Positronium image of the human brain in vivo.

Positronium is abundantly produced within the molecular voids of a patient's body during positron emission tomography (PET). Its properties dynamically respond to the submolecular architecture of the tissue and the partial pressure of oxygen. Current PET systems record only two annihilation photons and cannot provide information about the positronium lifetime. This study presents the in vivo images of positronium lifetime in a human, for a patient with a glioblastoma brain tumor, by using the dedicated Jagiellonian PET system enabling simultaneous detection of annihilation photons and prompt gamma emitted by a radionuclide. The prompt gamma provides information on the time of positronium formation. The photons from positronium annihilation are used to reconstruct the place and time of its decay. In the presented case study, the determined positron and positronium lifetimes in glioblastoma cells are shorter than those in salivary glands and those in healthy brain tissues, indicating that positronium imaging could be used to diagnose disease in vivo.

Discrete symmetries tested at 10-4 precision using linear polarization of photons from positronium annihilations.

Discrete symmetries play an important role in particle physics with violation of CP connected to the matter-antimatter imbalance in the Universe. We report the most precise test of P, T and CP invariance in decays of ortho-positronium, performed with methodology involving polarization of photons from these decays. Positronium, the simplest bound state of an electron and positron, is of recent interest with discrepancies reported between measured hyperfine energy structure and theory at the level of 10-4 signaling a need for better understanding of the positronium system at this level. We test discrete symmetries using photon polarizations determined via Compton scattering in the dedicated J-PET tomograph on an event-by-event basis and without the need to control the spin of the positronium with an external magnetic field, in contrast to previous experiments. Our result is consistent with QED expectations at the level of 0.0007 and one standard deviation.

Feasibility of the J-PET to monitor the range of therapeutic proton beams.

PURPOSE: The aim of this work is to investigate the feasibility of the Jagiellonian Positron Emission Tomography (J-PET) scanner for intra-treatment proton beam range monitoring. METHODS: The Monte Carlo simulation studies with GATE and PET image reconstruction with CASToR were performed in order to compare six J-PET scanner geometries. We simulated proton irradiation of a PMMA phantom with a Single Pencil Beam (SPB) and Spread-Out Bragg Peak (SOBP) of various ranges. The sensitivity and precision of each scanner were calculated, and considering the setup's cost-effectiveness, we indicated potentially optimal geometries for the J-PET scanner prototype dedicated to the proton beam range assessment. RESULTS: The investigations indicate that the double-layer cylindrical and triple-layer double-head configurations are the most promising for clinical application. We found that the scanner sensitivity is of the order of 10-5 coincidences per primary proton, while the precision of the range assessment for both SPB and SOBP irradiation plans was found below 1 mm. Among the scanners with the same number of detector modules, the best results are found for the triple-layer dual-head geometry. The results indicate that the double-layer cylindrical and triple-layer double-head configurations are the most promising for the clinical application, CONCLUSIONS:: We performed simulation studies demonstrating that the feasibility of the J-PET detector for PET-based proton beam therapy range monitoring is possible with reasonable sensitivity and precision enabling its pre-clinical tests in the clinical proton therapy environment. Considering the sensitivity, precision and cost-effectiveness, the double-layer cylindrical and triple-layer dual-head J-PET geometry configurations seem promising for future clinical application.

Detection of range shifts in proton beam therapy using the J-PET scanner: a patient simulation study.

