Congenital Tufting Enteropathy Research Articles

Approach to Congenital Diarrhea and Enteropathies (CODEs).

Congenital diarrhea and enteropathies (CODEs) constitute a group of rare genetic disorders characterized by severe diarrhea and malabsorption in the neonatal period or early infancy. Timely diagnosis and treatment is essential to prevent life-threatening complications, including dehydration, electrolyte imbalance, and malnutrition. This review offers a simplified approach to the diagnosis of CODEs, with a specific focus on microvillus inclusion disease (MVID), congenital tufting enteropathy (CTE), congenital chloride diarrhea (CLD), and congenital sodium diarrhea (CSD).Patients with CODEs typically present with severe watery or occasionally bloody diarrhea, steatorrhea, dehydration, poor growth, and developmental delay. Therefore, it is crucial to thoroughly evaluate infants with diarrhea to rule out infectious, allergic, or anatomical causes before considering CODEs as the underlying etiology. Diagnostic investigations for CODEs encompass various modalities, including stool tests, blood tests, immunological studies, endoscopy and biopsies for histology and electron microscopy, and next-generation sequencing (NGS). NGS plays a pivotal role in identifying the genetic mutations responsible for CODEs.Treatment options for CODEs are limited, often relying on total parenteral nutrition for hydration and nutritional support. In severe cases, intestinal transplantation may be considered. The long-term prognosis varies among specific CODEs, with some patients experiencing ongoing intestinal failure and associated complications. In conclusion, the early recognition and accurate diagnosis of CODEs are of paramount importance for implementing appropriate management strategies. Further research and advancements in genetic testing hold promise for enhancing diagnostic accuracy and exploring potential targeted therapies for these rare genetic disorders.

Early-onset tufting enteropathy in HAI-2-deficient mice is independent of matriptase-mediated cleavage of EpCAM.

Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.

414 - Congenital tufting enteropathy a rare cause of failure to thrive in an infant - a case report

414 - Congenital tufting enteropathy a rare cause of failure to thrive in an infant - a case report

A novel compound-heterozygous EPCAM mutation in congenital tufting enteropathy.

A novel compound-heterozygous EPCAM mutation in congenital tufting enteropathy.

EpCAM Is Essential to Maintaining the Immune Homeostasis of Intestines via Keeping the Expression of pIgR in the Intestinal Epithelium of Mice.

EpCAM deficiency causes congenital tufting enteropathy (CTE) which is considered as one kinds of very early onset inflammatory bowel disease (IBD). However, functions of EpCAM on regulating the immunity of intestines are still unclear. To study the mechanism of EpCAM on maintaining the intestinal immune homeostasis, the intestines of WT and EpCAM-/- mice at E18.5, P0 and P3 stages were collected for morphological, histological and gene expression tests. Serious inflammation was detected in the small intestines of P3 EpCAM-/- mice. Compared to WT mice, genes related to inflammatory factors and immunity cells, including TNFα, IL-1β, IL-6, IL-8rb, MIP2, MCP1, Ly6d and Ly6g, were all significantly upregulated and the expression of intestinal abundance matrix metalloproteinases (MMPs) was also significantly increased in the intestines of EpCAM-/- mice at E18.5, P0 and P3 stages. Signals of p38, ERK1/2 and JNK were hyper-activated in the intestines of EpCAM-/- mice. The expression of pIgR was significantly decreased and the expression and activation of transcriptional factors which promote the expression of pIgR were also reduced in the intestines of EpCAM-/- mice compared to WT controls. In conclusion, EpCAM could maintain the immune homeostasis of intestines via keeping the expression of pIgR in the intestinal epithelium.

Homozygous Missense Epithelial Cell Adhesion Molecule Variant in a Patient with Congenital Tufting Enteropathy and Literature Review.

Congenital diarrheal disorders (CDDs) with genetic etiology are uncommon hereditary intestinal diseases characterized by chronic, life-threatening, intractable watery diarrhea that starts in infancy. CDDs can be mechanistically divided into osmotic and secretory diarrhea. Congenital tufting enteropathy (CTE), also known as intestinal epithelial dysplasia, is a type of secretory CDD. CTE is a rare autosomal recessive enteropathy that presents with intractable neonatal-onset diarrhea, intestinal failure, severe malnutrition, and parenteral nutrition dependence. Villous atrophy of the intestinal epithelium, crypt hyperplasia, and irregularity of surface enterocytes are the specific pathological findings of CTE. The small intestine and occasionally the colonic mucosa include focal epithelial tufts. In 2008, Sivagnanam et al. discovered that mutations in the epithelial cell adhesion molecule (EpCAM, MIM# 185535) were the genetic cause of CTE (MIM# 613217). More than a hundred mutations have been reported to date. Furthermore, mutations in the serine peptidase inhibitor Kunitz type 2 (SPINT2, MIM# 605124) have been linked to syndromic CTE. In this study, we report the case of a 17-month-old male infant with congenital diarrhea. Despite extensive etiological workup, no etiology could be established before admission to our center. The patient died 15 hours after being admitted to our center in a metabolically decompensated state, probably due to a delay in admission and diagnosis. Molecular autopsy with exome sequencing revealed a previously reported homozygous missense variant, c.757G>A, in EpCAM, which was confirmed by histopathological examination.

Congenital Diarrhea and Enteropathies in Infants: Approach to Diagnosis.

Congenital diarrhea and enteropathies (CODEs) are monogenic disorders causing early onset of intractable diarrhea. Their diagnosis and management are challenging. With the availability of commercial next generation genetic testing, we are now better able to classify and manage these disorders. The authors present their experience with 4 cases. Two patients had congenital tufting enteropathy (CTE) and 1 case each of microvillous inclusion disease (MVID) and trichohepatoenteric syndrome (THES). Age at onset varied from 3 to 38 d of life. Light microscopy and electron microscopy of duodenal and rectal endoscopic biopsies were consistent with the diagnosis. Genetic evaluation was possible in 3 cases indicating causative mutations. Two children (CTE and MVID) were alive at last follow-up. The authors suggest a stepwise approach to the diagnosis and management of these disorders in the Indian context.

KOCURIA SPECIES: EMERGING PATHOGENS IN CHILDREN WITH CENTRAL VENOUS CATHETER, A CASE REPORT AND REVIEW OF LITERATURE

Kocuria species (KS) are gram-positive, coagulase-negative coccoids. Reports about KS infection in children are limited. We performed a short-review of literature and described a case of catheter related-bloodstream infection (CR-BSI) caused by KS. Electronic search through PubMed was conducted using the keywords: “Kocuria” AND “children” AND “sepsis”. The earliest publication date was of 2008, the latest of 2017 We analyzed 7 articles with 15 cases described. KS were responsible of endocarditis in 1 child, CR-BSI in 13 children and spontaneous bacterial peritonitis in 2 children. Most were immunocompromised hosts and CVC-carriers (2 short-bowel syndrome, 2 tumor, 1 congenital tufting-enteropathy, 1 Hirschsprung's disease, 1 methylmalonic-aciduria, 6 prematurity). Only 1 was an otherwise healthy child. Case report: during the COVID-19 lockdown, a 17yeas-old boy affected by Hirschsprung's disease and CVC-carrier developed fever, chills, vomit and hematuria. Due to “hospital fear”, he began empirical therapy with ceftriaxone at home, without symptoms resolution. Once admitted to the department, blood exams showed: normal lymphocyte immunophenotyping, thrombocytopenia, increased CRP and hyperbilirubinaemia. Haematuria, proteinuria and glycosuria also occurred. Culturing blood samples showed KS infection. Echocardiography was normal. Basing on the antibiogram, we started treatment with vancomycin with rapid patient recovery. The use of CVCs in immunocompromised patients represents the most important risk factor for KS-BSI. Due to the limited number of reports about this infection, there are no guidelines to manage it.In patients with a chronic disease, opportunistic pathogens as KS must be considered, also because symptoms and complications are still unknown.

