The dissemination of circulating tumor cells (CTCs) requires the Epithelial-to-Mesenchymal transition (EMT), in which cells lose their epithelial characteristics and acquire more mesenchymal-like ...phenotypes. Current isolation of CTCs relies on affinity-based approaches reliant on the expression of Epithelial Cell Adhesion Molecule (EpCAM). Here we show EMT-induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy; however, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label-free microfluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and potentially miss critical subpopulations. Further characterization of CTCs, including low-EpCAM populations, using this technology may improve detection techniques and cancer diagnosis, ultimately improving cancer treatment.
Summary Catumaxomab is a trifunctional antibody (trAb) characterized by its unique ability to bind three different cell types: tumor cells, T-cells, and accessory cells. It has two different ...antigen-binding specificities: one for epithelial cell adhesion molecule (EpCAM) on tumor cells and one for the CD3 antigen on T-cells. Catumaxomab also binds to type I, IIa, and III Fcγ receptors (FcγR) on accessory cells, e.g. macrophages, dendritic cells, and natural killer cells, via its intact Fc region. Its anti-tumor activity results from T-cell-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis via activation of FcγR-positive accessory cells. Importantly, no additional activation of immune cells is necessary for effective tumor eradication by catumaxomab, which represents a self-supporting system. Catumaxomab’s efficacy and safety have been demonstrated in a pivotal phase II/III study and supporting phase I/II studies. It is administered as four intraperitoneal (i.p.) infusions on days 0, 3, 7, and 10 at doses of 10, 20, 50, and 150 μg, respectively. Catumaxomab has been approved in the European Union since April 2009 for the i.p. treatment of malignant ascites (MA) in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer feasible. Catumaxomab is the first trAb and the first drug worldwide to be approved specifically for the treatment of MA. It is in clinical trials in a number of other indications including ovarian and gastric cancer. Alternative routes of administration are also under evaluation to further exploit the therapeutic potential of catumaxomab in EpCAM-positive carcinomas.
A nanostructured platform that combines electrospun TiO2 nanofibers (TiNFs)‐deposited substrate and cell‐capture agent realizes significant capture of circulating tumor cells (CTCs). The enhanced ...local topographic interactions between the horizontally packed TiNFs deposited substrates and extracellular matrix scaffolds, in addition to anti‐EpCAM/EpCAM biological recognition, contributes to the significantly enhanced capture efficiency compared to flat surfaces.
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•Facile and environment-friendly synthesis of MoS2@rGO nanohybrid.•Electrophoretic deposition of MoS2@rGO nanohybrid onto ITO substrate enhances electrochemical performance.•The ...device is label free, ultra-sensitivity with good selective and stability.•Great potential for cancer biomarker detection in spiked samples.•Can be an alternate platform for early determination of cancer biomarkers.
An ultrasensitive, electrochemical biosensor has been fabricated by utilizing molybdenum disulfide (MoS2) grafted reduced graphene oxide (MoS2@rGO) nanohybrid as a sensing platform. Biomolecular-assisted synthetic method was adopted to synthesize MoS2@rGO nanohybrid, where L-cys was used to reduce GO. The MoS2@rGO nanohybrid exhibits improved electrochemical performance when it has been electrophoretically deposited onto the indium tin oxide (ITO) coated glass substrate. Further, epithelialcell adhesion moleculeantibodies (anti-EpCAM) specific to cancer biomarker has been covalently immobilized on the MoS2@rGO/ITO electrodes for label-free detection of EpCAM. Electrochemical results confirm that anti-EpCAM/MoS2@rGO/ITO based biosensor can detect EpCAM in the concentration range of 0.001–20 ng mL−1 with a detection limit of 44.22 fg mL−1 (S/N = 3). The biosensor's excellent analytical performance has been attributed to the efficient immobilization of EpCAM antibodies on the MoS2@rGO surface, which results in high specificity for EpCAM antigen. The fabricated biosensor showed good selectivity, reproducibility, and stability. The successful detection of EpCAM antigen in spiked samples (human saliva, serum and urine) makes this platform an alternative method for early screening of cancer biomarker.
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•The use of a DNA nanotetrahedron-assisted aptasensor greatly enhances the specificity of target detection.•The use of quasi-ZIF-67 greatly improved the MB loading efficiency for ...enhancing the detection sensitivity.•The aptasensor exhibited high detection sensitivity with a low detection limit (3 pg/mL) for EpCAM.•This electrochemical aptasensor offers a cost-effective alternative to flow cytometry.
