The Emerging Roles of Extracellular Vesicles in Osteosarcoma

Extracellular vesicles (EVs) are heterogeneous nanosized vesicles that are constitutively released by virtually all types of cells. They have been isolated in almost all body fluids. EVs cargo consists of various molecules (nucleic acids, proteins, lipids, and metabolites), that can be found on EVs surface and/or in their lumen. EVs structure confer stability and allow the transfer of their cargo to specific cell types over a distance. EVs play a critical role in intercellular communication in physiological and pathological settings. The broadening of knowledge on EVs improved our comprehension of cancer biology as far as tumor development, growth, metastasis, chemoresistance, and treatment are concerned. Increasing evidences suggest that EVs have a significant role in osteosarcoma (OS) development, progression, and metastatic process. The modulation of inflammatory communication pathways by EVs plays a critical role in OS and in other bone-related pathological conditions such as osteoarthritis and rheumatoid arthritis. In this review we describe the emerging data on the role of extracellular vesicles in osteosarcoma and discuss the effects and function of OS-derived EVs focusing on their future applicability in clinical practice

Extracellular nanovesicles secreted by human osteosarcoma cells promote angiogenesis

Angiogenesis involves a number of different players among which extracellular nanovesicles (EVs) have recently been proposed as an efficient cargo of pro-angiogenic mediators. Angiogenesis plays a key role in osteosarcoma (OS) development and progression. Acidity is a hallmark of malignancy in a variety of cancers, including sarcomas, as a result of an increased energetic metabolism. The aim of this study was to investigate the role of EVs derived from osteosarcoma cells on angiogenesis and whether extracellular acidity, generated by tumor metabolism, could influence EVs activity. For this purpose, we purified and characterized EVs from OS cells maintained at either acidic or neutral pH. The ability of EVs to induce angiogenesis was assessed in vitro by endothelial cell tube formation and in vivo using chicken chorioallantoic membrane. Our findings demonstrated that EVs derived from osteosarcoma cells maintained either in acidic or neutral conditions induced angiogenesis. The results showed that miRNA and protein content of EVs cargo are correlated with pro-angiogenic activity and this activity is increased by the acidity of tumor microenvironment. This study provides evidence that EVs released by human osteosarcoma cells act as carriers of active angiogenic stimuli that are able to promote endothelial cell functions relevant to angiogenesis

Pre-clinical Models for Studying the Interaction between Mesenchymal Stromal Cells and Cancer Cells and the Induction of Stemness

Mesenchymal stromal cells (MSC) have essential functions in building and supporting the tumour microenvironment, providing metastatic niches, and maintaining cancer hallmarks, and it is increasingly evident that the study of the role of MSC in cancer is crucial for paving the way to clinical opportunities for novel anti-cancer therapies. To date, the vast majority of preclinical models that have been used for studying the effect of reactive MSC on cancer growth, metastasis, and response to therapy has been mainly based on in vitro flat biology, including the co-culturing with cell compartmentalization or with cell-to-cell contact, and on in vivo cancer models with different routes of MSC inoculation. More complex in vitro 3D models based on spheroid structures that are formed by intermingled MSC and tumour cells are also capturing the interest in cancer research. These are innovative culture systems tailored on the specific tumour type and that can be combined with a synthetic extracellular matrix, or included in in silico technologies, to more properly mimic the in vivo biological, spatial, biochemical, and biophysical features of tumour tissues. In this review, we summarized the most popular and currently available preclinical models for evaluating the role of MSC in cancer and their specific suitability, for example, in assaying the MSC-driven induction of epithelial-to-mesenchymal transition or of stem-like traits in cancer cells. Finally, we enlightened the need to carefully consider those parameters that might unintentionally strongly affect the secretome in MSC-cancer interplay and introduce confounding variables for the interpretation of results

Endogenous extracellular matrix regulates the response of osteosarcoma 3D spheroids to doxorubicin

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients' bed.

BACKGROUND: Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H\u2009+-rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. METHOD: MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. RESULTS: Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. CONCLUSION: This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy

Exosomes: Novel effectors of human platelet lysate activity

Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies

Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry

Emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. The former was the only available intervention when the current unprecedented Ebolavirus (EBOV) outbreak in West Africa began. Prior to this, the development of EBOV vaccines and anti-viral therapies required time and resources that were not available. Therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of EBOV and could be used immediately. Here we test three such medicines and measure their ability to inhibit pseudotype viruses (PVs) of two EBOV species, Marburg virus (MARV) and avian influenza H5 (FLU-H5). We confirm the ability of chloroquine (CQ) to inhibit viral entry in a pH specific manner. The commonly used proton pump inhibitors, Omeprazole and Esomeprazole were also able to inhibit entry of all PVs tested but at higher drug concentrations than may be achieved in vivo. We propose CQ as a priority candidate to consider for treatment of EBOV

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points