheral blood mononuclear cells (PBMCs) could be exploit difference in cell sizes. CTCs in prostate cancer are observed. -us, isolation and enrichment are often the first divided into three categories based on size (diameter): very steps in CTC detection in laboratories and hospitals [10]. small nuclear CTCs (<8.54μm), small nuclear CTCs -ere are two classical approaches to separating CTCs from (8.54–14.99μm), and large nuclear CTCs (>14.99μm) [3]. blood samples: physical separation that exploits unique Jiang et al. selectively enlarged the size of tumor cells physical properties of CTCs (such as density and size) and covered with polystyrene microspheres and the modified immune adhesion, which depends on the high binding af- cells were clearly distinguishable from white blood cells. -e finity of receptors on CTCs to specific antibodies or modification method had no significant effect on cell sur- aptamers [11]. Compared with immune adhesion, physical vival and proliferation. Using this method, 15 CTC subtypes isolation is a simpler method as it obviates the need for cell were detected in 18 cases of colorectal cancer at a concen- labeling. However, the immune adhesion method achieves tration of 4–72 CTCs/mL. -us, this method has great higher purity in CTC isolation. potential in the early diagnosis and individualized treatment Due to technical limitations, few studies have in- of cancer [15]. Zavridou et al. directly compared two dif- vestigated the precision of CTC detection methods. Keller ferent methods of isolating CTCs from head and neck and Pantel discussed how CTC analysis at single-cell res- squamous cell carcinoma: a size-dependent microfluidic olution provides unique insights into tumor heterogeneity system and epithelial cell adhesion molecule (EpCAM)- [12]. Martin et al. reviewed preclinical and clinical data on dependent positive selection. -e results showed that, in the cancer treatment, CTC mobilization, and other factors that same blood sample, the label-freesize-dependent CTC may promote metastasis, establishing that advanced thera- separation system had higher sensitivity than the EpCAM- peutic strategies could benefit patients with locally advanced dependent CTC enrichment system [16]. cancer [13]. However, a systematic review of the occurrence, development, and outcome of CTCs in metastatic cancer is largely lacking. In this review, we present an overview of the 2.2. Cellular Density. Density is the physical property biological characteristics of CTCs, current CTC detection exploited in traditional separation and enrichment methods techniques, and principles and methods of CTC isolation. for CTCs [17]. In Ficoll density gradient centrifugation, Finally, potential applications of CTCs in the treatment of CTCs, plasma, and monocytes remain in the upper layer, metastatic cancer are proposed. whereas erythrocytes and polymorphonuclear leukocytes Journal of Oncology 3 metastasis [32–34]. In this process, cancer cells lose polarity settle in the bottom layer. CTCs may occur in both plasma and separation fluid. -us, some liquids above the red blood and their connection with ECM, transforming into fusiform mesenchymal cells, which are easily detached from the tu- cell layer should be collected for enrichment to prevent the loss of CTCs [5]. Huang et al. developed a new density mor cell population. Several ECM-degrading proteases are gradient centrifugation method that uses biodegradable upregulated in cancer cells with EMT, increasing their in- gelatin nanoparticles wrapped on silica beads for isolation, vasiveness [32–35]. As shown in Figure 2, the occurrence of release, and downstream analysis of CTCs from colorectal EMT in CTCs could result in the loss of specific molecular and breast cancer patients. -is method has remarkable CTC markers in epithelial cells such as EpCAM and cytokeratin capture efficiency (>80%), purity (>85%), high CTC-release and overexpression of specific molecular markers in in- efficiency (94%), and viability (92.5%) [18]. -us, this ap- terstitial cells such as vimentin and cadherin. -ese cells proach provides new opportunities for personalized cancer have a strong survival advantage and high metastasis and potential for transfer in the blood circulation [17, 