Lectins as potential tools for cancer biomarker discovery from extracellular vesicles.

Extracellular vesicles (EVs) have considerable potential as diagnostic, prognostic, and therapeutic agents, in large part because molecular patterns on the EV surface betray the cell of origin and may also be used to "target" EVs to specific cells. Cancer is associated with alterations to cellular and EV glycosylation patterns, and the surface of EVs is enriched with glycan moieties. Glycoconjugates of EVs play versatile roles in cancer including modulating immune response, affecting tumor cell behavior and site of metastasis and as such, paving the way for the development of innovative diagnostic tools and novel therapies. Entities that recognize specific glycans, such as lectins, may thus be powerful tools to discover and detect novel cancer biomarkers. Indeed, the past decade has seen a constant increase in the number of published articles on lectin-based strategies for the detection of EV glycans. This review explores the roles of EV glycosylation in cancer and cancer-related applications. Furthermore, this review summarizes the potential of lectins and lectin-based methods for screening, targeting, separation, and possible identification of improved biomarkers from the surface of EVs.

Aberrant glycosylation of α3 integrins as diagnostic markers in epithelial ovarian cancer.

BACKGROUND: Glycans are strongly involved in stability and function of integrins (ITG) and tetraspanin protein CD63 and their respective interaction partners as they are dysregulated in the tumorigenic processes. Glycosylation changes is a universal phenomenon of cancer cells. In this study, glycosylation changes in epithelial ovarian cancer (EOC) are explored using tetraspanin and integrin molecules. METHODS: ITG and CD63 were immobilized from 10 EOC and 5 benign ovarian cyst fluid on microtiter wells and traced with 3 glycan binding proteins (STn, WGA, UEA) conjugated on europium nanoparticles. Total protein measurements (ITG & CD63 immunoassays) were also performed. The most promising glycovariant candidates identified were then clinically evaluated on the whole cohort of 77 ovarian cyst fluids. Additional testing was performed in ascites fluid samples of liver cirrhosis (n = 2) and EOC (n = 4). RESULTS: Sialylated Tn antibody based glycovariants of ITGα3 (ITGα3STn) and CD63 (CD63STn) performed better than corresponding protein epitope-based immunoassays, ITGα3IA and CD63IA respectively. Combined ITGα3 based assays (ITGα3IA + ITGα3STn) detected 49 out of 55 malignant & borderline cases without detecting any of the 22 benign and healthy cysts. CONCLUSION: Our findings indicate the potential diagnostic application of ITGα3STn along with total ITGα3IA, which could help reduce the unnecessary surgeries. The results encourage studying further the potential use of these novel assays to detect EOC at earlier clinical stages.

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer.

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15-3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15-3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16MGL , MUC16STn , MUC1STn and MUC1Tn provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15-3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early-stage disease. Serum MUC16STn performed best at high specificity (90%-99%), but sensitivity was also improved by the other glycovariants and CA15-3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.

Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum.

BACKGROUND: Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. AIM: Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. METHODS AND RESULTS: The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3 -doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. CONCLUSIONS: These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.

Nanoparticle-Aided Detection of Colorectal Cancer-Associated Glycoconjugates of Extracellular Vesicles in Human Serum.

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.

Detection of bladder cancer with aberrantly fucosylated ITGA3.

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins.

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78-115 ng/mL.

Glycovariant-based lateral flow immunoassay to detect ovarian cancer-associated serum CA125.

Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35-200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

Development of anti-immunocomplex specific antibodies and non-competitive time-resolved fluorescence immunoassay for the detection of estradiol.

Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from synthetic antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from synthetic antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL-1 and 32.4 pg mL-1 respectively. Graphical abstract.

Noncompetitive Chromogenic Lateral-Flow Immunoassay for Simultaneous Detection of Microcystins and Nodularin.

Cyanobacterial blooms cause local and global health issues by contaminating surface waters. Microcystins and nodularins are cyclic cyanobacterial peptide toxins comprising numerous natural variants. Most of them are potent hepatotoxins, tumor promoters, and at least microcystin-LR is possibly carcinogenic. In drinking water, the World Health Organization (WHO) recommended the provisional guideline value of 1 µg/L for microcystin-LR. For water used for recreational activity, the guidance values for microcystin concentration varies mostly between 4-25 µg/L in different countries. Current immunoassays or lateral flow strips for microcystin/nodularin are based on indirect competitive method, which are generally more prone to sample interference and sometimes hard to interpret compared to two-site immunoassays. Simple, sensitive, and easy to interpret user-friendly methods for first line screening of microcystin/nodularin near water sources are needed for assessment of water quality and safety. We describe the development of a two-site sandwich format lateral-flow assay for the rapid detection of microcystins and nodularin-R. A unique antibody fragment capable of broadly recognizing immunocomplexes consisting of a capture antibody bound to microcystins/nodularin-R was used to develop the simple lateral flow immunoassay. The assay can visually detect the major hepatotoxins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R) at and below the concentration of 4 µg/L. The signal is directly proportional to the concentration of the respective toxin, and the use of alkaline phosphatase activity offers a cost efficient alternative by eliminating the need of toxin conjugates or other labeling system. The easy to interpret assay has the potential to serve as a microcystins/nodularin screening tool for those involved in water quality monitoring such as municipal authorities, researchers, as well as general public concerned of bathing water quality.

Role of lectin microarrays in cancer diagnosis.

The majority of cell differentiation associated tumor markers reported to date are either glycoproteins or glycolipids. Despite there being a large number of glycoproteins reported as candidate markers for various cancers, only a handful are approved by the US Food and Drug Administration. Lectins, which bind to the glycan part of the glycoproteins, can be exploited to identify aberrant glycosylation patterns, which in turn would help in enhancing the specificity of cancer diagnosis. Although conventional techniques such as HPLC and MS have been instrumental in performing the glycomic analyses, these techniques lack multiplexity. Lectin microarrays have proved to be useful in studying multiple lectin-glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycomic profiling of cancers in a fast and efficient manner.

Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole.

The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 µg L(-1), which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds.

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.

Multiresidue detection of fluoroquinolones: specificity engineering of a recombinant antibody with oligonucleotide-directed mutagenesis.

Screening of a group of antibiotics from foodstuffs has traditionally relied on sophisticated chemical or physical analysis methods, such as liquid chromatography and mass spectrometric applications. The equipment for these techniques is expensive and not always applicable for high throughput screening. There is a need for an easy and cost efficient detection method for simultaneous screening of structurally similar compounds. Here we describe the engineering of a recombinant antibody which was subjected to oligonucleotide targeted random mutagenesis to emphasize the generic specificity of fluoroquinolone binding. Phage display together with small sized fluoroquinolone derivatives was used to find antibodies of high affinity and generic specificity. The most improved antibody was used to develop a time-resolved fluorescence immunoassay which was further optimized and applied for the detection of fluoroquinolone residues from spiked whole milk samples. The assay can be used to efficiently screen all European Agency for the Evaluation of Medicinal Products (EMEA) controlled fluoroquinolones from whole milk samples with detection levels ranging from 0.2 to 68 µg L(-1).

Engineering of a broad-specificity antibody: detection of eight fluoroquinolone antibiotics simultaneously.

Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider specificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody.