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Titanium implants undergo tribocorrosion processes releasing particles that interact with several cells at the implant–bone interface. Osteoblasts-derived exosomal proteins reduce osteogenic differentiation of HMSCs contributing to joint failure.

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Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. http://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/., Objective: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. Design: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). Results: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. Conclusion: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance., The work was supported by NORTE-01–0145-FEDER-000029, Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and national funds through FCT—Foundation for Science and Technology POCI-01–0145-FEDER-32189. Programa Operacional Regional do Norte and co-financed by European Regional Development Fund under the project "The Porto Comprehensive Cancer Center" with the reference NORTE-01-0145-FEDER-072678 - Consórcio PORTO.CCC – Porto.Comprehensive Cancer Center. CFR is supported by FCT (SFRH/BD/131461/2017), NB by (SFRH/BD/130801/2017), IB by FCT (SFRH/BD/144854/2019), and BA by FCT (PD/BD/135546/2018). DG’s contribution was supported by the NCI (R21 CA179907). We acknowledge the support of the i3S Scientific Platforms: Translational Cytometry, Animal Facility, Bioimaging and Histology and Electron Microscopy are members of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01–0145-FEDER-022122). Proteomics was performed at the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC), ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.

The clinical management of patients with pancreatic cystic lesions is of utmost importance to identify those at high risk for pathological progression. Current recommendations are guided by clinical presentation and radiologic criteria, but the results fall short for a disease that the only curative option is surgical resection. There is an urgent need for the introduction of biomarkers that can help in risk assessment of such lesions. We report a case of a pancreatic cystic lesion without imagiological findings suggestive of advanced disease, and high levels of a circulating biomarker, glypican-1 (GPC-1), which parallel those of patients with pancreatic cancer. One year after, the patient revealed malignant progression at follow-up. Our report is unprecedented in the literature. It describes a clinical case in which a biomarker was positive for a patient that only showed progression one year after its detection. This clinical information goes beyond the current knowledge in the field because it shows that the introduction of liquid biopsy and biomarkers is a highly promising clinical tool for the non-invasive assessment of pancreatic cancer precursor lesions, ultimately increasing the rate of patients eligible for surgical resection.

Individuals within specific risk groups for pancreatic ductal adenocarcinoma (PDAC) [mucinous cystic lesions (MCLs), hereditary risk (HR), and new-late onset diabetes mellitus (NLOD)] represent an opportunity for early cancer detection. Endoscopic ultrasound (EUS) is a premium image modality for PDAC screening and precursor lesion characterization. While no specific biomarker is currently clinically available for this purpose, glypican-1 (GPC1) is overexpressed in the circulating exosomes (crExos) of patients with PDAC compared with healthy subjects or those harboring benign pancreatic diseases.To evaluate the capacity of GPC1This cross-sectional study with a prospective unicentric cohort included 88 subjects: 40 patients with MCL, 20 individuals with HR, and 20 patients with NLOD. A control group (CG) was submitted to EUS for other reasons than pancreatic pathology, with normal pancreas and absence of hereditary risk factors (Half of MCLs harbored worrisome features (WF) or high-risk stigmata (HRS). Pancreatic abnormalities were detected by EUS in 10.0% and 35.0% in HR and NLOD individuals, respectively, all considered non-malignant and "harmless." Median levels of GPC1GPC1

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumor types, being the sixth leading cause of mortality worldwide and the fourth in Europe. Globally, it has a mortality/incidence ratio of 98%, and the 5‑year survival rate in Europe is only 3%. Although risk factors, such as obesity, diabetes mellitus, smoking, alcohol consumption and genetic factors, have been identified, the causes of PDAC remain elusive. Additionally, the only curative treatment for PDAC is surgery with negative margins. However, upon diagnosis, ~30% of the patients already present with locally advanced disease. In these cases, a multidisciplinary approach is required to improve disease‑related symptoms and prolong patient survival. In the present article, a comprehensive review of PDAC epidemiology, physiology and treatment is provided. Moreover, guidelines on patient treatment are suggested. Among the different available therapeutic options for the treatment of advanced PDAC, results are modest, most likely due to the complexity of the disease, and so the prognostic remains poor. Molecular approaches based on multi‑omics research are promising and will contribute to groundbreaking personalized medicine. Thus, economic investment that promotes research of pancreatic cancer will be critical to the development of more efficient diagnostic and treatment strategies.

