Peripheral Blood Exosomes Research Articles

Advances in Clinical Application of Liquid Biopsy in Non-small Cell Lung Cancer

肺癌是我国发病率最高的癌症，也是癌症患者死亡的首要病因。其中，约85%是非小细胞肺癌（non-small cell lung cancer, NSCLC）。肺癌患者的诊断方法和治疗措施一直是重中之重。近年来，随着分子生物学检测技术的快速进步以及精准治疗和个体化治疗的发展，液体活检因其具有传统侵入性活检所不具备的优点而成为关注的焦点，如安全、便捷、重复性、低创伤性等。液体活检作为一种新兴技术，其检测对象主要包括外周血中的循环肿瘤DNA（circulating tumor DNA, ctDNA）、循环肿瘤细胞（circulating tumor cells, CTCs）、外泌体等。本文将对ctDNA、CTCs和外泌体的检测及其在NSCLC患者中的临床应用作一简要综述。

Atherosclerotic Plaque Fissuration and Clinical Outcomes in Pre-Diabetics vs. Normoglycemics Patients Affected by Asymptomatic Significant Carotid Artery Stenosis at 2 Years of Follow-Up: Role of microRNAs Modulation: The ATIMIR Study.

Atherosclerotic plaque instability and rupture in patients with asymptomatic carotid artery stenosis (ACAS) is a leading cause of major adverse cardiac events (MACE). This could be mainly evidenced in patients with pre-diabetes. Indeed, the altered glucose homeostasis and insulin resistance could cause over-inflammation of atherosclerotic plaque, favoring its conversion to unstable phenotype with rupture and MACE. Notably, metformin therapy reducing the metabolic distress and the inflammatory burden could reduce MACE in ACAS patients with pre-diabetes. In this setting, the microRNAs (miRs) could be used as molecular biomarkers of atherosclerosis progression, plaque rupture, and worse prognosis in normoglycemics (NG) versus pre-diabetics metformin users (PDMU) versus pre-diabetics non-metformin users (PDNMU). However, our study aimed to investigate a wide miRNA panel in peripheral blood exosomes from patients with ACAS divided in NG versus PDMU versus PDNMU, and to associate the circulating miRNA expression profiles with MACE at 2 years of follow-up after endarterectomy. The study included 234 patients with ACAS divided into NG (n = 125), PDNMU (n = 73), and PDMU (n = 36). The miRs’ expression profiles of circulating exosomes were determined at baseline and at 2 years of follow-up by Affymetrix microarrays from the patients’ plasma samples from any study cohort. Then we collected and analyzed MACE at 2 years of follow-up in NG versus PDMU versus PDNMU. Prediabetics versus NG had over-inflammation (p < 0.05) and over expressed miR-24 and miR-27 at baseline. At 2 years of follow-up, PDNMU versus NG, PDMU versus NG, and PDNMU versus PDMU over-expressed inflammatory markers and miR-24, miR-27, miR-100, miR-126, and miR-133 (p < 0.05). Finally, at the end of follow-up, we observed a significant difference about MACE comparing PDNMU versus NG (n = 27 (36.9%) versus n = 8 (6.4%); p < 0.05), PDNMU versus PDMU (n = 27 (36.9%) versus n = 6 (16.6%); p < 0.05); and PDMU versus NG (n = 6 (16.6%) versus n = 8 (6.4%); p < 0.05). Admission glucose values (HR (hazard ratio) 1.020, CI (confidence of interval) 95% (1.001–1.038), p = 0.029), atheromatous carotid plaque (HR 5.373, CI 95% (1.251–11.079), p = 0.024), and miR-24 (HR 3.842, CI 95% (1.768–19.222), p = 0.011) predicted MACE at 2 years of follow-up. Specific circulating miRs could be over-expressed in pre-diabetics and specifically in PDNMU versus PDMU after endarterectomy. MiR24, hyperglycemia, and atheromatous plaque could predict MACE at 2 years of follow-up.

Expression of inflammatory interleukins and selected miRNAs in non-small cell lung cancer

Tumours are characterised by an ability to avoid immune destruction and the presence of cancer-associated inflammation. Better understanding of the link between lung cancer and such inflammation is vital for early detection and personalized treatment. Thus, we examined the mRNA expression of interleukins IL-1β, IL-6, IL-17 and miR-9, miR-122 as potential useful biomarkers of NSCLC. Tumour tissues, non-cancerous tissue and blood samples were collected from 39 patients with primary NSCLC undergoing surgical treatment. The selected RNA was isolated from tissue samples and selected miRNAs from peripheral blood exosomes. This RNA was transcribed to cDNA and quantified using RT-qPCR. Significantly higher expression of the selected interleukins was observed in non-cancerous than tumour tissue, and IL-6 was significantly higher in the tumour tissue of patients with a history of ≤ 40 pack-years (PYs) (2.197, IQR: 0.821–4.415) than in those with > 40 PYs (0.461, IQR: 0.372–0.741; p = 0.037). It is clear that inflammatory processes play a role in NSCLC, as indicated by the upregulation of IL-1β and IL-6 in tumour and adjacent tissue, and that smoking has a strong influence on inflammation in tumourigenesis, demonstrated by the upregulation of IL-6 in tumour samples among patients with ≤ 40 PYs compared to > 40 PYs.

