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Type: Thesis
Title: Modulation of BRAFV600E Signalling in Melanoma and Colorectal Cancer: Consequences for Tumour Immune Cell Infiltration
Author: Massy-Westropp, Madeleine Louisa
Issue Date: 2020
School/Discipline: School of Biological Sciences
Abstract: The mitogen-associated protein kinase (MAPK) pathway regulates cell growth and proliferation; activating mutations in constituent kinases Ras and BRAF promote excessive MAPK signalling and tumourigenesis. BRAF containing the V600E mutation (BRAFV600E) is present in 50% of melanomas and 10% of colorectal cancers (CRCs), spurring the development of small-molecule BRAFV600E inhibitors. These compounds are initially effective in treatment of advanced melanoma, the most responsive tumour thus far tested, but treatment resistance develops. Results in CRC are even less promising. Combination of BRAFi with other therapies could increase response rates and delay treatment resistance in both melanoma and CRC. Immune-based therapies are an attractive proposition, as several studies associated BRAFi treatment of melanoma with enhanced anti-tumour immunity. This includes increased proportions of CD8+ tumour-infiltrating lymphocytes (TILs), which can recognise and kill tumour cells. The mechanism underlying this enhanced CD8+ T cell recruitment has not yet been uncovered, possibly due to this area of research being hampered by the lack of well characterised BRAFV600E-driven mouse melanoma and CRC models. However, pharmacological inhibition of BRAFV600E could lead to increased levels of tumour derived CXCR3 ligands, which attract CXCR3+ CD8 T cells to the tumour. There are two major aims for the project: firstly, to utilise existing models or establish new models for BRAFV600E-driven melanoma and CRC, and secondly, to study how BRAFV600E inhibition affects tumour CXCR3 ligand expression and CD8+ T cell recruitment. An in vitro model for BRAFV600E-driven CRC was developed. This involved co-culture of colorectal organoids with inducible BRAFV600E expression and activated OT-I cells, which are CD8+ T cells which recognise SIINFEKL peptide. Methods were optimised to generate OT-I cells capable of migrating towards organoid-derived CXCR3 ligands, and to track the movement and proliferation of the cells during co-culture. The colorectal organoids could also be supplied with SIINFEKL peptide to present on MHC Class I, which would allow recognition by OT-I cells. However, BRAFV600E induction in the organoids induced a stress state involving crypt structure disintegration, cell death and CXCL10 production. Thus, these organoids did not represent BRAFV600E-driven CRC and further development was required, but due to time constraints was not pursued. The existing 4434 melanoma model is derived from mice with melanocyte-specific expression of BrafV600E from the endogenous Braf gene. When treated with vemurafenib in vitro, these cells upregulated CXCL10 expression. SM1 WT1, another existing model derived from mice with melanocyte specific transgenic BrafV600E expression, was also characterised. These cells did not upregulate CXCL10 expression after vemurafenib treatment, suggesting this response is cell-line dependent. Unfortunately, immune-mediated rejection of 4434 and SM1 WT1 cells in C57BL/6 mice limited in vivo analysis. Thus, a third model was established involving B16F10 melanoma cells, normally BRAF wild type, with inducible BRAFV600E expression. BRAFV600E induction did elevate MAPK signalling but did not increase cell proliferation or sensitivity to BRAFi, suggesting that these cells do not model BRAFV600E-driven melanoma. Due to limitations of the models used, the relationship between BRAFV600E signalling and CXCR3-dependent CD8+ T cell tumour recruitment could not be fully investigated. This study emphasises the need for better mouse models for BRAFV600E-driven melanoma and CRC.
Advisor: McColl, Shaun
Comerford, lain
Brummer, Tilman
Dissertation Note: Thesis (MPhil) -- University of Adelaide, School of Biological Sciences, 2020
Keywords: BRAF
colorectal cancer
tumour immunology
Provenance: This thesis is currently under Embargo and not available.
Appears in Collections:Research Theses

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