On Whether Ca-125 Is the Answer for Diagnosing Overhydration, Particularly in End-Stage Kidney Disease Patients-A Systematic Review.

Overhydration (OH) is a prevalent medical problem that occurs in patients with kidney failure, but a specific marker has still not been found. Patients requiring kidney replacement therapy suffer from a water imbalance, which is correlated with mortality rates in this population. Currently, clinicians employ techniques such as bioimpedance spectroscopy (BIS) and ultrasound (USG) markers of overhydration or markers of heart and kidney function, namely NT-pro-BNP, GFR, or creatinine levels. New serum markers, including but not limited to Ca-125, galectin-3 (Gal-3), adrenomedullin (AMD), and urocortin-2 (UCN-2), are presently under research and have displayed promising results. Ca-125, which is a protein mainly used in ovarian cancer diagnoses, holds great potential to become an OH marker. It is currently being investigated by cardiologists as it corresponds to the volume status in heart failure (HF) and ventricular hypertrophy, which are also associated with OH. The need to ascertain a more precise marker of overhydration is urgent mainly because physical examinations are exceptionally inaccurate. The signs and symptoms of overhydration, such as edema or a gradual increase in body mass, are not always present, notably in patients with chronic kidney disease. Metabolic disruptions and cachexia can give a false picture of the hydration status. This review paper summarizes the existing knowledge on the assessment of a patient's hydration status, focusing specifically on kidney diseases and the role of Ca-125.

Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker.

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging.

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

Salt-induced gold nanoparticles aggregation lights up fluorescence of DNA-silver nanoclusters to monitor dual cancer markers carcinoembryonic antigen and carbohydrate antigen 125.

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA1-AgNCs-apta1) and red-emitting DNA-AgNCs with CA125 aptamer (rDNA2-AgNCs-apta2) in the ratio of 1:1 in volume. Upon addition of AuNPs, gDNA1-AgNCs-apta1 and/or rDNA2-AgNCs-apta2 are flexibly adsorbed onto the surface of AuNPs by terminal aptamer(s), which prevents salt-induced AuNPs aggregation under high salt condition and results in fluorescence quenching based on surface plasmon enhanced energy transfer (SPEET). With the addition of CEA and/or CA125, the target(s) and corresponding aptamer(s) coordinate to form the complex, keeping DNA-AgNCs-apta(s) far away from the surface of AuNPs and making AuNPs aggregated in high salt medium. The AuNPs aggregation leads to the recovery of fluorescence signals of DNA-AgNCs-apta(s) due to weakened SPEET. Utilizing the fluorescence aptasensor system, the limit of detection of CEA and CA125 are as low as 7.5 pg·mL-1 and 0.015 U·mL-1, respectively. The proposed method can be applied to the selective and simultaneous determination of CEA and CA125 in human serum.

Radiopharmacologic screening of antibodies to the unshed ectodomain of MUC16 in ovarian cancer identifies a lead candidate for clinical translation.

INTRODUCTION: Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. METHODS: Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [89Zr]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). RESULTS: In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [89Zr]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. CONCLUSION: Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharmacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation.

Molecularly imprinted polymer SPE sensor for analysis of CA-125 on serum.

Considering the high incidence level and mortality rate of ovarian cancer, particularly among the European female population, the carbohydrate antigen 125 (CA-125) was selected as the protein target for this study for the development of a MIP-based biosensor. This work presents the development of molecular imprinting polymers (MIPs) on gold electrode surface for CA-125 biomarker recognition. The preparation of the CA-125 imprinting was obtained by electropolymerization of pyrrole (Py) monomer in a gold electrode using cyclic voltammetry (CV) in order to obtain highly selective materials with great molecular recognition capability. The quantification of CA-125 biomarker was made through the comparison of two methods: electrochemical (square wave voltammetry -SWV) and optical transduction (surface plasmon resonance -SPR). SWV has been widely used in biological molecules analysis since it is a fast and sensitive technique. In turn, SPR is a non-destructive optical technique that provides high-quality analytical data of CA-125 biomarker interactions with MIP. Several analytical parameters, such as sensitivity, linear response interval, and detection limit were determined to proceed to the performance evaluation of the electrochemical and optical transduction used in the development of the CA-125 biosensor. The biosensor based in the electrochemical transduction was the one that presented the best analytical parameters, yielding a good selectivity and a detection limit (LOD) of 0.01 U/mL, providing a linear concentration range between 0.01 and 500 U/mL. This electrochemical biosensor was selected for the study and it was successfully applied in the CA-125 analysis in artificial serum samples with recovery rates ranging from 91 to 105% with an average relative error of 5.8%.