BackgroundThe inflammatory response to titanium implant-derived wear particles is considered as the hallmark of periprosthetic osteolysis, an event that cause pain, reduce patient motility, ultimately leading to the need of a revision surgery. Although macrophages are major cell players, other cell types such as bone cells can indirectly contribute to periprosthetic osteolysis, however the mechanisms are not fully understood. Exosomes (Exos) has been related with several bone pathologies, with growing body of literature recognizing them as actively shuttle molecules through the body, with their cargo being completely dependent of external stimuli (e.g. chemicals and metals ions and particles). Till the moment, the role of wear debris on osteoblasts exosomes biogenesis is absent and the possible contribution of Exos to osteoimmune communication and periprosthetic osteolysis is still in its infancy. Taking that in consideration, in this work we investigate the effect of wear debris on Exo biogenesis, where two bone cell models were exposed to titanium dioxide nanoparticles (TiO2 NPs) similar in size and composition to wear debris associated with prosthetic implants. The contribution of Exos to periprosthetic osteolysis was evaluated performing functional tests stimulating primary human macrophages with bone-derived Exos.ResultsFor the first time, we report that TiO2 NPs enter in multivesicular bodies, the nascent of Exos and altered osteoblasts derived exosomes secretion and cargo. No significant differences were observed in Exos morphology and size, however mass spectrometry analysis identified urokinase-type plasminogen activator (uPA), specifically enriched in Exos derived from bone cells pre-incubated with TiO2 NPs. Functional tests confirmed the activation of human macrophages towards a mixed phenotype with consequent secretion of pro and anti-inflammatory cytokines. ConclusionsThe external stimuli of osteoblasts to TiO2 NPs induced a dose dependent secretion of Exos, suggesting alterations in their biogenesis as well as in their cargo. Functional tests reveal that enriched uPA exosomal cargo is stimulating macrophages towards a mixed M1 and M2 phenotype inducing the release of pro-and anti-inflammatory signals that are characteristic of periprosthetic osteolysis. Interestingly, uPA may be proposed, in the future, as a possible candidate biomarker to early diagnose particle induced periprosthetic osteolysis, since uPA was also detected in the pseudocapsular interface around implants of patients with loosening of total hip prosthesis and joint replacement surgery, suggesting their active role in disease progression.

Exosomes are described as central players in a myriad of biological processes. However, the available methodologies to study their function in complex biological systems in vivo are still very limited. The biodistribution of endogenously produced exosomes, the ability to trace their spontaneous flow in order to identify the cell types they interact with, remains a major challenge. New tools to identify comprehensive networks of communication established by exosomes originated in distinct cell types in vivo, are fundamental for a better understanding of their biology. Here, we describe the development of a genetically engineered mouse model that allows the expression of the mouse CD63 exosomal marker fused with one (monocolor) or up to four fluorescent proteins (multireporter), the ExoBow. The genetic design of the ExoBow transgene allows the conditional expression of the reporters in any tissue/cell-type in an inducible or non-inducible fashion. In addition, communication mediated by CD63 positive (CD63+) exosomes can be identified amongst the same tissue/cell types using the multireporter version of the model, in order to map intra-organ/tissue communication. We demonstrate the applicability of the ExoBow transgene in normal physiological conditions and in the context of cancer, using pancreas as a working model. The ExoBow comprises a unique strategy to identify intra- and inter-organ/cell-type communication mediated by CD63+ exosomes. We believe this tool will contribute for a better understanding of the complex interactions occurring in vivo that underly the biology of exosomes in health and disease.