OBJECTIVE: The Jagiellonian PET (J-PET) technology, based on plastic scintillators, has been proposed as a cost effective tool for detecting range deviations during proton therapy. This study investigates the feasibility of using J-PET for range monitoring by means of a detailed Monte Carlo simulation study of 95 patients who underwent proton therapy at the Cyclotron Centre Bronowice (CCB) in Krakow, Poland. Approach: Discrepancies between prescribed and delivered treatments were artificially introduced in the simulations by means of shifts in patient positioning and in the Hounsfield unit to the relative proton stopping power calibration curve. A dual-layer, cylindrical J-PET geometry was simulated in an in-room monitoring scenario and a triple-layer, dual-head geometry in an in-beam protocol. The distribution of range shifts in reconstructed PET activity was visualised in the beam's eye view. Linear prediction models were constructed from all patients in the cohort, using the mean shift in reconstructed PET activity as a predictor of the mean proton range deviation. Main results: Maps of deviations in the range of reconstructed PET distributions showed agreement with those of deviations in dose range in most patients. The linear prediction model showed a good fit, with coefficient of determination r^2 = 0.84 (in-room) and 0.75 (in-beam). Residual standard error was below 1 mm: 0.33 mm (in-room) and 0.23 mm (in-beam). Significance: The precision of the proposed prediction models shows the sensitivity of the proposed J-PET scanners to shifts in proton range for a wide range of clinical treatment plans. Furthermore, it motivates the use of such models as a tool for predicting proton range deviations and opens up new prospects for investigations into the use of intra-treatment PET images for predicting clinical metrics that aid in the assessment of the quality of delivered treatment. .

3D melanoma spheroid model for the development of positronium biomarkers.

It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes the properties of positronium atoms, which are built from the electrons and positrons produced in the body during PET examinations. We hypothesized that positronium imaging would be sensitive to the in vitro discrimination of tumor-like three-dimensional structures (spheroids) built of melanoma cell lines with different cancer activities and biological properties. The lifetime of ortho-positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered parameters such as the cell number, spheroid size and melanoma malignancy to evaluate their relationship with the o-Ps lifetime. We demonstrate pilot results for o-Ps lifetime measurement in extracellular matrix-free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells, the shorter the lifetime of ortho-positronium. In particular, we observed the following indications encouraging further research: (i) WM266-4 spheroids characterized by a higher proliferation rate and malignancy showed a shorter o-Ps lifetime than WM115 spheroids characterized by a lower growth rate. (ii) Both cell lines showed a decrease in the lifetime of o-Ps after spheroid generation on day 8 compared to day 4 in culture, and the mean o-Ps lifetime was longer for spheroids formed from WM115 cells than for those formed from WM266-4 cells, regardless of spheroid age. The results of this study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy.

Extracellular vesicles in vascular pathophysiology: beyond their molecular content.

Extracellular vesicles (EVs) are released by all cells, both in physiological and pathological conditions. Their molecular charge and composition emerge as possible biomarkers, but EVs may also be considered for other clinical applications. This review discusses the role of other features of EVs, such as their lipid components or composition of glycans that form the EV corona and regulate EV biodistribution and uptake by target cells. The importance of EV electric charge has been discussed as a new insight into EV fate and destination.

Developing a novel positronium biomarker for cardiac myxoma imaging.

PURPOSE: Cardiac myxoma (CM), the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. The diagnosis of CM is often elusive, especially in young stroke survivors and transthoracic echocardiography (TTE) is the initial technique for the differential diagnostics of CM. Less invasive cardiac computed tomography (CT) and magnetic resonance imaging (MRI) are not available for the majority of cardiac patients. Here, a robust imaging approach, ortho-Positronium (o-Ps) imaging, is presented to determine cardiac myxoma extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. We aimed to assess if the o-Ps atom, produced copiously in intramolecular voids during the PET imaging, serves as a biomarker for CM diagnosing. METHODS: Six perioperative CM and normal (adipose) tissue samples from patients, with primary diagnosis confirmed by the histopathology examination, were examined using positron annihilation lifetime spectroscopy (PALS) and micro-CT. Additionally, cell cultures and confocal microscopy techniques were used to picture cell morphology and origin. RESULTS: We observed significant shortening in the mean o-Ps lifetime in tumor with compare to normal tissues: an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Microscopic differences between tumor samples, confirmed in histopathology examination and micro-CT, did not influenced the major positronium imaging results. CONCLUSIONS: Our findings, combined with o-Ps lifetime analysis, revealed the novel emerging positronium imaging marker (o-PS) for cardiovascular imaging. This method opens the new perspective to facilitate the quantitative in vivo assessment of intracardiac masses on a molecular (nanoscale) level.

miRNA Signature of Urine Extracellular Vesicles Shows the Involvement of Inflammatory and Apoptotic Processes in Diabetic Chronic Kidney Disease.

