Microbiome impacts cancer development and therapeutic efficacy. In addition to the guts, microbes reside in
different tissues influencing the pathophysiology of the tissue microenvironment. These tissue-resident
microbes are largely attributed to translocation of gut microbes. In the breast, such passage is termed ‘gut-
breast axis’, helping establish microbiotas of breast tissue and milk. Nevertheless, gut-breast axis has been
mostly conceptualized around pregnancy, and it is completely unknown whether this axis indeed exists outside
pregnancy to impact breast health and carcinogenesis. Our long-term goal is to dissect how microbiome
contributes to breast pathophysiology. Especially, the objectives of the present study are to determine i)
whether gut-breast axis occurs on a regular basis; ii) whether this involves discrete sets of bacteria for healthy
cohorts vs. cancer patients, and iii) what are their roles. Our central hypothesis is that gut-breast axis takes
place on a regular basis, involving distinct sets of bacteria to confer anti-tumor effects on healthy cohorts vs.
pro-tumor effects on cancer patients. The proposed research is based on our preliminary studies allowing us to
harvest specific gut microbiotas from tumor-protected or -susceptible animals. We reported that supplementing
sepiapterin (SEP)—the endogenous precursor of tetrahydrobiopterin (the cofactor of nitric oxide (NO)
synthase)—normalized arginine metabolism and improved the immunogenicity of HER2-positive mammary
tumors. We then orally applied SEP to mice prone to HER2-positive mammary tumors and saw strong tumor
prevention. These mice also showed increases in NO levels and NO-producing bacteria in the guts. Besides,
extracts of these gut bacteria activated innate immune cells, suggesting the roles of these gut bacteria in anti-
tumor immunity. Here, we will determine whether these gut bacteria physically translocate to the breast to exert
tumor preventative effects. Our hypothesis will be tested through two SPECIFIC AIMS: 1) Determine whether
gut microbiotas of a) tumor-protected vs. b) -susceptible mice exert anti-tumor vs, pro-tumor effects; and 2)
determine whether distinct sets of gut microbes are translocated to mammary glands to exert anti-tumor vs.
pro-tumor effects. In Aim 1, we will transplant gut microbiota of a) tumor-protected (SEP-treated) or b) -
susceptible (DMSO-treated) HER2 mice into recipients and give the inverse drug treatments. We will test
whether the transplanted microbiotas antagonize the treatments. In Aim 2, gut microbiota of a) tumor-protected
(SEP) vs b) -susceptible (DMSO) mice are differentially labeled, and the 50:50 mixture is given to the recipients
undergoing SEP or DMSO treatment. Labeled microbes are analyzed for their gut-breast translocation; their
ratios in the breast; and the contributions of breast microbiota to the drug effects. The proposed study is
innovative because this is the first time to corroborate gut-breast axis and its contributions to breast
pathophysiology. The study is significant because it will have a positive translational impact by justifying the
development of a new breast cancer treatment or prevention strategy focused on breast microbiota.