NGS Gene Panel Analysis Revealed Novel Mutations in Patients with Rare Congenital Diarrheal Disorders.

Congenital diarrheal disorders (CDDs) are early-onset enteropathies generally inherited as autosomal recessive traits. Most patients with CDDs require rapid diagnosis as they need immediate and specific therapy to avoid a poor prognosis, but their clinical picture is often overlapping with a myriad of nongenetic diarrheal diseases. We developed a next-generation sequencing (NGS) panel for the analysis of 92 CDD-related genes, by which we analyzed patients suspect for CDD, among which were (i) three patients with sucrose-isomaltase deficiency; (ii) four patients with microvillous inclusion disease; (iii) five patients with congenital tufting enteropathy; (iv) eight patients with glucose-galactose malabsorption; (v) five patients with congenital chloride diarrhea. In all cases, we identified the mutations in the disease-gene, among which were several novel mutations for which we defined pathogenicity using a combination of bioinformatic tools. Although CDDs are rare, all together, they have an incidence of about 1%. Considering that the clinical picture of these disorders is often confusing, a CDD-related multigene NGS panel contributes to unequivocal and rapid diagnosis, which also reduces the need for invasive procedures.

EpCAM (CD326) Regulates Intestinal Epithelial Integrity and Stem Cells via Rho-Associated Kinase.

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy.

Background & AimsCongenital tufting enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutations of epithelial cell adhesion molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis.MethodsIntestine and colon from mice expressing a CTE-associated mutant form of EpCAM (mutant mice) were evaluated for specific markers by quantitative real-time polymerase chain reaction, Western blotting, and immunostaining. Body weight, blood glucose, and intestinal enzyme activity were also investigated. Enteroids derived from mutant mice were used to assess whether the decreased census of major secretory cells could be rescued.ResultsMutant mice exhibited alterations in brush-border ultrastructure, function, disaccharidase activity, and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in mutant mice led to decreased enteroendocrine cells and increased numbers of nonsecretory cells, though the hypertrophied absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with the Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in mutant enteroids (graphical abstract 1).ConclusionsAlterations in intestinal epithelial cell differentiation in mutant mice favor an increase in absorptive cells at the expense of major secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients.

Cover 2 - Disrupted Brush Border in Congenital Tufting Enteropathy

Cover 2 - Disrupted Brush Border in Congenital Tufting Enteropathy

Congenital Tufting Enteropathy: Biology, Pathogenesis and Mechanisms.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease of infancy that causes severe intestinal failure with electrolyte imbalances and impaired growth. CTE is typically diagnosed by its characteristic histological features, including villous atrophy, crypt hyperplasia and focal epithelial tufts consisting of densely packed enterocytes. Mutations in the EPCAM and SPINT2 genes have been identified as the etiology for this disease. The significant morbidity and mortality and lack of direct treatments for CTE patients demand a better understanding of disease pathophysiology. Here, the latest knowledge of CTE biology is systematically reviewed, including clinical aspects, disease genetics, and research model systems. Particular focus is paid to the pathogenesis of CTE and predicted mechanisms of the disease as these would provide insight for future therapeutic options. The contribution of intestinal homeostasis, including the role of intestinal cell differentiation, defective enterocytes, disrupted barrier and cell–cell junction, and cell-matrix adhesion, is vividly described here (see Graphical ). Moreover, based on the known dynamics of EpCAM signaling, potential mechanistic pathways are highlighted that may contribute to the pathogenesis of CTE due to either loss of EpCAM function or EpCAM mutation. Although not fully elucidated, these pathways provide an improved understanding of this devastating disease.

Tufting Enteropathy: A Review of Clinical and Histological Presentation, Etiology, Management, and Outcome.

