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

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease. Understanding its biology is crucial for finding effective therapies. Exosomes have been implicated in cancer, but their behavior in living systems remains poorly understood. We aimed to map the spatiotemporal distribution of exosomes from both healthy pancreas and PDAC to determine their biological significance. To achieve this, we developed a genetically engineered mouse model (ExoBow) to trace spontaneous exosomes communication. Within the PDAC microenvironment, cancer cells establish preferential communication routes with cancer associated fibroblasts and endothelial cells. The latter is a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios significantly enhanced angiogenesis, underscoring the contribution of exosomes to the vascularization of the organ and to cancer. Inter-organ communication is significantly increased in PDAC, and the thymus is a sustained target in both contexts. PDAC cells also communicate with the kidneys and lungs, and the healthy pancreas with bone-marrow, brain, and intestines. In sum, we found that exosomes mediate an organized communication network in vivo that controls angiogenesis locally, both in the healthy pancreas and PDAC, and that targets the thymus also in both conditions, unravelling their potential role in central immune surveillance and anti-tumor immune response.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Pancreas

DISEASE(S): Pancreatic Cancer

SUBMITTER: Hugo Osorio

LAB HEAD: Sonia A. Melo

PROVIDER: PXD047009 | Pride | 2023-12-12

REPOSITORIES: Pride

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Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates M-BM-1 SD. [mRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol . The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis (n=4). p0739_E1, p0739_ E2, p0739_E3 and p0739_E4 for the exosomes samples and p0739_C1, p0739_C2, p0739_C3, and p0739_C4 for the HMC-1 cells. [miRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol followed by RNeasy clean-up. The microarray experiments were performed by Exiqon.

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

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