Photovoltaics, plasmonics, plastic antibodies and electrochromism combined for a novel generation of self-powered and self-signalled electrochemical biomimetic sensors.

This work describes further developments into the self-powered and self-signalled biosensing system that merges photovoltaic cells, plastic antibodies and electrochromic cells into a single target. Herein, the plasmonic effect is introduced to improve the photoanode features of the photovoltaic cell, a dye sensitized solar cell (DSSC), and better electrocatalytic features are introduced in the electrode containing the sensing element. In brief, the DSSC had a counter-electrode of poly(3,4-ethylenedioxythiophene) on an FTO glass modified by a plastic antibody of 3,4-ethylenedioxythiophene and pyrrol. The photoanode had dye sensitized TiO2 modified with gold nanoparticles (AuNPs) to increase the cell efficiency, aiming to improve the sensitivity of the response of hybrid device for the target biomarker. The target biomarker was carcinoembryonic antigen (CEA). The response of the hybrid device evidenced a linear trend from 0.1 ng/mL to 10 µg/mL, with an anionic slope of 0.1431 per decade concentration. The response of the plastic antibody for CEA revealed great selectivity against other tumour markers (CA 15-3 or CA 125). The colour response of the electrochromic cell was also CEA concentration dependent and more sensitive when the hybrid device was set-up with a photoanode with AuNPs. A more intense blue colour was obtained when higher concentrations of CEA were present. Overall, this improved version of the self-powered and self-signalled set-up has zero-requirements and is particularly suitable for point-of-care analysis (POC). It is capable of screening CEA in real samples and differentiating clinical levels of interest. This concept opens new horizons into the current cancer screening approaches.

Development of a multiplexed giant magnetoresistive biosensor array prototype to quantify ovarian cancer biomarkers.

In this work, we developed benchtop and handheld Giant Magnetoresistive (GMR) biosensing systems that serve as platforms for detecting a wide variety of protein biomarkers for human diseases. System development included spintronic and nanomagnetic materials, biomolecular chemistry, electronic circuitry, analog and digital signal processing, firmware programming, user interface programming on both PC and Android smartphone, communications over both USB and Bluetooth, and mechanical integration. In this work, we demonstrated the benchtop GMR biosensing system in the context of ovarian cancer assay development. The prototype system delivered the required performance in terms of high-sensitivity multiplex assays in a portable format with enough flexibility to serve as a platform for ovarian cancer and many other diseases. We achieved multiplex detection of cancer antigen 125 (CA125 II), human epididymis protein 4 (HE4), and interleukin 6 (IL6), with limits of detection (LOD) as low as 3.7 U/mL, 7.4 pg/mL, and 7.4 pg/mL, respectively.

Focused Glycomic Profiling With an Integrated Microfluidic Lectin Barcode System.

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, the CA125 protein purified from ovarian cancer cell lines, and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glycobiomarker development.

Facile synthesis of polyaniline-polythionine redox hydrogel: Conductive, antifouling and enzyme-linked material for ultrasensitive label-free amperometric immunosensor toward carcinoma antigen-125.

