Date of Award

2015

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

M. Rita Young

Second Advisor

Carl Atkinson

Third Advisor

Terry Day

Fourth Advisor

Sebastiano Gattoni-Celli

Fifth Advisor

Amanda C. LaRue

Abstract

Head and neck squamous cell carcinoma (HNSCC) is characterized by immunosuppression, a state in which the established tumor evades the immune response. This presents a significant obstacle for treatment and the five-year survival rate for head and neck cancer patients remains at approximately 50%. While HNSCC causes profound immune inhibition, the immune changes that occur before HNSCC becomes established, at the premalignant lesion stage, are unknown. This study used the 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis to investigate how the immune environment changes as premalignant lesions progress to tumor. Premalignant lesion-bearing tongue tissue was found to release increased levels of Th1- and Th17-associated cytokines, including IL-2, IFN-γ, TNF-α and IL-17A, compared to control and HNSCC-bearing tongue tissue. Spleen cells and cervical lymph node cells produced increased levels of Th1- and Th2-associated cytokines in the presence of premalignant lesion-bearing tongue tissue compared to HNSCC-bearing tongue tissue. Premalignant lesion cells secreted significantly increased levels of proinflammatory G-CSF, RANTES, MCP-1, and PGE2 compared to HNSCC cells. Premalignant lesion cell supernatant elicited increased production of innate proinflammatory mediators and Th1- and Th17-associated cytokines from spleen cells compared to HNSCC cell supernatant or media alone. An increased percentage of spleen cells expressed markers of activation in the presence of premalignant lesion cell supernatant compared to HNSCC cell supernatant or media alone. This data shows that premalignant lesion cells release increased levels of proinflammatory mediators compared to HNSCC cells, and furthermore, elicit increased production of Th1- and Th17-associated cytokines and expression of activation markers in spleen cells compared to HNSCC cells. Compared to the HNSCC environment, the premalignant lesion environment is significantly more immune stimulatory, supporting a robust Th1- and Th17-associated immune response. Immunosuppression is a significant obstacle to treatment for advanced HNSCC patients. The tumor itself plays a role in establishing this local and systemic immunosuppression, by secreting mediators such as PGE2 that decrease T cell-mediated immunity and support immunosuppressive cells at the tumor site. This study aims to determine if inhibition of PGE2 production at an earlier stage of carcinogenesis, the premalignant lesion stage, can restore a beneficial immune response and slow progression to tumor, in the 4-NQO mouse model of oral carcinogenesis. Initial studies showed that inhibition of PGE2 production by premalignant lesion cells (but not HNSCC cells) induced increased production of the Th1-associated cytokine IL-2 and IFN-γ by spleen cells in vitro. To monitor the effects of inhibiting PGE2 production in vivo, indomethacin, a COX (cyclooxygenase) inhibitor, was administered to mice bearing 4-NQO-induced premalignant lesions and progression to tumor was monitored by endoscopy. Mice receiving indomethacin at the premalignant lesion stage had significantly improved clinical outcomes compared to mice receiving diluent control treatment, as defined by lower lesion scores. Cervical lymph node analysis showed an increased percentage of CD8+ T cells expressing IFN-γ at the endpoint of the study (20 weeks) in mice that had received indomethacin compared to mice that had received diluent control treatment, although the difference was not significant. Spleen cells from mice that had received indomethacin secreted increased levels of IFN-γ at 6 weeks and 20 post-treatment compared to mice that had received control treatment, suggesting that indomethacin treatment induced an increased systemic Th1-type immune response. The data suggests that the premalignant state is characterized by an immune stimulatory and inflammatory milieu and that inhibiting PGE2 production stimulates immune reactivity and slows progression to advanced HNSCC in the 4-NQO model.

Rights

All rights reserved. Copyright is held by the author.

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