Cancer is a lethal disease and the second most common cause of death worldwide. It is estimated that the next decades will bring a rise of about 50% in cancer incidence and mortality. At present, immunotherapy has shown the best results for cancer treatment. Because of this, the study of the anti-tumor immune response has gained a significant importance in cancer research. Many are the factors described to be involved in this process. Exosomes are extracellular vesicles that shape the cellular program of the recipient cells and are involved in cancer progression. Our review explores the link between exosomes and natural killer (NK) cells, the possible consequences in cancer progression, and the enclosed potential to identify new immunotherapy targets within this crosstalk. Natural killer (NK) cells are innate lymphoid cells involved in tumor surveillance. These immune cells have the potential to fight cancer growth and metastasis, as such, their deregulation can result in tumor immune escape. Recently exosomes were described as mediators of intercellular communication between cancer and NK cells. The exact role of this subclass of extracellular vesicles (EVs), which transport genetic and molecular material to recipient cells, in NK cell biology in the context of cancer, is still an open question. Several reports have demonstrated that tumor-derived exosomes (TDEs) can exert immunomodulatory activities, including immunosuppression, thus promoting cancer progression. Some reports demonstrate that the interplay between cancer exosomes and NK cells allows tumors to escape immune regulation. On the other hand, tumor exosomes were also described to activate NK cells. Additionally, studies show that NK cell exosomes can modulate the immune system, opening up their potential as an immunotherapeutic strategy for cancer treatment. Our review will focus on the reprogramming effect of cancer exosomes on NK cells, and the immunotherapeutic potential of NK cells-derived exosomes. The laboratory is supported by European Regional Development Fund (ERDF) through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, by FCT-Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project POCI-01-0145-FEDER-032189 and by AstraZeneca Foundation 2017 FAZ Ciência Award. IB is supported by FCT (SFRH/BD/144854/2019).

Liquid biopsy represents the analysis of tumor-derived material in the blood and other body fluids of cancer patients. This portrays a minimally invasive detection tool for molecular biomarkers. Liquid biopsy has emerged as a complementary or alternative method to surgical biopsy. This non-invasive detection tool overcomes the recurrent problems in the clinical assessment of tumors that stem from the lack of accessibility to the tumor tissue and its clonal heterogeneity. Moreover, body fluid-derived components have shown to reflect the genetic profile of both primary and metastatic lesions and provide a real-time monitoring of tumor dynamics, representing a great promise for personalized medicine. This review will highlight the latest breakthroughs and the current applications of several tumor-derived biomarkers that can be found in body fluids. The authors will focus on tumor-derived exosomes, tumor-educated platelets, and circulating tumor miRNAs and mRNAs, and how these can be used for tumor detection.

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

Metastasis is the leading cause of cancer mortality. Cancer cells must adapt to colonize and thrive at the metastatic site. The modulation of the receptive organ microenvironment is a key event in the adaptation process and is partially accomplished at a distance by the primary tumor. Exosomes, a subclass of extracellular vesicles (EVs), are distal mediators of communication that carry genetic and molecular information to neighboring and distant cells. Cancer exosomes have been involved in restructuring metastatic sites to support cancer cell colonization. In this article, we discuss the role of exosomes in the metastatic process.

Targeted therapies almost universally fail due to the development of resistance. EGFR-mutant non-small cell lung cancer (NSCLC) is a well characterized model of such mechanism. In this model, treatment with anti-EGFR therapy leads to resistance driven by the presence of the EGFR T790M mutation in about 50% of the cases. However, clinical data shows the EGFR resistance mutation is present only in a subpopulation of the relapsing cells. How cancer cells devoid of the EGFR T790M mutation become resistant is not known. The aim of this study is to explore if T790M-mediated resistance to anti-EGFR therapy can spread horizontally, through a mitosis-independent mechanism, among NSCLC cells. Immunodeficient mice (Rag2-/-Il2rg-/-) were engrafted with NSCLC HCC827 cells, harboring the Erlotinib-sensitive mutation EGFR E746-A750del, and H1975 cells, carrying the EGFR T790M Erlotinib-resistant mutation. These animals received a single inoculation of HCC827 cells in a single flank (n=10), or dual inoculation of HCC827 and H1975 cells in opposite flanks (n=20). Erlotinib was orally administered three times a week (25 mg/Kg) and tumors were collected at humane endpoint for genetic and transcriptomic analysis by next generation sequencing and digital PCR. T790M-driven resistance to Erlotinib was transferred in vivo from resistant to sensitive cells in dual inoculated animals, resulting in a significant decrease of relapse-free survival. Short tandem repeat profiling of the tumors showed absence of H1975 cells in sensitive tumors in dual inoculated animals, ruling out cell migration as the mechanism of resistance. This result can also not be explained by transfer of mutated DNA from resistant to sensitive cells because high-end sequencing detected the presence of the T790M mutation only in few sensitive tumors from dual inoculated mice and with an extremely low allele frequency ( Citation Format: Susana Junqueira-Neto, Ana Rita Oliveira, Joana Marques, Sónia Melo, José Luís Costa, José Carlos Machado. Resistance to anti-EGFR therapy can spread horizontally among cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1099.

