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

Integrins are enriched on aberrantly fucosylated tumour‐derived urinary extracellular vesicles

Urinary extracellular vesicles (uEVs) are enriched with glycosylated proteins which have been extensively studied as putative biomarkers of urological cancers. Here, we characterized the glycosylation and integrin profile of EVs derived from urological cancer cell lines. We used fluorescent europium-doped nanoparticles coated with lectins and antibodies to identify a biomarker combination consisting of integrin subunit alpha 3 (ITGA3) and fucose. In addition, we used the same cancer cell line-derived EVs as analytical standards to assess the sensitivity of the ITGA3-UEA assay. The clinical performance of the ITGA3-UEA assay was analysed using urine samples of various urological pathologies including diagnostically challenging benign prostatic hyperplasia (BPH), prostate cancer (PCa) and bladder cancer (BlCa). The assay can significantly discriminate BlCa from all other patient groups: PCa (9.2-fold; p = 0.00038), BPH (5.5-fold; p = 0.004) and healthy individuals (and 23-fold; p = 0.0001). Our results demonstrate that aberrantly fucosylated uEVs and integrin ITGA3 can be detected with fucose-specific lectin UEA in a simple bioaffinity assay for the detection of BlCa directly from unprocessed urine.</p

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression

Integrins are enriched on aberrantly fucosylated tumour‐derived urinary extracellular vesicles

Abstract Urinary extracellular vesicles (uEVs) are enriched with glycosylated proteins which have been extensively studied as putative biomarkers of urological cancers. Here, we characterized the glycosylation and integrin profile of EVs derived from urological cancer cell lines. We used fluorescent europium‐doped nanoparticles coated with lectins and antibodies to identify a biomarker combination consisting of integrin subunit alpha 3 (ITGA3) and fucose. In addition, we used the same cancer cell line‐derived EVs as analytical standards to assess the sensitivity of the ITGA3‐UEA assay. The clinical performance of the ITGA3‐UEA assay was analysed using urine samples of various urological pathologies including diagnostically challenging benign prostatic hyperplasia (BPH), prostate cancer (PCa) and bladder cancer (BlCa). The assay can significantly discriminate BlCa from all other patient groups: PCa (9.2‐fold; p = 0.00038), BPH (5.5‐fold; p = 0.004) and healthy individuals (and 23‐fold; p = 0.0001). Our results demonstrate that aberrantly fucosylated uEVs and integrin ITGA3 can be detected with fucose‐specific lectin UEA in a simple bioaffinity assay for the detection of BlCa directly from unprocessed urine