P64.01 MiRNAs in Exosomes Isolated From the Blood of Non-Small Cell Lung Cancer Patients: Biomarkers for Lung Cancer Prediction and Prognosis

Non-small cell lung cancer accounts for about 80% of lung cancer, and about 75% of patients have been found in advanced stages, and their survival rate is very low. However, sensitive and simple diagnostic tests for NSCLC and highly-targeted therapeutic biomarkers are still clinically unusable. The aim of this study was to investigate the expression profiles and clinical significance of miRNAs in platelet-derived exosomes in peripheral blood as biomarkers for NSCLC. The fasting blood samples of 102 patients with NSCLC and 89 healthy people were collected. The exosomes were obtained from platelets using ExoQuick Precipitation Kit. The expression of miR-223 in exosomes from platelets were determined by QRT-PCR. Furthermore, we explored its mechanism in the tumor progress by cell migration assay. First, we found that the levels of miR-223 in platelets and P-MVs were significantly up-regulated in lung cancer patients compared with the non-cancer control group. Second, in vitro assays using qRT-PCR showed that platelet miR-223 was rapidly delivered into cancer cells by P-MVs. Third, platelet-derived exogenous miR-223 reduces the protein level of the tumor suppressor EPB41L3, thereby promoting the invasion of lung cancer cells. The effect of P-MV on the reduction and enhancement of tumor cell invasion by EPB41L3 was largely eliminated by consumption of miR-223, indicating that miR-223 plays a key role in these processes. This study provides additional evidence that functional miRNAs in MVs secreted by cells can serve as biomarkers for lung cancer prediction and prognosis.

Effect of plasma exosomes on endothelial cell tight junction proteins in SLE patients with immune thrombocytopenia.

To study the expression of exosomes in peripheral blood of systemic lupus erythematosus (SLE) immune thrombocytopenia patients with and without hemorrhage symptoms, respectively, and the effect of exosomes on endothelial cell tight junction proteins (TJs) in vitro, so as to investigate the related mechanisms involved in the occurrence of hemorrhage symptoms. Patients diagnosed with SLE and immune thrombocytopenia (<50x109/L) were divided into 2 groups according to the presence or absence of hemorrhage symptoms. Plasma exosomes were isolated, and observed by transmission electron microscopy. The exosomes were co-cultured with endothelial cells in vitro. The permeability of umbilical vein endothelial cells (HUVECs) was measured by transendothelial electrical resistance (TEER). The mRNA and protein expression of tight junctions (occludin and claudin-5) were detected by RT-PCR and Western blot, respectively. Plasma exosomes were increased in the group without hemorrhage symptoms. The TEER value of HUVECs after adding plasma exosomes of hemorrhage group in vitro was not significantly changed compared to the control while increased after adding exosomes of non-hemorrhage group. Plasma exosomes of the non-hemorrhage group could increase both the mRNA and protein expression of TJs in vitro, while exosomes of the hemorrhage group could decrease the expression, the difference was statistically significant (p < 0.05). Plasma exosomes may influence the hemorrhage symptoms of SLE patients with immune thrombocytopenia by regulating the expression of endothelial tight junction proteins. Key Points • The role of exosomes in SLE immune thrombocytopenia is first reported in this study. • We have explored the mechanism that exosomes may participate in hemorrhage, which will facilitate individualized treatment of SLE immune thrombocytopenia.

Exosomal mRNAs and lncRNAs involved in multiple myeloma resistance to bortezomib.

