In both normal turnover of the hepatic tissue and acute hepatic injury, the liver predominantly activates terminally differentiated hepatocytes to proliferate and repair. However, in chronic and ...severe chronic injury, this capacity fails, and liver progenitor cells (LPCs) can give rise to hepatocytes to restore both hepatic architecture and liver metabolic function. Although the promotion of LPC-to-hepatocyte differentiation to acquire a considerable number of functional hepatocytes could serve as a potentially new therapeutic option for patients with end-stage liver disease, its development first requires the identification of the molecular mechanisms driving this process. Here, we found that the epithelial cell adhesion molecule (EpCAM), a progenitor cell marker, regulates the differentiation of LPCs into hepatocytes through Notch1 signaling pathway. Western blotting (WB) revealed a consistent expression pattern of EpCAM and Notch1 during LPC-to-hepatocyte differentiation in vitro. Additionally, overexpression of EpCAM blocked LPC-to-hepatocyte differentiation, which was in consistent with the repressive role of Notch signaling during hepatic differentiation. WB and immunofluorescence data also showed that the upregulation of EpCAM expression increased the generation of Notch intracellular domain (N1ICD), indicating the promotion of Notch1 activity. Our results established the EpCAM-Notch1 signaling axis as an inhibitory mechanism preventing LPC-to-hepatocyte differentiation in vitro.
•A novel molecular mechanism for regulation of LPC-to-hepatocyte differentiation is provided.•EpCAM-Notch1 signaling axis is an inhibitory mechanism preventing LPC-to-hepatocyte differentiation in vitro.•EpCAM blocks LPC-to-hepatocyte differentiation by positively regulating Notch1 activity.
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Circulating tumor cells (CTCs) are tumor cells present in the blood. CTCs have attracted much attention as a new tumor marker, because their analysis provides useful information for ...monitoring cancer progress. In this study, we developed cell-capture and release methods using three-dimensional (3D) microfiber fabrics without damaging the cells. Using functional peptides containing sequences from a polystyrene-binding site and a cleavable site for collagenase type IV, immobilized antibodies on the peptides were able to specifically capture MCF-7 cells in a few minutes and release the captured cells from 3D microfiber fabrics incorporating a vacuum system. The efficiency of cell capture was around 80% and that of the cell release was over 90%. The released cells proliferated normally in culture medium, suggesting that our system will be applicable for the culture and analysis of CTCs.
In this paper, we report cell-capture and release methods using enzyme-cleavable peptides immobilized on microfiber fabrics which has microporous polymeric three-dimensional structures. Detachment and collection of the selectively captured cancer cells are required for ex vivo culture and their further analysis, whereas the cell detachment methods developed so far might cause cell damage, even if cell viability is high enough. Therefore, specific attachment and gentle detachment from the device are required for the accurate analysis of cells. In this study, for capture and release of cancer cells we designed the peptide cleavable by collagenase type IV, which has no target molecule in cells. Our system will be useful for further CTC analysis and might lead to more accurate cancer diagnosis.
Insufficient intratumoral T-cell infiltration and lack of tumor-specific immune surveillance in tumor microenvironment (TME) hinder the progression of cancer immunotherapy. In this study, we explored ...a recombinant vaccinia virus encoding an EpCAM BiTE (VV-EpCAM BiTE) to modulate the immune suppressive microenvironment to enhance antitumor immunity in several solid tumors. VV-EpCAM BiTE effectively infected, replicated and lysed malignant cells. The EpCAM BiTE secreted from infected malignants effectively mediated the binding of EpCAM-positive tumor cells and CD3ϵ on T cells, which led to activation of naive T-cell and the release of cytokines, such as IFN-γ and IL-2. Intratumoral administration of VV-EpCAM BiTE significantly enhanced antitumor activity in malignancies with high other than with low EpCAM expression level. In addition, immune cell infiltration was significantly increased in TME upon VV-EpCAM BiTE treatment, CD8
+
T cell exhaustion was reduced and T-cell-mediated immune activation was markedly enhanced. Taken together, VV-EpCAM BiTE sophistically combines the antitumor advantages of bispecific antibodies and oncolytic viruses, which provides preclinical evidence for the therapeutic potential of VV-EpCAM BiTE.
