Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation

Abstract

The recent introduction of the “precision medicine” concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326− EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…Last but not least, flow cytometry (FC), commonly used for the analysis of cells, is being actively developed for EV analysis [146,147] and is adopted by an increasing number of research groups, mainly to study the larger EVs [148,149]. Direct FC analysis using tracers that stain the whole vital EV compartment, such as lipophilic carbocyanine dyes, combined with phalloidin, that selectively binds to F-actin, accurately discriminates EVs from artifacts [42][43][44]150]. Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2).…”

“…Direct FC analysis using tracers that stain the whole vital EV compartment, such as lipophilic carbocyanine dyes, combined with phalloidin, that selectively binds to F-actin, accurately discriminates EVs from artifacts [42][43][44]150]. Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2). Imaging flow cytometers combining conventional FC with fluorescence provide new, highly sensitive tools for EV studies [153,154].…”

“…Due to their substantial stability, EVs circulate systemically and have been detected in basically all body fluids, including blood, urine, cerebrospinal fluid, saliva, milk, and tears [16,[42][43][44][45][46][47][48]. Moreover, there is clear indication that EVs can cross multiple biological barriers, as demonstrated by the finding of glial/neuronal EVs in the cerebrospinal fluid, blood, tears, and urine [44].…”

Extracellular Vesicles in Feto–Maternal Crosstalk and Pregnancy Disorders

“…Combining such a detection methodology with the use of a tracer for the staining of the whole EV circulating population may allow the placement of the trigger threshold on a fluorescent channel, and therefore the identification of the smallest EVs become possible [45]. It also has been purposed to stain EV samples with phalloidin, in order to exclude damaged EVs from the flow cytometry analysis, allowing a more accurate discrimination of intact vesicles [6,19,20,46]. Finally, the use of the Rosetta Calibration system, which permits the identification of the EV compartment on the basis of the particle size values, combined to a flow cytometry standardized procedure, significantly enhance flow cytometry sensitivity [47,48].…”

“…In any case, EVs are released into the extracellular milieu and finally they could reach the blood circulation. EVs were identified in many different body fluids, such as cerebrospinal fluid, tears, saliva, urine, milk, and peripheral blood [1,6,8,[19][20][21][22][23]. For all of these reasons, EVs were pointed out as a reliable source of biomarkers.…”