Pancreatic cancer presents with a dismal mortality rate and is in urgent need of methods for early detection with potential for timely intervention. All living cells, including cancer cells, generate exosomes. We previously discovered double stranded genomic DNA in exosomes derived from the circulation of pancreatic cancer patients, which enabled the detection of prevalent mutations associated with the disease. Here, we report a proof-of-concept study that demonstrates the potential clinical utility of circulating exosomal DNA for identification of KRASG12D and TP53R273H mutations in patients with pancreas-associated pathologies, including pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP) and intraductal papillary mucinous neoplasm (IPMN), and in healthy human subjects. In 48 clinically annotated serum samples from PDAC patients, digital PCR analyses of exosomal DNA identified KRASG12D mutation in 39.6% of cases, and TP53R273H mutation in 4.2% of cases. KRASG12D and TP53R273H mutations were also detected in exosomal DNA from IPMN patients (2 out of 7 with KRASG12D, one of which also co-presented with TP53R273H mutation). Circulating exosomal DNA in 5 out of 9 CP patients enabled the detection of KRASG12D mutation. In 114 healthy subject-derived circulating exosomal DNA, 2.6% presented with KRASG12D mutation and none with TP53R273H mutation. This study highlights the value of circulating exosomal DNA for a rapid, low-cost identification of cancer driving mutations. The identification of mutations in IPMN patients and healthy subjects suggests that liquid biopsies may allow potential assessment of cancer risk but with a cautionary note that detection of clinical cancer cannot be assumed.

Genome-wide chromatin studies identified the tumor suppressor p53 as both a promoter and an enhancer-binding transcription factor. As an enhancer factor, p53 can induce local production of enhancer RNAs, as well as transcriptional activation of distal neighboring genes. Beyond the regulation of protein-coding genes, p53 has the capacity to regulate long intergenic noncoding RNA molecules (lincRNAs); however, their importance to the p53 tumor suppressive function remains poorly characterized. Here, we identified and characterized a novel p53-bound intronic enhancer that controls the expression of its host, the lincRNA00475 (linc-475). We demonstrate the requirement of linc-475 for the proper induction of a p53-dependent cell cycle inhibitory response. We further confirm the functional importance of linc-475 in the maintenance of CDKN1A/p21 levels, a cell cycle inhibitor and a major p53 target gene, following p53 activation. Interestingly, loss of linc-475 reduced the binding of both p53 and RNA polymerase II (RNAPII) to the promoter of p21, attenuating its transcription rate following p53 activation. Altogether, our data suggest a direct role of p53-bound enhancer domains in the activation of lincRNAs required for an efficient p53 transcriptional response.Oncogene advance online publication, 18 January 2016; doi:10.1038/onc.2015.502.

Exosomes are lipid bilayer extracellular vesicles (EVs) of 50-150nm in size, which contain nucleic acids (mRNA, ncRNAs and DNA), proteins and lipids. They are secreted by all cells and circulate in all body fluids. Exosomes are key mediators of several processes in cancer that mediate tumor progression and metastasis. These nano-vesicles, when secreted from cancer cells, are enriched in non-coding RNAs (e.g. microRNAs) complexed with the RNA-Induced Silencing Complex (RISC), that mediate an efficient and rapid silencing of mRNAs at the recipient cell, reprogramming their transcriptome. MicroRNAs in circulation encapsulated in exosomes are protected from degradation by a lipid bilayer and might serve as potential non-invasive diagnostic and screening tools to detect early stage cancer, to facilitate treatment options and possible help in curative surgical therapy decisions. Additionally, engineered exosomes can be used as therapy vehicles for targeted delivery of RNAi molecules, escaping the immune system detection.

Exosomes are a specific population of extracellular vesicles (EVs) that originate from an endocytic process. Virtually every cell type secretes exosomes and their size ranges from 40 to 150 nm. Exosomes are surrounded by a lipid bilayer and contain functional cargo that comprises proteins, lipids and genetic material such as protein, RNA and DNA. In the recent years, several studies have reported the role of exosomes as mediators of intercellular communication. Exosomes serve as vehicles used by cancer cells and stromal cells to influence both local and distant metastatic sites, by reprogramming recipient cells. This chapter will focus on the mechanisms underlying the role of exosomes in tumor development, metastasis, immune escape, therapy resistance, microenvironment reprogramming and angiogenesis. Furthermore, we will also discuss the potential to target exosomes as a new therapeutic strategy in cancer.

