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Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(-) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.
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BACKGROUND: Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and ß-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity. METHODS: Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC. RESULTS: EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the ß-Catenin destruction complex (GSK3ß and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC. CONCLUSIONS: This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.
Subject(s)
Colorectal Neoplasms , Epithelial Cell Adhesion Molecule , Wnt Signaling Pathway , Humans , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/genetics , Epithelial Cell Adhesion Molecule/metabolism , Epithelial Cell Adhesion Molecule/genetics , Mice , Animals , Cell Line, Tumor , Disease ProgressionABSTRACT
BACKGROUND: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging. METHODS: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment. RESULTS: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm. CONCLUSION: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches.
Subject(s)
Endometriosis , Epithelial Cell Adhesion Molecule , Folate Receptor 1 , Poly (ADP-Ribose) Polymerase-1 , Endometriosis/metabolism , Endometriosis/surgery , Endometriosis/genetics , Endometriosis/diagnosis , Endometriosis/pathology , Female , Humans , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Folate Receptor 1/genetics , Folate Receptor 1/metabolism , Epithelial Cell Adhesion Molecule/genetics , Epithelial Cell Adhesion Molecule/metabolism , Adult , Biomarkers/metabolism , Immunohistochemistry , Endometrium/metabolism , Endometrium/pathology , Endometrium/surgeryABSTRACT
Background and Objectives: Hepatocellular carcinoma (HCC) is a prevalent form of malignancy that is characterized by high mortality rates and prognosis that remain suboptimal, largely due to treatment resistance mechanisms. Recent studies have implicated cancer stem cells (CSCs), particularly those expressing epithelial cell adhesion molecule (EpCAM), in HCC progression and resistance. In the present study, we sought to assess EpCAM expression in HCC patients and its correlation with various clinicopathological parameters. Materials and Methods: Tissue samples from 42 HCC patients were subjected to immunohistochemical staining to evaluate EpCAM expression. Clinicopathological data were obtained including the size, grade and stage of tumors, vascular invasion status, alpha-fetoprotein levels, and cirrhosis status. The Chi square and Fisher's exact tests were employed to assess the association between categorical groups. Independent Student-t test or Mann-Whitney U test was used to investigate the association between continuous patient characteristics and survival. Results: Immunohistochemical analysis revealed EpCAM expression in 52.5% of HCC cases. EpCAM-positive tumors exhibited characteristics indicative of aggressive disease, including larger tumor sizes (p = 0.006), greater tumor multiplicity (p = 0.004), higher grades (p = 0.002), more advanced stages (p = 0.003), vascular invasion (p = 0.023), elevated alpha-fetoprotein levels (p = 0.013), and cirrhosis (p = 0.052). Survival analysis demonstrated that EpCAM expression was significantly associated with lower overall rates of survival and higher rates of recurrence in HCC patients. Conclusions: Our findings suggest that EpCAM expression may serve as a prognostic biomarker for HCC with a potential role in patient management. Targeting EpCAM-positive CSCs may represent a promising approach to overcome treatment resistance and improve clinical outcomes in HCC. However, further investigation into the molecular mechanisms underlying EpCAM's role in HCC progression is warranted to facilitate the development of personalized therapeutic interventions.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Epithelial Cell Adhesion Molecule , Liver Neoplasms , Neoplastic Stem Cells , Humans , Carcinoma, Hepatocellular/pathology , Epithelial Cell Adhesion Molecule/analysis , Liver Neoplasms/metabolism , Male , Female , Middle Aged , Neoplastic Stem Cells/metabolism , Biomarkers, Tumor/analysis , Aged , Adult , Immunohistochemistry , Prognosis , alpha-Fetoproteins/analysis , alpha-Fetoproteins/metabolismABSTRACT
Platinum-resistant high-grade serous carcinoma (HGSC) is an incurable disease, so biomarkers that could help with timely treatment adjustments and personalized approach are extensively being sought. Tumor-derived extracellular vesicles (EVs) that can be isolated from ascites and blood of HGSC patients are such promising biomarkers. Epithelial cell adhesion molecule (EpCAM) expression is upregulated in most epithelium-derived tumors; however, studies on prognostic value of EpCAM overexpression in ovarian carcinoma have shown contradictory results. The aim of our study was to evaluate the potential of total and EpCAM-positive EVs as prognostic and predictive biomarkers for advanced HGSC. Flow cytometry was used to determine the concentration of total and EpCAM-positive EVs in paired pretreatment ascites and plasma samples of 37 patients with advanced HGSC who underwent different first-line therapy. We found that higher EpCAM-positive EVs concentration in ascites is associated with shorter progression-free survival (PFS) regardless of treatment strategy. We also found a strong correlation of EpCAM-positive EVs concentration between ascites and plasma. Our findings indicate that EpCAM-positive EVs in ascites of patients with advanced HGSC have the potential to serve as prognostic biomarkers for predicting early recurrence and thereby likelihood of more aggressive tumor biology and development of chemoresistance.
