Simona Taverna

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

Our study is designed to assess if exosomes released from chronic myelogenous leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM‐1 and VCAM‐1 cell adhesion molecules and interleukin‐8 expression. The stimulation of cell‐cell adhesion molecules was paralleled by a dose‐dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE‐cadherin and β‐catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.

An active form of sphingosine kinase-1 is released in the extracellular medium as component of membrane vesicles shed by two human tumor cell lines.

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

carboxyamidotriazole-orotate inhibits the growth of imatinib resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.

Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice

Exosomes are nano-sized vesicles of endolysosomal origin, released by several cytotypes in physiological and pathological conditions. Tumor derived exosomes, interacting with other cells of the tumor microenvironment, modulate tumor progression, angiogenic switch, metastasis, and immune escape. Recently, extracellular vesicles were proposed as excellent biomarkers for disease monitoring and prognosis in cancer patients. Non-small cell lung cancer (NSCLC) has a poor 5-year survival rate due to the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Exosomes might be promising beneficial tools as biomarker candidates in the scenario of NSCLC, since they contain both, proteins and miRNAs. The clinical case reported in this manuscript is a proof of concept revealing that NSCLC exosomes and sorted miRNAs might constitute, in a near future, novel biomarkers. This review summarizes the role of exosomes in NSCLC, focusing on the importance of exosomal microRNAs in lung cancer diagnosis and prognosis.

Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype, via exosomal miR-21

Tumor derived exosomes are vesicles which contain proteins and microRNAs that mediate cell-cell communication and are involved in angiogenesis and tumor progression. Curcumin derived from the plant Curcuma longa, shows anticancer effects. Exosomes released by CML cells treated with Curcumin contain a high amount of miR-21 that is shuttled into the endothelial cells in a biologically active form. The treatment of HUVECs with CML Curcu-exosomes reduced RhoB expression and negatively modulated endothelial cells motility. We showed that the addition of CML control exosomes to HUVECs caused an increase in IL8 and VCAM1 levels, but Curcu-exosomes reversed these effects thus attenuating their angiogenic properties. This antiangiogenic effect was confirmed with in vitro and in vivo vascular network formation assays. SWATH analysis of the proteomic profile of Curcu-exosomes revealed that Curcumin treatment deeply changes their molecular properties, in particular, Curcumin induces a release of exosomes depleted in pro-angiogenic proteins and enriched in proteins endowed with anti-angiogenic activity. Among the proteins differential expressed we focused on MARCKS, since it was the most modulated protein and a target of miR-21. Taken together our data indicated that also Curcumin attenuates the exosomes ability to promote the angiogenic phenotype and to modulate the endothelial barrier organization.

Increased expression of interleukin-32 in the inflamed ileum of ankylosing spondylitis patients

OBJECTIVE To study the mRNA expression and protein tissue distribution of IL-32 in ileal biopsy specimens from patients with AS. METHODS Quantitative gene expression analysis, by real-time PCR, of IL-32, IL-1β, IL-10, TNF-α and IFN-γ was performed on ileal biopsies of 15 AS and 15 Crohns disease (CD) patients and 10 healthy subjects (HSs). IL-32 tissue distribution was evaluated by immunohistochemistry. The effect of IL-32 on the production of IL-10 by intestinal epithelial cell lines was also evaluated. RESULTS In the ileal specimens of patients with AS and intestinal chronic inflammation, significant up-regulation of IL-32 at both the mRNA and protein levels was found as compared with non-inflamed AS patients and controls. IL-32 over-expression in AS was accompanied by a significant increase of IL-10 but not of cytokines involved in IL-32 induction. IL-32 stimulates intestinal epithelial cell lines in vitro to produce IL-10. CONCLUSION Our findings suggest IL-32 as an important cytokine probably involved in the innate immune response occurring in early phases of intestinal inflammation, where it seems to play a prevalent protective role.

Membrane vesicles containing matrix metalloproteinase‐9 and fibroblast growth factor‐2 are released into the extracellular space from mouse mesoangioblast stem cells

Certain proteins, including fibroblast growth factor‐2 (FGF‐2) and matrix metalloproteinase‐9 (MMP‐9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF‐2 and MMP‐9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblasts may themselves secrete paracrine signals and factors that make damaged tissues more amenable to cell therapy through the release of membrane vesicles. J. Cell. Physiol. 224:144–151, 2010

Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity

The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.

Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis.

Intracellular trafficking of endogenous fibroblast growth factor-2.

We have previously reported how the release of fibroblast growth factor‐2 (FGF‐2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF‐2 is targeted to the budding vesicles. Considering that in vitro cultured Sk‐Hep1 hepatocarcinoma cells release FGF‐2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF‐2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in FGF‐2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF‐2 granules moving to the cell periphery and FM4‐64‐labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF‐2 release, were analyzed for their effects on FGF‐2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF‐2 movements toward the cell periphery. The FGF‐2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF‐2 clustered at the cell periphery, but the rate of its release decreased. FGF‐2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF‐2 translocation to the nucleus and nucleolus. FGF‐2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms.