Investigating the role of tumour-derived Interleukin-35 in the regulation of M2 macrophage polarisation in head and neck squamous cell carcinomas

Ali, Richard 2022. Investigating the role of tumour-derived Interleukin-35 in the regulation of M2 macrophage polarisation in head and neck squamous cell carcinomas. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

HNSCC is the 6th most common cancer worldwide. Despite advances in treatment, responses to immunotherapies and overall survival remain unsatisfactory. Immunity in the tumour microenvironment of inflamed HNSCC is often dysfunctional. Studies have shown that macrophages therein exist preferentially in the M2 activation state, which potently suppresses anti-tumour immunity and promotes tumour aggressiveness. To improve anti-tumour immunity, it is important to identify novel regulators of M2 macrophage polarisation, which could potentially be targeted for immunotherapy. Interleukin-35 (IL-35) is an immunosuppressive cytokine overexpressed in cancers that has been shown to reprogram immune cells to immunosuppressive phenotypes. Limited information is present on expression of IL-35 in HNSCC and its role in M2 macrophage polarisation. Preliminary studies indicated that gene expression of IL-35 subunits (EBI3 and p35) is low in HNSCC cell lines but becomes upregulated in response to stimulation with pro-inflammatory cytokines, suggesting a potential feedback response to anti-tumour immunity. This study aimed to further evaluate the upregulation of IL-35 expression in HNSCC cells in response to inflammatory stimuli, and investigate the potential role of HNSCC-derived IL-35 in the repolarisation of M1 macrophages to the prevalent M2 phenotype. Stimulation of the hypopharyngeal carcinoma (FaDu) and oral carcinoma (H357) cell lines, with IFNγ and TNFα, elevated gene expression of IL-35. These cytokines concurrently upregulated expression of IL-35 receptors in FaDu cells. IL-10 nor IL-35 stimulation affected IL-35 endogenous gene expression. Using conditioned medium (CM) from transfected FaDu cells that overexpress IL-35 (FaDu-IL-35), and mixed culture with M1 macrophages, HNSCC-derived IL-35 was found to suppress TNFα secretion. CM from EBI3-overexpressing FaDu cells showed similar effects. IL-35 overexpression did not downregulate expression of the M1 markers HLA-DR (antigen presentation) CD80 or CD86 (T cell activation), nor did it upregulate M2 cytokines (IL-10, VEGF-A), surface markers (CD206, CD163) or candidate M2-TAM markers (PD-L1, CD204, B7-H4). Notably, FaDu-IL-35 CM downregulated PD-L1 expression in M1 macrophages, which may have implications in responses to immunotherapy. CM from p35-overexpressing FaDu cells upregulated IL-10 secretion. These studies suggest that inflammation may induce IL-35 expression in hypopharyngeal carcinoma cells, and may also prime them to respond to exogenous IL-35 to further increase expression. IL-35 produced may not repolarise M1 macrophages to M2, but may suppress inflammation and affect responses to anti-PD 1/PD-L1 therapy. Further studies are required to confirm these findings, and to evaluate potential roles of individual IL-35 subunits in immunoregulation.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Dentistry
Subjects:	Q Science > QM Human anatomy Q Science > QR Microbiology R Medicine > R Medicine (General) R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Funders:	Cancer Research Wales
Date of First Compliant Deposit:	22 February 2023
Last Modified:	23 Feb 2023 10:59
URI:	https://orca.cardiff.ac.uk/id/eprint/157090

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