Johnson, James E. C.
(2023)
Investigating the role of tumour-deposited extracellular matrix in medulloblastoma progression.
PhD thesis, University of Nottingham.
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Abstract
Introduction: The extracellular matrix (ECM) is a web of macromolecules which under normal conditions provides structural support to tissues, as well as regulating cellular differentiation, motility, migration and survival. In the past decade emerging evidence has demonstrated that tumours are able to extensively modify the composition and mechanical properties of their surrounding ECM. Tumour ECM has been shown to be pro- or anti-tumorigenic and can therefore sometimes be used to predict patient prognosis. Medulloblastoma is the most common malignant paediatric brain tumour, accounting for 10% of all cancer related deaths in children, and is the focus of this thesis. The role of tumour ECM in medulloblastoma progression is currently poorly understood. However, a recent study by our group identified ECM sub-types within the medulloblastoma molecular sub-groups, characterised by the expression of specific ECM proteins. One of these ECM sub-types was defined based on the expression of laminin in SHH and Group 4 medulloblastoma patients. High expression of laminin (LAMhigh) was shown to be associated with more favourable overall survival in SHH medulloblastoma patients, whereas low laminin expression (LAMlow) was associated with poorer survival. The opposite trend was observed in Group 4 patients. Furthermore, it was shown that SHH medulloblastoma cell lines formed a sub-group specific laminin shell-like structure when grown in long-term 3D cell culture. This study aims to delineate the underlying molecular mechanisms which potentiate this pattern of survival conveyed by laminin.
Methods and Results: Differential gene expression analysis between LAMhigh and LAMlow SHH and Group 4 medulloblastoma tumours in genomic patient data identified candidate ‘laminin regulated genes’. However, initial validation of candidate genes by qRT-PCR analysis of SHH and Group 4 medulloblastoma cell lines grown in 2D and 3D hydrogel in vitro cell culture models, with and without laminin, was inconclusive.
CRISPR-Cas9 genetic editing technology was therefore utilised to create a stable laminin knockout in a SHH medulloblastoma cell line to study the effect of laminin in vitro more accurately. From this point onwards the focus of the study shifted towards just SHH medulloblastoma. Using this laminin knockout cell line, 3D cell culture assays were employed to assess the role of laminin in key aspects of tumour progression: migration, invasion, apoptosis evasion and therapy resistance. The findings of these assays demonstrated that laminin promotes cell migration and maintains sensitivity to apoptosis and the standard of care therapeutic vincristine in SHH medulloblastoma cells. To assess unbiased global changes in protein expression caused by laminin in SHH medulloblastoma cells, liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis was conducted on the laminin knockout SHH medulloblastoma cell line. This identified that estrogen signalling was upregulated by laminin, whereas collagen-integrin signalling was downregulated. Consultation of the literature confirmed that these pathways could underpin laminin’s role in migration and maintaining apoptotic and therapeutic sensitivity, respectively.
In support of the above, integrated bioinformatics analysis of SHH medulloblastoma genomic and proteomic patient data, and subsequent immunohistochemical validation in long-term 3D cell culture models, revealed that SHH medulloblastoma cells form a multicomponent ECM shell-like structure, the components of which were all associated with a more favourable patient prognosis.
Conclusions: This study demonstrated the importance laminin in cellular migration, as well as maintaining sensitivity to apoptosis and vincristine in SHH medulloblastoma, and that these phenotypes may be mediated by the up and down regulation of estrogen and collagen-integrin signalling, respectively. Our investigations also revealed a multicomponent ECM shell-like structure in SHH medulloblastoma, the components of which could represent potential biomarkers of more favourable overall survival in patients.
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