A Multifunctional Aptamer Decorated Lipid Nanoparticles for the Delivery of EpCAM-targeted CRISPR/Cas9 Plasmid for Efficacious In Vivo Tumor Regression.

Epithelial cell adhesion molecule (EpCAM) gene encodes a type-I trans-membrane glycoprotein that is overexpressed in many cancerous epithelial cells and promotes tumor progression by regulating the expression of several oncogenes like c-myc and other cyclins. Because of this tumorigenic association, the EpCAM gene has been a potential target for anti-cancer therapy in recent days. Herein, it is attempted to knockout the proto-oncogenic EpCAM expression by efficiently delivering an all-in-one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) plasmid via a lipid nanoparticle system made out of synthetic stimuli-sensitive lipids. The plasmid possesses the necessary information in the form of a guide RNA targeted to the EpCAM gene. The aptamer decorated system selectively targets EpCAM overexpressed cells and efficiently inhibits the genetic expression. It has explored the pH-responsive property of the developed lipid nanoparticles and monitored their efficacy in various cancer cell lines of different origins with elevated EpCAM levels. The phenomenon has further been validated in vivo in non-immunocompromised mouse tumor models. Overall, the newly developed aptamer decorated lipid nanoparticle system has been proven to be efficacious for the delivery of EpCAM-targeted CRISPR/Cas9 plasmid.

Salmonella inhibits tumor metastasis by downregulating epithelial cell adhesion molecules through the protein kinase-B/mammalian target of rapamycin signaling pathway.

Epithelial cell adhesion molecules (EpCAM) are highly expressed in many carcinomas and regulate the epithelial-mesenchymal transition, which is required for tumor metastasis. Furthermore, EpCAM overexpression induces tumor cells to develop a stem cell-like phenotype and promotes tumor progression. Targeting EpCAM may be a promising approach for inhibiting tumor metastasis and progression. Salmonella treatment suppresses tumor growth and reduces metastatic nodules in tumor-bearing mice. Based on these results, we hypothesized that Salmonella-based treatments could inhibit the expression of metastasis-associated proteins. The dose-dependent Salmonella treatment significantly downregulated the levels of EpCAM and decreased the phosphorylation of protein kinase-B (AKT)/mTOR (mammalian target of rapamycin) pathway, as shown by immunoblotting. In addition, Salmonella treatment increased the levels of epithelial markers and decreased the levels of mesenchymal markers in a dose-dependent manner. Wound-healing and Transwell assays showed that Salmonella treatment significantly reduced tumor cell migration. The mice were intravenously injected with B16F10 and CT26 cells pre-incubated with or without Salmonella, and the survival of tumor-bearing mice in the Salmonella group increased, indicating an antimetastatic effect. Our findings demonstrate that Salmonella plays a role in inhibiting tumor metastasis by downregulating EpCAM via the AKT/mTOR signaling pathway and has great potential for cancer therapy.

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma.

OBJECTIVES: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. MATERIALS AND METHODS: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). RESULTS: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. CONCLUSION: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

Comprehensive epithelial biomarker analysis of malignant mesothelioma: EpCAM positivity is a potential diagnostic pitfall.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently used to distinguish carcinoma from background mesothelial cells during cytologic examination of body cavity fluids. Previously, the authors identified one malignant mesothelioma case with strong and diffuse membranous EpCAM staining, making it indistinguishable from carcinoma. METHODS: In this study, the authors evaluated all available effusion specimens from patients with malignant mesothelioma, including the above-mentioned index case, obtained at Stanford Health Care, from 2011 to 2021 (N = 17) as well as control cases (N = 5). Analyses included an immunohistochemistry (IHC) assay for EpCAM and claudin-4, a multiplexed immunofluorescent (IF) assay for EpCAM, and an RNA in situ hybridization assay targeting EpCAM. RESULTS: The authors detected EpCAM positivity of variable intensity and percentage in four malignant mesothelioma cases (23.5%; although only two showed positivity for the epithelial-specific IHC marker MOC31 in ≥40% of cells) and claudin-4 negativity in all cases, with two cases displaying focal and weak claudin-4 staining in <1% of cells. Multiplexed IF staining on the cases with EpCAM IHC positivity showed strong, membranous EpCAM staining in one of four cases. RNA in situ hybridization also was used to assess the correlation between EpCAM positivity by IHC/IF and RNA expression levels. Strong EpCAM RNA expression was detected in the three malignant mesothelioma cases. CONCLUSIONS: The current findings revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit the immunophenotypic features of carcinoma when evaluating for EpCAM only. Additional biomarker testing, such as claudin-4, may help avoid this potential pitfall to yield accurate diagnoses.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells.