Sensitivity enhancement and proteins adsorption are the common challenges faced in protein immunoassays. In this work, an ultrasensitive and protein-resistant label-free amperometric immunosening platform for carcinoma antigen-125 (CA125) based on redox polyaniline-polythionine hydrogel (PANI-PThi gel) was developed. The as-prepared hydrogel, which was facilely synthesized by electropolymerization, exhibited good conductivity and strong hydrophilicity while the sensitivity and specificity of the immunosensor can be enhanced. Furthermore, the as-prepared AuNPs functionalized PANI-PThi gel exhibited strong current signal and H2O2 electrocatalytic ability, which guaranteed a large current variable range. Based on these, the prepared immunosensor revealed a wide linear range from 0.0001 U mL-1 to 1 kU mL-1, a limit of detection of 0.00125 U mL-1 and its sensitivity was at least three-fold higher than previous works. More importantly, the prepared immunosensor exhibited excellent specificity, making it capable of assaying CA125 in human serum.

Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16.

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

Antibodies Against Specific MUC16 Glycosylation Sites Inhibit Ovarian Cancer Growth.

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.

A Nanoparticle-Lectin Immunoassay Improves Discrimination of Serum CA125 from Malignant and Benign Sources.

BACKGROUND: Measurement of serum cancer antigen 125 (CA125) is the standard approach for epithelial ovarian cancer (EOC) diagnostics and follow-up. However, the clinical specificity is not optimal because increased values are also detected in healthy controls and in benign diseases. CA125 is known to be differentially glycosylated in EOC, potentially offering a way to construct CA125 assays with improved cancer specificity. Our goal was to identify carbohydrate-reactive lectins for discriminating between CA125 originating from EOC and noncancerous sources. METHODS: CA125 from the OVCAR-3 cancer cell line, placental homogenate, and ascites fluid from patients with cirrhosis were captured on anti-CA125 antibody immobilized on microtitration wells. A panel of lectins, each coated onto fluorescent europium-chelate-doped 97-nm nanoparticles (Eu(+3)-NPs), was tested for detection of the immobilized CA125. Serum samples from high-grade serous EOC or patients with endometriosis and healthy controls were analyzed. RESULTS: By using macrophage galactose-type lectin (MGL)-coated Eu(+3)-NPs, an analytically sensitive CA125 assay (CA125(MGL)) was achieved that specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125(MGL) measurement better discriminated patients with EOC from endometriosis compared to conventional immunoassay. The discrimination was particularly improved for marginally increased CA125 values and for earlier detection of EOC progression. CONCLUSIONS: The new CA125(MGL) assay concept could help reduce the false-positive rates of conventional CA125 immunoassays. The improved analytical specificity of this test approach is dependent on a discriminating lectin immobilized in large numbers on Eu(+3)-NPs, providing both an avidity effect and signal amplification.

In vitro selection of RNA aptamers against CA125 tumor marker in ovarian cancer and its study by optical biosensing.

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays.

Identification of Anti-CA125 Antibody Responses in Ovarian Cancer Patients by a Novel Deep Sequence-Coupled Biopanning Platform.

High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides. After two rounds of biopanning, we analyzed the selectant population of MS2-VLPs by Ion Torrent deep sequencing. One of the top 25 most abundant peptides identified (DISGTNTSRA) had sequence similarity to cancer antigen 125 (CA125/MUC16), a well-known ovarian cancer-associated antigen. Mice immunized with MS2-DISGTNTSRA generated antibodies that cross-reacted with purified soluble CA125 from ovarian cancer cells but not membrane-bound CA125, indicating that the DISGTNTSRA peptide was a CA125/MUC16 peptide mimic of soluble CA125. Preoperative ovarian cancer patient plasma (n = 100) was assessed for anti-DISGTNTSRA, anti-CA125, and CA125. Patients with normal CA125 (<35 IU/mL) at the time of diagnosis had significantly more antibodies to DISGTNTSRA and to CA125 than those patients who had high CA125 (>35 IU/mL). A statistically significant survival advantage was observed for patients who had either normal CA125 and/or higher concentrations of antibodies to CA125 at the time of diagnosis. These data show the feasibility of using deep sequence-coupled biopanning to identify TAA autoantibody responses from cancer patient plasma and suggest a possible antibody-mediated mechanism for low CA125 plasma concentrations in some ovarian cancer patients.

