Muscadine Grape Extract Reduces Lung and Liver Metastasis in Mice with Triple Negative Breast Cancer in Association with Changes in the Gut Microbiome (P05-017-19)

Abstract

Objectives The goal of this study was to determine if a proprietary muscadine grape seed and skin extract (MGE) inhibits triple negative breast cancer (TNBC) metastasis and alters the gut microbiota. Methods 4T1 TNBC cells were injected into the mammary fat pad of 6-week-old female Balb/c mice. After 2 weeks, tumors were surgically removed and mice were placed into a control group (n = 8) or a treatment group that received 0.1 mg/mL total phenolics MGE (Piedmont R&D) in the drinking water (n = 8). Mice were sacrificed after 4 weeks; tissues and fecal samples were collected for analysis. Immunohistochemistry (Ki67, α-SMA) and hemotoxylin and eosin staining were used to quantify metastases using the inForm© 2.2 software. Gut microbial composition was determined by 16S rRNA sequencing and short chain fatty acids were detected by gas chromatography (Microbiome Insights). Data are expressed as means ± SEM using student's t-test. Results MGE reduced Ki67 cell positivity in the lungs and livers of mice, indicating reduced metastatic proliferation (9.3 ± 0.9% vs 6.2 ± 0.7% and 5.0 ± 1.5% vs 0.77 ± 0.2% cells, respectively; P < 0.01), and decreased cancer associated fibroblasts in the lungs (5.3 ± 1.0% vs 3.0 ± 0.5% cells; P < 0.05), which are associated with metastasis. MGE significantly reduced the number (4.7 ± 0.7 vs 2.2 ± 0.4 tumors/field; P < 0.01) and size (1358 ± 48 vs 1121 ± 47 pixels; P < 0.01) of liver metastases, resulting in decreased metastatic tumor burden (6656 ± 1220 vs 3096 ± 644 total area in pixels; P < 0.01). Attenuated TNBC metastasis correlated with MGE-induced changes in gut microbiota. Alpha diversity (4.15 ± 0.10 vs 4.51 ± 0.13 Shannon index; P < 0.05) and the Firmicutes to Bacteroidetes ratio (0.37 ± 0.07 vs 0.76 ± 0.12; P < 0.05) were significantly increased in MGE-treated mice, indicating enhanced microbial richness and increased energy harvest by the gut microbiome. Butyrate-producing bacteria, such as Ruminococcus, Butyricicoccus and Lachnospiraceae, were increased with MGE (P < 0.05) as well as the anti-inflammatory compound butyrate relative to other short-chain fatty acids (25.0 ± 2.7% vs 75.3 ± 15.5%; P < 0.01). Conclusions These data show that MGE attenuates TNBC metastasis in association with alterations in the gut microbiome, suggesting that MGE may be an effective treatment against TNBC metastatic progression. Funding Sources Chronic Disease Research Fund.

tRNA Queuosine Modification Enzyme Modulates the Growth and Microbiome Recruitment to Breast Tumors

“…Our data on the reduced butyrate-producing genera Lachnospiraceae and Ruminococcaceae in the mice with tumor growth support the role of intestinal dysbiosis in promoting breast cancer. Bacteria that enhance producing butyric acid are known to protect the body from colon cancer [22] and triple-negative breast cancer (TNBC) [23]. Intestinal Lactobacillus are frequently correlated with diseases, like Crohn's disease [24], obesity, type 2 diabetes, cancer [25], and breast cancer tumor development [26].…”

“…Several core bacteria were markedly changed in mice post cell injection, such as Lachnospiraceae, Lactobacillus, and Alistipes (Figure 4e; Figures S5 and S6). The butyrate-producing genera Lachnospiraceae and Ruminococcaceae, where the genus could enhance producing butyric acid to protect the body from colon cancer [22] and triple-negative breast cancer (TNBC) [23], were found suppressed in the mouse model post breast cancer cell injection. The proportions of Lactobacillus, one of the common colonic microbiota, are frequently correlated with intestinal diseases like Crohn's disease [24], obesity, type 2 diabetes, and cancer [25], and breast cancer tumor development [26] was found decreased as well.…”

tRNA queuosine modification enzyme modulates the growth and microbiome recruitment to breast tumors

“…Bacteria which enhance producing butyric acid are known to protect the body from colon cancer [19] and triple negative breast cancer (TNBC) [20], Our data on the reduced butyrate-producing genera Lachnospiraceae and Ruminococcaceae in the mice with breast cancer cells support the role of intestinal dysbiosis in promoting breast cancer. Intestinal Lactobacillus are frequently correlated with intestinal diseases, like Crohn's Disease (CD), obesity, type 2 diabetes and cancer [21], and breast cancer tumor development [22].…”

“…Several core bacteria were markedly changed in mice post cell injection, such as Lachnospiraceae, Lactobacillus and Alistipes (Figure 4e; Figure S5-6). The butyrate-producing genera Lachnospiraceae and Ruminococcaceae which may enhance producing butyric acid to protect the body from colon cancer [19] and triple negative breast cancer (TNBC) [20], was found suppressed in the mouse model post breast cancer cells injection. The proportions of Lactobacillus, one of the common colonic microbiota, are frequently correlated with intestinal diseases, like Crohn's Disease (CD), obesity, type 2 diabetes and cancer [21], and breast cancer tumor development [22], were found decreased as well.…”

Phase I Study of Muscadine Grape Extract for Patients With Advanced Cancer

“…18 In addition, exposure to MGE reduced the migration of breast cancer cells, 19 and in animal models of breast cancer, reduced the number and size of liver and lung metastases. 20 Further, MGE in combination with trastuzumab inhibited the proliferation of trastuzumab-sensitive breast cancer cells to a greater extent than either agent alone. 21 This work is consistent with other studies using varied muscadine grape preparations showing reduction in tumor proliferation, 22 increase in tumor cell cycle arrest and apoptosis, 3,4,6 and decreased cell migration 23 in preclinical studies.…”