BACKGROUND: The aim of this study was to investigate the role of urine-derived extracellular vesicles (uEVs) in diabetic kidney disease (DKD) in patients diagnosed with type 2 diabetes mellitus (T2DM). METHODS: UEVs were characterized by size distribution and microRNA content by next-generation small RNA sequencing and quantitative reverse transcription PCR. RESULTS: A subset of sixteen miRNAs enriched in T2DM patients with DKD, including hsa-miR-514a-5p, hsa-miR­451a, hsa-miR-126-3p, hsa-miR-214, or hsa-miR­503 was identified. Eight miRNAs as hsa-miR-21-3p, hsa-miR-4792, hsa-miR­375, hsa-miR-1268a, hsa-miR-501-5p, or hsa-miR-582 were downregulated. Prediction of potential target genes and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed possible functions related to cellular processes such as apoptosis, inflammation, and tissue remodeling, that promote diabetic complications, such as DKD. Among them, hsa-miR-375, hsa-miR-503, and hsa-miR-451a make important contribution. Additionally, downregulated hsa-miR-582-5p has not been reported so far in any diabetes-related pathways. CONCLUSIONS: This study revealed the most significant miRNAs in uEVs of patients with T2DM. However, as this is a bioinformatic prediction that we performed based on the putative targets of the identified miRNAs. Thus, further in vitro functional studies are needed to confirm our findings. Knowing the fact that EVs are crucial in transferring miRNAs, there is a great need toto discover their involvement in the pathomechanism of T2DM-related kidney disease.

Ticagrelor effectively inhibits platelet aggregation in comatose survivors of cardiac arrest undergoing primary percutaneous coronary intervention treated with mild therapeutic hypothermia.

BACKGROUND: Mild therapeutic hypothermia (MTH) is believed to reduce the effectiveness of antiplatelet drugs. Effective dual-antiplatelet therapy after percutaneous coronary intervention (PCI) is mandatory to avoid acute stent thrombosis. The effectiveness of ticagrelor in MTH-treated out-of-hospital cardiac arrest (OHCA) survivors is still a matter of debate. The aim of the study was to evaluate the impact of MTH on the platelet-inhibitory effect of ticagrelor in comatose survivors of OHCA treated with primary PCI. METHODS: Eighteen comatose survivors of OHCA with acute coronary syndrome undergoing immediate PCI treated with MTH were compared with 14 patients with uncomplicated primary myocardial infarction after PCI, matched for gender and age, in a prospective, single-center, observational study. Platelet aggregation was evaluated using VerifyNow P2Y12 point-of-care testing at 3 time points: admission (T0), during MTH (T1), and 48-72 h after rewarming (T2). RESULTS: Ticagrelor effectively inhibits platelet aggregation in OHCA patients subjected to MTH and in all patients in the control group. The effectiveness of ticagrelor did not differ between the MTH group and the control group (p = 0.581). In 2 cases in the MTH population, the platelet response to ticagrelor was inadequate, and in one of them it remained insufficient during the re-warming phase. There was no stent thrombosis in these patients. CONCLUSIONS: The present study confirmed the effectiveness of ticagrelor to inhibit platelets in myocardial infarction patients after OHCA treated with primary PCI undergoing hypothermia. The use of cooling was not associated with an increased risk of stent thrombosis.

Radiovesicolomics-new approach in medical imaging.

This review introduce extracellular vesicles (EVs) to a molecular imaging field. The idea of modern analyses based on the use of omics studies, using high-throughput methods to characterize the molecular content of a single biological system, vesicolomics seems to be the new approach to collect molecular data about EV content, to find novel biomarkers or therapeutic targets. The use of various imaging techniques, including those based on radionuclides as positron emission tomography (PET) or single photon emission computed tomography (SPECT), combining molecular data on EVs, opens up the new space for radiovesicolomics-a new approach to be used in theranostics.

