Jackson, Hannah K.
(2021)
Analysing the role of medulloblastoma exosomes as biomarkers and their functional contribution to metastasis.
PhD thesis, University of Nottingham.
Abstract
Introduction: Recurrent/metastatic medulloblastoma is a devastating disease with an abysmal prognosis of less than 10% five-year survival. Whilst primary tumours can be classified based on epigenetic and transcriptomic features, there is very little information regarding molecular signatures of metastatic tumours. The secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. The most investigated EVs are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. The role of exosomes as vehicles for cell-to-cell communication between a tumour and its microenvironment is a relatively new concept, with only limited study of this potential mechanism in medulloblastoma. This study aims to gain insight into the differences between exosomes of primary and metastatic medulloblastoma cell lines to better understand how exosomal cargo may account for differences seen between these phenotypes and to identify their applicability as a prognostic biomarker of increased metastatic potential.
Methods and Results: Metastatic medulloblastoma cells were found to secrete markedly more exosomes compared to non-metastatic primary cells. Additionally, metastatic exosomes significantly enhanced the migration and invasiveness of primary and non-malignant cells in a transwell migration assay. Moreover, we demonstrated that the pro-migratory function of metastatic exosomes was, in part, due to tumour-promoting proteins EMMPRIN and MMP-2 enriched on their external surface. Furthermore, using zymography assay, metastatic exosomes were shown to potentiate medulloblastoma migration resulting in degradation of extracellular matrix components, via the active protease, MMP-2, on their surface. In support of this, stable genetic knockdown of MMP-2 or EMMPRIN antagonised the pro-migratory function of exosomes.
Next generation sequencing was utilised to characterise exosomal RNA cargo (mRNA and miRNA). Hierarchical clustering, demonstrated that metastatic exosomes had
distinct genetic cargoes compared to primary and normal exosomes. Further characterisation of the RNA delineated target pathways of importance for medulloblastoma progression and interaction with the surrounding microenvironment, in agreement with the pro-migratory phenotype conferred by metastatic exosomes.
Differential gene expression analysis of mRNA sequencing data, identified PCAT1, PAPPA2 and POU5F1B as highly enriched in metastatic exosomes compared to their matched primary counterpart. These genes had previously been implicated in cancer progression in other cancer types, and in this study their high expression correlated with poor overall survival and tumour metastasis in medulloblastoma. A number of miRNAs were also identified which were differentially secreted in metastatic exosomes relative to primary and normal exosomes, yielding a candidate set of exosomal miRNAs as potential metastatic medulloblastoma biomarkers. Notably, several of the candidate miRNAs (miR-382-5p, miR-381-3p, miR-346, miR-628-5p) were detected at substantially higher levels in metastatic disease CSF samples, compared to matched CSF samples from their primary tumour.
Conclusion: This study demonstrates the importance of exosomes in creating a favourable environment to drive medulloblastoma metastasis, either through extracellular matrix signalling, via surface-bound proteins, or through intracellular delivery of their RNA cargo to recipient cells. Our observations have also highlighted the possibility of CSF-derived exosomal miRNAs as biomarkers for metastatic medulloblastoma.
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