BACKGROUND: Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. METHODS: In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a "safe harbour" for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. RESULTS: We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. CONCLUSION: Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Functional Implications of the Dynamic Regulation of EpCAM during Epithelial-to-Mesenchymal Transition.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein expressed in epithelial tissues. EpCAM forms intercellular, homophilic adhesions, modulates epithelial junctional protein complex formation, and promotes epithelial tissue homeostasis. EpCAM is a target of molecular therapies and plays a prominent role in tumor biology. In this review, we focus on the dynamic regulation of EpCAM expression during epithelial-to-mesenchymal transition (EMT) and the functional implications of EpCAM expression on the regulation of EMT. EpCAM is frequently and highly expressed in epithelial cancers, while silenced in mesenchymal cancers. During EMT, EpCAM expression is downregulated by extracellular signal-regulated kinases (ERK) and EMT transcription factors, as well as by regulated intramembrane proteolysis (RIP). The functional impact of EpCAM expression on tumor biology is frequently dependent on the cancer type and predominant oncogenic signaling pathways, suggesting that the role of EpCAM in tumor biology and EMT is multifunctional. Membrane EpCAM is cleaved in cancers and its intracellular domain (EpICD) is transported into the nucleus and binds ß-catenin, FHL2, and LEF1. This stimulates gene transcription that promotes growth, cancer stem cell properties, and EMT. EpCAM is also regulated by epidermal growth factor receptor (EGFR) signaling and the EpCAM ectoderm (EpEX) is an EGFR ligand that affects EMT. EpCAM is expressed on circulating tumor and cancer stem cells undergoing EMT and modulates metastases and cancer treatment responses. Future research exploring EpCAM's role in EMT may reveal additional therapeutic opportunities.

EpCAM expression in esophageal cancer and its correlation with immunotherapy of solitomab.

BACKGROUND: Recurrence of esophageal cancer (EC) after chemotherapy may mainly be explained by the existence of chemotherapy-resistant cells, and an effective drug against chemotherapy-resistant cells is highly sought. The aim of this study was to investigate the cytotoxicity of bispecific antibody solitomab combined with γ δ T cells on Eca109 cell spheres. METHODS: We cultured Eca109 cell spheres in serum-free medium, and the morphological differences between wild-type Eca109 cells and Eca109 cell spheres were compared by microscope and flow cytometry. Different concentrations of nanoparticle albumin-bound paclitaxel (Nab-PTX) and cisplatin were used to treat the two groups of cells and compare their drug resistance. Flow cytometry was then used to detect the expression level of epithelial cell adhesion molecule (EpCAM) and the cytotoxicity of γ δ T cells combined with bispecific antibody solitomab on the two groups. RESULTS: Flow cytometry analysis showed that Eca109 cell spheres were smaller in size and had less cytoplasmic granules and CCK-8 assay showed that the viability of Eca109 cell spheres treated with different concentrations of Nab-PTX and cisplatin was significantly higher than that of wild-type Eca109 cells (P<0.05). Flow cytometry also showed that the expression level of EpCAM on Eca109 cell spheres was higher than that of wild-type Eca109 cells. Co-culture experiment showed that there was no significant difference in the cytotoxicity of γ δ T cells to wild-type Eca109 cells and Eca109 cell spheres without solitomab. However, after adding solitomab, the cytotoxicity of γ δ T cells to Eca109 cell spheres was significantly higher than that of wild-type Eca109 cells (P<0.05). CONCLUSIONS: EC Eca109 cell spheres have strong stem cell characteristics such as multidrug resistance and may contain a high proportion of EC stem cells. Further, EC Eca109 cell spheres have a high expression level of EpCAM, and EpCAM may be one of the markers of EC stem cells. Therefore, EpCAM could be used as a potential molecular target of immunotherapy for EC, and solitomab may become an effective immunotherapeutic drug for chemotherapy-resistant EC cells.