Congenital tufting enteropathy (CTE), also named intestinal epithelial dysplasia, is a rare, autosomal recessive enteropathy with persistent and life-threatening intractable diarrhea early in life. Intractable diarrhea is present independent of breast or formula feeding. Most CTE patients require total parenteral nutrition (TPN), and in severe cases, small bowel transplantation is needed. In the last decade, we have seen remarkable progress in certain aspects, such as the pathogenesis and diagnostic methods of the disease. Rapidly developing molecular analysis techniques have improved the diagnostic methods for CTE and reduced invasive and expensive procedures. Mutations in the gene encoding human epithelial cell adhesion molecule (EpCAM) were identified in the typical form of CTE, which usually exhibits isolated refractory diarrhea. Moreover, the syndromic form of CTE features anal and choanal atresias as well as ophthalmologic signs, which are associated with mutations in the gene encoding Serine Peptidase Inhibitor Kunitz Type 2 (SPINT2). This article reviews CTE disease based on its clinical and histological presentation, etiology and pathogenesis, and management and outcome.

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells.

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule (EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers (Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors (Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease.NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule (EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

118 – Activation of the Unfolded Protein Response in the Murine Model of Congenital Tufting Enteropathy

118 – Activation of the Unfolded Protein Response in the Murine Model of Congenital Tufting Enteropathy

EpCAM Mutant Enteroids Show Alterations in Differentiation and Permeability

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is expressed in the basolateral cell surface of most of the epithelial tissues with the highest expression in the gut. Apart from its role in homotypic cellular adhesion and tight junctions, EpCAM is known to be involved in cellular signaling that promotes proliferation, migration, and maintenance of the undifferentiated state of pluripotent stem cells. Several tumors and carcinomas have been linked with EpCAM overexpression, while the mutations in EpCAM is directly associated with a severe diarrheal disease of infancy known as Congenital Tufting Enteropathy (CTE). Alterations in intestinal cell types and barrier permeability have been observed in the patient and the murine model with CTE. We hypothesize that EpCAM plays a pivotal role in intestinal cell type differentiation and intestinal barrier function. In the current study, we evaluate whether the mutation of EpCAM has an effect on the differentiation and permeability of the intestinal epithelial cells (IECs).We have developed an inducible EpCAM mutant enteroid model based on mutations found in CTE patients (deletion of EpCAM exon 4) to allow for ex vivo study of the consequences of EpCAM mutation on all types of IECs. We first established the mutant enteroid model by confirming the deletion of EpCAM exon 4. Immuno‐fluorescence study demonstrates the mislocalization of mutant EpCAM expression away from the cell surface. The different IECs markers were studied by RNA and protein. The gene expression of different transcription factors that are involved in the secretory pathway of intestinal cell differentiation were analyzed. We observe that mutant enteroids have significantly decreased (n=3; p<0.0001) gene expression of the differentiated goblet and paneth cells markers (Muc2, Lysozyme respectively) than the control enteroids. The expression of other cell types and differentiation markers (enteroendocrine cell marker ChgA, intestinal stem cell marker Lgr5, intestinal differentiation marker Krt20) are also decreased in the mutant model (n=3) as shown by qPCR. Moreover, the gene expression of Hnf1b and ATOH1, two early transcription factors for secretory pathway differentiation, are significantly decreased (n=3; p< 0.05) in mutant enteroids compared with controls. The enteroids were polarized onto monolayers to check the trans‐epithelial resistance and barrier integrity with FITC‐dextran flux assay. Trans‐epithelial resistance values remained intact between the control and mutant enteroid derived monolayer, while the epithelial permeability was significantly impaired in the EpCAM mutant enteroid model compared to the control (n=3; p<0.0001).In this study, we develop the first EpCAM mutant enteroid model. With this model, we note the role of EpCAM in the differentiation of IECs from intestinal crypt progenitor/stem cells. The involvement of EpCAM in barrier permeability is also confirmed. This study further establishes a new facet of EpCAM biology which will help in understanding the pathophysiology CTE and other EpCAM associated diseases.Support or Funding InformationNIH R01 DK107764This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Shedding light on the EpCAM: An overview.

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.