36–38]. diagnosis and treatment and may also be useful in de- veloping drug treatment guidelines for cancer. Based on the EMT stage, CTCs are divided into E-type, M- type, E/M-type, and N (null)-type. Several studies have shown that E/M-type CTCs have enhanced epithelial cell 2.3. Heteromorphy in CTCs. Marrinucci et al. conducted adhesion and extravasation capacity, representing more cellular morphological evaluation of circulating components aggressive subtype of cancer cells with the highest metastatic of highly metastatic breast cancer. -ey found highly capacity [39–41]. Additionally, M-type CTCs exhibit en- polymorphic CTCs in breast cancer patients, including hanced resistance to clinically relevant chemotherapeutics. CTCs with high and low nuclear-to-cytoplasmic ratios and early and late apoptotic changes. In addition, compared with 3. Separation and Enrichment of CTCs tumor cells in other sites, the complete morphologic spec- trum of cancer cells in primary and metastatic tumors was -ere are two major approaches based on the principle of also present in peripheral blood circulation [19]. Several CTC separation and enrichment: physical property sepa- studies have found various forms of CTCs in peripheral ration and affinity-based identification [42, 43]. For the blood existing either independently or in clusters, with some physical property separation method, tumor cells are sep- CTCs even interacting with platelets to form a shell around arated from other cells based on differences in size [44, 45], them [20–23]. Aceto et al. found that CTC clusters in breast density [46], deformability, and adhesion between tumor cancer-bearing mice were shed as whole oligomeric colonies cells and normal blood cells [18, 47]. -e affinity-based rather than as simply a group of CTCs aggregating in the identification method involves identification of the spe- bloodstream [24, 25]. cific antigen on the surface of cancer cells using antibodies [44, 48], aptamer [49, 50], or E-selectin [51, 52]. Additional details are shown in Figure 3. 2.4. Proliferation and Apoptosis of CTCs. For tumor cells in circulation, only a few CTCs with high viability and potent metastatic potential survive and colonize distant organs to 3.1. Gradient Density Centrifugation. Two centrifugation- develop into metastatic foci. CTCs entering the circulatory based systems are available in the market today: Onco- system have very short survival times, typically less than Quick and AccuCyte [43, 53]. After isolation and enrich- 24 h, and vary in their indices of proliferation [26]. Driemel ment with Ficoll-Paque separation fluid, 24 CTCs were et al. found that the high expression of EpCAM was common detected in fifty-eight 1 mL blood samples from colorectal in cancer cells in the proliferation stage, while the low ex- cancer patients using real-time reverse transcription- pression of EpCAM inhibited the proliferation of CTCs [27]. polymerase chain reaction (RT-PCR) [53]. Rosenberg Studies have reported low levels of expression of pro- et al. reported that using a new OncoQuick system to isolate liferating nuclear antigen Ki-67 in CTCs, suggesting that cancer cells had a 632-fold enrichment effect compared with most CTCs may remain in a dormant state without entering less than 4-fold enrichment effect using Ficoll-Paque [53]. In the cell division cycle [28, 29]. It has also been found that, addition, 11 CTCs were detected in 37 samples of gastric several years after primary tumorigenesis, dispersed CTCs cancer patients using a combination of OncoQuick and and micrometastasis niche can remain dormant for a long RT-PCR [53]. In another study, 5 and 25 CTCs were de- time during resection of primary tumors [30]. -ese results tected in 60 cases of early breast cancer patients after im- suggest that dormant CTCs can be attached to tissues or cell munofluorescence and 63 samples of patients with advanced clusters until their activation or that of a certain factor in the breast cancer, respectively [2]. Although density-gradient isolation procedures. -e specific mechanism may be related centrifugation is uncomplicated, it lacks specificity and can to the body’s immune response. easily lead to loss of tumor cells without corresponding density. 2.5. 