Investigating the contribution of tumour cells to the vasculature of glioblastoma

Maddison, Kelsey

Title: Investigating the contribution of tumour cells to the vasculature of glioblastoma
Creator: Maddison, Kelsey
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2024
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: Glioblastoma is an aggressive and invasive adult primary brain tumour with poor survival outcomes. After tumour resection and treatment with radiotherapy and temozolomide (TMZ) chemotherapy, glioblastoma progresses as a treatment-resistant recurrent tumour. At recurrence, glioblastoma may be treated with bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody targeting tumour angiogenesis. The plasticity of glioblastoma cells, and their interactions with the complex brain tumour microenvironment, promotes resistance to bevacizumab and other anti-angiogenic therapies through several mechanisms. One such method of anti-angiogenic resistance is the development of tumour cell-derived vasculature to compensate for the loss of angiogenic vessels. This may occur through the development of tumour cell-lined vascular structures lacking an endothelium, known as vasculogenic mimicry (VM), and/or through the transdifferentiation of glioma stem cells (GSCs) into endothelial cells that incorporate into the tumour vasculature. Additionally, prostate-specific membrane antigen (PSMA) is expressed by the neovasculature of multiple solid tumours, including glioblastoma. Whether PSMA is expressed by VM vessels in addition to abnormal angiogenic vessels in glioblastoma has not previously been determined. This thesis primarily aimed to investigate the extent to which tumour cell-derived vessels contribute to the glioblastoma vasculature and further explore the characteristics of these vessels. In glioblastoma, VM and tumour to endothelial transdifferentiation have been reported both as separate mechanisms of tumour vascularisation and as components of a single process. A systematic review was conducted to determine the methods and markers used to describe VM and endothelial transdifferentiation in the glioblastoma literature. In studies that specifically described VM, absence of the endothelial cell markers CD31 and/or CD34 in vascular structures was the most common definition. In contrast, studies defining tumour to endothelial transdifferentiation described tumour cell-derived vessels as expressing endothelial markers, including CD31 and/or CD34, in addition to tumour or stem cell markers. The results of this systematic review were used to inform the methods of subsequent studies, which investigated glioblastoma cell-derived vessel formation in patient tissue and patient-derived cell lines. To determine the extent to which VM occurs in glioblastoma, and the type of tumour vessels expressing PSMA, patient tissue from 35 matched pairs of primary and recurrent glioblastoma was assessed by immunohistochemistry. Sections from each tumour were labelled for PSMA and CD34 and stained with periodic acid-Schiff (PAS). As described by previous studies of VM in glioblastoma tissue, blood vessels were categorised as either endothelial vessels (CD34+/PAS+) or VM (CD34–/PAS+). The majority of blood vessels in both primary and recurrent tumours were endothelial vessels, with VM accounting for a small proportion of the vasculature overall. In this cohort of patient tumours, PSMA labelling was detected only in endothelial vessels, as determined by assessment of serial sections labelled for PSMA and CD34. Interestingly, the density of endothelial vessels and the expression of PSMA were significantly decreased in recurrent glioblastoma. Though VM was observed less frequently in recurrent (28.57%) compared to primary (42.86%) glioblastoma, the density of VM vessels did not differ between groups. To the best of my knowledge, this is the first report of VM in recurrent glioblastoma tissue. The ability of patient-derived glioblastoma cell lines to form vessel-like structures was investigated using the tube formation assay (TFA), a common method of assessing the in vitro angiogenic ability of endothelial cells, and the VM capability of tumour cells. Though the TFA is the most frequently used method of assessing VM in glioblastoma in vitro, as determined by the systematic review performed as part of this thesis, there is a large amount of variation in the methods used in relation to cell seeding densities, basement membrane matrix, media composition, use of serum-cultured cells, and incubation time. Therefore, 12 patient-derived glioblastoma cell lines were assayed under consistent TFA conditions, using growth factor-reduced Matrigel, serum-free media, and optimised seeding densities, and tube formation was assessed at regular intervals over 48 hours. Three of the patient-derived cell lines consistently formed tube-like structures under the TFA conditions tested. These three glioblastoma cell lines were subsequently treated with a clinically relevant concentration of bevacizumab (260µg/mL) to assess the effects of the anti-angiogenic treatment used in glioblastoma on the tumour cell-derived vessel-like structures. Bevacizumab appeared not to inhibit glioblastoma cell tube formation, however, there were a number of limitations associated with the treatment assay that prevented further data analysis. This thesis supports previous reports of the presence of VM in a subset of glioblastomas. Furthermore, the quantification of VM in patient tissue, investigation of the presence of VM in recurrent tumours, and the assessment of a relatively large selection of patient-derived glioblastoma cell lines suggest that VM may make only a minor contribution to the tumour vasculature of glioblastoma. Although PSMA was not observed in VM, its expression by endothelial cells is of interest for future studies of tumour-specific vascular targets in glioblastoma.
Subject: brain cancer; glioblastoma; tumour microenvironment; tumour vasculature; vasculogenic mimicry; transdifferentiation
Identifier: http://hdl.handle.net/1959.13/1511442
Identifier: uon:56498
Language: eng
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