Author: Azam, Md Zulfikar
Title: Glioblastoma pathogenesis and diagnosis
Advisors: To, Shing-shun Tony (HTI)
Degree: Ph.D.
Year: 2021
Subject: Gliomas
Glioblastoma multiforme -- Pathogenesis
Glioblastoma multiforme -- Diagnosis
Nervous system -- Tumors
Hong Kong Polytechnic University -- Dissertations
Department: Department of Health Technology and Informatics
Pages: xviii, 164 pages : color illustrations
Language: English
Abstract: Glioblastoma (GBM) is the deadliest incurable malignant primary brain tumour. Despite decades of research, GBM is invariably fatal and the median survival rate remains poor. A number of intracellular signalling pathways are dysregulated in GBM and the phosphoinositide-3-kinase (PI3K) pathway is one of the most prominent. The roles of Class IA PI3K isoforms (PIK3CA, PIK3CB) in GBM have been extensively studied. However, the third isoform (PIK3CD), preferentially expressed in leukocytes, is now considered to play important roles in solid tumours including GBM. We and others previously reported the involvement of PIK3CD in GBM, but its molecular mechanism remains unclear. Extracellularly, GBM is known to alter its microenvironment by secreting extracellular vesicles to mediate exchange of proteins and RNA, ultimately changes the surrounding microglia towards tumour enhancing molecular phenotypes. The mechanism by which these vesicles mediate such changes is still elusive. There are numerous genetic aberrations in GBM and one of the most common is epidermal growth factor receptor (EGFR) amplification/mutations. The genetic changes in EGFR confer drug resistance during disease progression; therefore, it is now considered one of the biomarkers for GBM targeted therapy. Disappointingly, targeting EGFR amplification/mutations (e.g. EGFRvIII) has been unsuccessful so far and it is believed to be due to the heterogeneous nature of EGFR mutations. On this account, a sensitive diagnostic tool to identify the EGFR mutational spectrum in GBM would be most beneficial. In this thesis, the pathogenic mechanisms of PIK3CD and GBM derived extracellular vesicles were investigated. In addition, a sensitive molecular assay was developed to allow precise detection and monitoring of EGFR mutations in GBM patients.
We first applied CRISPR-Cas9 gene knockout strategy to completely abrogate the activity of the PIK3CD gene in the human GBM cell line U87-MG. As expected, the migration, invasion, and proliferation capacities were reduced in the knockout cells. Their tumourigenic ability was also substantially decreased as shown by the in vitro clonogenic assay and in nude mice tumour implantation. These changes are mediated by much reduced activity of downstream signalling molecules, especially PAK3 and PLEK2, at both protein and mRNA levels. Bioinformatics­based RNAseq analysis revealed that PI3K/Akt signalling and epithelial–mesenchymal transition related processes are dysregulated. Using mouse cell lines, we were able to show that GBM derived extracellular vesicles could activate microglial cells, characterized by high levels of allograft inflammatory factor 1 and major histocompatibility complex II; as well as elevated microglia matrix metalloproteinase (MMP2, MMP9) activity. Cytokine expression analysis showed that mixed M1 (high iNOS activity) and M2 (high IL10 activity) features developed in microglia and this reprogramming might happen through IL10-mediated STAT3 signalling. To achieve precise and comprehensive detection of EGFR mutations, we utilised the highly sensitive droplet digital polymerase chain reaction (ddPCR) platform to develop a multiplex assay. Making use of the drop-phase, milepost and drop-off probe designs, our assay can simultaneously detect EGFR amplification, EGFRvIII mutations and the rare but more aggressive EGFR ectodomain missense A289 mutation respectively. Most importantly, our assay was able to detect EGFRvIII in DNA extracted from circulating EVs of GBM patients, thus providing a tool for non-invasive monitoring of GBM progression.
Rights: All rights reserved
Access: open access

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