Subject(s)
Ascites , Biomarkers, Tumor , Cystadenocarcinoma, Serous , Epithelial Cell Adhesion Molecule , Extracellular Vesicles , Ovarian Neoplasms , Progression-Free Survival , Humans , Epithelial Cell Adhesion Molecule/metabolism , Extracellular Vesicles/metabolism , Female , Ascites/metabolism , Ascites/pathology , Middle Aged , Aged , Biomarkers, Tumor/metabolism , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/mortality , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovarian Neoplasms/mortality , Prognosis , Adult , Neoplasm GradingABSTRACT
BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. METHODS: The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages' inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. RESULTS: Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient's samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. CONCLUSIONS: Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.
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INTRODUCTION: Morphology is routinely used for detecting malignant cells in body fluids, but it has limitations. Recently, flow cytometry (FCM) is used as an effective tool for studying non-haematological malignancies. The main objective of this study is to standardize a simple and rapid FCM test for the detection of malignant epithelial cells in body fluids. MATERIALS AND METHODS: Body fluids that had been processed for cytology/cytology and FCM were enrolled in this prospective study. We developed a fluorescent-labelled, monoclonal antibody panel composed of cell surface markers for this FCM assay. We compared the results of cytology/cell block and FCM. RESULTS: A total of 121 fluid samples were studied. Comparing the diagnostic performance of cytology/cell block and FCM, 52 (43%) cases were positive and 60 (49.5%) cases were negative for carcinoma cells by both techniques. Nine cases showed discordant results between the two techniques. Six cases were cytology+ but FCM- and three cases were FCM+ cytology-. Clustered Epithelial Cell Adhesion Molecule (EpCAM)-positive events with high scatter properties were definitive for positive diagnosis by FCM. We studied PD-L1 expression in 13 cases by FCM. Six cases were reported as false negative by this FCM assay due to hypocellularity and lack of EpCAM expression in malignant cells. CONCLUSIONS: This FCM assay is simple, easier and cost-effective yielding sensitive results with no inter-observer variability. FCM would become a valuable tool to complement routine diagnostic cytology and reduces misdiagnosis.
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Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.
Subject(s)
CD3 Complex , Colorectal Neoplasms , Epithelial Cell Adhesion Molecule , Animals , Humans , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/immunology , Colorectal Neoplasms/therapy , Epithelial Cell Adhesion Molecule/metabolism , CD3 Complex/immunology , Mice , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , HCT116 Cells , Xenograft Model Antitumor Assays , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Female , Cell Line, Tumor , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Mice, Inbred BALB C , T-Lymphocytes/immunology , T-Lymphocytes/drug effects , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Immunotherapy/methodsABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.
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EpCAM is expressed in many epithelial tumors and is used for the distinction of malignant mesotheliomas from adenocarcinomas and as a surrogate pan-epithelial marker. A tissue microarray containing 14,832 samples from 120 different tumor categories was analyzed by immunohistochemistry. EpCAM staining was compared with TROP2 and CKpan. EpCAM staining was detectable in 99 tumor categories. Among 78 epithelial tumor types, the EpCAM positivity rate was ≥90% in 60 categories-including adenocarcinomas, neuroendocrine neoplasms, and germ cell tumors. EpCAM staining was the lowest in hepatocellular carcinomas, adrenocortical tumors, renal cell neoplasms, and in poorly differentiated carcinomas. A comparison of EpCAM and CKpan staining identified a high concordance but EpCAM was higher in testicular seminomas and neuroendocrine neoplasms and CKpan in hepatocellular carcinomas, mesotheliomas, and poorly differentiated non-neuroendocrine tumors. A comparison of EpCAM and TROP2 revealed a higher rate of TROP2 positivity in squamous cell carcinomas and lower rates in many gastrointestinal adenocarcinomas, testicular germ cell tumors, neuroendocrine neoplasms, and renal cell tumors. These data confirm EpCAM as a surrogate epithelial marker for adenocarcinomas and its diagnostic utility for the distinction of malignant mesotheliomas. In comparison to CKpan and TROP2 antibodies, EpCAM staining is particularly common in seminomas and in neuroendocrine neoplasms.