A novel monoclonal antibody to a defined peptide epitope in MUC16.

The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.

Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi/post-Golgi compartments.

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is up regulated in multiple malignancies and is associated with poor prognosis. While the pro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associated carboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavage generating MUC16-Cter has remained unknown. Using different lengths of dual-epitope (N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, we demonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretch of twelve amino acids that generates a ~17 kDa cleaved product and is distinct from the predicted sites. This was further corroborated by domain swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi/post-Golgi compartments and is dependent on the acidic pH in the secretory pathway. A similar pattern of ~17 kDa cleaved MUC16 was observed in multiple cell types eliminating the possibility of cell type specific phenomenon. MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds to the chromatin suggesting its involvement in regulation of gene expression. Taken together, we demonstrate for the first time the oft-predicted cleavage of MUC16 that is critical in designing successful therapeutic interventions based on MUC16.

Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion.

The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1ß, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken ß actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.

Ultrasensitive simultaneous detection of four biomarkers based on hybridization chain reaction and biotin-streptavidin signal amplification strategy.

A sandwich-type electrochemical immunosensor based on redox probe tags identification technology for ultrasensitive simultaneous detection of four antigens was proposed. In this project, well-distributed graphene/gold (GR-Au) hybrid film was acquired through one-step codeposition in an electrode surface and served as the base substrate for immobilizing capture antibodies (Ab1). Hybridization chain reaction (HCR) and biotin/streptavidin (B/SA), combining with gold magnetic nanoparticles were applied to increase the immobilization amount of signal tags in detection antibody (Ab2) bioconjugates. To verify this strategy, four representative biomarkers, a-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen (CA)125 and prostate special antigen (PSA), were used as model analytes. The resulting immunosensor could simultaneously detect four antigens in single-pass differential pulse voltammetry (DPV) scan, and exhibited obviously improved senstivity compared to previous similar immunosensors, displayed good linear relationships in the ranges from 0.2 to 800 pg/mL for AFP, 0.2 to 600 pg/mL for CEA, 0.2 to 1000 pg/mL for CA125, 0.2 to 800 pg/mL for PSA and with detection limits of 62, 48, 77 and 60 fg/mL, respectively.

Transformation of NIH3T3 mouse fibroblast cells by MUC16 mucin (CA125) is driven by its cytoplasmic tail.

MUC16 (CA125) is a transmembrane mucin that contributes to the progression of epithelial ovarian cancer (EOC). Expression of MUC16 is not detectable in the epithelial surface of normal ovaries. MUC16 expression is, however, common in serous EOC as well as in metastatic and recurrent tumors. Despite these observations, its contribution to the development of EOC is unknown. We stably expressed either empty vector, MUC16 C-terminal domain (MUC16 CTD) or MUC16 TMU (a construct that lacks the cytoplasmic tail) in NIH3T3 mouse fibroblast cells. In this study, we provide evidence for the role of MUC16 CTD in oncogenic transformation. We show that ectopic expression of MUC16 CTD enhances the growth of NIH3T3 cells under normal and low serum conditions, and promotes anchorage-dependent colony formation. The deletion of the cytoplasmic tail abrogated these effects. MUC16 CTD expression in NIH3T3 cells also enhances the formation of colony in soft agar as compared to MUC16 TMU. MUC16 CTD expression enhances tumor formation in nude mice. Our findings provide the first evidence that MUC16 induces the transformation of NIH3T3 cells and indicate that MUC16 functions as an oncogene. Furthermore, our data suggest that the cytoplasmic tail is critical for MUC16 oncogenic properties.