Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

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Liquid biopsy has become a useful alternative in metastatic colorectal cancer (mCRC) patients when tissue biopsy of metastatic sites is not feasible. In this study we aimed to investigate the clinical utility of circulating exosomes DNA in the management of mCRC patients. Exosomes level and KRAS mutational status in exosomal DNA was assesed in 70 mCRC patients and 29 CRC primary tumor and were analysed at different disease steps evaluating serial blood samples (240 blood samples). There was a significant correlation between the extension of disease and exosomes level and the resection of primary localized tumor was correlated with a decrease of KRAS G12V/ D copies and fractional abundance in metastatic disease. CEA expression and liver metastasis correlated with a higher number of KRAS G12V/D copies/ml and a higher fractional abundance; in the subgroup of mCRC patients eligible for surgery, the size of tumor and the radiological response were related to exosomes level but only the size was related to the number of KRAS WT copies; both KRAS wild-type and mutated levels were identified as a prognostic factor related to OS. Finally, we found that 91% of mutated mCRC patients became wild type after the first line chemotherapy but this status reverted in mutated one at progression in 80% of cases. In a prospective cohort of mCRC patients, we show how longitudinal monitoring using exosome-based liquid biopsy provides clinical information relevant to therapeutic stratification.

nano-sized (20-1000 nm) membranous structures released from cells, play critical roles in both physiological and pathological processes [1]. Extracellular vesicles can be classified based on their size into small (sEVs; <200 nm, also known as exosomes) and medium (mEVs; >200 nm, also known as microvesicles) [1]. Extracellular vesicles might be a target for personalised medicine, given their content and biological origin [2]. Indeed, extracellular vesicles detected in various biological fluids, including sputum supernatants, mucus, epithelial lining fluid, the pulmonary circulation and bronchoalveolar lavage fluid represent a unique tool for both investigating the pathophysiology of respiratory disease and for biomarker discovery [2,3]. Micro (mi)RNAs, mostly in a membrane-encapsulated form, have previously been detected in exhaled breath condensate (EBC) [4], a noninvasive technique for collecting aerosol particles generated in the airways [5]. However, no studies have tried to detect, isolate and quantify EBC sEVs and mEVs. Respiratory tract lining fluid proteins could be detected in endogenous particles in exhaled breath [6]. We aimed to investigate whether extracellular vesicles are present and detectable in EBC and to perform a preliminary comparison of their concentrations in severe asthma, COPD and healthy control subjects. We also compared the nature and quantity of extracellular vesicles isolated from EBC with those isolated from induced sputum supernatants [7]. Our results strongly suggest that extracellular vesicles can be isolated from both fluids.We isolated and characterised sEVs and mEVs following international guidelines [1]. Briefly, 1 mL EBC or sputum supernatant samples were washed with PBS and centrifuged at low speed (750×g for 15 min and 1500×g for 5 min). Next, samples were centrifuged at 17 000×g for 30 min and at 120 000×g for 1.5 h to isolate mEVs and sEVs, respectively. The isolated fractions were resuspended in 100 μL PBS and were characterised using dynamic light scattering for size (figure 1a) and by scanning electron microscope for morphology (figure 1b). In addition, we performed a Western blot on proteins extracted from sEVs (figure 1c) using an anti-CD63 antibody (dilution 1:500, sc-5275; Santa Cruz Biotechnology, Dallas, TX, USA). CD63 is a tetraspanin mostly expressed on the surface of nano-sized (20-120 nm) extracellular vesicles of endocytic origin [8]. The results confirmed that we had indeed isolated sEVs and mEVs from both EBC and sputum supernatant. Next, both extracellular vesicle fractions were bound to 4 µm aldehyde sulfate latex beads and examined by cytofluorometry (figure 1d). They were then stained with calcein, a dye commonly used to label extracellular vesicles because it is converted to green-fluorescent calcein by intracellular esterases which are not present in damaged vesicles and debris [9]. Both extracellular vesicle subpopulations demonstrated enzymatic activity and were stained by calcein (figure 1d). Moreover, flow cytometry confirmed the expression of ex...