A Modified Method to Isolate Circulating Tumor Cells and Identify by a Panel of Gene Mutations in Lung Cancer.

The CellSearch system is the only FDA approved and successful used detection technology for circulating tumor cells(CTCs). However, the process for identification of CTCs by CellSearch appear to damage the cells, which may adversely affects subsequent molecular biology assays. We aimed to explore and establish a membrane-preserving method for immunofluorescence identification of CTCs that keeping the isolated cells intact. 98 patients with lung cancer were enrolled, and the efficacy of clinical detection of CTCs was examined. Based on the CellSearch principle, we optimized an anti-EpCAM antibody and improved cell membrane rupture. A 5 ml peripheral blood sample was used to enrich CTCs with EpCAM immunomagnetic beads. Fluorescence signals were amplified with secondary antibodies against anti-EpCAM antibody attached on immunomagnetic beads. After identifying CTCs, single CTCs were isolated by micromanipulation. To confirm CTCs, genomic DNA was extracted and amplified at the single cell level to sequence 72 target genes of lung cancer and analyze the mutation copy number variations (CNVs) and gene mutations. A goat anti-mouse polyclonal antibody conjugated with Dylight 488 was selected to stain tumor cells. We identified CTCs based on EpCAM+ and CD45+ cells to exclude white blood cells. In the 98 lung cancer patients, the detection rate of CTCs (≥1 CTC) per 5 ml blood was 87.76%, the number of detections was 1-36, and the median was 2. By sequencing 72 lung cancer-associated genes, we found a high level of CNVs and gene mutations characteristic of tumor cells. We established a new CTCs staining scheme that significantly improves the detection rate and allows further analysis of CTCs characteristics at the genetic level.

First time intravesically administered trifunctional antibody catumaxomab in patients with recurrent non-muscle invasive bladder cancer indicates high tolerability and local immunological activity.

Transurethral resection of the tumor (TUR-B) followed by adjuvant intravesical treatment with cytostatic drugs or Bacillus Calmette-Guérin (BCG) as standard therapy of non-muscle-invasive bladder cancer (NMIBC) is associated with a high recurrence rate of about 60-70%, considerable side effects and requires close monitoring. Alternative treatment options are warranted. Two patients with epithelial cell adhesion molecule (EpCAM)-positive recurrent non-muscle invasive bladder cancer were treated the first time by an intravesical administration of the trifunctional bispecific EpCAM targeting antibody catumaxomab (total dosage of 470 and 1120 µg, respectively). The binding and killing activity of catumaxomab in urine milieu was evaluated in vitro. In contrast to its previous systemic application catumaxomab was well tolerated without any obvious signs of toxicity. Relevant cytokine plasma levels were not detected and no significant systemic drug release was observed. The induction of a human anti-mouse-antibody (HAMA) reaction was either absent or untypically weak contrary to the high immunogenicity of intraperitoneal applied catumaxomab. Tumor cells that were detectable in urine patient samples disappeared after catumaxomab therapy. Endoscopically confirmed recurrence-free intervals were 32 and 25 months. Our data suggest that intravesical administration of catumaxomab in NMIBC is feasible, safe and efficacious, thus arguing for further clinical development of catumaxomab in this indication.

Comparison of circulating tumor cell (CTC) detection rates with epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) antibodies in different solid tumors: a retrospective study.

