Aldighieri, Macha
(2020)
Identifying factors that control medulloblastoma cell migration using a model of brain-CSF dissemination.
PhD thesis, University of Nottingham.
Abstract
Introduction
Medulloblastoma is the most common type of malignant paediatric brain tumour, accounting for 10% of cancer deaths in children. While survival rates have improved in recent years, metastatic spread poses a significant challenge to therapy success. Current therapies also cause irreparable damage to the surrounding brain leading to a decreased quality of life in surviving patients, consequently there is a need for new targeted therapy options. One of the routes for dissemination of metastatic cells is invasion into the leptomeninges, a process during which cells cross basement membrane barriers as they travel from the primary tumour niche into the metastatic niche in the cerebrospinal fluid (CSF). The pathways involved in dissemination and metastasis remain elusive, and a better understanding is crucial to developing targeted therapies. This project aimed to develop an in vitro model system to mimic key characteristics of cell migration from the brain to the CSF, and identify novel candidate genes driving medulloblastoma metastasis.
Methods
An in vitro culture model representing certain features of the basement membrane interface between the brain parenchyma and the CSF was developed in order to isolate cells for transcriptomic analysis from three compartments: (a) non-metastatic suspension cells, (b) early metastatic adherent cells, and (c) metastatic migrated cells. Differentially expressed genes in these three compartments were identified by Next Generation 3ˡ UPX RNA Seq. The prognostic role of candidates was assessed by analysis of a large medulloblastoma gene expression dataset (Cavalli et al., 2017) on the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). SORCS2 expression was validated in medulloblastoma cell lines by qRT-PCR and western blotting, and a possible function assessed in vitro after siRNA knockdown.
Results
A trans-well migration assay format was modified to represent certain features of the basement membrane, collagen IV and laminin 111 were used as a basement membrane-like coating, and cells were induced to migrate towards artificial CSF supplemented with FBS. The transcripts SORCS2, CORO2B and LMNB1-DT were identified as candidates that may drive the process of migration. The gene SORCS2 demonstrated a significant increase in expression as Group 3 medulloblastoma cells progressed through the stages of migration, from the suspension compartment to the adherent compartment and then to the migration compartment. The transcripts CORO2B and LMNB1-DT were upregulated in cells in the adherent compartment. The expression of SORCS2, CORO2B and LMNB1-DT correlated with poor patient outcome and metastatic recurrence in medulloblastoma patients. SORCS2 was selected for further in vitro investigation as it most significantly correlated with an early poor survival trend in patients with metastatic tumours. SORCS2 gene and protein expression was confirmed in Group 3 and Group 4 cell lines. The results from in vitro experiments did not allow significant conclusions relating to SORCS2 function in migration to be drawn, however they offer preliminary data encouraging further investigation.
Conclusions
This non-biased, model-led, target identification approach identified genes of interest SORCS2 and CORO2B, and lncRNA LMNB1-DT, in migrating medulloblastoma cells. SORCS2 encodes a brain-specific transmembrane receptor in the VPS10 family of sorting proteins previously linked to actin dynamics in neuronal growth cones, and was implicated in the migratory behaviour of basal-like breast carcinoma. The analyses in this project suggested a link between SORCS2, metastasis, and tumour recurrence in medulloblastoma. The other candidates identified by these analyses warrant further validation and may shed light on the metastatic process in medulloblastoma. A better understanding of the processes and pathways that drive malignancy is a starting point for a push towards improving efficacy of our treatment options while reducing toxicity.
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