Estrogen receptor alpha positive (ERα+) breast cancers are refractory to immune checkpoint blocker (ICB) monotherapy, while ICBs are part of a standard of care for triple negative breast cancers (TNBCs). Besides tumor ERα expression, another difference between the two types of breast cancers is that only ERα+ patients exhibit elevated tumor estradiol (E2) levels, compared with surrounding normal tissue. Recent evidence suggests that inhibition of ERα or activation of ERβ or G protein-coupled estrogen receptor (GPER) in immune cells in the tumor microenvironment (TME) increases tumor CD8+ T cell infiltration and boosts cancer ICB response. Ovarian and adipose-produced estrogens activate all three ERs equally, but plant estrogens (phytochemicals) preferentially activate ERβ or GPER. The gut microbiota is a key player in determining response to ICBs, and high abundance of Firmicutes and high fecal levels of short chain fatty acids (SCFAs) that are mainly produced by Firmicutes, are linked to improved effectiveness of ICB therapy. Interestingly, the gut microbiota of ERα+ breast cancer patients contain significantly lower abundance of Firmicutes species than the gut microbiota of TNBC patients. Many factors modify the gut microbiota, especially diet. The gut microbiota altering diets include (i) foods high in ERβ and GPER activating plant phytochemicals or (ii) SCFAs producing fiber that also reduces circulating estrogen levels, (iii) estrogen levels reducing fasting/caloric restriction, or (iv) ketogenic diet which reduces fecal SCFA levels but increases hepatic production of SCFA receptor activating ketone bodies. It is thus possible that certain foods or dietary patterns can modify both the gut microbiota and activation of the estrogen receptors in the tumor immune cells, and consequently regulate the effectiveness of ICB therapy against cancers.

Inadequate social contacts and loneliness, often referred to as social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients have been identified as feeling socially isolated. Moreover, socially isolated breast cancer survivors have a 43% higher risk of recurrence than socially integrated survivors. To prevent increased mortality, biological mechanisms which mediate the effects of SI on cancer need to be identified. One unexplored, but possible mechanism is through the gut microbiota. Through bidirectional interactions, the gut is affected by stress and the gut microbiota in turn can modulate stress response, host immunity and metabolism. Here we tested the hypothesis that SI induces gut dysbiosis. In our study, repeated in four separate experiments, adult female mice were divided into two groups – those kept group housed (GH, 4 mice per cage) and those housed singly in SI for 4 weeks. Several differences in the gut microbial family, genus and species levels were seen, but the differences were mostly unique to each of the four experiment. Beta-diversity was increased in three of the four studies in SI mice. Since beta-diversity is increased by aging, SI may accelerate the aging process. At the genus level, SI significantly suppressed the abundance of Akkermansia in all four studies and increased Acetatifactor in three studies. These two bacterial changes are expected to disrupt mitochondrial oxidative phosphorylation (OXPHOS), most likely by suppressing the short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. In a separate study, we discovered that SI impaired OXPHOS and activated inflammatory pathways in the mammary gland. We also have assessed immune cells in the spleen. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg (CD4+Foxp3+) and PMN-MDSCs cells. In addition, SI increased PD1 expression in Foxp3+ cells, suggesting that anti-PD1 therapy might adversely affect socially isolated breast cancer patients by invigorating Treg cells. We are currently studying if the changes in the gut microbiota in SI mice are causally linked to their impaired mitochondrial metabolism, immunosuppression and increased mammary cancer mortality. We also plan to investigate if dietary modifications can reverse gut dysbiosis in SI mice and prevent their increased mortality from mammary cancer. Citation Format: Fabia de Oliveira Andrade, Lu Jin, Vivek Verma, Maddie McDermott, Chris Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: do they explain enhanced mammary tumorigenesis? [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-05-16.

Social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients are estimated to feel socially isolated when assessed between 6 months and 2 years following their cancer diagnosis. Since socially isolated breast cancer survivors are at a 64% higher risk of breast cancer-specific mortality than socially integrated survivors, the biological mechanisms which mediate the effects of SI need to be identified to prevent increased mortality. In a preclinical model, we found that SI caused recurrence of mammary cancers which had been treated with hormone therapy and thus led cancer cells to emerge from dormancy. We further found that mammary glands of SI rats exhibited upregulation of IL6/JAK/STAT3 signaling pathway and impaired oxidative phosphorylation (OXPHOS), compared with group housed (GH) rats. We investigated if these changes could be mediated through the gut-brain-axis. Stress can affect the gut microbiota, and the gut microbiota is critical for regulating the host immunity. In four separate experiments, the effect of SI on the gut microbiota composition was assessed in C57BL/6 mice that were either group housed or housed singly in SI for 4 weeks. In each study replicate, several differences in the gut microbiota composition were seen, but these differences were mostly unique to each experiment. Beta-diversity was increased in three of the four experiments in SI mice. Nevertheless, at the genus level, SI suppressed the abundance of Akkermansia in all four replicates and increased Acetatifactor in three of the four replicates. These two bacterial changes are expected to disrupt OXPHOS, perhaps by resulting a suppression in short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. Thus, it is possible that the changes in the composition of the gut microbiota of SI animals explain an increase in IL6 signaling and a suppression in OXPHOS in their mammary gland. We also have studied immune cells in the spleen in SI and GH mice. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg cells (CD4+Foxp3+) and myeloid derived suppressor cells (PMN-MDSCs). SI also increased PD1 expression in Foxp3+ cells. In an on-going study, we found that fecal microbiota transplant (FMT) from SI mice increased E0771 mammary tumor growth in antibiotic treated GH recipient mice, compared with mice receiving FMT from GH mice. Whether the FMT also altered IL6 and OXPHOS signaling in the mammary glands and tumors of recipient mice is currently being investigated. Our findings suggest that social isolation may increase breast cancer through inducing gut dysbiosis. Citation Format: Fabia de Oliveira Andrade, Maddie McDermott, Lu Jin, Vivek Verma, Karla Andrade de Oliveira, Christopher Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4237.