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Investigating the HPV-Positive & HPV-Negative Oropharyngeal Cancer Tumour Microenvironment in vivo and in vitro

Al-Sahaf, Sarmad (2018) Investigating the HPV-Positive & HPV-Negative Oropharyngeal Cancer Tumour Microenvironment in vivo and in vitro. PhD thesis, University of Sheffield.

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Abstract

Human papillomavirus (HPV) is now recognised as a major aetiological agent in the pathogenesis of oropharyngeal carcinoma (OPC). HPV-positive tumours are associated with better outcomes compared to HPV-negative tumours, possibly due to differences in their aetiology, immune responses and/or the tumour microenvironment. Increased numbers of tumour-associated leukocytes have been observed in many cancers including OPC, with variable influence on prognosis depending on the leukocyte subpopulation investigated. Whether, HPV-status influences leukocyte recruitment to OPC remains unknown. The main aim of this study was to examine if differences exist in the immune responses between HPV-positive compared to HPV-negative OPC by examining levels of infiltrated leukocyte sub-populations, and to further determine the molecular mechanisms driving leukocyte recruitment into these two distinct forms of tumours. Immunohistochemical staining of HPV-positive (n=40) and HPV-negative (n=19) OPC biopsies, followed by survival analysis showed that individuals with HPV displayed a distinct survival advantage. In terms of leukocyte abundance, HPV-negative OPC contained significantly (P<0.05) more neutrophils than HPV-positive tumours, whilst there was no difference in macrophage or T cell abundance between the two tumours except in the low expressive cases. A subsequent in vitro study examined differences in the chemoattractant capacity of HPV-positive and HPV-negative OPC cell lines to determine if these factors were implicated in the neutrophil, macrophage and T cell recruitment observed in vivo. Gene and protein expression analysis demonstrated that both monocultures of HPV-positive and HPV-negative cell lines, along with normal tonsillar fibroblasts (NTF), expressed low chemokine levels, whilst NTF cultured with conditioned medium from HPV-negative OPC cells expressed significantly higher levels of all chemokines tested compared to NTF incubated with the medium from HPV-positive OPC cell lines. HPV-negative OPC cell lines expressed the pro-inflammatory cytokines IL-1α & β mRNA whereas HPV-positive cells did not, and NTF constitutively expressed IL-1R. Pre-treatment with the IL-1R antagonist, Anakinra, or siRNA to IL-1R1 significantly reduced chemokine secretion from NTF stimulated with conditioned medium from HPV-negative tumour cells or recombinant IL-1β (P<0.05). Similar data was obtained when cells were cultured all together in a 3D-model of OPC. In addition, in a 3D co-culture model, treatment with Anakinra significantly reduced the chemokines and number of infiltrating leukocytes into the model compared to untreated 3D cultures. Taken together, these data suggest that secretion of chemokines is driven by the interaction between HPV-negative OPC cells and stromal tonsillar fibroblasts through an IL-1/IL-1R-mediated mechanism that is less prominent within the HPV-positive tumour microenvironment. These observations may explain differences in leukocyte sub-populations recruited to HPV-positive versus negative OPC and indicate that HPV-status is a key determinant in controlling the inflammatory tumour microenvironment. Controlling leukocyte infiltration into HPV-negative OPC using Anakinra may be of potential clinical benefit.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Dentistry (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
Depositing User: Dr Sarmad Al-Sahaf
Date Deposited: 07 Jan 2019 12:22
Last Modified: 07 Jan 2019 12:22
URI: http://etheses.whiterose.ac.uk/id/eprint/22533

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