Mitoko, Christine Angeline
(2021)
Development of a magnetic resonance imaging biomarker to improve prognostication in metastatic medulloblastoma.
PhD thesis, University of Nottingham.
Abstract
Introduction
Metastatic medulloblastoma (MB) manifests in one third of paediatric patients at diagnosis, conferring a poor prognosis. Our limited capacity to detect dissemination at diagnosis and disease recurrence however, results in the erroneous risk stratification of paediatric patients with MB, with serious subsequent prognostic implications.
Hypothesis
Significantly correlated with metastasis and poor prognosis in other solid cancers, tumour-derived enzymes that facilitate their dissemination known as matrix metalloproteinases (MMPs) and specifically MMP-2 and MMP-9, were postulated to be potential biomarkers of metastasis in MB. We hypothesised that differential levels of functionally active MMP-2 and MMP-9, as expressed and secreted by a panel of primary and metastatic sub-grouped MB cell lines and patient tumours, would correlate with their propensity to metastasise. Once biologically characterised, these findings would be translatable, using our locally synthesised 19Fluorine-MMP-MRI biosensor, which would demonstrate an increase in the fluorine signal proportional to the concentration of the MMP-2/MMP-9 within the sample, as its substrate linker is cleaved from a paramagnetic gadolinium III-DOTA complex over time.
Methods
Protein microarrays, western blotting and qRT-PCR were performed to determine the levels of MMP-2 and MMP-9 protein and gene expression in nine sub-grouped primary and metastatic MB cell lines. Levels of secreted functionally active MMP-2 and MMP-9 from conditioned supernatants from the same MB cell lines and cerebrospinal fluid (CSF) from six paediatric patients with MB were assayed using gelatin zymography. Functional migration and invasion assays of 3-D cultured MB were performed to determine whether the detected levels of functionally active MMP-2 and MMP-9, would correlate to their migratory capacity and expected metastatic potential. The same samples were further examined using fluorescence resonance energy transfer (FRET) assays, to simulate and optimise the conditions for the biosensor experiments, prior to their imaging.
Results Our data strongly suggests that MMP-2 gene and protein is upregulated in metastatic MB cell lines. This was most evident in a matched pair of Group 4 MB where the recurrent cell line CHLA-01R-MED consistently expressed higher MMP-2 at all regulatory levels in comparison to its primary counterpart, CHLA-01-MED. This finding was also reflected in their increased migratory capacity in invasion assays. Zymography of MB patient CSF demonstrated significantly increased levels of functionally active MMP-2 in the recurrent samples; a finding resonated in our FRET assay experiments. 19Fluorine-MRI-MMP biosensor imaging demonstrated increased signal from the Fluorine moiety, following enzymatic degradation of the sensor by recombinant MMP-2 protein, demonstrating its capacity to detect the levels of functionally active MMP-2 and MMP-9 in MB patient cerebrospinal fluid and its potential as a clinically relevant imaging biomarker, once optimised.
Conclusions
Levels of functionally active MMP-2 could prove to be a sensitive marker of metastasis in paediatric patients with medulloblastoma, at their initial diagnosis and ongoing disease surveillance. With further optimisation, we believe that our MMP-2 biosensor could become a useful adjunct as an intra-operative marker of residual metastatic cells, to enhance our limits of safe maximal resection.
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