The bone marrow microenvironment plays an essential role in multiple myeloma (MM) progression. We aimed to explore the alterations of levels of long noncoding RNAs and messenger RNAs (mRNAs), derived from exosomes in peripheral blood, in resistance to bortezomib (Btz) of MM patients. Peripheral blood samples were collected from five Btz‐resistant and five Btz‐sensitive MM patients. Exosomes in patients' peripheral blood were enriched, and the profiles of long noncoding RNAs (lncRNAs) and mRNAs in exosomes were determined using deep sequencing. Bioinformatics analysis was performed to explore biological function. MTS was employed to determine the viability of Roswell Park Memorial Institute (RPMI) 8226 and LP‐1 cells incubated with exosomes derived from Btz‐resistant patients. Quantitative polymerase chain reaction (qPCR) was used to evaluate the levels of exosomal FFAR1, SP9, HIST1H2BG, and ITIH2. Incubation with Btz‐resistant patient‐derived exosomes significantly increased the viability of Btz‐treated RPMI 8226 and LP‐1 cells in a dose‐dependent manner. We identified 482 lncRNAs and 2099 mRNAs deregulated in exosomes of the Btz‐resistance group; and 78 mRNAs were enriched in DR‐related pathways, including mammalian target of rapamycin, platinum drug resistance, and the cAMP and phosphoinositide 3‐kinase–Akt signaling pathways. qPCR results verified the increases in FFAR1 and SP9 and decreases in HIST1H2BG and ITIH2 in Btz‐resistant patient‐derived exosomes. Moreover, exosomal FFAR1 and SP9 exhibited potential as independent prognostic indicators of survival of MM patients. Our study reveals significant dysregulation of exosomal RNA components in the Btz‐resistant group of MM patients as well as several mRNAs that may be used as biomarkers of prognosis of MM patients that are resistant to Btz.

Novel 2 Marker Sandwich Immunoassay for the Detection of Precursor- and Mature Granulocyte-Derived Exosomes in Peripheral Blood: A Preliminary Case Study

Introduction: In order to assess hematopoietic precursor cells, bone marrow biopsies are routinely conducted, which is painful for the patient and is not applicable for frequent testing. However, precursor, maturating, and matured cells are known to produce exosomes, which are released into peripheral blood. By analyzing these exosomes in plasma, bone marrow condition may be assessed noninvasively. Myeloid precursor cells express different surface markers than when they have matured. It is reasonable to speculate that these markers would be incorporated into exosomes. In this study, we developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) by using a combination of monoclonal antibodies against 2 different leukocyte antigens, to account for the number of exosomes in plasma. Methods In 6 patients undergoing chemotherapy, venous blood was drawn at regular intervals before, during, and after respective chemotherapies was given. Various clones of monoclonal antibodies against CD11b, CD33, CD34, CD35, CD81, and CD117 were obtained, and antibodies were biotinylated by EZ link Sulfo-NHS-LC-Biotin. Anti-brain antibodies were switched with anti-leukocyte antibodies, and exosome activity was trended. Results CD11b+ and CD 117+ plates showed small changes during chemotherapy (within +/- 50% of the initial value), while plasma WBC count showed a dramatic decrease during chemotherapy. In the recovery phase of WBC after chemotherapy, CD11b+ and CD16+showed a greater than 2-fold increase, with the up-trend taking place earlier than its plasma WBC counterparts. CD 117+ CD33+ also showed an up-trend, but was slower than their CD11b+ and CD16+ counterpart. Finally, each exosome value was divided by other markers to calculate the ratio, and compared between pre and post chemotherapy blood. In half of the cases, there was not a difference, versus an almost double premature/mature ratio in post-chemotherapy blood in the other half of patients. Conclusion From data collected from pre-chemotherapy studies, the lack of correlation between ELISA data compared to plasma WBC data, indicates that plasma exosome analysis might not be suitable for the monitoring of acute bone marrow suppression. Additionally, in the recovery phase, surface markers correlation with WBC was present in only some of the markers, indicating that the assay sensitivity is dependent on the marker combinations. Overall, both myeloid precursor- and mature granulocyte-derived exosomes were increased during the recovery from chemotherapy in 3 out of 5 patients, and exosome profiles were different after chemotherapy in 2 out of 5 patients. Disclosures Mitsuhash: NanoSomiX:Current Employment.

MicroRNA Derived from Circulating Exosomes as Noninvasive Biomarkers for Diagnosing Renal Cell Carcinoma.

