Size-dependent internalization of exomemes

Exosomes are biologically active extracellular vesicles, whose size range from 30 to 100nm, which are released by many cell types in various body fluids. These particles have been proved to be strong players in cell-cell communication, thus reaching interest especially about their involvement in cancer progression, invasion and metastasis. For example, our group has already investigated the tumor supporting function of exosomes released by Glioma-Associated Stem Cells (GASC). Considering the mechanism of action of exosomes, besides the undeniable role exerted by the message they deliver, much interest is now focused on to their physical properties, since they could also influence the biological function observed, as shown for bioengineered nanoparticles, and consequently their therapeutic potential. As exosomal size is nanoscopic, the estimation of particle size and density has been elusive, and the use of several techniques has been applied, such as Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS), each displaying strength and weaknesses. Moreover, another extremely relevant matter, for which it has not yet reached a consensus, regards the ideal method to isolate exosomes, as it could lead to different exosomal populations with different features. In this thesis, we compared the exosomes preparations obtained by two different enrichment methods, wondering if this could affect the exosome uptake by cells and their ability to alter the biological functions of target cells. Therefore, we isolated exosomes released by patient-derived high-grade GASC and human glioblastoma cell line A172 in cell supernatants using both ultracentrifugation (UC) and ExoQuick (EQ) precipitation methods. Then we assessed the purified particles both for their physical properties (characterizing particle size and particle density) using AFM and NTA, and molecular properties (evaluating total protein content and a specific exosomal marker) using Bradford, BiCinchoninic Acid Assay (BCA) and CD9 ELISA assay. Then we performed cell uptake assays to observe the differential internalization of the preparations, and finally we evaluated whether this differential internalization could influence glioma cell motility. Our results demonstrate that polymer-based precipitation results in particles that have a size distribution smaller than that of ultracentrifuge-isolated ones. Moreover, we further established that smaller exosomes are better uptaken by the receiving cells and, furthermore, this affects cell motility. These data suggest that the isolation method could profoundly affect the size distribution of the obtained exosomal preparation and this is associated with differences in their physical and biological properties, thus improving or decreasing their potential therapeutic capability

Role of microenvironment in glioma invasion: What we learned from in vitro models

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used

Circulating methylated DNA to monitor the dynamics of RAS mutation clearance in plasma from metastatic colorectal cancer patients

The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1 ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2 ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3 50% of wild-type-RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples

Molecular profiling of follicular fluid microRNAs in young women affected by Hodgkin lymphoma.

Research question Treatments for Hodgkin lymphoma have improved but one of their common effects is gonadal toxicity, which contributes to fertility damage of patients and induces temporary or irreversible loss of fertility. Could micro-RNA (miRNA) expression profiles in follicular fluid be influenced by Hodgkin lymphoma? Could their alteration affect molecular pathways involved in follicle growth and oocyte maturation? Design miRNA expression profile was investigated in follicular fluid samples from young women affected by Hodgkin lymphoma compared with healthy controls by NanoString technology. Bioinformatic analysis was used to verify miRNA involvement in follicle development and miRNA deregulation with Hodgkin lymphoma in a larger cohort of follicular fluid samples was confirmed by real-time quantitative polymerase chain reaction. Results Thirteen miRNAs are deregulated in Hodgkin lymphoma samples compared with controls and are involved in molecular pathways related to cancer, gametogenesis and embryogenesis. Among them, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are implicated in biological processes related to follicle development and oocyte maturation. Let-7b-5p holds the central position in the regulatory network of miRNA-mRNA interactions, has the highest number of mRNA target genes shared with the other differentially expressed miRNAs and is significantly downregulated in Hodgkin lymphoma follicular fluid samples. Conclusions These data led us to question the potential influence of miRNA deregulation on oocyte quality. Further studies are needed to verify the reproductive potential of young patients with Hodgkin lymphoma before starting chemotherapy protocols and an adequate protocol of fertility preservation needs to be guaranteed

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

Semaphorin-7A on Exosomes: A Promigratory Signal in the Glioma Microenvironment

Exosomes are one of the most important mediators of the cross talk occurring between glioma stem cells (GSCs) and the surrounding microenvironment. We have previously shown that exosomes released by patient-derived glioma-associated stem cells (GASC) are able to increase, in vitro, the aggressiveness of both GSC and glioblastoma cell lines. To understand which molecules are responsible for this tumour-supporting function, we performed a descriptive proteomic analysis of GASC-exosomes and identified, among the others, Semaphorin7A (SEMA7A). SEMA7A was described as a promigratory cue in physiological and pathological conditions, and we hypothesised that it could modulate GSC migratory properties. Here, we described that SEMA7A is exposed on GASC-exosomes' surface and signals to GSC through Integrin \u3b21. This interaction activates focal adhesion kinase into GSC and increases their motility, in our patient-based in vitro model. Our findings suggest SEMA7A-\u3b21-integrin as a new target to disrupt the communication between GSCs and the supporting microenvironment