2e Metastatic Portent of Circulating Tumor Cells. -erefore, density gradient centrifugation is often used EMT is a biological process by which epithelial cells acquire as the first step to separate CTCs and then combined with a mesenchymal phenotype through a series of biochemical other methods to specifically bind and separate CTCs. For changes [31]. In recent years, accumulating evidence sug- example, Hu et al. used density gradient centrifugation and gests EMT phenomena in the process of cancer cell magnetic separation based on CD45 antibody to separate 4 Journal of Oncology TGF, EGF, IGF, FGF, HGF, PDGF, Hypoxia … Snail1/2 Mesenchymal traits Epithelial traits Elf5 Zeb1/2 Grhl2 Twist miR-200 FOXC2 miR-34 Six1 EMT Deceased cell-cell adhesion Cell detachment & extrusion Vessels Dissemination Figure 2: Induction of epithelial-mesenchymal transition (EMT) by various cytokines. It is generally assumed that the metastatic spread of epithelial tumors depends on EMT, a process in which cancer cells lose their polarity and cell-cell adhesion to acquire ‰broblast-like features such as migration and invasion. CTCs collection Centrifugation Filtration Ficoll centrifugation (a) (b) Adhesion Microfluids (c) (d) CTC Immunocytes Red blood cells Figure 3: Circulating tumor cell (CTC) enrichment technologies. (a) Density gradient centrifugation. (b) Micro¥uidic-based separation technology. (c) Di‘erent ‰ltration systems depending on the size of blood cells used for separation and enrichment of CTC. (d) Adhesion is dependent on the a¦nity and speci‰c binding of antibodies or aptamers (e.g., immune adhesion) to the CTC receptor. Magnetic beads Journal of Oncology 5 hepatocellular carcinoma with conventional methods is CTCs [46]. Different from the traditional negative enrich- ment, Hu et al. applied the subtraction enrichment and significantly limited, leading to false-negative detection of such CTCs. Wang et al. reported for the first time the ex- immunostaining fluorescence in situ hybridization (SE- iFISH) strategy to detect CTCs, which effectively removed istence of small-sized CTCs (<5μm WBC) with cytogenetic red blood cells by centrifugation rather than using hypotonic abnormalities in aneuploid chromosome 8, which is pre- injury [54]. dominantly detected in hepatocellular carcinoma (HCC) patients [69]. 3.2. Method for Separation and Capture Based on Cell Size. -e method takes advantage of the larger size of CTCs 3.3.ImmunomagneticBeads. Almost all cells in the blood are compared with erythrocytes [55]. Isolation by size of epi- diamagnetic or weakly magnetic [70]. -erefore, tumor cells thelial tumor cells (ISET) and ScreenCell systems have been are usually labeled with antibody-conjugated magnetic used for clinical trials in melanoma, breast, lung, and beads or nanoparticles. -ese antibodies bind primarily to pancreatic cancers [56, 57]. For the first time, Zheng et al. tumor-cellsurface-specific antigens, including some in- used parylene-C to make circular and oval microporous tracellular antigens [71]. -e number of CTCs counted using filters, achieving a CTC capture efficiency of 90% [58]. A cell search has been used for prognosis of some cancers after model for gene analysis and analysis of cells after chip metastasis [72–74]. Wu et al. developed a magnetic cell electrolysis developed by Birkhahn et al. [59] was sub- centrifugation platform (MCCP) combining the separation sequently applied to the detection of exfoliated cells from mechanism of magnetically labeled cells with the size-based urinary bladder cancer. Hosokawa et al. integrated nickel method and obtained target cells with 97% purity, high microporous sieves made from micro-electroforming into throughput of 2μL/s, and a sample enrichment factor of a microfluidic chip. -e team also applied the improved 66 times [75]. Overall, the performance of the immuno- nickel microporous sieves to the detection of CTCs in the magnetic particle separation method mainly includes the blood of patients with small-cell lung cancer [60]. following factors: (1) the expression level and specificity of To improve the capture efficiency of CTCs, Coumans the target antigen and the binding ability of the corre- et al. studied factors affecting the trapping of filter cells [61]. sponding antibody