PurposeRenal cell carcinoma (RCC) is the most common type of kidney cancer in adults. Exosomes are membrane-enclosed extracellular vesicles, and exosomal RNA can be a biomarker for cancer diagnosis and prognosis in RCC patients. We aim to identify differences in miRNA expression profiles in peripheral blood exosomes between RCC patients and healthy subjects as well as to investigate novel markers of RCC.MethodsWe performed exosomal miRNA sequencing of plasma samples obtained from five RCC patients and five control subjects, subsequently 22 RCC patients and 16 control subjects were investigated using qPCR to confirm the differential miRNA which from plasma exosomal RNA sequencing. ROC curves were constructed to assess the diagnostic accuracy of exosomal miRNAs as diagnostic biomarkers of RCC.ResultsExosomes were isolated with the exoeasy maxi kit and confirmed using TEM and NTA. They have a spherical structure with a diameter of approximately 40–180 nm. The exosomal miRNA sequence results showed that a total of 2357 miRNAs were detected, and 245 miRNAs were differentially expressed between RCC patients and healthy controls (p<0.001, average counts >5, log|fc|>1). Further analysis revealing that, versus the control, 17 miRNAs are up-regulated and 5 miRNAs are down-regulated under selection conditions with average miRNAs counts >100. qPCR was performed using 38 subjects—the results showed that the expression levels of hsa-mir-149-3p and hsa-mir-424-3p were upregulated; the expression levels of hsa-mir-92a-1-5p were significantly downregulated in the plasma exosomes of RCC. For diagnosis of RCC, the AUC of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p was 0.8324, 0.7188 and 0.7727, with the sensitivity of 0.875, 0.750 and 0.750, and the specificity of 0.773,0.727 and 0.818, respectively, at the best cutoff value.ConclusionOur study revealed that the expression levels of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p were significantly abnormal in RCC patients, which may be novel biomarkers for RCC diagnosis.

A Strong Decrease in TIMP3 Expression Mediated by the Presence of miR-17 and 20a Enables Extracellular Matrix Remodeling in the NSCLC Lesion Surroundings.

Background: Lung cancer is one of the most common causes of death worldwide with a relatively high fatality rate and a mean 5-years survival of about 18%. One of the hallmarks of cancer is the extracellular matrix (ECM) remodeling, which is crucial for metastasis. This process may be regulated by miRs targeting metalloproteinases (MMPs) associated with the ECM breakdown and metastatic process or blocking the action of tissue inhibitors of metalloproteinases (TIMPs). Search for early biomarkers is essential in detecting non-small cell lung cancer (NSCLC) and distinguishing its subtypes: Adenocarcinoma (AC) from Squamous Cell Carcinoma (SCC), enabling targeted chemotherapy.Methods: MiR-17 and miR-20a targeting MMP2 and TIMP3 were selected by TCGA data analysis with further validation using miRTarBase and literature. The study group comprised 47 patients with primary NSCLC (AC and SCC subtypes). RNA was isolated from the tumor and normal-looking neighboring tissue (NLNT) free of cancer cells. MiRs from peripheral blood exosomes were extracted on admission and 5–7 days after surgery. Gene and miRs expression were assessed in qPCR using TaqMan probes.Results: The MMP2 has been expressed on a similar level in NLNT, as in cancer. While, TIMP3 expression was decreased both in cancer tissue and NLNT, with significantly lower expression in cancer. TIMP3 downregulation in NLNT and in SCC subtype correlated negatively with miR-20a. The preoperative miR-17 expression was significantly higher among patients with SCC compared to AC. Receiver operating characteristic (ROC) analysis of miR-17 as AC subtype classifier revealed 90% specificity and 48% sensitivity in optimal cut-off point with area under ROC curve (AUC): 0.71 (95%CI: 0.55–0.87). Within NSCLC subtypes: a strong negative correlation between pack-years (PY) and TIMP3 expression was observed for NLNT in the SCC group.Conclusion: The TIMP3 silencing observed in the NLNT and its negative correlation with presurgical expression of miR-20a (from serum exosomes), suggest that miRs can influence ECM remodeling at a distance from the center of the lesion. The miRs expression pattern in serum obtained before surgery significantly differs between AC and SCC subtypes. Moreover, decreased TIMP3 expression in NLNT (in SCC group) negatively correlates with the amount of tobacco smoked in a lifetime in PY.

Expression Level of MiRNA-126 in Serum Exosomes of Allergic Asthma Patients and Lung Tissues of Asthmatic Mice.

To investigate MiRNA-126 amounts in serum exosomes from allergic asthma patients as well as lung tissues of asthmatic mice, evaluating the expression of its target gene DNMT1 in mouse specimens. MiRNA-126 amounts in serum exosomes from asthmatic patients were detected by real-time PCR. The mouse model of allergic asthma was established by OVA-sensitization, and allergic symptoms were recorded; serum IL-4 and sIgE level evaluation (ELISA), broncho alveolar lavage fluid (BALF) cell count and H&E staining were performed to assess airway inflammation. MiRNA-126 and DNMT1 levels in the lung of asthmatic and control mice were detected by real-time PCR; DNMT1 protein levels were detected by immunoblot. MiRNA-126 amounts in peripheral blood exosomes from patients with allergic asthma were significantly higher than that of healthy volunteers (P<0.05). The frequencies of scratching of both sides of the nose and sneezing were elevated within 10 min of excitation in asthmatic rats compared with controls. Meanwhile, OVA-sIgE and IL-4 levels were significantly higher in asthmatic animals than controls (P<0.05). In the asthma group, narrowed bronchial lumen and thickened wall were observed, and bronchial and peripheral vessels showed overt inflammatory cell infiltration. Eosinophil, neutrophil and mast cell amounts in the BALF of asthmatic mice were significantly higher than control values. Furthermore, lung miRNA-126 expression in asthmatic mice was significantly higher than that of controls. Finally, DNMT1 mRNA and protein levels were significantly lower in asthmatic animals compared with controls (P < 0.01). MiRNA-126 is highly expressed in serum exosomes from allergic asthma patients and lung tissues of asthmatic mice, suggesting that it may be involved in the pathogenesis of bronchial asthma.

