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Development of cutaneous melanoma spheres for drug testing

Mukhtar, Abdullahi 2021. Development of cutaneous melanoma spheres for drug testing. PhD Thesis, Cardiff University.
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Abstract

Metastatic melanoma is a therapy resistant tumour with conventional chemotherapy having little or no impact on survival leading to high mortality rate. The incidence of Melanoma has been increasing over decades. Among the various agents licensed in treating the disease, clinicians are often faced with challenges of non-response, ineligibility, toxicity or secondary resistance. Thus there remains an unmet clinical need for new therapies. Melanoma Cancer stem cells were shown to be responsible for the resistance of MM to therapies. A number of cell surface proteins (ABCB5,CD133, CD271, and CD20) are expressed by these stem-like cells in Melanoma, of which CD20 is potentially targetable. We have developed an easily reproducible sphere formation (melanosphere) cell culture assay in which six of seven human melanoma cell lines studied demonstrated melanosphere under non-adherent 3D culture conditions, with reproducible sphere forming efficiency when cells were plated at low density. Of these previously identified melanoma cancer stem cell markers, enrichment in 3D non-dherent culture was consistently observed with CD20 but not CD133 or CD271 markers. The expression of High Molecular Weight Melanoma Associated Antigen (HMWA) was also dependent upon non-adherent conditions. Consistent with transformation features, healthy human melanocytes studied neither forms spheres in 3D non-adherent culture nor expresses CD20 in 2D or 3D culture conditions. Mutations resulting in activation of RAS-MAPK pathway were defined in our melanoma cell lines using PCR and sanger sequencing. The BRAF mutant cell lines in culture demonstrated higher sphere forming efficiency (p>0.05), and were not killed with BRAF inhibitor (Vemurafenib) treatment. Rituximab, a CD20 targeting antibody, at licensed dose was able to disrupt colony formation. Furthermore, combination therapy with vemurafenib led to significant loss in cell viability of CD20+ BRAF mutant cells. However, this combination was less effective in killing CD20 negative BRAF mutant and CD20 negative wild type BRAF melanoma cell lines. Flow cytometric analysis for apoptosis, showed that combination therapy was associated with a 3-fold increase in early apoptosis of BRAF-mutant CD20 expressing melanoma cell line. In the more aggressive NF1-mutant melanoma cell line, pigment epithelium derived factor (PEDF) loss was observed to be associated with increased proliferation, migration and invasiveness. Additional studies including In-vivo assays are required to further explore the role and applicability of CD20 and PEDF in melanoma. Thus, our findings demonstrate the utility of our melanospheres in recreating in-vivo differentiation to determine cell biology and test therapeutic targeting, notably of putative CD20 expressing cancer stem cells.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 30 June 2021
Last Modified: 30 Jun 2022 01:30
URI: https://orca.cardiff.ac.uk/id/eprint/142266

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