Exosomes are man-sized vesicles shed by all cells, including cancer cells. Exosomes can serve as novel liquid biopsies for diagnosis of cancer with potential prognostic value. The exact mechanism/s associated with sorting or enrichment of cellular components into exosomes are still largely unknown. We reported Glypican-1 (GPC1) on the surface of cancer exosomes and provided evidence for the enrichment of GPC1 in exosomes from patients with pancreatic cancer1. Several different laboratories have validated this novel conceptual advance and reproduced the original experiments using multiple antibodies from different sources. These include anti-GPC1 antibodies from ThermoFisher (PA5-28055 and PA-5-24972)1,2, Sigma (SAB270028), Abnova (MAB8351, monoclonal antibodies clone E9E)3, EMD Millipore (MAB2600-monoclonal antibodies)4, SantaCruz5, and R&D Systems (BAF4519)2. This report complements such independent findings and report on the specific detection of Glypican-1 on the exosomes derived from the serum of pancreas cancer patients using multiple antibodies. Additionally, the specificity of the antibodies to GPC1 was determined by western blot and Protein Simple analyses of pancreatic cancer cells and their exosomes. Interestingly, our results highlight a specific enrichment of high molecular weight GPC1 on exosomes, potentially contributed by heparan sulfate and other glycosylation modifications.

Exosomes are small extracellular vesicles with a significant role in most processes associated with cancer. On one hand, exosomes role in the different hallmarks of cancer has been widely described, highlighting the urge to understand the potential to target communication mediated by exosomes as a novel therapeutic approach in cancer. On the other hand, exosomes stability in circulation and tumor-targeting capacity shows their applicability in the delivery of anti-cancer molecules. This review will discuss the dual applicability of exosomes in cancer focusing on their usage for therapy improvement, or their targeting to block their supportive role in tumor progression and response to therapy. We highlight the current developments and the strategies used to enhance the potential of exosomes to become clinical partners in the treatment of cancer.

Exosomes are a subclass of extracellular vesicles involved in intercellular communication that are released by all cell types, including cancer cells. Cancer exosomes carry malignant information in the form of proteins, lipids, and nucleic acids that can reprogram recipient cells. Exosomes have emerged as putative biological mediators in cancer contributing to major steps of disease progression. A leading role exists for cancer exosomes in specific aspects of tumor progression: modulation of immune response, tumor microenvironment reprogramming, and metastasis. This review will address the functions attributed to cancer exosomes in these three aspects of cancer biology, highlighting recent advances and potential limitations. Finally, we explore alternative strategies to develop better models to study cancer exosomes biology. Cancer Res; 77(23); 6480–8. ©2017 AACR.

SummaryExosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

Hypoxia is associated with tissue injury and fibrosis but its functional role in fibroblast activation and tissue repair/regeneration is unknown. Using kidney injury as a model system, we demonstrate that injured epithelial cells produce an increased number of exosomes with defined genetic information to activate fibroblasts. Exosomes released by injured epithelial cells promote proliferation, α-smooth muscle actin expression, F-actin expression, and type I collagen production in fibroblasts. Fibroblast activation is dependent on exosomes delivering TGF-β1 mRNA among other yet to be identified moieties. This study suggests that TGF-β1 mRNA transported by exosomes constitutes a rapid response to initiate tissue repair/regenerative responses and activation of fibroblasts when resident parenchyma is injured. The results also inform potential utility of exosome-targeted therapies to control tissue fibrosis.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Orientadora: Profª. Drª. Margaret Cristina da Silva Boguszewski Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Saúde da Criança e do Adolescente. Defesa: Curitiba, 19/12/2017 Inclui referências : f.111-119 Resumo: Introdução: Os Meios de Comunicação Social (MCS) estão sendo cada vez mais utilizados no âmbito da saúde. Sites, blogs e perfis em redes sociais que abordam temas de saúde são criados diariamente, tanto por profissionais de saúde como por pacientes ou indivíduos interessados, abordando doenças, conselhos de tratamento e até possíveis curas. O Diabetes Mellitus Tipo 1 (DM1), embora em extensão menor que o Diabetes Mellitus Tipo 2, vem ganhando destaque nos MCS, especialmente nas redes sociais. Entidades, pesquisadores e até instituições de apoio aos pacientes portadores do DM1 apresentam, em diversos espaços da Internet, sua forma própria de lidar com a doença. Justificativa: Em um cenário onde são utilizadas cada vez mais as Mídias Sociais para interagir entre si, e onde um número crescente de organizações busca se aproximar de seu público-alvo usando tais ferramentas, é fundamental que a pesquisa científica também esclareça o significado deste processo, a fim de que possa desenvolver estratégias de comunicação mais eficientes e seguras, aproximação e fortalecimento dos laços com os indivíduos ou grupos envolvidos. Objetivos: Oferecer informações e um ambiente de discussão por meio de visitas às comunidades e do uso de ferramentas da Internet para aumentar o conhecimento sobre o DM1, incentivar o compromisso com o tratamento e apoiar a melhoria da qualidade de vida de crianças, adolescentes e adultos com a doença e suas famílias. Material e Métodos: Estudo de caráter populacional. População: moradores da Região Metropolitana de Curitiba (RMC), portadores de DM1 e internautas de modo geral. Delineamento: O projeto somou três ações: trabalho presencial nas comunidades carentes; trabalho remoto, pela Internet; e a capacitação de multiplicadores voluntários. A intervenção se deu com a participação do pesquisador em grupos comunitários, postagens em Redes Sociais, elaboração de um site, contatos via Facebook e whatsapp, com ênfase nos elementos necessários para um maior conhecimento do DM1 e para reforçar a importância do tratamento e acompanhamento com profissionais de saúde. Foram utilizadas técnicas de comunicação, apoiadas em práticas de boa saúde. Para as dúvidas e questões pontuais, foram realizadas duas enquetes populares. Além disso, o pesquisador abriu um canal de diálogo com portadores da doença e familiares, através das Redes Sociais, denominado "DiabeteStation". Resultados: No total, 537 moradores da RMC participaram de reuniões no período de julho de 2015 a dezembro de 2016. Duas enquetes populares foram realizadas. Na primeira, sobre alimentação saudável e atividade física, participaram 198 indivíduos. Na segunda, sobre DM1 e MCS, participaram 256 indivíduos, sendo 78 portadores de DM1. As enquetes foram realizadas de forma presencial e via eletrônica. 254 indivíduos afirmaram que o projeto ajudou a aumentar o conhecimento sobre o DM1. 75 portadores de DM1 afirmaram ter adquirido um maior senso crítico em relação aos MCS e 63 disseram que o projeto ajudou na perseverança ao tratamento. Conclusão: Com medidas simples e utilização de ferramentas de comunicação, foi possível aumentar o conhecimento sobre o DM1; enfatizar a importância do tratamento; ampliar o canal de discussão; e estabelecer novos laços de apoio entre comunidades, famílias e pacientes. Palavras-chave: Diabetes Tipo 1. Comunicação e Saúde. Educação Comunitária em Saúde. Abstract: Introduction: The Means Social Comunication Media (MCS) is being progressively used in health. Websites, blogs and profiles on social networks that address health issues are created daily by health professionals such as patients or interested individuals, lifting up about raising t diseases, treatment advice and even possible cures. Type 1 Diabetes Mellitus (DM1), although to a lesser extent than Type 2 Diabetes Mellitus, has been prominent in MCS, especially in social networks. Entities, researchers and institutions that support patients with DM1 have their own way of dealing with the disease in various areas of the Internet. Justification: In a context where social media are increasingly used for interaction and where more and more organizations approach their target audience using these tools, it is crucial that scientific research also clarifies the meaning of this process, with the purpose of developing more efficient and secure communication strategies, approaching and strengthening the ties with the individuals or groups involved. Objectives: To provide information by creating a discussion environment through visits to communities and the use of Internet tools to increase knowledge about DM1, encourage commitment to treatment and support the improvement of the quality of life of children, adolescents and adults with disease and their families. Material and Methods: Population study. Population: Residents of the Metropolitan Region of Curitiba (MRC), DM1 and Internet users in general. Planning: The project added three actions: presential work in poor communities; remote work over the Internet; and the training of volunteer multipliers. The intervention took place with the participation of the researcher in community groups, postings in Social Networks, elaboration of a website, contacts via Facebooke, whatsapp, with emphasis on the elements necessary for a greater knowledge of DM1 and to reinforce the importance of treatment and follow-up with professionals of health. Communication techniques were used, based on good health practices. For doubts and specific questions, two popular questions were asked. In addition, the researcher opened a channel of dialogue with disease carriers and family members, through Social Networks, called "DiabeteStation". Results: In total, 537 residents of the MRC participated in meetings from July 2015 to December 2016. Two popular questions were asked. In the first one, about healthy eating and physical activity, 198 individuals participated. In the second one, about DM1 and MCS, 256 individuals participated, being 78 DM1 carriers. The questionings were carried out in person and electronically. 254 individuals stated that the project helped increase awareness about DM1. 75 patients with DM1 reported having acquired a greater critical sense regarding MCS and 63 said that the project helped in perseverance to treatment. Conclusion: With simple measures and use of communication tools, it was possible to increase knowledge about DM1; emphasize the importance of treatment; broadening the means of discussion; and establish new support ties between communities, families and patients. Keywords: Type 1 Diabetes. Communication and Health. Community Health Education.