Large extracellular vesicles do not mitigate the harmful effect of hyperglycemia on endothelial cell mobility.

Extracellular vesicles, especially the larger fraction (LEVs - large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs.

Study of the influence of hyperglycemia on the abundance of amino acids, fatty acids, and selected lipids in extracellular vesicles using TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) with the Bi3+ liquid metal ion gun was used to investigate the content of lipids and amino acids (AAs) in extracellular vesicles (EVs). We induced metabolic changes in human pancreatic ß-cells by stimulation with high glucose concentrations (35 mM) and tested the hypothesis of hyperglycemia (HG) has a detrimental effect on lipids and AAs in released EV subpopulations: ectosomes and exosomes. As a result of HG treatment, selected fatty acids (FAs) such as arachidonic, myristic and palmitic acids, changed their abundance in ectosomes and exosomes. Also, intensities of the characteristic peaks for cholesterol (m/z 95.09; 147.07; 161.11; 369.45) along with the molecular ion m/z 386.37 [C27H46O+] under HG conditions, both for ectosomes and exosomes, have changed significantly. Comparative analysis of HG EVs and normoglycemic (NG) ones showed statistically significant differences in the signal intensities of four AAs: valine (m/z 72.08 and 83.05), isoleucine (m/z 86.10), phenylalanine (m/z 120.08 and 132.05) and tyrosine (m/z 107.05 and 136.09). We confirmed that ToF-SIMS is a useful technique to study selected AAs and lipid profiles in various EV subpopulations. Our study is the first demonstration of changes in FAs and AAs in exosomes and ectosomes derived from ß-cells under the influence of HG.

Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review.

The progressively increasing use of nanomaterials (NMs) has awakened issues related to nanosafety and its potential toxic effects on human health. Emerging studies suggest that NMs alter cell communication by reshaping and altering the secretion of extracellular vesicles (EVs), leading to dysfunction in recipient cells. However, there is limited understanding of how the physicochemical characteristics of NMs alter the EV content and their consequent physiological functions. Therefore, this review explored the relevance of EVs in the nanotoxicology field. The current state of the art on how EVs are modulated by NM exposure and the possible regulation and modulation of signaling pathways and physiological responses were assessed in detail. This review followed the manual for reviewers produced by The Joanna Brigs Institute for Scoping Reviews and the PRISMA extension for Scoping Reviews (PRISMA-ScR): checklist and explanation. The research question, "Do NMs modulate cellular responses mediated by EVs?" was analyzed following the PECO model (P (Population) = EVs, E (Exposure) = NMs, C (Comparator) = EVs without exposure to NMs, O (Outcome) = Cellular responses/change in EVs) to help methodologically assess the association between exposure and outcome. For each theme in the PECO acronym, keywords were defined, organized, and researched in PubMed, Science Direct, Scopus, Web of Science, EMBASE, and Cochrane databases, up to 30 September 2021. In vitro, in vivo, ex vivo, and clinical studies that analyzed the effect of NMs on EV biogenesis, cargo, and cellular responses were included in the analysis. The methodological quality assessment was conducted using the ToxRTool, ARRIVE guideline, Newcastle Ottawa and the EV-TRACK platform. The search in the referred databases identified 2944 articles. After applying the eligibility criteria and two-step screening, 18 articles were included in the final review. We observed that depending on the concentration and physicochemical characteristics, specific NMs promote a significant increase in EV secretion as well as changes in their cargo, especially regarding the expression of proteins and miRNAs, which, in turn, were involved in biological processes that included cell communication, angiogenesis, and activation of the immune response, etc. Although further studies are necessary, this work suggests that molecular investigations on EVs induced by NM exposure may become a potential tool for toxicological studies since they are widely accessible biomarkers that may form a bridge between NM exposure and the cellular response and pathological outcome.

Raman spectroscopy of urinary extracellular vesicles to stratify patients with chronic kidney disease in type 2 diabetes.