and (2) the efficiency of immuno- In microfluidic chips, the precise fabrication of shapes and magnetic particle labeling. Immunomagnetic particles used microstructures in microchannels makes it possible to for cell separation have high recovery and purity and even separate and enrich tumor cells on a size-by-size basis [62]. detect CTCs in one step [76–78]. Erythrocytes have stronger deformability and smaller vol- ume, thus can easily cross various microstructures [63]. Nicinski ´ et al. proposed a new tool that uses microfluidic 3.4. Chip Technology. In the 1990s, Manz et al. proposed devices, photovoltaic (PV)-based SERS activity platform, a microfluidic chip technology [79]. Commonly used CTC and shell separation nanoparticles (shins) for simultaneous antibodies include human EpCAM and leukocyte common separation and unlabeled analysis of circulating tumor cells antigen CD45 [80]. Affinity sorting includes two types of in blood samples. -e results demonstrated the potential of capture methods. -e first type is the positive sorting SERS-based tools for isolating tumor cells from whole blood method, which directly targets and specifically captures samples in a simple and minimally invasive way in a scaled- target cells. -e second type is the negative sorting method, up detection and molecular identification pipeline [64]. which involves the capture nontarget cells, which are then Ohnaga et al. used a microchannel to capture circulating discarded. A schematic diagram of the working principle is tumor cells in esophageal and breast cancers [65]. Zeinali shown in Figure 4. et al. used a sensitive microfluidic CTC capture device to analyze circulating epithelium and EMT-like CTCs in 3.4.1. Positive Sorting Methods. -e traditional affinity pancreatic cancer [66]. To capture CTCs larger than the maximum pore size sorting method involves direct binding of the antibody to the microfluidic chip channel [81]. Sequist et al. developed the regardless of cell surface expression, blood is filtered through pores (usually 8μm in diameter). However, the success of second-generation CTC chip called herringbone (HB)-chip this process depends on many factors, including blood flow [82]. Compared with first-generation CTC chips, the rate, pore size uniformity, and membrane stiffness. High second-generationHB-chip is easy to use and more efficient, flow velocity will cause CTC to “squeeze” through pores, providing comprehensive and easy access to data. Hughes causing membrane distortion. A very slow flow rate will lead et al. integrated halloysite nanotubes into this chip [83] by to excessive accumulation of white blood cells, blood co- immobilizing E-selectin and anti-EpCAM on nanotubes. In agulation, and prolonged processing time [67]. Moreover, this design, E-selectin captures rapidly moving CTCs, tumor cells undergoing epithelial-mesenchymal transition whereas anti-EpCAM specifically captures CTCs [84, 85], increasing the purity of the captured CTCs. To simplify the (EMT) were smaller than those without EMT characteristics [68]. -erefore, CTCs receiving EMT may not be detected experimental procedures, Stott et al. designed a fishbone- based affinity sorting chip for direct analysis of whole blood using these technologies. Due to the inherent heterogeneity and dynamic expression of EpCAM and the degradation of samples with a sorting speed of up to 1 mL/h. Captured cytokeratin during the transformation of epithelial cells into circulating tumor cells could also be used for other assays or mesenchymal cells, the detection of circulating tumor cells in cell culture [86]. Sheng et al. optimized the fishbone 6 Journal of Oncology Blood sample from patients Red blood cells Immunocytes CTCs Negative sorting method Positive sorting method Microfluidic chip Target cells Non-target cells Figure 4: Peripheral blood samples from patients with non-small-cell lung cancer were obtained before any treatment and immediately processed in the circulating tumor cell (CTC) herringbone (HB)-chip that captures anti-EP-CAM-coated column epithelial cell adhesion molecules (left). Negative consumption of untargeted cells by a negative consumption method, including red blood cells and immune cells. Targeted cells such as CTCs were left in the chip