PURPOSE: Status of epithelial-mesenchymal transition (EMT) varies from tumors to tumors. Epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) are the most common used targets for isolating epithelial and mesenchymal CTCs, respectively. This study aimed to identify a suitable CTC capturing antibody for CTC enrichment in each solid tumor by comparing CTC detection rates with EpCAM and CSV antibodies in different solid tumors. METHODS: Treatment-naive patients with confirmed cancer diagnosis and healthy people who have performed CTC detection between April 2017 and May 2018 were included in this study. CTC detection was performed with CytoSorter® CTC system using either EpCAM or CSV antibody. In total, 853 CTC results from 690 cancer patients and 72 healthy people were collected for analysis. The performance of CTC capturing antibody was determined by the CTC detection rate. RESULTS: EpCAM has the highest CTC detection rate of 84.09% in CRC, followed by BCa (78.32%). CTC detection rates with EpCAM antibody are less than 40% in HCC (25%), PDAC (32.5%) and OC (33.33%). CSV has the highest CTC detection rate of 90% in sarcoma, followed by BC (85.71%), UC (84.62%), OC (83.33%) and BCa (81.82%). CTC detection rates with CSV antibody are over 60% in all 14 solid tumors. Except for CRC, CSV has better performances than EpCAM in most solid tumors regarding the CTC detection rates. CONCLUSION: EpCAM can be used as a target to isolate CTCs in CRC, LC, GC, BCa, EC, HNSCC, CC and PCa, especially in CRC, while CSV can be used in most solid tumors for isolating CTCs.

Expression level of epithelial cell adhesion molecule (EpCAM) of circulating tumor cells (CTCs) of patients with NSCLC as an early indicator to monitor the effects of postoperative adjuvant chemotherapy.

BACKGROUND: An early indicator for monitoring the effect of adjuvant treatment after lung cancer surgery is urgently needed. The study was to explore the effects of epithelial cell adhesion molecule (EpCAM) of circulating tumor cells (CTCs) in NSCLC patients with postoperative adjuvant chemotherapy. METHODS: Two drugs (platinum-containing chemotherapeutics + platinum-free chemotherapeutics) first-line chemotherapy regimen were given after surgery. MRNA of EpCAM was detected. Chest computed tomography, head computed tomography and abdominal B-ultrasound were reviewed before the first and third chemotherapy. RESULTS: EpCAM in CTCs from peripheral blood between the recurrent group and the non-recurrent group at 1 day before surgery, first, second and third adjuvant chemotherapy were no significant differences (P>0.05). Only one day before the fourth adjuvant chemotherapy treatment, it showed significant difference between the recurrent group and the non-recurrent group (P=0.008). There was a significant difference between the time of imaging diagnosis of recurrence or metastasis and the time of monitoring the expression level of EpCAM in CTCs from peripheral blood (P<0.0001). CONCLUSIONS: EpCAM in CTCs from peripheral blood during postoperative adjuvant chemotherapy was related to recurrence or metastasis of NSCLC patients.

Development of a novel mammalian display system for selection of antibodies against membrane proteins.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

Functional reprogramming of Candida glabrata epithelial adhesins: the role of conserved and variable structural motifs in ligand binding.

For host-cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.

Carcinogenetic initiation contributed by EpCAM+ cancer cells in orthotopic HCC models of immunocompetent and athymic mice.