Background Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. Results We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients’ overall survival. Conclusions The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1224-0) contains supplementary material, which is available to authorized users.

In line with their broad-based effects, microRNAs (miRNAs), small non-coding RNA molecules ∼22 nucleotides long that silence target mRNAs, are thought to act as oncogenes or tumor suppressor genes based on their inhibition of tumor-suppressive and oncogenic mRNAs, respectively. We and others previously showed that global downregulation of miRNAs, a common feature of human tumors, is functionally relevant to oncogenesis as impairment of miRNA biogenesis enhanced transformation in both cancer cells and a K-Ras-driven model of lung cancer. The dysregulation of miRNA biosynthesis in cancer emerges as a cancer-specific mechanism that enhances its tumorigenic capacity. These observations are further supported by the fact that frameshift mutations of TARBP2 occur in sporadic and hereditary carcinomas with microsatellite instability and that DICER1 mutations are associated with familial pleuropulmonary blastoma. Accordingly, it was reported that reduced expression of miRNA-processing factors is associated with poor prognosis in lung cancer and ovarian cancer. Recently we have also demonstrated the presence of Exportin 5 (XPO5) inactivating mutations in tumors with microsatellite instability. This observed genetic defect is responsible for nuclear retention of pre-miRNAs, thereby reducing miRNA processing. The characterized mutant form of the XPO5 protein lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and the protein itself, as well as pre-miRNAs accumulating in the nucleus of cancer cells. Most importantly, the restoration of XPO5 function reverses the impaired export of pre-miRNAs and has tumor suppressor features. Our data suggest a cancer-specific mechanism to guide the subcellular distribution of miRNA precursors and prevent them from being processed to the active mature miRNA. The control of the miRNA biosynthesis pathway is emerging as an important mechanism in defining the spatiotemporal pattern of miRNA expression in cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, associated with a late diagnosis and a five-year survival rate of 8%. Currently available treatments fall short in improving the survival and quality of life of PDAC patients. The only possible curative option is still the surgical resection of the tumor. Exosomes are extracellular vesicles secreted by cells that transport proteins, lipids, and nucleic acids to other cells, triggering phenotypic changes in the recipient cells. Tumor cells often secrete increased amounts of exosomes. Tumor exosomes are now accepted as important players in the remodeling of PDAC tumor stroma, particularly in the establishment of an immunosuppressive microenvironment. This has sparked the interest in their usefulness as mediators of immunomodulatory effects for the treatment of PDAC. In fact, exosomes are now under study to understand their potential as nanocarriers to stimulate an immune response against cancer. This review highlights the latest findings regarding the function of exosomes in tumor-driven immunomodulation, and the challenges and advantages associated with the use of these vesicles to potentiate immunotherapy in PDAC. The laboratory is supported by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by FCT—Fundacao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Inovacao in the framework of the projects “Institute for Research and Innovation in Health Sciences” POCI-01-0145-FEDER-007274 and POCI-01-0145-FEDER-32189; and by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), in the framework of the project NORTE-01-0145-FEDER-000029; and Fundacao AstraZeneca 2017 FAZ Ciencia Award. SM is supported by FCT (IF/00543/2013).