for further analysis (right). structure to achieve a CTC capture e¦ciency and sorting Containing Anti-CD56 to collect CTCs in peripheral blood, purity higher than 90% and 84%, respectively [87]. ‚ese was used to monitor the occurrence and disease response to micro¥uidic chip technologies have shown good CTC treatment at di‘erent time points [91]. capture capability. However, releasing CTCs from micro- Unlike traditional negative enrichment, researchers ¥uidic chips for subsequent analysis is challenging. ‚ere- utilize subtraction enrichment (SE), independent of cell size, fore, researchers have introduced magnetic materials into cluster, or surface anchor protein expression. ‚e immu- micro¥uidic chips for CTC sorting [88]. nostained proteins were proved to be free from the re- striction of antigen epitopes inside and outside cells and membrane-related tumor biomarkers. With the clinical 3.4.2. Negative Sorting Methods. EpCAM-based a¦nity application of SE-iFISH, in addition to the traditional tumor separation cannot be applied to CTCs with weakly expressed cell types, there are more and more accidental discoveries of or nonexpressed EpCAM in the process of tumor cell me- various phenotypes of CTCs [54]. Zhang et al. have shown − + tastasis, which leads to the signi‰cant decrease or even loss of that aneuploidy CD31 CTC and CD31 CTEC may be used EpCAM expression. For example, Lee designed a chip called as a pair of biomarkers for circulating cell tumors to predict “μ-MixMACS”, which greatly increased the number of CTCs patients with non-small-cell lung cancer receiving anti- detected [89]. Sajay et al. designed a two-step negative CTC angiogenesis combined therapy [40]. Based on the SE-iFISH sorting platform where the recovered cells remain bioactive strategy, Yang et al. demonstrated that patients with early and can be further analyzed for protein or nucleic acid bladder cancer had more triploid CTCs, tetraploid CTCs, content [90]. A CTC-negative enrichment scheme, which and total circulation endothelial cells (CECs). Various CTC/ utilized the RosetteSep CTC Enrichment Cocktail CEC subtypes may have di‘erent potential function to guide ™ Journal of Oncology 7 the diagnosis, prognosis prediction, and treatment decision tests and the specificity of series tests [116]. -e molecular of bladder cancer [92]. Li et al. found that the presence of spectrum of four genes, including CK20, CK19, CEA, and circulating tumor-cell-associated white blood cell (CTC- GCC, identified 87.7% of tumor metastases with a false- WBC) clusters is an independent prognostic factor for positive rate of only 2.2% [117]. advanced non-small-cell lung cancer [93]. 5. Clinical Applications of CTCs 4. Detection of CTCs CTCs are a promising biomarker for early disease diagnosis, treatment response and disease progression evaluation, re- 4.1. Immunocytochemistry. Immunochemistry is a modern currence monitoring, and therapeutic target identification technology that binds specific monoclonal antibodies with for drug development [118]. Detection of CTCs has been CTCs, followed by conjugation of a chromogenic reagent to widely used in the diagnosis of early and metastatic cancers visualize CTCs. -e most commonly used monoclonal (Table 1). antibody are anti-CK antibodies, such as epithelial-specific markers (CK) [94], interstitial cell surface markers (Snail1, E47, and Twist) [95], E-cadherin antagonist [96], stem cell 5.1. Early Diagnosis and Staging of Cancer. Traditional im- markers (CD133+, CD44+, and CD24−), aldehyde de- aging methods cannot effectively detect early tumor lesions. hydrogenase 1 (ALDH1) [97–100], special marker Survivin CTC detection approaches can detect tumor earlier than [101, 102], estrogen receptor (ER) [103], and progesterone imaging or clinical manifestations when the lesion is<1 cm, receptor (PR) [104]. EpCAM and CK may be lost during hence facilitate early diagnosis. Besides its role in early epithelial-mesenchymal transition (EMT), leading to the tumor diagnosis, CTC is also correlated with tumor grade failure of EpCAM- and CK-dependent strategies to detect and TNM stage. Santos et al. found that CTCs have great CTCs. -erefore, Li et al. used SET-iFISH technology to potential in the early diagnosis of colorectal cancer