Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9

Supplementary material for: [https://doi.org/10.1186/s12934-017-0856-9]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2069

Identification of tumorigenesis-related mRNAs associated with RNA-binding protein HuR in thyroid cancer cells

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior

Systemic T cells immunosuppression of glioma stem cell-derived exosomes is mediated by monocytic myeloid-derived suppressor cells

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression

MCM5 as a target of BET inhibitors in thyroid cancer cells

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC

Characterization of the Proinflammatory Profile of Synovial Fluid-Derived Exosomes of Patients with Osteoarthritis

The purpose of this study is to characterize synovial fluid- (SF-) derived exosomes of patients with gonarthrosis comparing two methods of isolation and to investigate their immune regulatory properties. Extracellular vesicles (EVs) have been isolated from inflamed SF by polymer precipitation method and quantified by Exocet kit and by nanoparticle tracking analysis. Vesicles expressed all the specific exosomal markers by immunoblot and FACS. After isolation with Exoquick, a relevant contamination by immune complexes was detected, which required further magnetic bead-based purification to remove. SF-derived exosomes significantly stimulated the release of several inflammatory cytokines and chemokines and metalloproteinases by M1 macrophages but did not influence the expression of CD80 and CD86 costimulatory molecules. In conclusion, we characterized purified exosomes isolated from inflamed SF and demonstrate that purified exosomes are functionally active in their ability to stimulate the release of proinflammatory factors from M1 macrophages. Our data indicate that SF-derived exosomes from gonarthrosis patients play a role in disease progression

Biologically driven cut-off definition of lymphocyte ratios in metastatic breast cancer and association with exosomal subpopulations and prognosis

High neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte ratio (MLR) are respectively associated with systemic inflammation and immune suppression and have been associated with a poor outcome. Plasmatic exosomes are extracellular vesicles involved in the intercellular communication system that can exert an immunosuppressive function. Aim of this study was to investigate the interplay between the immune system and circulating exosomes in metastatic breast cancer (MBC). A threshold capable to classify patients according to MLR, NLR and PLR, was computed through a receiving operator curve analysis after propensity score matching with a series of female blood donors. Exosomes were isolated from plasma by ExoQuick solution and characterized by flow-cytometry. NLR, MLR, PLR and exosomal subpopulations potentially involved in the pre-metastatic niche were significantly different in MBC patients with respect to controls. MLR was significantly associated with number of sites at the onset of metastatic disease, while high levels of MLR and NLR were found to be associated with poor prognosis. Furthermore, exosomal subpopulations varied according to NLR, MLR, PLR and both were associated with different breast cancer subtypes and sites of distant involvement. This study highlights the nuanced role of immunity in MBC spread, progression and outcome. Moreover, they suggest potential interaction mechanisms between immunity, MBC and the metastatic niche

In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis

Background: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. Methods: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. Results: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. Conclusion: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies

Serological and cellular response to mRNA-SARS-CoV2 vaccine in patients with hematological lymphoid malignancies: Results of the study “Cervax”

messenger RNA (mRNA)-Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) vaccines such as BNT162b2 became available in late 2020, but hematological malignancy patients (HM pts) were not evaluated in initial registration trials. We hereby report the results of a prospective, unicentric, observational study Response to COVID-19 Vaccination in hEmatological malignancies (CERVAX) developed to assess the postvaccine serological and T-cell-mediated response in a cohort of SARS-CoV2-negative HM pts vaccinated with BNT162b2. Patients with lymphomas [non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL)], chronic lymphocytic leukemia (CLL), and multiple myeloma (MM); off-therapy for at least 3 months; in a watch-and-wait program; or in treatment with ibrutinib, venetoclax, and lenalidomide were included. Different time points were considered to assess the serological response to the vaccine: before the second dose (T1), at 3–6–12 months after the first dose (T2–3–4, respectively). Since March 2021, 39 pts have been enrolled: 15 (38%) NHL, 12 (31%) CLL, and 12 (31%) MM. There were 13 of the 39 pts (33%) seroconverted at T1; an increase of the serological response was registered after the second dose (T2) (22/39 pts, 56%) and maintained after 6 months (22/39 pts, 56%) and 12 months (24/39 pts, 61%) from the first dose (T3–T4, respectively). Non-serological responders at T4 were 7/39 (18%): 0/15 NHL, 1/12 MM (8%), and 6/12 CLL (50%). All of them were on therapy (one lenalidomide, three ibrutinib, and three venetoclax). SARS-CoV2-reactive T-cell analysis (interferon gamma release assays) was available since June 2022 and was evaluated at 12 months (T4) from the first dose of vaccine in 31/39 pts (79%). T-cell-mediated-responders were 17/31 (55%): most of them were NHL and MM (47%, 41% and 12% for NHL, MM, and CLL, respectively). Both serological and T-cell non-responders were represented by pts on active therapy (venetoclax/ibrutinib). During the period of observation, eight (20.5%) pts developed mild SARS-CoV2 infection; no coronavirus disease 19 (COVID-19)-related deaths or hospitalizations were registered. In conclusion, in our cohort of lymphoproliferative pts receiving BNT162b2, CLL diagnosis and venetoclax/ibrutinib seem to be related with a lower humoral or T-mediated response. Nevertheless, the efficacy of mRNA vaccine in HM pts and the importance to continue the vaccine program even in non-responders after the first dose are supported in our study by demonstrating that a humoral and T-cell-mediated seroconversion should be observed even in the subsets of heavily immunocompromised pts

MOESM4 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Additional file 4: Figure S4. Anti-exosome nanobodies bind EV-fractions separated by chromatography. Flow cytometry experiments show that both H1 and H6 strongly bind to exosomes present in the fraction 1 separated by IEX chromatography