Integrated analysis of exosomal lncRNA and mRNA expression profiles reveals the involvement of lnc-MKRN2-42:1 in the pathogenesis of Parkinson's disease.

BackgroundParkinson's disease (PD) is a common movement disorder for which diagnosis mainly depends on the medical history and clinical symptoms. Exosomes are now considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material. Long noncoding (lnc) RNA in exosomes plays a critical role in many diseases, including neurodegenerative disease.AimTo study expression differences for lncRNAs in peripheral blood exosomes of PD patients compared with healthy individuals and to look for lncRNAs that might be related to the pathogenesis of PD.Materials and MethodsWe recruited PD patients along with age‐ and sex‐matched healthy individuals as healthy controls and evaluated levels of lncRNAs extracted from exosomes in plasma samples via next‐generation sequencing and real‐time quantitative PCR. Correlation analysis was conducted for the clinical characteristics of PD patients and the expression of selected lncRNAs.ResultsWe found 15 upregulated and 24 downregulated exosomal lncRNAs in the PD group. According to bioinformatics analyses, we chose lnc‐MKRN2‐42:1 for further study. Interestingly, lnc‐MKRN2‐42:1 was positively correlated with the MDS‐UPDRS III score for PD patients.ConclusionOur study suggested that lnc‐MKRN2‐42:1 may be involved in the occurrence and development of PD.

Common molecular markers between circulating tumor cells and blood exosomes in colorectal cancer: a systematic and analytical review.

Nearly half of patients with colorectal cancer (CRC), the third leading cause of cancer deaths worldwide, are diagnosed in the late stages of the disease. Appropriate treatment is not applied in a timely manner and nearly 90% of the patients who experience metastasis ultimately die. Timely detection of CRC can increase the five-year survival rate of patients. Existing histopathological and molecular classifications are insufficient for prediction of metastasis, which limits approaches to treatment. Detection of reliable cancer-related biomarkers can improve early diagnosis, prognosis, and treatment response prediction and recurrence risk. Circulating tumor cells (CTCs) and exosomes in peripheral blood can be used in a liquid biopsy to assess the status of a tumor. Exosomes are abundant and available in all fluids of the body, have a high half-life and are released by most cells. Tumor-derived exosomes are released from primary tumors or CTCs with selective cargo that represents the overall tumor. The current systematic review highlights new trends and approaches in the detection of CRC biomarkers to determine tumor signatures using CTC and exosomes. When these are combined, they could be used to guide molecular pathology and can revolutionize detection tools. Relevant observational studies published until July 24, 2019 which evaluated the expression of tumor markers in CTCs and exosomes were searched in PubMed, Scopus, Embase, and ISI Web of Science databases. The extracted biomarkers were analyzed using String and EnrichR tools.

CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival.

PF539 MIR‐223 IS SUPPRESSED IN EXOSOMES OF PATIENTS WITH HIGH‐RISK MYELODYSPLASTIC SYNDROMES AND MAY BE A MARKER OF RESIDUAL GRANULOCYTIC‐MONOCYTIC DIFFERENTIATION

