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.

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.

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.

Plasmatic miR-210, miR-221 and miR-1233 profile: potential liquid biopsies candidates for renal cell carcinoma.

Renal cell carcinoma (RCC) represents a challenge for clinicians since the nonexistence of screening and monitoring tests contributes to the fact that one-third of patients are diagnosed with metastatic disease and 20-40% of the remaining patients will also develop metastasis. Modern medicine is now trying to establish circulating biomolecules as the gold standard of biomarkers. Among the molecules that can be released from tumor cells we can find microRNAs. The aim of this study was to evaluate the applicability of cancer-related miR-210, miR-218, miR-221 and miR-1233 as prognostic biomarkers for RCC. Patients with higher levels of miR-210, miR-221 and miR-1233 presented a higher risk of specific death by RCC and a lower cancer-specific survival. The addition of miR-210, miR-221 and miR-1233 plasma levels information improved the capacity to predict death by cancer in 8, 4% when compared to the current variables used by clinicians. We also verified that hypoxia stimulates the release of miR-210 and miR-1233 from HKC-8, RCC-FG2 and 786-O cell lines. These results support the addition of circulating microRNAs as prognostic biomarkers for RCC.

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.

miRNAs: mediators of ErbB family targeted therapy resistance.

The ErbB/HER tyrosine kinase receptors family plays a key regulatory role in different cellular processes by activating several signaling pathways. In different tumor types, mutations or overexpression of the ErbB family members are a common feature, which led to the development of targeted therapies against this receptors. Although with this kind of treatment we are heading to a more personalized medicine, the development of acquired resistance is still an issue, therefore, several studies focused on discovering the mechanisms behind it. More recently, miRNAs have been described as important mediators of acquired resistance, specifically, acquired resistance to ErbB family targeted therapies. Ultimately, miRNA-based therapeutics using exosomes as a drug delivery model can revolutionize today's approach of cancer treatment.