Genomic landscape and survival analysis of ctDNA “neo-RAS wild-type” patients with originally RAS mutant metastatic colorectal cancer

Background: The term “neo-RAS wild-type” refers to the switch to RAS wild-type disease in plasma circulating tumor DNA (ctDNA) from originally RAS mutant colorectal cancers. Consistently, the hypothesis to re-determine RAS mutational status in ctDNA at disease progression in RAS mutant mCRC opened to a new perspective for clinically-based selection of patients to be treated with EGFR inhibitors. Currently, the genomic landscape of “neo-RAS wild-type” is unknown. This is a prospective study aimed to investigate clinical and genomic features associated with RAS mutation clearance in a large cohort of RAS mutant mCRC patients who converted to RAS wild- type in liquid biopsy at failure of first-line treatments. Secondary aim was to investigate the long term prognostic significance of “true neo-RAS wild- type”. Patients and methods: 70 patients with stage IV RAS mutant colorectal cancer were prospectively enrolled. Plasma samples were collected at progression from first-line treatment. RAS/BRAF mutations in plasma were assessed by RT-PCR. In RAS/BRAF wild-type samples, ctDNA was used to generate libraries using a 17 genes panel whose alteration has clinical relevance. To investigate the prognostic significance of RAS mutation clearance, test curves for PFS and OS were represented by Kaplan-Meier estimator plot and Log-rank test. Results: The most commonly detected actionable mutations in “neo-RAS wild-type” were: PIK3CA (35.7%); RET (11.9%); IDH1 (9.5%); KIT (7%); EGFR (7%); MET (4.7%); ERBB2 (4.7%); FGFR3 (4.7%). Both OS and post-progression survival were longer in patients with “neo-RAS wild-type” compared to those who remained RAS mutant (p<0.001 for both). Conclusions: De-novo-targetable mutations occured in a large percentage of “neo-RAS wild-type”, being PIK3CA the most commonly detected. RAS mutation clearance in ctDNA is associated with long- term improvement of overall survival

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

Size-dependent cellular uptake of exosomes

The ability of exosomes to elicit specific cellular responses suggests that they may be increasingly used as therapeutics. Their vesicular nature makes them suitable as potential nanocarriers for drugs or nucleic acids delivery. Here we address the question whether the method of preparation of enriched exosomal fractions can affect their uptake by cells and their ability to trigger a response. We compared ultracentrifugation and polymer-based precipitation methods on supernatants of glioma-associated stem cells isolated from a high-grade glioma patient. We determined particle size distributions after purification and their correlation with uptake, proliferation and migration in glioblastoma cell cultures. Our findings indicate that polymer-based precipitation leads to smaller particle size distributions, faster uptake by target cells and increased cellular motility. The different effect that isolation method-dependent populations of particles have on cell motility suggests their size distribution could also profoundly affect exosome therapeutic potential

Exploratory pilot study of circulating biomarkers in metastatic renal cell carcinoma

With the introduction of immune checkpoint inhibitors (ICIs) and next-generation vascular endothelial growth factor receptor–tyrosine kinase inhibitors (VEGFR–TKIs), the survival of patients with advanced renal cell carcinoma (RCC) has improved remarkably. However, not all patients have benefited from treatments, and to date, there are still no validated biomarkers that can be included in the therapeutic algorithm. Thus, the identification of predictive biomarkers is necessary to increase the number of responsive patients and to understand the underlying immunity. The clinical outcome of RCC patients is, in fact, associated with immune response. In this exploratory pilot study, we assessed the immune effect of TKI therapy in order to evaluate the immune status of metastatic renal cell carcinoma (mRCC) patients so that we could define a combination of immunological biomarkers relevant to improving patient outcomes. We profiled the circulating levels in 20 mRCC patients of exhausted/activated/regulatory T cell subsets through flow cytometry and of 14 immune checkpoint-related proteins and 20 inflammation cytokines/chemokines using multiplex Luminex assay, both at baseline and during TKI therapy. We identified the CD3+CD8+CD137+ and CD3+CD137+PD1+ T cell populations, as well as seven soluble immune molecules (i.e., IFNγ, sPDL2, sHVEM, sPD1, sGITR, sPDL1, and sCTLA4) associated with the clinical responses of mRCC patients, either modulated by TKI therapy or not. These results suggest an immunological profile of mRCC patients, which will help to improve clinical decision-making for RCC patients in terms of the best combination of strategies, as well as the optimal timing and therapeutic sequence