Background:Quantification of exosomal miRNAs in body fluids represents a prominent source of biomarkers for a variety of conditions. Several miRNAs have been described as potential predictors of severity and progression in neoplastic diseases, pointing to a class of less invasive and readily accessible prognostic tools. Among them, mir‐223 has been studied with contradictory findings: mir‐223 has been shown to play an important role in quimiorresistence in solid tumors (Cheng Q, et al., Mol Med Rep. 2017;15:597–604); however, suppression of mir‐223 in Acute Myeloid Leukemia (AML) is associated with lack of differentiation and adverse cytogenetic profile (Akhter A, et al., Appl Immunohistochem Mol Morphol, 2015;23:733–9).Aims:The aim of this study was to evaluate the expression of mir‐223 in exosomes extracted from serum of patients with myelodysplastic syndromes (MDS), AML and healthy blood donors and to explore the role of mir‐223 in the differentiation of human hematopoietic stem cells.Methods:Samples of venous blood were obtained from 14 patients with AML, 28 patients with MDS (prior to any treatment), and 19 healthy controls. Exosomes were isolated using the ExoQuick® precipitation solution; their concentration and size distribution were characterized by Nanoparticle Tracking Analysis (NanoSight, UK). Exosomal RNA was prepared using miRNeasy Micro Kit and submitted to reverse transcription with miScript II RT kit (QIAGEN, USA) and expression levels of mir‐223 and mir‐192 (endogenous control) were assessed by RT‐PCR using specific primers. CD34+ progenitors were purified from human umbilical cord blood using positive immunomagnetic selection and expanded in culture medium supplemented with G‐CSF, IL‐3 and SCF. The expression levels of mir‐223 were analyzed in cultured cells on days 0, 7, and 14; expression of CD34, CD14 and CD15 was verified by flow cytometry and morphologic changes were observed by examining Wright‐Giemsa‐stained cytospin smears.Results:The concentration of exosomes in serum samples ranged from 5.6 x 1011 to 3.3 x 1013 particles per μl of liquid suspension, with significantly lower concentrations in the patient samples in relation to healthy individuals (p = 0.004). A statistically significant decrease in the expression of mir‐223 was observed in exosomes from individuals with high‐risk MDS compared to healthy subjects (p = 0.002) and to patients with low‐risk MDS (p = 0.012). Interestingly, in the total group of patients, there was a statistically significant positive correlation between the expression of mir‐223 in the exosomes and countings of: total leukocytes (Spearman's r s = 0.37, p = 0.003), neutrophils (r s = 0.392, p = 0.002), monocytes (r s = 0.484, p &lt; .001), lymphocytes (r s = 0.442, p &lt; .001) and platelets (r s = 0.387, p = 0.002) in peripheral blood. During granulocytic‐monocytic differentiation of normal human CD34+ cells, we observed a 4‐fold increase in mir‐223 expression in cultured cells after 7 days, reaching almost 6‐fold increase after 14 days of culture, with granulocytic‐monocytic differentiation demonstrated by cytology and immunophenotyping with CD14 and CD15 markers.Summary/Conclusion:The results here obtained showed that levels of mir‐223 in peripheral blood exosomes are suppressed in patients with high‐risk MDS and correlate with cell counts in both MDS and AML. As mir‐223 levels progressively increase during normal granulocytic‐monocytic differentiation, we speculate that mir‐223 may represent a marker of residual differentiation in MDS and AML.

Peripheral blood exosomes from patients with multiple myeloma mediate bortezomib resistance in cultured multiple myeloma cells

To investigate the role of exosome in mediating bortezomib (Btz) resistance in multiple myeloma cells in vitro and explore the underlying mechanisms. Peripheral blood samples were collected from 15 patients with multiple myeloma with Btz tolerance, and serum exosomes were isolated by ultracentrifugation and identified with electron microscopy, NTA and Western blotting. In vitro cultured multiple myeloma cells were treated with gradient concentrations of Btz to determine the optimal drug concentration for subsequent experiment. The cells were pretreated with different concentrations of exosomes, and their sensitivity to BTZ was assessed using MTS assay. We searched the exosome database Exocarta and used STRING to generate the network map and the protein interaction graph. The diameters of the vesicles isolated from the peripheral blood of the patients were mostly below 200 nm with a mean particle size of 153 nm and a mode of 140.1 nm. The results of Western blotting showed that the isolated exosomes expressed the marker proteins CD63, Tsg101 and Alix. In cultured multiple myeloma cells, pretreatment with exosomes resulted in a decreased sensitivity of the cells to bortezomib, and longer treatment durations and higher exosome concentrations consistently enhanced the resistance of the cells to the same Btz concentration. Analysis of the Exocarta database identified human serum exosomal proteins ABCB1, ABCB4, PDCD6IP, and EGFR, among which EGFR served as a network node. Exosome within a specific concentration range may serve as a signal carrier to mediate the resistance of multiple myeloma cells to Btz. EGFR likely plays a key role to promote exosome-mediated Btz resistance in myeloma cells.