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The epithelial cell adhesion molecule (EpCAM) is a single transmembrane protein on the cell surface. Given its strong expression on epithelial cells and epithelial cell-derived tumors, EpCAM has been identified as a biomarker for circulating tumor cells (CTCs) and exosomes and a target for cancer therapy. As a cell adhesion molecule, EpCAM has a crystal structure that indicates that it forms a cis-dimer first and then probably a trans-tetramer to mediate intercellular adhesion. Through regulated intramembrane proteolysis (RIP), EpCAM and its proteolytic fragments are also able to regulate multiple signaling pathways, Wnt signaling in particular. Although great progress has been made, increasingly more findings have revealed the context-specific expression and function patterns of EpCAM and their regulation processes, which necessitates further studies to determine the structure, function, and expression of EpCAM under both physiological and pathological conditions, broadening its application in basic and translational cancer research.
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To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFvs) incorporated into both glycoprotein H and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with herpesvirus entry mediator (HVEM), the second canonical HSV entry receptor, and is, therefore, recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an epithelial cell adhesion molecule (EpCAM)- and a human epidermal growth factor receptor 2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared with single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose seemed to be harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent, versatile, and systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.
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The majority of triple negative breast cancers (TNBCs) are basal-like breast cancers (BLBCs), which tend to be more aggressive, proliferate rapidly, and have poor clinical outcomes. A key prognostic biomarker and regulator of BLBC is the Forkhead box C1 (FOXC1) transcription factor. However, because of its functional placement inside the cell nucleus and its structural similarity with other related proteins, targeting FOXC1 for therapeutic benefit, particularly for BLBC, continues to be difficult. We envision targeted nonviral delivery of CRISPR/Cas9 plasmid toward the efficacious knockdown of FOXC1. Keeping in mind the challenges associated with the use of CRISPR/Cas9 in vivo, including off-targeting modifications, and effective release of the cargo, a nanoparticle with context responsive properties can be designed for efficient targeted delivery of CRISPR/Cas9 plasmid. Consequently, we have designed, synthesized, and characterized a zwitterionic amino phospholipid-derived transfecting nanoparticle for delivery of CRISPR/Cas9. The construct becomes positively charged only at low pH, which encourages membrane instability and makes it easier for nanoparticles to exit endosomes. This has enabled effective in vitro and in vivo downregulation of protein expression and genome editing. Following this, we have used EpCAM aptamer to make the system targeted toward BLBC cell lines and to reduce its off-target toxicity. The in vivo efficacy, biodistribution, preliminary pharmacokinetics, and biosafety of the optimized targeted CRISPR nanoplatform is then validated in a rodent xenograft model. Overall, we have attempted to knockout the proto-oncogenic FOXC1 expression in BLBC cases by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system derived from a designer lipid derivative. We believe that the nonviral approach for in vitro and in vivo delivery of CRISPR/Cas9 targeted toward FOXC1, studied herein, will greatly emphasize the therapeutic regimen for BLBC.
Subject(s)
Nanoparticles , Triple Negative Breast Neoplasms , Humans , CRISPR-Cas Systems , Phospholipids , Tissue Distribution , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/geneticsABSTRACT
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.
Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.
Subject(s)
Antibodies, Bispecific , Biological Products , Neoplasms , Animals , Humans , Epithelial Cell Adhesion Molecule , Antibodies, Bispecific/pharmacology , Immunotherapy , Neoplasms/therapy , Tumor MicroenvironmentABSTRACT
OBJECTIVE: Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where hopeless for effective therapies. Identification of more reliable biomarkers for early detection of HCC is urgently needed. Circulating tumor cells (CTCs) represent a unique liquid biopsy carrying comprehensive biological information of the primary tumor. Herein, we sought to develop a novel score based on the combination of the most significant CTCs biomarkers with routine laboratory tests for accurate detection of HCC. MATERIAL & METHODS: Epithelial cell adhesion molecule (EpCAM), α-fetoprotein, albumin, and platelets count were assayed in HCC patients (98), liver cirrhosis patients (77). Areas under receiving operating curve (AUCs) were calculated and used for construction on novel score. RESULTS: A novel score named EpCAM-HCC = AFP (U/L) × 0.11 - Albumin (g/dl) × 1.5 + EpCAM % × 2.9 - Platelets count (×109)/L× 0.75 - 93. EpCAM-HCC score produce AUC of 1 for differentiate patients with HCC from those with liver cirrhosis with sensitivity and specificity of a cut-off 1.7 (i.e., less than 1.7 the case is considered cirrhotic, whereas above 1.7 it is considered HCC. CONCLUSION: EpCAM-HCC score could replace AFP during screening of HCV patients and early detection of HCC.
Subject(s)
Carcinoma, Hepatocellular , Hepatitis C , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/pathology , Epithelial Cell Adhesion Molecule , alpha-Fetoproteins , Liver Neoplasms/diagnosis , Liver Neoplasms/pathology , Biomarkers , Liver Cirrhosis/diagnosis , Albumins , Hepatitis C/complications , Hepatitis C/diagnosis , Biomarkers, Tumor/metabolismABSTRACT
BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. METHODS: One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. RESULTS: There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). CONCLUSION: TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.
