Recent studies have shown that individuals with colorectal cancer have an altered gut microbiome compared to healthy controls. It remains unclear whether these differences are a response to tumorigenesis or actively drive tumorigenesis. To determine the role of the gut microbiome in the development of colorectal cancer, we characterized the gut microbiome in a murine model of inflammation-associated colorectal cancer that mirrors what is seen in humans. We followed the development of an abnormal microbial community structure associated with inflammation and tumorigenesis in the colon. Tumor-bearing mice showed enrichment in operational taxonomic units (OTUs) affiliated with members of the Bacteroides, Odoribacter, and Akkermansia genera and decreases in OTUs affiliated with members of the Prevotellaceae and Porphyromonadaceae families. Conventionalization of germfree mice with microbiota from tumor-bearing mice significantly increased tumorigenesis in the colon compared to that for animals colonized with a healthy gut microbiome from untreated mice. Furthermore, at the end of the model, germfree mice colonized with microbiota from tumor-bearing mice harbored a higher relative abundance of populations associated with tumor formation in conventional animals. Manipulation of the gut microbiome with antibiotics resulted in a dramatic decrease in both the number and size of tumors. Our results demonstrate that changes in the gut microbiome associated with inflammation and tumorigenesis directly contribute to tumorigenesis and suggest that interventions affecting the composition of the microbiome may be a strategy to prevent the development of colon cancer.

Inflammation is a critical player in the development of both colitis-associated and sporadic colon cancers. Several studies suggest that the microbiota contribute to inflammation and tumorigenesis; however, studies to understand the role of the microbiota in colon tumor development in germfree (GF) mice are limited. We therefore studied the effects of the microbiota on the development of inflammation and tumors in germfree and conventionally-raised specific pathogen-free (SPF) mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS). We discovered that GF mice developed significantly more and larger tumors compared to that in SPF mice after AOM and DSS treatment despite the lack of early acute inflammation in response to chemically-induced injury by DSS. Although the extent of intestinal epithelial damage and apoptosis was not significantly different in GF and SPF mice, there was a delay in intestinal epithelial repair to DSS-induced injury in GF mice resulting in a late onset of proinflammatory and protumorigenic responses and increased epithelial proliferation and microadenoma formation. Recolonization of GF mice with commensal bacteria or administration of LPS reduced tumorigenesis. Thus, although commensal bacteria are capable of driving chronic inflammation and tumorigenesis, the gut microbiota also have important roles in limiting chemically-induced injury and proliferative responses that lead to tumor development.

In the tumor microenvironment, CD11b+Gr1+ bone marrow-derived cells are a predominant source of pro-tumorigenic factors such as matrix metalloproteinases (MMPs), but how distal tumors regulate these cells in the bone marrow is unclear. Here we addressed the hypothesis that the parathyroid hormone-related protein (PTHrP) potentiates CD11b+Gr1+ cells in the bone marrow of prostate tumor hosts. In two xenograft models of prostate cancer, levels of tumor-derived PTHrP correlated with CD11b+Gr1+ cell recruitment and microvessel density in the tumor tissue, with evidence for mediation of CD11b+Gr1+ cell-derived MMP-9 but not tumor-derived VEGF-A. CD11b+Gr1+ cells isolated from mice with PTHrP-overexpressing tumors exhibited relatively increased pro-angiogenic potential, suggesting that prostate tumor-derived PTHrP potentiates this activity of CD11b+Gr1+ cells. Administration of neutralizing PTHrP monoclonal antibody reduced CD11b+Gr1+ cells and MMP9 in the tumors. Mechanistic investigations in vivo revealed that PTHrP elevated Y418 phosphorylation levels in Src family kinases in CD11b+Gr1+ cells via osteoblast-derived IL-6 and VEGF-A, thereby upregulating MMP-9. Taken together, our results showed that prostate cancer-derived PTHrP acts in the bone marrow to potentiate CD11b+Gr1+ cells, which are recruited to tumor tissue where they contribute to tumor angiogenesis and growth.

Prostate carcinoma frequently metastasizes to bone where the microenvironment facilitates its growth. Inhibition of bone resorption is effective in reducing tumor burden and bone destruction in prostate cancer. However, whether drugs that inhibit osteoclast function inhibit tumor growth independent of inhibition of bone resorption is unclear. Calcium is released during bone resorption and the calcium sensing receptor is an important regulator of cancer cell proliferation. The goal of this investigation was to elucidate the role of calcium released during bone resorption and to determine the impact of drugs which suppress bone resorption on tumor growth in bone. To compare tumor growth in a skeletal versus non-skeletal site, equal numbers of canine prostate cancer cells expressing luciferase (ACE-1luc) prostate cancer cells were inoculated into a simple collagen matrix, neonatal mouse vertebrae (vossicles), human de-proteinized bone, or a mineralized collagen matrix. Implants were placed subcutaneously into athymic mice. Luciferase activity was used to track tumor growth weekly and at one month tumors were dissected for histologic analysis. Luciferase activity and tumor size were greater in vossicles, de-proteinized bone and mineralized collagen matrix versus non-mineralized collagen implants. The human osteoblastic prostate carcinoma cell line C4-2b also grew better in a mineral rich environment with a greater proliferation of C4-2b cells reflected by Ki-67 staining. Zoledronic acid (ZA), a bisphosphonate, and recombinant OPG-Fc, a RANKL inhibitor, were administered to mice bearing vertebral implants (vossicles) containing ACE-1 osteoblastic prostate cancer cells. Vossicles or collagen matrices were seeded with ACE-1luc cells subcutaneously in athymic mice (2 vossicles, 2 collagen implants/mouse). Mice received ZA (5μg/mouse, twice/week), (OPG-Fc at 10mg/kg, 3 times/week) or vehicle, and luciferase activity was measured weekly. Histologic analysis of the tumors, vossicles and endogenous bones and serum biochemistry were performed. Antiresorptive administration was associated with decreased serum TRAP5b and reduced osteoclast numbers, increased tibia and vossicle bone areas. ZA significantly decreased bone marrow calcium concentrations without affecting serum calcium. ZA and OPG-Fc significantly inhibited tumor growth in bone but not in collagen implants. In conclusion, the inhibitory effects of ZA or OPG-Fc on prostate tumor growth in bone are mediated via blocking bone resorption and calcium release from bone.

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<p>PDF file - 7060K, Supplemental Fig. 1 Ectopic PTHrP increased CD11b+Gr1+ cell-dependent prostate tumors angiogenesis.</p>

<p>PDF file - 2044K, Supplemental Fig. 4 Anti-PTHrP monoclonal antibody suppress cellular proliferation.</p>

<p>PDF file - 5411K, Supplemental Fig. 2 PTHrP promoted tumorigenic function of CD11b+Gr1+ cells.</p>