•Our study reported a significantly greater binding valency to cancer cell lines with over-expressing EpCAM.•The results demonstrated a higher rate of binding to EpCAM-positive cell lines.•Findings ...of the histopathology assay indicated a higher degree of reaction to treatment in the anti-EpCAM receiving group.•The desired anti-EpCAM scFv could be a potential therapeutic agent to suppress tumor progression.
The utilization of monoclonal antibodies (moAbs), an issue correlated with the biopharmaceutical professions, is developing and maturing. Coordinated with this conception, we produced the appealingly modeled anti-EpCAM scFv for breast cancer tumors.
Afterward cloning and expression of recombinant antibody in Escherichia coli bacteria, the correctness of the desired antibody was checked by western blotting. Flow cytometry was utilized to determine the capacity of the recombinant antibody to append to the desired receptors in the malignant breast cancer (BC)cell line. The recombinant antibody (anti-EpCAM scFv) was examined for preclinical efficacy in reducing tumor growth, angiogenesis, and invasiveness (in vitro- in vivo).
A target antibody-mediated attenuation of migration and invasion in the examined cancer cell lines was substantiated (P-value < 0.05). Grafted tumors from breast cancer in mice indicated significant and compelling suppression of tumor growth and decrement in blood supply in reaction to the recombinant anti-EpCAM intervention. Evaluations of immunohistochemical and histopathological findings revealed an enhanced response rate to the treatment.
The desired anti-EpCAM scFv can be a therapeutic tool to reduce invasion and proliferation in malignant breast cancer.
Aptamers are short, single-stranded oligonucleotides that bind to their targets with high affinity and specificity. Usually, aptamers are selected experimentally using SELEX approach. Here, we ...describe a computational approach for selection of aptamers for proteins, which involves generation of a virtual library of sequences, modeling of their 3D-structures and selection of perspective aptamers through docking, molecular dynamics simulation, binding free energy calculations and finally estimating the experimental affinity. Using this method, a 15-mer RNA aptamer was designed for epithelial cell adhesion molecule. Flow cytometry and fluorescence microscopy results reviled that RNA1 aptamer interacts specifically with human cancer cells that express EpCAM, but not with the EpCAM negative cells. The binding affinity of the RNA1 aptamer to MCF-7 and MDA-MB-231 is approximately 21.8 and 96.9 nM respectively. This novel RNA aptamer will help in the future development of targeted therapeutics and molecular imaging.
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•Specific aptamer was identified for EpCAM by in silico approach.•The binding affinity and specificity of aptamer was verified in vitro.•Endocytosis inhibition confirmed that the RNA1 aptamer is efficiently internalized upon binding to EpCAM positive cells.•RNA1 aptamer can be used for diagnostic, imaging and for therapeutic purposes.
The circulating tumor cells (CTCs, the root cause of cancer metastasis and poor cancer prognosis) are very difficult to culture for scale-up
, which has hampered their use in cancer ...research/prognosis and patient-specific therapeutic development. Herein, we report a robust electromicrofluidic chip for not only efficient capture of heterogeneous (EpCAM+ and CD44+) CTCs with high purity but also glutathione-controlled gentle release of the CTCs with high efficiency and viability. This is enabled by coating the polydimethylsiloxane (PDMS) surface in the device with a 10 nm gold layer through a 4 nm titanium coupling layer, for convenient PEGylation and linkage of capture antibodies via the thiol-gold chemistry. Surprisingly, the percentage of EpCAM+ mammary CTCs can be as low as ∼35% (∼70% on average), showing that the commonly used approach of capturing CTCs with EpCAM alone may miss many EpCAM- CTCs. Furthermore, the CD44+ CTCs can be cultured to form 3D spheroids efficiently for scale-up. In contrast, the CTCs captured with EpCAM alone are poor in proliferation
, consistent with the literature. By capture of the CTC heterogeneity, the percentage of stage IV patients whose CTCs can be successfully cultured/scaled up is improved from 12.5% to 68.8%. These findings demonstrate that the common practice of CTC capture with EpCAM alone misses the CTC heterogeneity including the critical CD44+ CTCs. This study may be valuable to the procurement and scale-up of heterogeneous CTCs, to facilitate the understanding of cancer metastasis and the development of cancer metastasis-targeted personalized cancer therapies conveniently via the minimally invasive liquid/blood biopsy.
Objectives:
This study aims to investigate the diagnostic and prognostic values of EpCAM, TGM2, and HE4 in endometrial cancer (EC).