Subject(s)
Colorectal Neoplasms , Inflammatory Bowel Diseases , Microbiota , Humans , Colorectal Neoplasms/pathology , Toll-Like Receptor 4/genetics , Lipopolysaccharides , Epithelial Cell Adhesion Molecule/genetics , Inflammatory Bowel Diseases/metabolism , Immunoglobulin AABSTRACT
Background: Epithelial cell adhesion molecule (EpCAM), a well-established marker for circulating tumor cells, plays a crucial role in the complex process of cancer metastasis. The primary objective of this investigation is to study EpCAM expression in pan-cancer and elucidate its significance in the context of kidney renal clear cell carcinoma (KIRC). Methods: Data obtained from the public database was harnessed for the comprehensive assessment of the EpCAM expression levels and prognostic and clinicopathological correlations in thirty-three types of cancer. EpCAM was validated in our own KIRC sequencing and immunohistochemical cohorts. Subsequently, an in-depth exploration was conducted to scrutinize the interrelationship between EpCAM and various facets, including immune cells, immune checkpoints, and chemotherapy drugs. We employed Cox regression analysis to identify prognostic immunomodulators associated with EpCAM, which were subsequently utilized in the development of a prognostic model. The model was validated in our own clinical cohort and public datasets, and compared with 137 published models. The role of EpCAM in KIRC was explored by biological function experiments in vitro. Results: While EpCAM exhibited pronounced overexpression across a wide spectrum of cancer types, a notable reduction was observed in KIRC tissues. As grade increased, EpCAM expression decreased. EpCAM expression decreased in patients without metastasis. EpCAM mRNA and protein levels were used as independent, favorable prognostic factors in patients with KIRC in our own cohort. The expression of EpCAM exhibited strong associations with immune-related pathways, demonstrating an inverse correlation with the majority of immune cell types. Immune checkpoint inhibitors exert better therapeutic effects on patients with low EpCAM expression. In addition, EpCAM can be used as a drug resistance indicator and guide the clinical medication of patients with KIRC. A robust model, which had good predictive accuracy and applicability, showed significant superiority over other models. Importantly, EpCAM played the dual roles of promoting proliferation and resisting metastasis in KIRC. Conclusion: In the context of KIRC, EpCAM assumes a surprising dual role, where it not only facilitates cell proliferation but also exerts resistance against the metastatic process. EpCAM serves as a standalone prognostic marker for patients with KIRC, and related models can also effectively predict prognosis. These discoveries offer novel perspectives on the functional significance of EpCAM in the context of KIRC.
ABSTRACT
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.
Subject(s)
Cholangitis , Zebrafish , Animals , Humans , Epithelial Cell Adhesion Molecule/genetics , Epithelial Cell Adhesion Molecule/metabolism , Liver/metabolism , Bile Ducts, Intrahepatic/metabolism , Hepatocytes/metabolism , Epithelial Cells/metabolism , Cholangitis/pathology , Liver RegenerationABSTRACT
Small interfering RNAs (siRNAs) can be used as a powerful tool in gene therapy to downregulate the expression of specific disease related genes. Some properties however, such as instability, and low penetration into cells can limit their efficacy, and thus reduce their therapeutic potential. Metal-organic frameworks (MOFs) such as zeolitic imidazolate framework-8 (ZIF-8), which consist of organic bridging ligands and metal cations (Zn), have a very high binding affinity with nucleic acids including siRNAs. In this study, we designed a PEGylated ZIF-8 platform that was equipped with epithelial cell adhesion molecule (EpCAM) aptamer for the targeted delivery of siRNA molecules, in order to knockdown SNHG15 in both a prostate cancer (PC) cell line, and a human PC xenograft mouse model. SNHG15 is a long noncoding RNA, with oncogenic roles in different cancers including PC. The results indicated that the depletion of SNHG15 by Apt-PEG-siRNA@ZIF-8 nanoplatfrom inhibited cell proliferation and colony formation, and increased apoptosis in PC cells. This nanoparticle facilitated the release of siRNAs into the tumor environment in vivo, and subsequently reduced the tumor growth, with no side effects observed in vital organs. We have therefore developed a novel siRNA nano-delivery system for targeted prostate cancer treatment; however further studies are required before it can be tested in clinical trials.
Subject(s)
Prostatic Neoplasms , RNA, Long Noncoding , Zeolites , Male , Humans , Animals , Mice , RNA, Small Interfering , Zeolites/pharmacology , Cell Line, Tumor , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Cell Proliferation , Disease Models, Animal , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.