Exosomes define a local and systemic communication network in healthy pancreas and pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a lethal disease, requires a grasp of its biology for effective therapies. Exosomes, implicated in cancer, are poorly understood in living systems. Here we use the genetically engineered mouse model (ExoBow) to map the spatiotemporal distribution of exosomes from healthy and PDAC pancreas in vivo to determine their biological significance. We show that, within the PDAC microenvironment, cancer cells establish preferential communication routes through exosomes with cancer associated fibroblasts and endothelial cells. The latter being a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios enhances angiogenesis, underscoring their contribution to vascularization and to cancer. Inter-organ communication is significantly increased in PDAC with specific organs as most frequent targets of exosomes communication occurring in health with the thymus, bone-marrow, brain, and intestines, and in PDAC with the kidneys, lungs and thymus. In sum, we find that exosomes mediate an organized intra- and inter- pancreas communication network with modulatory effects in vivo.

SMC3 epigenetic silencing regulates Rab27a expression and drives pancreatic cancer progression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is expected to soon surpass colorectal cancer as a leading cause of cancer mortality in both males and females in the US, only lagging behind lung cancer. The lethality of PDAC is driven by late diagnosis and inefficient therapies. The complex biology of PDAC involves various cellular components, including exosomes that carry molecular information between cells. Thus, recipient cells can be reprogrammed, impacting tumorigenesis. Rab27a is a GTPase responsible for the last step of exosomes biogenesis. Hence, dissecting the mechanisms that regulate the expression of Rab27a and that control exosomes biogenesis can provide fundamental insights into the molecular underpinnings regulating PDAC progression. METHODS: To assess the mechanism that regulates Rab27a expression in PDAC, we used PDAC cell lines. The biological significance of these findings was validated in PDAC genetically engineered mouse models (GEMMs) and human samples. RESULTS: In this work we demonstrate in human PDAC samples and GEMMs that Rab27a expression decreases throughout the development of the disease, and that Rab27a knockout promotes disease progression. What is more, we demonstrate that Rab27a expression is epigenetically regulated in PDAC. Treatment with demethylating agents increases Rab27a expression specifically in human PDAC cell lines. We found that SMC3, a component of the cohesin complex, regulates Rab27a expression in PDAC. SMC3 methylation is present in human PDAC specimens and treatment with demethylating agents increases SMC3 expression in human PDAC cell lines. Most importantly, high levels of SMC3 methylation are associated with a worse prognosis in PDAC. Mechanistically, we identified an enhancer region within the Rab27a gene that recruits SMC3, and modulates Rab27a expression. CONCLUSION: Overall, we dissected a mechanism that regulates Rab27a expression during PDAC progression and impacts disease prognosis.

Titanium dioxide nanoparticles affect osteoblast-derived exosome cargos and impair osteogenic differentiation of human mesenchymal stem cells.

Titanium (Ti) and its alloys are the most widely used metallic biomaterials in total joint replacement; however, increasing evidence supports the degradation of its surface due to corrosion and wear processes releasing debris (ions, and micro and nanoparticles) and contribute to particle-induced osteolysis and implant loosening. Cell-to-cell communication involving several cell types is one of the major biological processes occurring during bone healing and regeneration at the implant-bone interface. In addition to the internal response of cells to the uptake and intracellular localization of wear debris, a red flag is the ability of titanium dioxide nanoparticles (mimicking wear debris) to alter cellular communication with the tissue background, disturbing the balance between osseous tissue integrity and bone regenerative processes. This study aims to understand whether titanium dioxide nanoparticles (TiO2 NPs) alter osteoblast-derived exosome (Exo) biogenesis and whether exosomal protein cargos affect the communication of osteoblasts with human mesenchymal stem/stromal cells (HMSCs). Osteoblasts are derived from mesenchymal stem cells coexisting in the bone microenvironment during development and remodelling. We observed that TiO2 NPs stimulate immature osteoblast- and mature osteoblast-derived Exo secretion that present a distinct proteomic cargo. Functional tests confirmed that Exos derived from both osteoblasts decrease the osteogenic differentiation of HMSCs. These findings are clinically relevant since wear debris alter extracellular communication in the bone periprosthetic niche, contributing to particle-induced osteolysis and consequent prosthetic joint failure.

Exosomal glypican-1 is elevated in pancreatic cancer precursors and can signal genetic predisposition in the absence of endoscopic ultrasound abnormalities.

