Date of Award

Summer 2019

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Maria Marjorette O. Pena

Abstract

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer related deaths. While CRC is initiated by mutations in critical tumor suppressor genes, its development and progression to malignancy is aided by host derived cells that are recruited to the tumor microenvironment (TME) and by cancer-associated microbiota that constitute the gut microbiome. Components of the gut microbiota have been shown to alter immune cell infiltration into tumors and influence response to therapy, disease stage, and patient survival. In previous studies, we found that ApcMin/+ mice, a model of early colon carcinogenesis, had reduced tumor burden when weaned to a folate free (FF) diet as compared to mice weaned to regular rodent chow (RC). When subjected to systemic therapy with 5-FU, tumor response in mice on the folate free diet was more cytotoxic as compared to mice on rodent chow, where tumor response was only cytostatic, with tumor burden rebounding to untreated levels upon removal of 5-FU.

The goal of this study was to identify alterations in the gut microbiome composition in ApcMin/+mice on the different diets, RC, FF, and FF+FA. The FF and FF+FA diets are both defined diets that contain the antibiotic succinyl sulfathiazole (SST), which prevents the production and absorption of folate. The distinguishing factor amongst these diets was that the FF diet contains no folic acid (FA) and the FF+FA diet contains 6 parts per million (ppm) FA. The FF+FA diet contains similar levels of FA that are present in the crude RC diet, which is a standard diet for mice.

The results showed significant changes in the gut microbiome composition between RC, FF, and FF+FA diets with additional changes upon treatment with 5-FU. The Bacteroidetes S24-7 population was nearly eliminated in mice fed the FF and FF+FA diets, while the Firmicutes Ruminococcus and Clostridiales populations expanded. In addition, the concentration and production of metabolites, specifically short chain fatty acids (SCFAs), were significantly reduced when mice were fed the FF and FF+FA diets, and these levels remained at low levels during treatment with 5-FU. This correlated with reduced infiltration of immune cells into the TME. Mast cells, macrophages, myeloid derived suppressor cells (MDSCs), T-regulatory cells, and CD8 cytotoxic T cells were significantly reduced in the TME when mice were fed the FF and FF+FA diets, and these levels remained low during treatment with 5-FU. Although similar results were observed in mice fed the FF and FF+FA diets, tumor burden was reduced by 70% when mice fed the FF diet were treated with 5-FU as compared to untreated controls. Mice fed the FF+FA diet that were treated with 5-FU only had a mild 30% reduction in tumor burden as compared to untreated controls. These data suggest that elimination of folate from dietary intake and the gut microbiota was essential to enhance the cytotoxicity of 5-FU.

However, because the FF and FF+FA diets are both defined diets and contain the antibiotic, SST, it was important to distinguish the effects of the removing dietary folate versus inhibiting gut microbe folate production. Mice were fed a basal and FF diet that were defined diets lacking SST, designated as basal and FF* diets, respectively, and then subjected to 5-FU therapy. The basal diet contained 6 ppm FA whereas the FF* diet contained no FA and no SST. The results showed that removal of SST reversed the previous effects on the gut microbiota, ie, the Bacteroidetes S24-7 population was restored and the Firmicutes Ruminococcus and Clostridiales populations returned to lower levels in mice fed the basal and FF* diets that was similar in abundance to that in mice fed the RC diet. However, the composition of SCFAs remained significantly lower in mice fed the basal and FF* diets, and remained low during treatment with 5-FU. The infiltration of mast cells, macrophages, MDSCs, and T-regulatory cells were significantly lower in the TME as compared to mice fed the FF and FF+FA diets containing SST. While tumor burden was 15% lower in mice fed the basal and FF* diets, treatment with 5-FU caused a modest 30% reduction in tumor burden as as compared to untreated controls and similar to that observed in mice fed RC diet. These results highlight the importance of inhibiting folate production by the gut microbiota in influencing the activation and infiltration of immune cells into the TME, and enhancing the cytotoxic effects of 5-FU.

These studies show that understanding the role of the gut microbiome in response to 5-FU could lead to the development therapeutic strategies targeting the gut microbiome to alter its metabolites in order to enhance the antitumor efficacy of 5-FU while minimizing its cytotoxic effects to the patient.

Rights

© 2019, Kristen Michele Hogan

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