Multiple myeloma patients-derived exosomes as a potential new clinical tool

Introduction: Multiple Myeloma (MM) is a highly heterogeneous neoplasia, usually preceded by a monoclonal-gammopathy-of-undetermined-significance (MGUS). MM has a diverse clinical presentation, variable response to treatment and outcome. Poor response to novel drugs or extramedullary-disease (EMD) are some of the parameters associated to poor prognosis. Exosomes (EXO) are extracellular vesicles implicated, among others, in promotion of supportive tumor microenvironments [1]. EXO in MM may act as biomarkers of disease aggressiveness [2] and potentially be used as “liquid biopsies” for better stratification of disease. Materials and methods: EXO were isolated from peripheral blood (PB) and bone marrow (BM) patient samples by sequential ultracentrifugation. Patient (MM, MGUS) and healthy donors (HD) were compared regarding EXO concentration (EXO/ml) and average protein cargo/EXO (µg/EXO), in PB and BM. Informed consent of the subjects and acceptance of the study protocol by a local ethics committee has been obtained. µg/EXO was determined by the ratio between total quantity of EXO proteins, measured by protein assay using bicinchoninic acid, and the total number of exosomes, measured by Nanosight. Statistical analysis was done with GraphPad. Results: There was no difference in EXO/ml in MM vs MGUS vs HD. However, a positive correlation in EXO/ml between PB and BM in MM and MGUS (Pearson R = 0.80; p < 0.0001) was found. MM patients (n = 10) had 2,8x more µg/EXO in PB, when compared to MGUS patients (n = 5) (p = 0.058); MM median 1.2 (0.50–3.33) µg/EXO versus MGUS median 0.43 (0.10–0.69) µg/EXO. In BM, µg/EXO was also 2,6x higher in MM than in MGUS: 0.92 (0.15–2.13) µg/EXO versus 0.36 (0.06–0.45) µg/EXO, respectively. Analyzing patients by clinical subgroups, we found that those with EMD had higher µg/EXO. Comparing µg/EXO pre-treatment (T1) and at response evaluation (T2), we found that patients with progressive disease had a lower decrease (48%) when compared to patients achieving response to treatment (90%): median T1 1.24 (1.12–3.45) µg/EXO and median T2 0.65 (0.5–0.72) µg/EXO vs median T1 3.29 (1.17–4.15) µg/EXO and median T2 0.35 (0.32–0.5) µg/EXO, respectively. Discussion and conclusions: 1-The positive correlation of the number of EXO in PB and BM samples in MM and MGUS patients may support the use of EXO as liquid biopsies for biomarkers of BM disease, as a less invasive method. 2-MM patients have higher total exosomal protein cargo in PB when compared to MGUS patients and HD and this finding is particularly striking in patients with extramedullary disease and more aggressive forms of MM. The longitudinal evaluation in 2 different time-points (T1 and T2) suggests a correlation between EXO protein load and response to treatment, to be confirmed in a larger cohort. Our work will further evaluate the profile of the proteins included in these EXO and its roles on disease progression, response to treatment and micro environment modification.

Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review).

In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. Medical actions include, but are not limited to, early diagnosis, staging, prognosis, anticipation (lead time) and the prediction of therapy responses, as well as follow-up. Historically, the applications of liquid biopsy in cancer have focused on circulating tumor cells (CTCs). More recently, this analysis has been extended to circulating free DNA (cfDNA) and microRNAs (miRNAs or miRs) associated with cancer, with potential applications for development into multi-marker diagnostic, prognostic and therapeutic signatures. Liquid biopsies avoid some key limitations of conventional tumor tissue biopsies, including invasive tumor sampling, under-representation of tumor heterogeneity and poor description of clonal evolution during metastatic dissemination, strongly reducing the need for multiple sampling. On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of cfDNA, the instability of RNA, the low concentrations of certain analytes in body fluids and the confounding presence of normal, as well as aberrant DNAs and RNAs. For these reasons, the analysis of cfDNA has been mostly focused on mutations arising in, and pathognomonicity of, tumor DNA, while the analysis of cfRNA has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression. This review lists some major applicative areas, briefly addresses how technology is bypassing liquid biopsy limitations, and places a particular emphasis on novel, PCR-free platforms. The ongoing collaborative efforts of major international consortia are reviewed. In addition to basic and applied research, we will consider technological transfer, including patents, patent applications and available information on clinical trials aimed at verifying the potential of liquid biopsy in cancer.