Methods:
In this study, 42 patients diagnosed with EC (EC group), ...41 patients diagnosed with myoma (benign group), and 43 healthy women (healthy group), who applied to Affiliated Hospital of Xuzhou Medical University between March 2018 - September 2019 were recruited. Serum EpCAM, TGM2, and IL-33 levels were measured by ELISA, while serum HE4 and CA-125 levels were measured by ECLIA. The serum markers listed above were also measured in 12 paired pre- and post-operative EC patients. The diagnostic and prognostic values of serum markers were analyzed.
Results:
The serum EpCAM, TGM2, HE4, CA-125, and IL-33 levels were significantly higher in the EC group. The sensitivity and specificity of combined detection of EpCAM and HE4 was 92.86 and 69.05%, which were significantly higher than using a single marker or other combinations. Among these markers, serum HE4 levels were significantly higher in patients with myometrial invasion, metastasis, and lymphovascular invasion (
p
= 0.006,
p
= 0.0004,
p
= 0.0004, respectively). And serum TGM2 levels were significantly decreased in post-operative than that of pre-operative EC patients (
p
< 0.001).
Conclusions:
The combination of EpCAM and HE4 showed the highest specificity and sensitivity in the diagnosis of EC. HE4 was successful in the detection of high-risk individuals preoperatively. Additionally, TGM2 might be a prognostic factor for EC.
Epithelial cell adhesion molecules (EpCAMs) have been identified as surface markers of proliferating ductal cells, which are referred to as liver progenitor cells (LPCs), during liver regeneration ...and correspond to malignancies. These cells can differentiate into hepatocytes and biliary epithelial cells (BECs) in vitro. EpCAM-positive LPCs are involved in liver regeneration following severe liver injury; however, the in vivo function of EpCAMs in the regenerating liver remains unclear. In the present study, we used a zebrafish model of LPC-driven liver regeneration to elucidate the function of EpCAMs in the regenerating liver in vivo. Proliferating ductal cells were observed after severe hepatocyte loss in the zebrafish model. Analyses of the liver size as well as hepatocyte and BEC markers revealed successful conversion of LPCs to hepatocytes and BECs in epcam mutants. Notably, epcam mutants exhibited severe defects in intrahepatic duct maturation and bile acid secretion in regenerating hepatocytes, suggesting that epcam plays a critical role in intrahepatic duct reconstruction during LPC-driven liver regeneration. Our findings provide insights into human diseases involving non-parenchymal cells, such as primary biliary cholangitis, by highlighting the regulatory effect of epcam on intrahepatic duct reconstruction.
•LPC-driven liver regeneration following severe liver injury results in a defect in bile duct morphogenesis in epcam mutants, indicating that epcam plays a fundamental role in the functioning of the intrahepatic biliary network.•EpCAMs are required for intrahepatic duct reconstruction during liver regeneration.
Forty percent of non-small cell lung cancer (NSCLC) patients develop brain metastases, resulting in a dismal prognosis. However, patients in an oligo-metastatic brain disease setting seem to have ...better outcomes. Here, we investigate the possibility of using circulating tumor cells (CTCs) as biomarkers to differentiate oligo-metastatic patients for better risk assessment. Using the CellSearch
system, few CTCs were detected among NSCLC patients with brain metastases (
= 52, 12.5% ≥ two and 8.9% ≥ five CTC/7.5 mL blood) and especially oligo-metastatic brain patients (
= 34, 5.9%, and 2.9%). Still, thresholds of both ≥ two and ≥ five CTCs were independent prognostic indicators for shorter overall survival time among all of the NSCLC patients (
= 90, two CTC ≥ HR: 1.629,
= 0.024, 95% CI: 1.137⁻6.465 and five CTC ≥ HR: 2.846,
= 0.0304, CI: 1.104⁻7.339), as well as among patients with brain metastases (two CTC ≥ HR: 4.694,
= 0.004, CI: 1.650⁻13.354, and five CTC ≥ HR: 4.963,
= 0.003, CI: 1.752⁻14.061). Also, oligo-brain NSCLC metastatic patients with CTCs had a very poor prognosis (
= 0.019). Similarly, in other tumor entities, only 9.6% of patients with brain metastases (
= 52) had detectable CTCs. Our data indicate that although patients with brain metastases more seldom harbor CTCs, they are still predictive for overall survival, and CTCs might be a useful biomarker to identify oligo-metastatic NSCLC patients who might benefit from a more intense therapy.
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