Roper, Sophie Jayne
(2020)
Development of three-dimensional spheroid models for the analysis of medulloblastoma drug response and metastatic dissemination.
PhD thesis, University of Nottingham.
Abstract
Introduction: Medulloblastoma, the most common malignant paediatric brain tumour, is a heterogeneous disease with tumours classified based on their histological and molecular profiles. Studying medulloblastoma in vitro is challenging, as traditional two-dimensional (2D) cell culture models fail to recapitulate the multi-dimensional growth, cell-cell interactions, and complex microenvironment that exists in vivo and in patient tumours. This project therefore aimed to respond to the need for a robust, reliable, and optimised three-dimensional (3D) in vitro culture method for medulloblastoma, which could be applied to study drug response and metastatic dissemination.
Methods: A panel of medulloblastoma cell lines representing three of the four molecular subgroups (SHH, Group 3, and Group 4) were tested for use in the 3D spheroid model. Standardised growth conditions, including spheroid size and culture medium, were adopted to allow direct comparisons across cell lines and treatments. Spheroids were characterised by imaging analysis, luminescence-based assays, and immunohistochemical staining. Drug response of 3D spheroids was compared to 2D monolayer culture, with additional long-term and dual-inhibitor studies conducted to exemplify the use of the 3D spheroid model in drug screening approaches. SHH medulloblastoma spheroids were applied to migration and invasion models using basement membrane matrices and hyaluronan (HA) hydrogels containing additional extracellular matrix (ECM) components. Next-generation sequencing (NGS) technologies were adopted to gain an insight into the genes and pathways involved in SHH medulloblastoma leptomeningeal metastatic dissemination using the HA hydrogel model.
Results: Medulloblastoma cell lines formed spheroids of different morphologies. The SHH subgroup cell lines (DAOY, ONS76, and UW228-3) formed tight, highly reproducible spheroids, and were therefore chosen as suitable models for downstream analysis. SHH medulloblastoma spheroids could be maintained for up to 3 weeks in culture, forming physiological gradients within the 3D structure over time. 3D spheroid culture increased resistance to standard-of-care chemotherapeutic drugs compared to 2D monolayer culture and a subpopulation of viable cells was present after treatment. Dual-inhibitor studies with vardenafil, an inhibitor of the multidrug transporter ABCB1, showed synergistic effects which could facilitate the use of lower doses of chemotherapy. The HA hydrogels, containing ECM components highly expressed in SHH patient tumours, were more suitable, physiologically-relevant models of SHH medulloblastoma metastasis than commercially-available basement membrane matrices. NGS revealed the downregulation of cell cycle genes and upregulation of cell movement genes in DAOY and ONS76 spheroids migrating on the HA hydrogel. Overexpression of the insulin pathway, consistent with in vivo SHH medulloblastoma metastasis models and patient tumours, was also observed. Thus our model allows us to recapitulate known, and identify novel, mechanisms of cell dissemination from the primary tumour.
Conclusion: The 3D spheroid model of medulloblastoma described in this study can be used to analyse drug response and model metastatic dissemination. It is a significant improvement over current in vitro techniques and represents an important breakthrough in medulloblastoma research.
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