Polyphenol-rich Muscadine Grape Extract Reduces Triple Negative Breast Cancer Metastasis in Mice with Changes in the Gut Microbiome

Triple negative breast cancer (TNBC) has a high propensity to metastasize and no treatments are available to slow or 3 prevent metastatic progression. The goal of this study is to determine whether a proprietary high-polyphenol content 4 muscadine grape extract (MGE) inhibits TNBC metastasis. 4T1 TNBC cells were injected into the mammary fat pad of 6-week-old female Balb/c mice. After 2 weeks, tumors 8 were surgically removed and mice were placed into a control (n=8) or treatment group that received 0.1 mg/mL total 9 phenolics MGE in the drinking water (n=8) for 4 weeks. Immunohistochemistry (Ki67, α-SMA) and hemotoxylin 10 and eosin staining were used to quantify metastases. Gut microbial composition was determined by 16S rRNA 11 sequencing and short chain fatty acids (SCFAs) were detected by gas chromatography. MDA-MB-231, BT-549 and 12 4T1 TNBC cell motility and cytoskeletal organization was assessed in vitr o by scratch wound migration and 13 confocal microscopy, respectively. Data were evaluated by student’s t -test. For the first time, we showed that MGE inhibits TNBC metastasis in mice concomitant with MGE-induced changes in TNBC cell morphology and migration as well as the gut microbiota, which may mediate anti-cancer effects of MGE. In an ongoing Phase I clinical trial in patients with solid tumors, MGE has a favorable safety 314 profile suggesting that the extract can be administered to patients at concentrations similar to those used in our 315 studies [80]. Since TNBC patients currently have no therapeutic options to reduce metastatic progression after 316 standard-of-care, MGE may be an effective adjuvant to reduce TNBC metastases.


Antibody verification information is found in Supplemental
. Negative controls with only the secondary 97 antibody were included to account for non-specific binding. Cell positivity was determined using inForm® software 98 (Perkin-Elmer).

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For each animal, five separate tissue sections from the formalin-fixed livers at least 50 μm apart were stained with 102 hemotoxylin and eosin (Newcomer Supply). Images of stained tissues were collected from four separate quadrants of each liver section using the Quantitative Pathology Imaging System Mantra™ microscope (Perkin-Elmer). Liver were monitored for proliferation with the IncuCyte® ZOOM System according to the manufacturer's protocol

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Abcam; Cambridge, UK) was diluted in 5% Blotting-Grade Blocker in Tris-buffered saline (TBS) with 0.1% 156 Tween ® and applied to membranes overnight at 4°C with gentle agitation. Antibody verification information is 157 found in Supplemental Table 1

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Short chain fatty acids (SCFA) were extracted from fecal samples collected from the distal colon of mice at the time 170 of sacrifice using the method previously described [34]. SCFAs were detected by gas chromatography (Trace 1310 171 Gas Chromatograph) using the Thermo TG-WAXMS A GC column (30 m, 0.32 mm, 0.25 µm) coupled to a flame 172 ionization detector (Thermo Fisher) by Microbiome Insights.

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indicating reduced tumor burden (5758 ± 1168 vs 2564 ± 628 pixels, P < 0.05; Fig. 2c, d). Metastases were 194 quantified using an automated analysis mask trained to detect healthy liver, metastases, or background from 195 hematoxylin and eosin stained liver tissues. Serum IL-6 was quantified because increased concentrations of IL-6 are 196 associated with breast cancer liver metastases in humans [36] and a grape powder reduced serum IL-6 in an 197 inflammatory mouse model [37]. Serum IL-6 was negligible in MGE-treated mice compared to serum from control 198 mice (53.6 ± 24 vs 0.23 ± 0.23 pg/mL, P = 0.06; Fig. 2e). Taken together, these results show that MGE reduces 199 TNBC metastasis in vivo.