BACKGROUND: 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. AIM: To evaluate the capacity of GPC1+ crExos to identify individuals at higher risk within these specific groups, all characterized by EUS. METHODS: This 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 (n = 8). The inclusion period was between October 2016 and January 2019, and the study was approved by the Ethics Committee of Centro Hospitalar Universitário de São João, Porto, Portugal. All patients provided written informed consent. EUS and blood tests for quantification of GPC1+ crExos by flow cytometry and carbohydrate antigen 19-9 (CA 19-9) levels by ELISA were performed in all subjects. EUS-guided tissue acquisition was done whenever necessary. For statistical analysis, SPSS® 27.0 (IBM Corp., Armonk, NY, United States) version was used. All graphs were created using GraphPad Prism 7.00 (GraphPad Software, San Diego, CA, United States). RESULTS: 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 GPC1+ crExos were statistically different: MCL [99.4%, interquartile range (IQR): 94.9%-99.8%], HR (82.0%, IQR: 28.9%-98.2%), NLOD (12.6%, IQR: 5.2%-63.4%), and CG (16.2%, IQR: 6.6%-20.1%) (P < 0.0001). Median levels of CA 19-9 were within the normal range in all groups (standard clinical cut-off of 37 U/mL). Within HR, individuals with a positive history of cancer had higher median levels of GPC1+ crExos (97.9%; IQR: 61.7%-99.5%), compared to those without (59.7%; IQR: 26.3%-96.4%), despite no statistical significance (P = 0.21). Pancreatic cysts with WF/HRS were statistically associated with higher median levels of GPC1+ crExos (99.6%; IQR: 97.6%-99.8%) compared to those without (96.5%; IQR: 81.3%-99.5%) (P = 0.011), presenting an area under the receiver operating characteristic curve value of 0.723 (sensitivity 75.0% and specificity 67.7%, using a cut-off of 98.5%; P = 0.012). CONCLUSION: GPC1+ crExos may act as biomarker to support the diagnosis and stratification of PDAC precursor lesions, and in signaling individuals with genetic predisposition in the absence of EUS abnormalities.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

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.

Treatment optimization of locally advanced and metastatic pancreatic cancer (Review).

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 5­year 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 disease­related 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 multi­omics 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.

The Interplay of Exosomes and NK Cells in Cancer Biology.

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.

Exosomal glypican-1 for risk stratification of pancreatic cystic lesions: A case of pathological progression in the absence of any suspicious imaging finding.

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.

Decoding the Biology of Exosomes in Metastasis.

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.

Liquid Biopsy beyond Circulating Tumor Cells and Cell-Free DNA.

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.

Exosomes and the Future of Immunotherapy in Pancreatic Cancer.

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.

Exosomes and Immune Response in Cancer: Friends or Foes?

Exosomes are a type of extracellular vesicle whose study has grown exponentially in recent years. This led to the understanding that these structures, far from being inert waste by-products of cellular functioning, are active players in intercellular communication mechanisms, including in the interactions between cancer cells and the immune system. The deep comprehension of the crosstalk between tumors and the immune systems of their hosts has gained more and more importance, as immunotherapeutic techniques have emerged as viable options for several types of cancer. In this review, we present a comprehensive, updated, and elucidative review of the current knowledge on the functions played by the exosomes in this crosstalk. The roles of these vesicles in tumor antigen presentation, immune activation, and immunosuppression are approached as the relevant interactions between exosomes and the complement system. The last section of this review is reserved for the exploration of the results from the first phase I to II clinical trials of exosomes-based cell-free cancer vaccines.

Quantitative Analysis of Precursors MicroRNAs and Their Respective Mature MicroRNAs in Cancer Exosomes Overtime.

Exosomes are a cell-to-cell communication system that transports information to neighbor and/or distant cells. Their content is composed of lipids, proteins, RNAs, and DNA. The role of exosomes released by cancer cells in disease progression has been widely studied. Here, we report a detailed protocol to quantify precursor microRNAs and mature microRNAs overtime in exosomes of cancer cells. Those precursor microRNAs that are packaged together with RISC proteins will get further processed in the exosomes overtime and an accumulation of the mature form of the microRNAs will occur.

Exosomes in cancer: Use them or target them?

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.

Pancreatic Cancer Diagnosis and Management: Has the Time Come to Prick the Bubble?

Pancreatic cancer (PC) is associated with poor prognosis and very dismal survival rates. The most effective possibility of cure is tumor resection, which is only possible in about 15% of patients diagnosed at early stages of disease progression. Recent whole-genome sequencing studies pointed genetic alterations in 12 core signaling pathways in PC. These observations hint at the possibility that the initial mutation in PC might appear nearly 20 years before any symptoms occur, suggesting that a large window of opportunity may exist for early detection. Biomarkers with the potential to identify pre-neoplastic disease or very early stages of cancer are of great promise to improve patient survival. The concept of liquid biopsy refers to a minimally invasive sampling and analysis of liquid biomarkers that can be isolated from body fluids, primarily blood, urine and saliva. A myriad of circulating molecules may be useful as tumor markers, including cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating tumor cells (CTC), circulating tumor proteins, and extracellular vesicles, more specifically exosomes. In this review, we discuss with more detail the potential role of exosomes in several aspects related to PC, from initiation to tumor progression and its applicability in early detection and treatment. Exosomes are small circulating extracellular vesicles of 50-150 nm in diameter released from the plasma membrane by almost all cells and exhibit some advantages over other biomarkers. Exosomes are central players of intercellular communication and they have been implicated in a series of biological process, including tumorigenesis, migration and metastasis. Several exosomal microRNAs and proteins have been observed to distinguish PC from benign pancreatic diseases and healthy controls. Besides their possible role in diagnosis, understanding exosomes functions in cancer has clarified the importance of microenvironment in PC progression as well as its influence in proliferation, metastasis and resistance to chemotherapy. Increasing knowledge on cancer exosomes provides valuable insights on new therapeutic targets and can potentially open new strategies to treat this disease. Continuous research is needed to ascertain the reliability of using exosomes and their content as potential biomarkers, so that, hopefully, in the near future, they will provide the opportunity for early diagnosis, treatment intervention and increase survival of PC patients.

The Biology of Cancer Exosomes: Insights and New Perspectives.

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.

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

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.

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration.

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.

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression.

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 TGFß, 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 TGFß-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 TGFß are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFß-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. ©2017 AACR.