Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer

Abstract

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…In conclusion, EVs represent an ideal source of diagnostic and prognostic biomarkers for liquid biopsy [277,278]. Given their ability to pass through biological barriers, EVs, which are easily obtainable from accessible biological fluids, such as tears, blood, and/or urine, can provide valuable information about pathophysiological conditions that affect organs or systems that are inaccessible or not easily accessible to direct biological sampling, such as CNS, kidneys, and embryo-fetal placental tissues.…”

Impact of Extracellular Vesicle Isolation Methods on Downstream miRNA Analysis in Semen: A Comparative Study

“…SP (200 µL) was first passed through a 0.22 μm filter to enrich for sEVs [ 6 ]. The resulting filtrate was processed by either ultracentrifugation, three commercial exosome-EV isolation reagents [ miRCURY ® Exosome Cell/Urine/CSF kit ( Qiagen , NV; Germany), miRCURY ® Exosome Serum/Plasma kit ( Qiagen , NV; Germany), ExoQuick ® ULTRA EV Isolation kit for Serum and Plasma ( System Biosciences, Palo Alto, CA, USA )] or a non-commercial exosome-EV isolation reagent [ ExoGAG (NasasBiotech, Santiago de Compostela, Spain)] previously tested for isolating plasma EV from endometrial cancer patients [ 22 ]. They all were precipitation-based methods, although ExoQuick ® ULTRA EV Isolation kit for Serum and Plasma can also include a final solid extraction phase (precipitation + column-based method).…”

“…A suitable volume of precipitation reagent A of ExoGAG kit was added to 0.2 mL of filtered seminal plasma (reagent A/sample ratio 2:1) [ 22 ]. The mixture was incubated for 5 min at 4 °C and subsequently centrifuged at either 1500× g or 3500× g for 30 min at 4 °C.…”

“…An additional flow cytometry analysis (BD FACSAria TM IIu, BDbiosciences, SanJosé, CA, USA) was performed and tetraspanins CD63, and CD81 protein levels were analysed as the most usually employed markers for EV characterization [ 19 , 24 ]; their presence demonstrates the lipid-bilayer structure specific of EVs. Semen EVs isolated by UC , ExoGAG or miRCURY cell/urine/CSF were incubated for 1 h with antiCD81 (1:50, sc7637, Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiCD63 (1:50, sc5275, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiIgG (1:50, 400120, BioLegend) as negative control, followed by anti-mouse Alexa488 (1:1000, ab150113, Abcam, Cambridge, UK) as the secondary antibody as described [ 22 ]. Quantification of fluorescence was calculated as the fold-change of fluorescence intensity median with respect to IgG (immunoglobulin G) negative control.…”

Circulating Extracellular Vesicles in Gynecological Tumors: Realities and Challenges

“…Importantly, in this work, we identified higher levels of ANXA2 in cEVs isolated from plasma samples of patients with EC than healthy controls. Furthermore, the analysis of ANXA2 in plasma EVs had favorable specificity and sensitivity as an EC biomarker (AUROC = 0.74) and was correlated with tumors with high risk of recurrence and non-endometrioid histology, thus indicating the potential of cEV-based ANXA2 levels as a promising diagnostic and prognostic liquid biomarker in EC (48).…”