MicroRNAs [1] have emerged as key post-transcriptional regulators of gene expression, involved in various physiological and pathological processes. It was found that several miRNAs are directly involved in human cancers, including lung, breast, brain, liver, colon cancer and leukemia. In addition, some miRNAs may function as oncogenes or tumor suppressors in tumor development. Furthermore, a widespread down-regulation of miRNAs is commonly observed in human cancers and promotes cellular transformation and tumorigenesis [2–5]. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, frequently amplified or deleted in human cancer, suggesting an important role in malignant transformation. A better understanding of the miRNA regulation and misexpression in cancer may ultimately yield further insight into the molecular mechanisms of tumorigenesis and new therapeutic strategies may arise against cancer. Here, we discuss the occurrence of the deregulated expression of miRNAs in human cancers and their importance in the tumorigenic process.

Exosomes are lipid bilayer-enclosed extracellular vesicles (EVs) that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer cell-derived exosomes in circulation is currently lacking. Using mass spectrometry analyses, we identified a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of cancer patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreas cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreas disease from patients with early and late stage pancreas cancer. Levels of GPC1+ crExos correlate with tumor burden and survival in patients pre- and post-surgical tumor resection. GPC1+ crExos from patients and from mice with spontaneous pancreas tumors driven by oncogenic KRAS contained RNA with specific KRAS mutation, and it emerges as a reliable biomarker for the detection of PanIN lesions despite negative signal by MRI in mice. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreas cancer to facilitate possible curative surgical therapy.

Macrobrachium birai Lobão, Melo & Fernandes, 1986, espécie de camarão de água doce do Brasil, foi capturada no Rio Branco, Cananéia, Estado de São Paulo, Brasil. No início do desenvolvimento embrionário os ovos têm cor verde oliva e tamanho médio de 0,60 x 0,43 mm. As larvas foram criadas, individualmente, a temperatura média de 25ºC. O desenvolvimento larval de Macrobrachium birai é do tipo prolongado, constituído de 15 estágios morfológicos de zoea e o decapodito. A metamorfose foi atingida em 144 dias de cultivo, através de 44 ecdises. As mudas ocorreram em intervalos de dois a oito dias e em média com 3,3 dias. As larvas de M. birai possuem algumas características morfológicas que podem diferenciá-las das larvas de M. olfersii (Wiegmann, 1836).Macrobrachium birai Lobão, Melo & Fernandes, 1986, a native Brazilian species of freshwater shrimp, was captured along the coast of Rio Branco, Cananéia, state of São Paulo, Brazil. The eggs in early development were green and the average size was 0.60 x 0.43 mm. The larvae were reared in laboratory at average temperature of 25ºC. Macrobrachium birai has a prolonged larval development, with 15 morphological zoeal stages and the decapodito. The metamorphose was obtained in 144 days, through about 44 ecdysis. The molts occurred from two to eight days, in average of 3.3 days. The larvae of the M. birai present some morphological characteristics distinct from that of M. olfersii (Wiegmann, 1836).

RNA-induced silencing complex is the cytoplasmic effector machine of the microRNA (miRNA) pathway and contains a single-stranded miRNA guiding it to its target mRNAs. The biogenesis of mature miRNAs is a regulatory process achieved by complex machinery composed of few protein components, among which the ribonuclease III Dicer plays a central role. Dicer is essential for miRNA maturation and catalyzes one of the rate-limiting steps of miRNA production. In this chapter, we describe a protocol to study the catalytic activity of Dicer in cell extracts by measuring their ability to process precursor RNA (pre-miRNAs) into functional mature miRNAs. Impairment of the miRNA biogenesis machinery due to loss-of-function mutations in effectors of the pathway such as Dicer has been demonstrated before. Dicing assay can be used in cancer to assess Dicer enzymatic activity compared with healthy controls. Therefore, this experimental approach is likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases.

Non-coding RNAs have been commanding increasingly greater attention in recent years as the few that have been functionalized to date play important roles in key cellular processes. Here we show that N-BLR, a ~900 bp non-coding RNA, modulates the epithelial-to-mesenchymal transition, increases colorectal cancer invasion, and functions as a migration enabler by affecting the expression of ZEB1 and E-cadherin. In patients with colorectal cancer, N-BLR expression associates with tumor stage and invasion potential. As N-BLR contains several instances of a category of DNA motifs known as pyknons, we also designed a custom-made array to investigate the possibility that other pyknon loci may be transcribed. For several of the loci probed by the array we found that the corresponding pyknons are differentially expressed between cancer and normal tissue samples. Taken together the data suggest that a systematic study of other pyknon-containing non-coding RNAs like N-BLR may be warranted in the context of colorectal cancer.