Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER (-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor and post-tumor in both BE and LC groups. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia genus and S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER (-) breast cancer prevention.

This combination treatment administered in a breast cancer xenograft mouse model also resulted 96 in significant inhibition of tumor growth when compared with singly administrated compounds 97 [25]. In addition, our recent study reported that the prenatal or maternal consumption of BSp has 5 98 more protective effects on tumor development than postnatal or adulthood administration of BSp 99 in SV40 transgenic and Her2/neu transgenic mouse models [26]. 100 The human gastrointestinal tract harbors trillions of microorganisms (≥10 14 ) that are 101 reported to be at an approximate ratio of 1:1 with the human cells [27]. The gut microbiota plays 102 an important role in regulation of human metabolic and physiological functions by production of 103 crucial metabolites such as short chain fatty acids (SCFAs) [28,29]. SCFAs are produced from 104 the fermentation of non-digestible carbohydrates by gut microbiota and the major SCFAs include 105 butyrate, acetate and propionate [30]. These microbial-produced metabolites actively participate 106 in epigenetic modulations in the host cells, that in turn can have profound effects on the inhibition 107 of cancer [31]. 108 Several studies have attempted to investigate the link between dietary compounds, gut 109 microbiota and breast cancer [32,33]. However, few studies have explored the impact of BSp or 110 GTPs on the gut microbiota in relation to the breast cancer. Some studies have shown that BSp or 111 GTPs can have a significant impact on gut microbial diversity and metabolite production in 112 humans and animals [34][35][36][37]. BSp are rich in glucosinolates, which are metabolized by gut 113 microbiota into isothiocyanates. Further, dietary supplementation with broccoli was reported to 114 lead to alterations in cecal microbiota composition, metabolism and intestinal morphology in an 115 inflammatory disease mouse model [38]. A recent study focused on the impact of broccoli 116 ingestion on gut microbiota of C57BL/6 mice found that increased levels of Clostridiaceae, 117 Lachnospiraceae and Porphyromonadaceae and abundance in gut microbiota diversity was 118 associated with broccoli consumption [34]. EGCG can be degraded by microbial enzymes 119 produced in the digestive tract [39]. Previous studies focused on the consumption of GTPs on 120 intestinal microbiota of healthy humans have found a significant decrease in Clostridium spp. and 6 121 an increase in Bifidobacterium spp. resulting in significantly high levels of acetate and propionate 122 [40]. Others have demonstrated that supplementation of green tea extract resulted in an increase 123 of Bifidobacterium species in calves and humans. Bifidobacterium is a beneficial bacterial species 124 that possess prebiotic properties and can lead to improvement in colon environment [41,42]. A  Here we investigated the impact of the dietary botanicals, BSp or GTPs and their 129 combination, on the gut microbiota of Her2/neu mice when these compounds were administered 130 lifelong from conception and from the beginning of early life. We evaluated the impact of these 131 dietary treatments on ER (-) mammary cancer prevention in these mice by assessing the tumor 132 volume percentage and average tumor latency. Gut microbial communities are known for direct 133 interaction with the host as well as indirect interactions via production of diverse metabolites in 134 the host [43]. In this study, we performed 16S rRNA gene sequencing to investigate the gut 135 microbial composition of Her2/neu mice before and after tumor onset, and identified key bacterial 136 phylotypes that were significantly altered with dietary treatment. We also studied the impact of 137 BSp, GTPs and the combination diet on the plasma levels of SCFAs, which are gut microbial-138 produced metabolites. The animal study was reviewed and approved by Institutional Animal Use and Care through adulthood until termination (Fig 1).  Lifelong from conception (LC) group: 40 Her2/neu female mice were randomly divided 186 into four dietary treatments groups (10 mice/group) as the following: control or BSp or GTPs or 187 combination upon pregnancy. The dietary treatments were continued throughout the gestation and 188 lactation periods. After the lactation period, their female offspring mice were weaned at 3 wks of 189 age. Twenty offspring female mice were then randomly selected from each group and fed the same 190 diet as their mother throughout the study until termination. The quantification of purified PCR products was carried out by PICO green dsDNA Reagent.