since enrich and characterize CTCs in advanced gastric cancer they can be detected in the peripheral blood of patients with (AGC) and obtained a higher positive detection rate than early-stage colorectal cancer. -erefore, the CTC test may be that obtained using EpCAM-dependent detection strategies applied to the diagnosis of colorectal cancer [123]. Clinical (CellSearch) [105]. In addition, Li et al. captured CTCs in staging of colorectal cancer is often based on anatomical AGC through SE-iFISH and found that cHER2 phenotype is alterations of the intestine; however, it is difficult to accu- useful to understanding the treatment resistance of AGC rately identify micrometastasis during the prognosis and patients [106]. Subsequently, scientists used this method to treatment of patients [124]. Detection of CTCs in the blood characterize the markers of CTC, such as EpCAM [41], does not necessarily indicate the occurrence of metastasis. PD-L1 [107], vimentin [40], and CD44 [108]. However, several studies have shown the value of detection of CTCs in the staging of colorectal cancer in clinical practice [125]. Using an advanced CanPatrol CTC enrich- 4.2. RT-PCR. Currently, RT-PCR is considered the gold ment technique and in situ hybridization to sort and classify standard in the detection of some viruses because of its high CTCs in blood samples, 90.18% of hepatocellular carcinoma sensitivity [109, 110]. RT-PCR is also widely used in tumor (HCC) patients were found to be CTC positive, even at the detection [111, 112]. However, selecting optimal RNA early stage of HCC [126]. CTCs were also detected in 2 of 12 markers can be challenging, limiting its efficacy. An ideal patients with hepatitis B virus (HBV) infection, with both RNA marker should have the following characteristics: all patients developing small HCC tumors in less than five types of tumor cells are expressed in peripheral blood months. Another study by Wang et al. implicated CTCs in leukocytes, nontumor epithelial cells are not expressed, and tumor staging [127]. Recent studies have shown that CTCs no illegitimate transcription events [113]. Using qualitative also put into good use in hematologic malignancies. Primary RT-PCR, Wang et al. found that the expression of androgen plasma cell leukemia (pPCL) is clinically distinguishable receptor variant 7 (AR-V7) in CTCs from patients with from newly diagnosed multiple myeloma (NDMM) based prostate cancer was associated with drug resistance. -e on the proportion of circulating tumor cells of 20% [128]. upregulation of AR-V7 led to the enhancement of cancer cell Zhang et al. also used a technology based on oncolytic proliferation, suggesting poor patient prognosis [114]. Wei herpes-simplex-virus-1 to detect CTCs in non-Hodgkin’s et al. recruited 78 patients with stage I –II cervical cancer A2 A1 lymphoma [129]. who had undergone radical hysterectomy by laparotomy or laparoscopy and selected 34 uterine fibroid patients and 32 5.2. Treatment Evaluation and Recurrence Monitoring. healthy subjects as the positive control group and negative control group, respectively. RT-PCR was used to amplify Treatment evaluation and recurrence monitoring of CTCs peripheral blood CK19, CK20, and SCC-Ag from total RNA. has been extensively studied. Lin et al. measured the number -e results showed that CTCs were highly expressed in both of peripheral blood CTCs before and after NK cell immu- the open surgery group and the laparoscopic radical mas- notherapy in stage IV non-small-cell lung cancer (NSCLC) tectomy group, with no significant difference between the patients, providing a useful reference for monitoring any two groups [115]. Using Survivin, hTERT and hMAM change in NK cell therapeutic effect [130]. Nagrath et al. markers to detect CTC in breast cancer patients, Shen et al. detected and monitored CTCs using a CTC chip and found found that these markers improved the sensitivity of parallel that the CTC count of patients with lung and prostate cancer 8 Journal of Oncology Table 1: Clinical applications of CTCs Patient Detection Clinical trial Cancer type Marker Significance Reference number methods no. EpCAM, In breast cancer patients with first-line chemotherapy, CTC Breast cancer 549 CellSearch NCT00382018 Paoletti