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Establishing a flow-based system for repressing target gene expression in a microfluidic device using a glioblastoma spheroid model

Hosni Ep Ben Daoud, Ines


Ines Hosni Ep Ben Daoud


John (Professor of tumour immunology) Greenman


Glioblastoma (GBM) is a deadly disease with poor prognosis. The gold standard treatment is surgery followed by chemoradiotherapy almost always including Temozolomide. Despite extensive research for new therapeutic approaches relapse after treatment still occurs in approximately 80% of patients. The median survival, after first diagnosis, remains at 15 months, with a 5-year survival rate still less than 5%. Translationally-relevant models are urgently needed to allow reliable identification and validation of new potential therapeutic targets. The current study aims to establish a microfluidic flow-based system for identifying targets via gene knockdown in a GBM spheroid model.
Two genes, PRMT2 and RAB21, were chosen as suitable targets for doing knockdown experiments, following a review of the literature and demonstration that both were expressed in the U87 GBM cell line. Gene knockdown was initially undertaken using lipofectamine based siRNA transfection of U87 cultured in monolayer, 3D static and 3D flowing systems. Gene expression was assessed by qPCR whereas western blotting and MTS assays were used to measure protein expression and cell proliferation, respectively. Accel™ siRNA, a chemically modified form of siRNA, were used to try and improve the gene repression levels. Finally, a comparison of the whole transcriptome profile between spheroids from three, independent, GBM cell lines was conducted both pre- and post-maintenance on the microfluidic device.
The microfluidic chip was demonstrated to successfully maintain GBM spheroids in a viable state for up to 7 days, during which the RNA yield was significantly higher compared to the 3D static conditions for three independent genes (PRMT2, RAB21 and FUS). Respectively, PRMT2 and RAB21 siRNA silencing levels showed a gradual decrease from monolayer (75.66±5.13% vs 86.33±9.01%), to 3D static (43.00±24.75% vs 59.00±4.58%) and 3D flow system (26.33±2.51% vs 19.67±8.02%) using 5nM of siRNA. Gene repression of PRMT2 and RAB21 had no effect on the protein expression or on the proliferation rate. This study also showed that Accell siRNA was effective in inducing gene silencing in U87 spheroids maintained in static conditions (with 1%FBS, 49±0.09% of FUS knockdown level and 29.71±0.03% of protein depletion using 0.5µM of Accell siFUS).
Next Generation Sequencing analysis of the transcriptome of three GBM cell lines showed that the correlation in gene expression pattern was higher between two patient derived cells (PDCs) than between them and U87. Furthermore, PDCs presented a heterogeneity in activated and deactivated pathways similar to developing tumour tissues, compared to U87 which was characterised by a predominance of pro-proliferative pathways. In addition, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis demonstrated that GBM spheroids when cultured in the microfluidic system compared to the static conditions, exhibited a more in vivo-like profile by activating pathways considered as hallmarks of cancer. These included pathways and cellular functions involved in: proliferation, inflammation, metabolism dysregulation, senescence, necrosis, and invasion.
In conclusion, this study showed that siRNA mediated gene knockdown was successful in the 3D flowing microfluidic platform, however no effects were observed on protein expression or cell proliferation; this may be due to the half-life of mRNA and will require additional study. Furthermore, PDCs spheroids cultured in this flow system compared to static state, exhibited properties more comparable to that of in vivo tumour tissues. Further investigation using a range of other genes or comparing spheroids with primary human tissue would be likely to give additional evidence about the effectiveness of the current flow system for mimicking the in vivo microenvironment.


Hosni Ep Ben Daoud, I. (2022). Establishing a flow-based system for repressing target gene expression in a microfluidic device using a glioblastoma spheroid model. (Thesis). University of Hull. Retrieved from

Thesis Type Thesis
Deposit Date Jan 5, 2023
Publicly Available Date Feb 24, 2023
Keywords Biomedical sciences
Public URL
Additional Information Department of Biomedical Sciences, The University of Hull
Award Date Jun 1, 2022


Thesis (7.6 Mb)

Copyright Statement
© 2022 Hosni Ep Ben Daoud, Ines. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

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