PURPOSE: Overexpression of epithelial cell adhesion molecule (EpCAM) correlates with poor prognosis, therapeutic failure and early tumor recurrence in hepatocellular carcinoma (HCC) patients. The tumor microenvironment dictates the fate of tumor-initiating cancer stem cells (CSCs); however, very limited studies were attempted to evaluate CSC tumorigenesis in the liver microenvironment. Here, we have systemically investigated the role of EpCAM+ cancer cells in tumor initiation in orthotopic HCC models. RESULTS: Control mice and the mice with bland steatosis failed to develop tumors. In the mice with steatohepatitis, EpCAM+ CSCs have shown significantly increased ability in terms of tumor initiation and growth, compared to that with EpCAM- non-CSCs inoculation (p < 0.005). For Hep3B inoculation, EpCAM-High group has shown significantly higher tumor growth compared with EpCAM-Low (p < 0.005). For HepG2 inoculation, both EpCAM-High and EpCAM-Low groups confirmed similar tumor incidence and growth. METHODS: Diet-induced compromised microenvironments were established to mimic clinical fatty liver and non-alcoholic steatohepatitis (NASH) patients and the tumorigenic capabilities of Hepa1-6 cells were evaluated. CSCs were enriched by spheroid culture and labeled with copGFP for EpCAM+ CSCs and with mCherry for non-CSCs. FACS-sorted cells were inoculated into left liver lobes, and tumor growth was monitored by high-frequency ultrasound. The subpopulations of Hep3B and HepG2 cells in terms of EpCAM-Low and EpCAM-High were evaluated in the orthotopic model of athymic mice. CONCLUSIONS: NASH microenvironment promotes the EpCAM+ CSCs initiated tumorigenesis in immunocompetent mouse model. Differential EpCAM expression demonstrates distinct tumor biology in athymic mouse models.

An Immune-Magnetophoretic Device for the Selective and Precise Enrichment of Circulating Tumor Cells from Whole Blood.

Here, we validated the clinical utility of our previously developed microfluidic device, GenoCTC, which is based on bottom magnetophoresis, for the isolation of circulating tumor cells (CTCs) from patient whole blood. GenoCTC allowed 90% purity, 77% separation rate, and 80% recovery of circulating tumor cells at a 90 µL/min flow rate when tested on blood spiked with epithelial cell adhesion molecule (EpCAM)-positive Michigan Cancer Foundation-7 (MCF7) cells. Clinical studies were performed using blood samples from non-small cell lung cancer (NSCLC) patients. Varying numbers (2 to 114) of CTCs were found in each NSCLC patient, and serial assessment of CTCs showed that the CTC count correlated with the clinical progression of the disease. The applicability of GenoCTC to different cell surface biomarkers was also validated in a cholangiocarcinoma patient using anti-EPCAM, anti-vimentin, or anti-tyrosine protein kinase MET (c-MET) antibodies. After EPCAM-, vimentin-, or c-MET-positive cells were isolated, CTCs were identified and enumerated by immunocytochemistry using anti-cytokeratin 18 (CK18) and anti-CD45 antibodies. Furthermore, we checked the protein expression of PDL1 and c-MET in CTCs. A study in a cholangiocarcinoma patient showed that the number of CTCs varied depending on the biomarker used, indicating the importance of using multiple biomarkers for CTC isolation and enumeration.

Predictors of recurrence and survival of hepatocellular carcinoma: A prospective study including transient elastography and cancer stem cell markers.

BACKGROUND AND STUDY AIMS: To investigate whether the measurement of liver stiffness (LSM) using fibroscan and the serum Cancer Stem Cells (CSC): Ep-CAM and cytokeratin-19, could predict the recurrence of hepatocellular carcinoma (HCC) and their impact on clinical outcome and overall survival. PATIENTS AND METHODS: This is a prospective study, including 179 HCV-related HCC patients. All patients were treated following the BCLC guidelines. All HCC patients had transient elastography, measurements of Ep-CAM and cytokeratin-19 before and six months post-treatment. We looked for predictors of recurrence and performed a survival analysis using Kaplan-Meier estimates. RESULTS: TACE was the most common procedure (77.1%), followed by microwave ablation (15.6%). Complete ablation was achieved in 97 patients; 55 of them developed HCC recurrence. After treatment, LSM increased significantly with a significant reduction in CSCs levels in complete and partial response groups. The median time to observe any recurrence was 14 months. LSM increased significantly post-treatment in patients with recurrence versus no recurrence. Higher levels of CSCs were recorded at baseline and post-treatment in patients with recurrence but without statistical significance. We used univariate analysis to predict the time of recurrence by determining baseline CK-19 and platelet levels as the key factors, while the multivariate analysis determined platelet count as a single factor. The univariate analysis for prediction of overall survival included several factors, LSM and EpCAM (baseline and post-ablation) among them, while multivariate analysis included factors such as Child score B and incomplete ablation. CONCLUSION: Dynamic changes were observed in LSM and CSCs levels in response to HCC treatment and tumour recurrence. Child score and complete ablation are factors that significantly affect survival.