et al. [119] CK counts were associated with mortality. A transcriptional profile detectable in CTCs can serve as an NCT01942837, Prostate cancer 147 mCRPC VERSA EpCAM Sperger et al. [120] independent prognostic marker in mCRPC. NCT01942837 Pancreatic CTC-positive preoperatively (≥1 CTC/7.5 mL) showed 209 patients CellSearch EpCAM NCT01919151 Hugenschmidt et al. [121] cancer a detrimental outcome despite successful tumor resections. Baseline CTCs≥ 3 were detected in 19% of the patients. CTC≥ 3 Colorectal EpCAM, 153 CellSearch at baseline and 4 weeks after therapy showed shorter overall NCT01442935 Bidard et al. [122] cancer CK survival. Journal of Oncology 9 decreased significantly before and after chemotherapy and the detection of CTCs will become a powerful and in- endocrine therapy. Although the changes in the CTC count dispensable tool for the diagnosis of circulating tumor cells (tumor DNA) with its advantages of repeatability, mutation due to treatment are affected by the differences between individual patients, they can still be used as a reference for detection at the molecular level, noninvasive diagnosis and evaluating the efficacy of tumor treatment [131–133]. In broad application potential in targeted therapy, efficacy some cases, CTCs are more sensitive than imaging, thus they testing, postoperative prognosis, radiotherapy and chemo- are included in efficacy evaluations [134]. In recent years, therapy strategy guidance, as well as in differential diagnosis. several detection techniques have been developed for CTC genotyping as well as detection of crucial gene mutations, Data Availability such as ER [135], HER2 [136], and TP53 [137]. -us, these techniques can help clinicians in treatment evaluation and -e data used to support the findings of this study are in- monitoring tumor recurrence. Zhou et al. used PCR and cluded within the article. fluorescence-activatedsingle-cell sorting (FACS) to detect + + + + levels of EpCAM mRNA CTCs and CD4 CD25 Foxp3 Conflicts of Interest Treg cells in 49 HCC patients before surgery. -e data showed that CTC/Treg levels were positively correlated with -e authors declare that they have no conflicts of interest. the risk of postoperative recurrence [138]. Due to differences in tumor type and stage among cancer Authors’ Contributions patients and occult, it is difficult to detect metastatic tumor relapse within five years of primary tumor resection. Cancer JX and DC conceived the research topic. SW and SZ con- that persists despite treatment and cannot be detected by ducted literature review, drafted the manuscript, and current medical imaging modalities is defined as minimal designed the figures and tables. JX, DC, and SW revised the residual disease (MRD), which is in the occult stage of cancer manuscript. All the authors approved the submitted version. progression. Liquid biopsy methods based on detection of Siwen Wu and Shubi Zhao contributed equally to this work. small amounts of circulating tumor cells (CTCs) or trace amounts of circulating cell-free tumor DNA (ctDNA) are Acknowledgments now available for MRD detection in patients with various malignant tumors. Monitoring CTCs and ctDNA during -e work was supported by the National Natural Science postoperative follow-up assessments can detect disease re- Foundation of China (Grant Nos. 82172335, 81971994, and currence months earlier than other medical imaging 91846103), Zhejiang Provincial Key Research and Devel- methods. Further characterization of CTCs and ctDNA opment Program (Grant No. 2020C03032), and Technology could provide insights into the molecular evolution of MRD Project of Zhejiang Provincial Health Commission (Grant during tumor progression, which has important implica- No. 2022KY155). tions for treatments that delay or even prevent metastatic recurrence. -erefore, the detection of CTCs has become the References main method for the assessment of minimal residual disease (MRD) [139]. [1] V. J. Hofman, M. I. Ilie, C. Bonnetaud et al., “Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method,” American Journal Of 6. 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Journal of OncologyHindawi Publishing Corporation

Published: Sep 2, 2022

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