Abstract 5596: Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients

Abstract Liquid biopsy represents a powerful clinical tool that makes use of the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes in peripheral blood. Each different analysis is complementary to each other and can give additional valuable clinical information. These biomarkers are susceptible to quick degradation, presenting a challenge in a clinical environment and may require different blood collection tubes. The aim of this CANCER-ID study was to analyze the influence of different blood tubes on quantity and quality of CTCs, ctDNA and total and exosomal miRNA isolated from the same blood tube. After confirming that plasma removal does not influence CTC recovery rates, peripheral blood from 20 cases of metastatic melanoma patients was collected in EDTA, Streck and Transfix tubes. CTC isolation from the PBMC fraction was performed by the ClearCell device (Clearbridge BioMedics). CTCs were identified by immunofluorescence staining. CtDNA was extracted from Streck and EDTA plasma samples, quantity (Qubit) and quality (Tapestation) measurements were performed before analyzing 86 hot-spot mutations in 13 genes by the UltraSEEK chemistry (Agena Bioscience). Ten of the 20 metastatic melanoma patients and 5 healthy donors were chosen for miRNA analysis. MiRNAs were extracted with miRNeasy Serum/Plasma Advanced Kit and miRNeasy Serum/Plasma Kit (QIAGEN) from total plasma and from extracellular vesicles (EVs), respectively. EVs were isolated by ultracentrifugation. QIAseq miRNA libraries were produced and sequenced (Illumina NextSeq 550). The reads were mapped to miRBase and normalized (geNorm). Volcano plots of fold change versus p-value were used to display the miRNAs that are significantly regulated. The CTC enrichment results showed that 3/20 of the EDTA blood samples were positive, whereas samples from Streck and Transfix tubes were negative. The quantity or quality of the ctDNA did not significantly differ between the EDTA and Streck tubes. Mutation analysis of ctDNA performed so far showed similar detection sensitivities between the two tubes. The morphology, particle concentration and size distribution of EVs did not either differ between the two tubes. MiRNA-NGS analyses from plasma revealed that seven (EDTA) and four (Streck) miRNAs are significantly differentially expressed in patients compared with healthy donors. No overlap of these miRNAs was found between the two tubes. In the EV fraction 24 and 31 miRNAs were found significantly differentially expressed in the EDTA and Streck tubes, respectively. Here, one EV miRNA is up-regulated and six are down-regulated in both tubes. In conclusion, the CTC recovery rates and minimal overlap of circulating miRNAs indicate that the different tubes affect CTC, ctDNA and miRNA results. Thus, the outcome of liquid biopsy analyses strongly depends on the choice of blood collection tubes as important pre-analytical variable. Citation Format: Svenja Schneegans, Lelia Lück, Leonie Bluhm, Janina Staub, Rüdiger Greinert, Beate Volkmer, Alexander Sartori, Darryl Irwin, Taija af Hallstrom, Melanie Hussong, Jonathan Shaffer, Markus Sprenger-Haussels, Stefan W. Schneider, Peter Mohr, Klaus Pantel, Harriet Wikman. Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5596.

Increasing circulating exosomes-carrying TRPC5 predicts chemoresistance in metastatic breast cancer patients.

Chemoresistance, the major obstacle in breast cancer chemotherapy, results in unnecessary chemotherapy and wasting of medical resources. No feasible method has been available to predict chemoresistance before chemotherapy. In our previous study, elevated expression of transient receptor potential channel TRPC5 was found to be an essential element for chemoresistance in breast cancer cells, and it was determined that it could be transferred to chemosensitive breast cancer cells through releasing extracellular vesicles (EV) containing TRPC5 from chemoresistant cells, resulting in acquired chemoresistance. Exosomes, a type of EV, are secreted membrane‐enclosed vesicles of 50–150‐nm diameter. In this study we found that circulating exosomes in peripheral blood from breast cancer patients carried TRPC5. In the present study, circulating exosome‐carrying TRPC5 (cirExo‐TRPC5) level was significantly correlated with TRPC5 expression level in breast cancer tissues and tumor response to chemotherapy. Furthermore, increased cirExo‐TRPC5 level after chemotherapy preceded progressive disease (PD) based on imaging examination and strongly predicted acquired chemoresistance. Taken together, our study demonstrated that cirExo‐TRPC5 might act as a noninvasive chemoresistance marker and might serve as an adjuvant to the current imaging examination‐based chemoresistance.

Circulating MicroRNAs as Novel Biomarkers of Stenosis Progression in Asymptomatic Carotid Stenosis

Progression of asymptomatic carotid artery stenosis (ACAS) in patients with >50% luminal narrowing is considered a potential risk factor for ischemic stroke; however, subclinical molecular biomarkers of ACAS progression are lacking. Recent studies suggest a regulatory function for several microRNAs (miRNAs) on the evolution of carotid plaque, but its role in ACAS progression is mostly unknown. The aim of our study was to investigate a wide miRNA panel in peripheral blood exosomes from patients with ACAS to associate circulating miRNA expression profiles with stenosis progression. The study included 60 patients with ACAS carrying >50% luminal narrowing. First, miRNA expression profiles of circulating exosomes were determined by Affymetrix microarrays from plasma samples of 16 patients from the cohort. Second, those miRNAs among the most differentially expressed in patients with ACAS progression were quantified by real-time polymerase chain reaction in a separate replication cohort of 39 subjects within the patient sample. Our results showed that ACAS progression was associated with development of stroke. MiR-199b-3p, miR-27b-3p, miR-130a-3p, miR-221-3p, and miR-24-3p presented significant higher expression in those patients with ACAS progression. In conclusion, our study supports that specific circulating miRNA expression profiles could provide a new tool that complements the monitoring of ACAS progression, improving therapeutic approaches to prevent ischemic stroke.