The cell surface protein epithelial cell adhesion molecule (EpCAM) has oncogenic effects, and its overexpression is strongly correlated with tumor development. Here, an electrochemical method is proposed to detect and identify EpCAM at the level of purified proteins and living cells. The core concept of this method is the use of DNA nanotetrahedron (NTH)-based capture probes and aptamer-modified quasi-ZIF-67@Au@methylene blue (QZIF-67@Au@MB@APT) as signal probes. In our design, NTH-assisted targeted immobilization can greatly improve the accessibility of nucleotide-containing artificial aptamers to suspended targets and the specificity of the target EpCAM. Moreover, following partial deligandization, ZIF-67 was converted to QZIF-67 with an extensively improved porous structure that significantly enhanced the loading efficiency of signaling molecules and thus led to a remarkable increase in detection sensitivity. QZIF-67@Au@MB can be integrated with an aptamer to act as a signal reporter with an electrochemical sensing platform for EpCAM detection. By tracing the electrochemical signals from the QZIF-67@Au@MB@APT, the method demonstrated the detection of targets as low as 3 pg/mL within a wide linear range from 0.005 to 100 ng/mL. We have successfully applied this aptasensor for in situ characterization of EpCAM on the cellular surface and monitoring alterations in EpCAM expression during drug treatment, which provides a low-cost but robust alternative to highly expensive and experience-dependent flow cytometry. Overall, electrochemical characterization may provide promising technical support for the precise management of tumors.
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This study proposes the fabrication of a highly sensitive electrochemical immunosensor for label-free detection of EpCAM antigen. MXenes, novel 2D materials have become popular owing ...to their unique electrochemical properties. Unlike conventional immunosensors, which are unable to detect the carcinoma at primary stage and also time consuming, the use of highly conducting MXene provides a label-free and highly sensitive immunosensor. Herein, we develop a unique immunosensor, which is based on the in-situ growth of 2D-TiO2 onto the novel 2D-Ti3C2Tx sheets by hydrothermal treatment. The 2D/2D TiO2/Ti3C2Tx hybrid provides a platform having a large effective surface area, and more number of electrochemically active sites to enhance the electron transfer rate through the redox probe. The designed sensing platform, BSA/anti-EpCAM/TiO2/Ti3C2Tx@ITO shows a broad linear range (1 ag/mL to 10 ng/mL) with high sensitivity (6.661 µA ag−1 mL cm−2), and low detection limit (0.7 ag/mL) for EpCAM antigen detection under optimized conditions. The proposed immunosensor possesses good reproducibility, long-term stability, and outstanding selectivity and specificity. Moreover, the clinical applicability of the novel immunosensor is tested in spiked human serum showing good recovery.
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)
redox couple using impedance technique. Based on the advantages of PAMAM nanomaterial and immune reaction, a linear response to HepG2 cells ranging from 1 × 10
to 1 × 10
cells mL
with a calculated detection limit of 2.1 × 10
cells mL
was obtained. We expect this method can provide a potential tool for cancer cell monitoring and protein expression analysis.
Background
The role of the epithelial cell adhesion molecule (EpCAM) in cancer is still unclear. EpCAM cleavage through regulated intramembrane proteolysis results in fragments which interact with ...both oncogenic and tumor suppressive pathways. Additionally, the EpCAM molecule itself is used as a descriptive therapeutic target in urothelial cancer (UC), while data on its actual tumor specificity remain limited.
Methods
Samples from diagnostic formalin-fixed paraffin-embedded (FFPE) UC tissue and fresh-frozen UC cells were immunoblotted and used for qualitative characterization of five different EpCAM fragments. These expression patterns were quantified across a cohort of 76 samples with 52 UC and 24 normal urothelial samples. Cell viability effects of the extracellular EpEX fragment were assessed in the UC cell lines T24 and HT1376.
Results
The proteolytic EpCAM fragments could be identified in clinical FFPE tissue specimens too. Neither overall nor fragment-specific EpCAM expression showed relevant tumor specificity. EpEX and its deglycosylated variant showed an inverse relationship across healthy and tumor tissue with a decrease of deglycosylated EpEX in tumors. However, extracellular EpEX did not show a relevant effect in vitro.
Conclusions
EpCAM should not be regarded as tumor-specific in UC without patient-specific predictive testing. EpCAM fragment patterns indicate cancer-specific changes and could be involved in its complex tumor-biological role.
Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and ...transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).
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