In this study, we verified the hypothesis that Raman signature of urinary extracellular vesicles (UEVs) can be used to stratify patients with diabetes at various stages of chronic kidney disease (CKD). Patients with type 2 diabetes diagnosed with different stages of CKD and healthy subjects were enrolled in the study. UEVs were isolated using low-vacuum filtration followed by ultracentrifugation. Correlation analysis, multiple linear regression and principal component analysis were used to find differences between spectral fingerprints of UEVs derived from both groups of patients. Electron microscopy and nanoparticle tracking analysis were applied to characterize the size and morphology of UEVs. We observed significant correlations between selected Raman bands measured for UEVs and clinical parameters. We found significant differences in the area under the specific bands originating mainly from proteins and lipids between the study groups. Based on the tryptophan and amide III bands, we were able to predict the estimated glomerular filtration rate (eGFR). Principal component analysis, partial least squares regression (PLSR) and correlation analysis of the UEV Raman spectra supported the results obtained from the direct analysis of Raman spectra. Our analysis revealed that PLSR and a regression model including tryptophan and amide III bands allows to estimate the value of eGFR.

Positronium imaging with the novel multiphoton PET scanner.

In vivo assessment of cancer and precise location of altered tissues at initial stages of molecular disorders are important diagnostic challenges. Positronium is copiously formed in the free molecular spaces in the patient's body during positron emission tomography (PET). The positronium properties vary according to the size of inter- and intramolecular voids and the concentration of molecules in them such as, e.g., molecular oxygen, O2; therefore, positronium imaging may provide information about disease progression during the initial stages of molecular alterations. Current PET systems do not allow acquisition of positronium images. This study presents a new method that enables positronium imaging by simultaneous registration of annihilation photons and deexcitation photons from pharmaceuticals labeled with radionuclides. The first positronium imaging of a phantom built from cardiac myxoma and adipose tissue is demonstrated. It is anticipated that positronium imaging will substantially enhance the specificity of PET diagnostics.

Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles.

For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.

Fourier-Transform InfraRed (FT-IR) spectroscopy to show alterations in molecular composition of EV subpopulations from melanoma cell lines in different malignancy.

BACKGROUND: Melanoma cells release extracellular vesicles (EVs) subpopulations which differ in size, phenotype and molecular content. Melanoma derived EVs play a role in the development and progression of cancer by delivering surface receptors and bioactive (proteins, lipids, nucleic acids) or signaling molecules to target cells. METHODS: We applied Fourier Transform Infrared Spectroscopy (FTIR) to compare infrared spectra of absorption for different subpopulations of EVs originating from normal human melanocytes, primary cutaneous melanoma (WM115) and metastatic cutaneous melanoma (WM266-4). RESULTS: FTIR results showed that exosome and ectosome populations differ in content of protein and lipid components. We obtained higher lipid to protein ratio for ectosomes in comparison with exosomes what confirms that exosomes are very densely packed with protein cargo. We identified the lowest value of saturated fatty acids/unsaturated fatty acids parameter in the metastatic WM266-4 cell line and ectosomes derived from WM266-4 cell line in comparison with normal melanocytes and the primary WM115 cell line. We identified the alterations in the content of secondary structures of proteins present in EV subpopulations originating from melanocytes and melanoma cells in different malignancy. CONCLUSIONS: Obtained results revealed differences in the molecular composition of melanoma derived EVs subtypes, including protein secondary structure, and showed progressive structural changes during cancer development.

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development.

Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvß3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.

Prospects and Clinical Perspectives of Total-Body PET Imaging Using Plastic Scintillators.

Total-body PET opens a new diagnostic paradigm with prospects for personalized disease treatment, yet the high cost of the current crystal-based PET technology limits dissemination of total-body PET in hospitals and even in research clinics. The J-PET tomography system is based on axially arranged low-cost plastic scintillator strips. It constitutes a realistic cost-effective solution of a total-body PET for broad clinical applications. High sensitivity of total-body J-PET and triggerless data acquisition enable multiphoton imaging, opening possibilities for multitracer and positronium imaging, thus promising quantitative enhancement of specificity in cancer and inflammatory disease assessment.

Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules.

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC-MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial-mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.