Bert Dhondt

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Function of extracellular vesicle-associated miRNAs in metastasis.

Extracellular RNA (exRNA) is functionally transferrable from donor to recipient cells and is protected from RNAses by electrostatic interactions with proteins or by membrane encapsulation. In addition to bioactive RNA, extracellular vesicles (EVs) contain intraluminal and membrane-associated proteins. The cellular context and fitness affect the composition of EVs and thus the outcome of the communication between the EV-producer and recipient cells. Adaptive communication through EVs is particularly important between cancer cells and their local and distant environment and drives life-threatening metastatic progression. Small noncoding RNAs (miRNAs) have been reported in EV isolations and play a role in local invasion, angiogenesis, immune modulation, metastatic niche preparation, colonization and dormancy. The metastasis-related functions attributed to EV-associated miRNAs are currently increasing exponentially in the scientific literature. We must be aware that the correct and efficient separation of non-vesicular entities (soluble proteins, RNA-protein complexes and RNA-lipoprotein complexes) from EVs is necessary to determine the true contribution of EVs in any experiment that describes the molecular content or the functional consequences of the isolated material.

Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

Urinary extracellular vesicle biomarkers in urological cancers : from discovery towards clinical implementation

Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect an individuals metabolic and pathophysiologic state. Despite intensive research into the discovery of urinary biomarkers to facilitate early diagnosis, accurate prognosis and prediction of therapy response in urological cancers, none of these markers has reached widespread use. Their implementation into daily clinical practice is hampered by a substantial degree of heterogeneity in performance characteristics and uncertainty about reliability, clinical utility and cost-effectiveness, in addition to several technical limitations. Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery because of their role in intercellular communication and the resemblance of their molecular content to that of the releasing cells. We review currently used urinary biomarkers in the clinic and attempts that have been made to identify EV-derived biomarkers for urological cancers. In addition, we discuss technical and methodological considerations towards their clinical implementation.

Confounding factors in extracellular vesicle ultrafiltration and protein analysis

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Prognostic and therapeutic implications of circulating androgen receptor gene copy number in prostate cancer patients using droplet digital polymerase chain reaction

&NA; Because resistance mechanisms in the androgen receptor (AR) pathway remain key drivers of progression, we assessed the AR gene copy number (CN) gain in a general population (n = 100) and its implications in prostate cancer. AR CN gain was found to be a promising biomarker anticipating faster progression to castration‐resistant prostate cancer (CRPC), a poor prognosis, and worse abiraterone and enzalutamide outcomes in CRPC. Background: Resistance mechanisms in the androgen receptor (AR) signaling pathway remain key drivers in the progression to castration‐resistant prostate cancer (CRPC) and relapse under antihormonal therapy. Materials and Methods: We evaluated the circulating AR gene copy number (CN) gain using droplet digital polymerase chain reaction in 21 control and 91 prostate cancer serum samples and its prognostic and therapeutic implications in prostate cancer. Results: In CRPC, AR CN gain was associated with faster progression to CRPC (P = .026), a greater number of previous treatments (P = .045), and previous chemotherapy (P = .016). Comparing patients with and without CN gain, the median progression‐free survival (PFS) in the abiraterone subgroup was 1.7 months versus not reached (P = .004), and the median overall survival (OS) was 7 months versus 20.9 months (P = .020). In the enzalutamide subgroup, PFS was 1.7 versus 10.8 months (P = .006), and OS was 6.1 versus 16.5 months (P = .042). In the taxane subgroup, PFS was 3.2 versus 6.5 months (P = .093), and OS was 3.9 months versus not reached (P = .026). The presence of more AR copies correlated with shorter androgen deprivation (P = .002), abiraterone (P = .022), enzalutamide (P = .008), and taxane (P = .039) therapy. Conclusion: Circulating AR CN gain predicts for a poor prognosis in CRPC. It is a promising biomarker predetermining rapid CRPC progression and predicting worse abiraterone and enzalutamide outcomes. Furthermore, it is associated with multiple previous treatments and previous chemotherapy.