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The methanol contained the internal standard 13 C 4 -butyric acid (0.5 mg/ml). The samples were     Similarly, hierarchical clustering using hclust package in R (v3.6.0) in the LC temporal 333 treatment group was performed and no outliers were identified (S2 Fig); thus, we included each 334 sample in this study. We observed an overall similar clustering as seen in the BE group, the 335 microbial composition of BSp-fed mice strongly overlapped with combination-fed mice and the 336 microbial composition of GTPs-fed mice strongly overlapped with control-fed mice (Fig 3b). This  (Fig 4a, S1 Table). Compared to the control diet, the relative 355 abundance of Firmicutes (f_Erysipelotrichaceae g_Allobaculum s_unclassified) was higher in the 356 BSp-fed and combination-fed mice. Family Bifidobacteriaceae and Lactobacillaceae showed 357 higher abundance in the control-fed and GTPs-fed when compared to BSp-fed and combination-358 fed diets. After investigation of significance of bacterial communities using false-discovery rate 359 (FDR) correction, numerous significantly different bacterial taxa (S1 File) were found. Table 1 17 360 shows the top bacterial taxa that were different between the BSp-fed, GTPs-fed and combination-361 fed as compared to control-fed mice groups, respectively. Allobaculum levels were found to be 362 significantly increased in the BSp-fed (5.5-fold)     the control diet (Fig 3d). The relative abundance of the top 25 bacterial taxonomic units are 383 depicted in the heatmap with clustering dendrogram (Fig 4b, S2 Table). Similar to the BE group, whether the gut microbiota was altered after the onset of tumor by our dietary treatments in mice.

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We sought to investigate changes in gut microbial composition with 3D PCoA plot distance metric 399 (Bray Curtis) and found a distinct clustering of microbial communities with the treatment of BSp,

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GTPs or combination as compared to the control diet in the BE group (Fig 5a). PERMANOVA The changes in phylum levels of microbial composition were also observed after tumor 420 onset for all dietary treatments of both groups. In the BE group, the mice on the BSp diet showed 421 a significant increase in Bacteroidetes (33% versus 18%, p < 0.01) and a significant decrease in 422 Actinobacteria (6% versus 11%, p = 0.02) and Proteobacteria (0.14% versus 12%, p < 0.01) as compared to mice on the control diet (Fig. 5c) abundance. The heat map (Fig 6a, S3 Table)  and control diet groups with each other. Red color depicts higher abundance; yellow color depicts 448 lower abundance. The details of abbreviated lineages have been provided in S1 and S2 Tables.

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The alterations in microbial compositions were also observed in the LC group after the 450 onset of tumors (Fig 5d). BSp treatment led to a significant decrease in levels of Proteobacteria with the control-fed mice. The heatmap (Fig 6b) indicated the augmentation of bacteria of genus 456 Allobaculum in BSp-fed and combination-fed diet groups when compared to the control group.

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There were several significantly different bacterial taxa between dietary treatments after correction 458 for multiple comparisons with the FDR correction (<0.05) (S1 File). Table 1  In the LC group (Fig 7b), the BSp-fed mice showed a no effect on the levels of SCFAs in

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Adlercreutzia is a gram-positive, strict anaerobic bacterium, which has been considered to exhibit 526 beneficial effects and possess immunoregulatory properties [64]. In addition, Adlercreutzia is an 527 equol-producing bacterium, which has been isolated from the gut of animals and humans [65]. In the LC group after the onset of tumor, we found a significant increase in S24-7 family,

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Lactococcus and Adlercreutzia genus at taxonomic levels induced by our dietary treatments. In and that may be potentially beneficial for health and in cancer prevention.

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The relationship between commensal communities residing in the gut and host physiology