Fusobacterium-Stromal Cell Interactions in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer related deaths among adults within the United States. The complexity of the CRC tumor microenvironment (TME), comprising immune cells and stromal cells, such as cancer-associated fibroblasts (CAFs), and microbiota, further complicate the challenge in developing effective therapeutic targets. Fusobacterium, a Gram-negative, anaerobic oral microbe that has been shown to invade human gingival fibroblasts and promote a pro-inflammatory environment in the mouth, has also been associated with worsened CRC patient prognosis, specifically in CRC patients displaying a stromal cell-rich microenvironment. Additionally, substantial evidence has established a positive correlation between Fusobacterium, stromal cells, and immune dysregulation during CRC development. Thus, understanding the multifactorial components of the CRC TME may reveal potential therapeutic targets. We hypothesized, thus, that Fusobacterium within the CRC TME similarly play a pivotal role in inducing a pro-inflammatory environment through its interactions with CAFs which exacerbates CRC development. We sought to investigate the interactions between Fusobacterium species (spp.) and fibroblasts by first developing optimized techniques for indirect immunofluorescence microscopy-based detection of Fusobacterium on Formalin-Fixed, Paraffin-Embedded (FFPE) sections. This was an important initial step, as Fusobacterium spp. have not been visually presented in FFPE of mouse colons within published works to date, however successful detection of Fusobacterium spp. within the colon allows us to further localize Fusobacterium in the gut and further evaluate its impact on normal tissue and in conditions of colonic pathology. In these experiments, bacterial cultures of Fusobacterium spp. (Fn7-1, Fn7-3, and Fn25586) were each incubated with resected colons of Germ-Free (GF) mice, which are completely devoid of microorganisms, to assess and ensure the validation of our antibody detection. Fusobacterium spp. presence was assessed using primary antibodies against each species, and a fluorescent secondary antibody specific to the host of the primary antibody. After successfully detecting and optimizing the detection of Fusobacterium on resected colons of GF mice, we used the optimized techniques to assess Fusobacterium localization in the colons of Altered-Schaedler Flora (ASF) mice following oral inoculation. These mice contain a well-defined, limited consortia of eight, non-pathogenic microorganisms, typically employed for the controlled study of physiologically relevant microorganisms and their impacts on a host. In assessment of the potential influence of Fusobacterium and colonic fibroblasts, we assessed the expression of CAF markers within the colons of Fusobacterium-inoculated ASF mice, which revealed a correlation between the presence of Fusobacterium and upregulated expression of two CAF markers, namely platelet-derived growth factor receptor alpha (PDGFR)-a and alpha smooth muscle actin (aSMA). This suggested that the presence of Fusobacterium may have influence on the transition between normal fibroblasts to a more CAF-like phenotype, validated by the upregulated expression of CAF-specific markers. Next, we sought to investigate these findings in Specific Pathogen-Free (SPF) mice, which contain a conventional microbiota composition devoid of a specific pathogen. A time-point experiment tracking the abundance of orally inoculated Fusobacterium showed that Fusobacterium abundance falls below the threshold of detection after 24-hours, and therefore does not stably colonize the colons of these mice for further investigation. Due to this result, we sought to implement in vitro experiments consisting of isolated mouse colonic fibroblasts in coculture with Fusobacterium or microbially associated elements. The in vitro co-culture studies revealed increases in interleukin (Il)-6, and Pdgfr-a. An increase in fibroblast secretion of IL-6 was also observed, further supporting our hypothesis that Fusobacterium induces a pro-inflammatory phenotype. We next investigated whether Fusobacterium presence may further promote a pro-inflammatory microenvironment within a mouse tumor model. SPF mice were orthotopically-injected with organoids harboring mutations in KRAS, p53, and NOTCH, which mimic a stromal-cell rich TME and result in primary tumor formation and metastasis to the liver. Mice were then tail-vein intravenously inoculated with Fusobacterium, and Fusobacterium presence within tumors was validated by qPCR. Immunofluorescence imaging on mouse colon tumors of Fusobacterium-inoculated mice revealed upregulation of CAF marker expression when compared to non-inoculated mice. Through immunofluorescence imaging, RNA expression analysis, in vitro coculture, and in vivo inoculation studies, this thesis aims to provide a deeper analysis of Fusobacterium and its potential impact on inducing a pro-inflammatory environment through its interaction with colon and colonic fibroblasts.