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Investigating the role of arginine methylation in glioblastoma using biomicrofluidics

Samuel, Sabrina Francesca


Sabrina Francesca Samuel


John (Professor of tumour immunology) Greenman


Despite intense research, the prognosis of patients with glioblastoma (GBM) remains extremely poor, with a median survival of 1.5 years after diagnosis. The need for new therapeutic options is therefore a vital step towards securing a better outlook for patients. Protein Arginine Methyltransferases (PRMTs) are novel targets in current oncology clinical trials due to their ability to augment a vast variety of cellular processes including growth factor signalling as well as Deoxyribonucleic acid (DNA) damage repair and proliferation. There are three known types of PRMT with varying target specificities and activities. All PRMT’s are able to transfer a single methyl group onto the guanidino nitrogen on the target arginine, producing the monomethyl-arginine (MMA) mark. Type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6 and PRMT8) then transfer a second methyl group on the same nitrogen to produce the asymmetrical dimethyl-arginine (ADMA) mark. Type II PRMTs (PRMT5 & PRMT9) transfer a second methyl group to the opposite nitrogen on the arginine functional group, making symmetrical dimethyl-arginine (SDMA). The type III PRMT, PRMT7, is only able to carry out the first methylation step.
The generally accepted view is that PRMT expression is increased in tumour cells, including GBM. Therefore, the overarching hypothesis here is that GBM cells are dependent on PRMT expression, and the inhibition of these enzymes will result in cell death or reduced proliferation. This research aims to determine the efficacy of PRMT inhibitors as a therapeutic avenue in GBM treatment by measuring the anti-proliferative effects of PRMT inhibiting drugs on GBM cells. A further aim of this research is to characterise an ex vivo microfluidic model that would allow for patient specific testing of pharmacological drugs in a simulated tumour microenvironment.
Using the U-87MG glioma cell line as a model system, differing levels of efficacy of PRMT inhibitors were observed. PRMT1 inhibition by Furamidine resulted in a reduction in cellular viability, however, inhibition of PRMT1 and other type I PRMTs by MS023 had minimal effect, as measured by MTS assays in Two-dimensional (2D) and Three-dimensional (3D) culture U-87MG models. Western blotting revealed an increase in the symmetrical dimethylation of a ~70 kDa protein following inhibition of Type I PRMTs with MS023, suggesting cross-talk between Type I and Type II PRMTs.
To further investigate the use of PRMT inhibitors and possible cross-talk mechanisms, an ex vivo microfluidic model was utilised, where patient biopsies were perfused with growth medium with and without MS023. Patient samples could be maintained in a viable state on the microfluidic device for up to 192 hr (8 days), as determined by the minimal release of the membrane contained enzyme lactate dehydrogenase into the effluent (LDH assay). However, a reduction in proliferation was observed in samples incubated on chip compared with the matched fresh biopsies. Treatment of patient samples with MS023 did not result in an increase in cell death as determined by LDH assay or a reduction in proliferation as determined by the quantification of ki-67 positivity by immunohistochemistry (IHC).
The cross-talk activity seen in the earlier cell line models was further demonstrated in the ex vivo microfluidic model. In patient biopsies six out of nine and four out of five patient samples showing increases in SDMA and MMA following MS023 treatment, respectively. To identify specific proteins that had undergone changes in methylation status, heavy methyl SILAC mass spectrometry was used. With this methodology, the RNA binding protein FUS was found to be methylated in the presence of MS023 only.
To further understand the effects of the ex vivo incubation of patient samples and explore the presence of intra-tumour heterogeneity, samples were treated with Temozolomide (TMZ), the current chemotherapy drug used is clinical practice, and cell death and proliferative activity determined by LDH assay and ki-67 positivity through IHC. Treatment with 10 μM TMZ did not result in an increase in cell death or a reduction in proliferation. However, a reduction in ki-67 was again seen in the samples incubated on chip compared with fresh biopsies. To explore intra-tumour heterogeneity, multiple slices from the same patient biopsy were ran in parallel. The slices appeared to have variation in ki-67 positivity, suggesting that the rate of proliferation is not uniform within the tumour mass and could perhaps mask any response to drug treatment during analysis.
In order to elicit a tumour response in GBM patient biopsies, MS023 was given in combination with TMZ, or GSK591, a PRMT5 inhibitor. Despite this aggressive approach, there were no significant changes in cell death or proliferative activity. It is hypothesised that the reduction in proliferative activity seen following incubation on the microfluidic device could reduce sensitivity to anti-tumour drugs and mute any changes that would otherwise be observed.
To conclude, this study has explored the feasibility of using a GBM-on-chip model to investigate emerging drugs, that is, PRMT inhibitors, towards the treatment of GBM. Further improvements to the microfluidic model, or broader analysis of the effect of TMZ and PRMT inhibitors on other cellular processes, such as apoptosis and senescence, are needed. This study has presented exciting cross- talk activity amongst PRMT enzymes not previously demonstrated in GBM tissue which warrants further investigation.


Samuel, S. F. (2021). Investigating the role of arginine methylation in glioblastoma using biomicrofluidics. (Thesis). University of Hull. Retrieved from

Thesis Type Thesis
Deposit Date Jan 26, 2022
Publicly Available Date Feb 24, 2023
Keywords Biomedical sciences
Public URL
Additional Information Department of Biomedical Sciences, The University of Hull
Award Date Apr 1, 2021


Thesis (6.7 Mb)

Copyright Statement
© 2021 Samuel, Sabrina Francesca. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

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