Development and characterisation of an osteoclastic in vitro model of multiple myeloma

Lewis, Thomas 2019. Development and characterisation of an osteoclastic in vitro model of multiple myeloma. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Multiple myeloma is an incurable malignancy of terminally differentiated B-cells – also known as plasma cells. These malignant cells are extremely reliant on the bone marrow microenvironment for their growth and survival, as well as their acquired ability to resist therapeutic intervention. Consequently, maintaining primary myeloma cells in vitro remains a challenge. Patients suffering from this incurable disease often develop osteolytic lesions, due to an imbalance between osteoblasts and osteoclasts, which cause bone pain and a high frequency of fractures. This project aimed to create a physiologically relevant in vitro model of myeloma, incorporating an osteoclast microenvironment. Osteoclasts normally work in concert with osteoblasts during bone remodelling. In myeloma their activity predominates and is intrinsic to disease progression. It is now clear that osteoclasts also contribute to the survival of myeloma cells but the precise mechanism(s) for this remain unresolved. The first aim of this research was to develop and characterise an in vitro osteoclastic model using the myelo-monocytic U937 cell line. Treatment with 100nM PMA and 10nM 1,25(OH)2D3 caused these cells to merge and form multi-nucleated, TRAP positive and RANK positive cells with bone resorbing capabilities. Culturing two different myeloma cell lines, H929 and JJN3, in co-culture with these osteoclast-like cells for a period of 48 hours resulted in the preferential expansion of a small subpopulation of myeloma cells that were CD138dim. RNA-sequencing was performed on cell sorted CD138bright and CD138dim, which revealed substantial differences in the transcriptomes of CD138dim and CD138bright cells. Comparative analysis inferred an activation of signalling pathways relating to adhesion, migration and survival in CD138dim cells. Phenotypic analysis showed that CD138dim cells had significantly higher expression of adhesion markers CXCR4, CD40, CD45 and CD49e, the angiogenesis inducer CXCL8 and the activation marker CD69 (p0.05) in both cell lines as a result of co-culture with differentiated U937 osteoclast-like cells. Functionally, CD138dim had greater migratory capacity and increased chemoresistance to bortezomib in comparison with CD138bright cells. These data could imply a role for CD138dim myeloma cells in disease propagation in both the pre and post-treatment settings. As a result, this model could provide a good foundation for future studies on the influence of the bone marrow microenvironment on resistance mechanisms in myeloma.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	5 May 2020
Last Modified:	05 May 2021 02:05
URI:	https://orca.cardiff.ac.uk/id/eprint/131446

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