Determining the role of CD200 in basal cell carcinoma

Gibbs, Alex 2024. Determining the role of CD200 in basal cell carcinoma. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

CD200 is an immunomodulatory protein that is expressed on the cell membrane of hair follicle bulge stem cells and basal cell carcinoma (BCC) cancer stem cells, consistent with the literature suggesting that human BCC arise from hair follicle bulge stem cells. The “driver” mutations in human BCC activate the hedgehog (Hh) signalling pathway. Similarly, growth factor pathway activation is common in cancer; notably Hh pathway activation is observed in one-quarter of all cancers. The recent development of targeted cancer therapies, typically small-molecule drugs that block growth factor activation, represents a great advance in cancer management. Yet targeted therapies, such as smoothened antagonists for the treatment of BCC, are beset by primary and secondary resistance, suggesting the presence of additional cell survival signalling pathways. Hypothesis Given primary and secondary resistance is seen with smoothened antagonists in BCC, I hypothesised that cancer stem cells facilitate this resistance via additional pathway activation. Aims 1. Determine whether CD200 is a functionally relevant marker for the isolation and function of BCC cancer stem cells. v 2. Use RNA sequencing to elucidate cell signalling pathways specific to the BCC cancer stem cell-enriched population. Methods To assess functional relevance of CD200 to CSCs, CD200-transduced HeLa and UWBCC1 cells were used in vivo to assess whether CD200 causes cancer immune cell evasion. Flow sorting followed by bulk and single-cell RNA sequencing approaches were used to define BCC cancer stem cells and signalling pathways in UW-BCC1 and primary human BCC samples. Immunohistochemistry was used to validate signalling pathway cascades identified by sequencing in primary human BCC. Results CD200 did not alter tumour growth rates but led to a significant reduction in tumour necrosis, immune cell infiltrate, and NK cell viability. Hence the CD200+ BCC subpopulation aids immune evasion and is therefore a functionally relevant cell surface marker to BCC CSCs. Bulk RNA sequencing comparing flow-sorted CD200+ and CD200- CD45- primary human BCC subpopulations revealed upregulation of endothelial cell processes, including VEGF signalling. The VEGF pathway differentially expressed genes included those predicated to be expressed on both tumour and endothelial cells, confirmed by VEGFR2 (KDR) immunohistochemical labelling. Single-cell RNA sequencing showed that up to 44% of flow-sorted CD200+ CD45- primary human BCC tumour dissociate were endothelial cells. Isolating the BCC keratinocytes, we identified 17 clusters based on transcriptomic similarities, which we were able to separate into three vi functional categories. All BCC keratinocytes expressed Hh pathway-regulated genes. The CD200 high expressing “basal” BCC population demonstrated active TGFβ signalling, which we validated by immunohistochemical labelling to identify phosphorylated SMAD3 nuclear translocation in BCC tissues. This suggests that BCC CD200+ CSCs upregulate the TGFβ pathway which may facilitate metastasis and treatment resistance. Conclusions CD200 functions to protect BCC cancer stem cells from immune attack. The BCC cancer stem cell-enriched CD200+ CD45- tumour population, which included endothelial cells, is enriched for VEGF-mediated angiogenesis. This enrichment, however, may be emanating from the endothelial cell contamination of the dataset. Specifically, BCC cancer stem cells may use TGFβ signalling for survival and intrinsic resistance to Hh pathway antagonists.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Funders:	KESS
Date of First Compliant Deposit:	17 May 2024
Last Modified:	23 May 2024 11:14
URI:	https://orca.cardiff.ac.uk/id/eprint/169002

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