A Polyamidoamine Dendrimer-Based Electrochemical Immunosensor for Label-Free Determination of Epithelial Cell Adhesion Molecule- Expressing Cancer Cells.

A new electrochemical immunosensor for cancer cell detection based on a specific interaction between the metastasis-related antigen of epithelial cell adhesion molecule (EpCAM) on the cell membrane and its monoclonal antibody (Anti-EpCAM) immobilized on a gold electrode has been developed. The amino-terminated polyamidoamine dendrimer (G6 PAMAM) was first covalently attached to the 3-mercaptopropionic acid (MPA)-functionalized gold electrode to obtain a thin film, and then completely carboxylated by succinic anhydride (SA). Next, the Anti-EpCAM was covalently bound with the G6 PAMAM to obtain a stable recognition layer. In the presence of the EpCAM expressing hepatocellular carcinomas cell line of HepG2, the specific immune recognition (Anti-EpCAM/EpCAM) led to an obvious change of the electron transfer ability. The properties of the layer-by-layer assembly process was examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The final determination of HepG2 cells was performed in the presence of the reversible [Fe(CN)6]3-/4- redox couple using impedance technique. Based on the advantages of PAMAM nanomaterial and immune reaction, a linear response to HepG2 cells ranging from 1 × 104 to 1 × 106 cells mL-1 with a calculated detection limit of 2.1 × 103 cells mL-1 was obtained. We expect this method can provide a potential tool for cancer cell monitoring and protein expression analysis.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.

Membrane-associated epithelial cell adhesion molecule is slowly cleaved by γ-secretase prior to efficient proteasomal degradation of its intracellular domain.

Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and ß-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aß-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.

Novel method to detect, isolate, and culture prostate culturing circulating tumor cells.

BACKGROUND: Effectively detecting and culturing circulating tumor cells (CTCs), is critical for diagnosis of early metastasis, monitoring anti-cancer therapeutic efficacy, and drug screening. However, most current FDA approved CTC detection methods are based on antibody binding, which has limitations due to the nature of variations in antibody preparation and antibody-CTC size mismatches. Thus, searching for alternative and advanced methods is urgent and necessary. METHODS: Prostate cancer tissue was digested by collagenase and cultured. Cancer stromal cells were identified and labelled with 4',6-diamidino-2-phenylindole (DAPI) before incubation with whole blood of cancer mice (bearing a later stage of prostate cancer). The attached blood CTCs on the DAPI-labeled cancer stromal cells were detected, isolated, cultured and produced into individual cancer cell lines. RESULTS: Five clones of prostate cancer cells isolated from cancer tissue were successfully cultured. One (Clone-1) of the five clones showed positive staining for all three cancer stromal cell markers (CD133, α2ß1 integrin and CD44). Clone-1 cells rich with epithelial cell adhesion molecule (EpCAM) on the cell surface were further identified. The Clone-1 stromal cells labeled as "bait" attracted and caught a trace number of CTCs from the whole blood of mice with advanced stage cancer. Efficient culturing of the caught CTCs from single cell to forming of individual cancer cell line(s) were established. CONCLUSIONS: We present a fundamental advancement of CTC detection and culturing using a different mechanism (cell-cell interaction) rather than the traditional antibody-based immune-binding, such as CellSearchTM system. This study has potential to be fully developed into a novel approach for early cancer metastasis detection, and chemotherapy efficacy monitoring. The efficiently cultured CTCs could be used for single-clone CTC analysis and anti-cancer drug screening to further advance the development of individualized medicine.