MGE reduces triple negative breast cancer cell migration and alters cytoskeletal arrangement in vitro 201
The effect of MGE on cell migration was assessed in vitro, as cell motility plays a critical role in the metastatic 202 cascade. Confluent monolayers of TNBC cells were scratched to create a denuded area and cell migration was 203 measured in MDA-MB-231 (Fig. 3a), BT-549 (Fig. 3c), and 4T1 ( Fig. 3e) cells. MGE inhibited migration of 108 ± 16 μm, P < 0.05) and 4T1 cells by 59.6% (203 ± 30.9 vs 82.1 ± 29.5 μm, P < 0.01) (Fig. 3b, d, f). The 206 reduction in migration suggests that MGE reduces TNBC cell motility.

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The effect of MGE on TNBC cell structure was assessed since cytoskeletal reorganization is crucial for cell 208 migration. MGE altered the shape of MDA-MB-231 cells compared to control cells. MGE increased cell area by 209 49%, as quantified by an increase in both cell length and width (P < 0.05; Fig. 4a, b, c), and increased cell 210 eccentricity, indicating that MGE-treated cells are less circular than untreated cells (P < 0.001; Fig. 4d).

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Representative images also show cell polarization in MDA-MB-231 cells (marked by the asterisk in Fig. 4e); in 212 contrast, MGE-treated cells lack directionality or polarity.

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Cell size was reduced by 60% in MGE-treated BT-549 cells compared to control cells, which correlated 214 with reduced cell length and width (P < 0.0001; Fig. 5a, b, c). MGE had no effect on BT-549 cell eccentricity (

MGE inhibits metastasis protein RHAMM in vitro 227
Receptor for hyaluronan-mediated motility (RHAMM), a prominent metastasis related protein that can associate 228 with microtubules and actin filaments to promote cell motility, is implicated in breast cancer cell migration [40,41].

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MGE increased the relative abundance of several genera belonging to the Firmicutes phylum. The feces of and a 2.5-fold increase in Butyricicoccus (P < 0.05) genera compared to the feces of control mice (Fig. 8e, g, h).

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Butyrate reduced 4T1 cell proliferation and migration in a dose-and time-dependent manner (P < 0.05; Fig. 9e,  (Supplementary Fig. 2). These results show that, at physiologically relevant concentrations, butyrate inhibited 4T1 TNBC cell proliferation and migration, which could contribute to the MGE-induced metastasis reduction in vivo.  [48][49][50]. The MGE-275 mediated differential effects on cytoskeletal organization may be regulated by the Rho family GTPase Cdc42, which 276 is a master regulator of cell polarity, or by the WAVE/WASP and Arp2/3 pathway, which participates in actin-277 mediated protrusions to confer changes in cell morphology [51,52]. In addition, punctuated actin staining may 278 result from effects of MGE on actin disassembly [53]. The precise molecular mechanism for the alterations in 279 cytoskeletal organization by MGE is currently under investigation.

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MGE reduced RHAMM and HMMR, the gene encoding RHAMM, in all three TNBC cell lines. RHAMM 281 is a multi-functional protein found on the cell surface, in the cytoplasm, or in the nucleus and participates in cell 282 motility regulation [41]. RHAMM overexpression in primary breast tumors is associated with a poor prognosis and 283 lymph node metastases, suggesting that RHAMM has an important role in breast cancer metastasis [42]. RHAMM 284 can associate with ERK1/2, Src, and FAK to promote cell migration [40,[54][55][56][57][58][59] and loss of RHAMM reduces focal adhesion turnover, filopodia, and cell locomotion [40,58,59]. Therefore, the reduction of RHAMM by MGE in 286 TNBC cells could lead to the reduction in cell migration and metastasis.

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MGE reduced Ki67 in the lungs and livers of mice with TNBC metastasis in association with effects on the 288 gut microbiota. Polyphenols alter the gut microbiome and are metabolized by the microbiota into distinct bioactive 289 compounds [21,60,61]. MGE increased alpha diversity, indicating that the extract improved gut health by 290 increasing robustness and functional capability [62,63]. Likewise, MGE increased the F/B ratio, which improves 291 the health of the mice by increasing the ability of the gut microbiota to extract energy from the diet to reduce weight 292 loss and increase overall fitness [45,[64][65][66].

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MGE also increased the relative abundance of butyric acid in fecal samples compared to control mice, 294 which may be more beneficial than changes in total SCFA [67][68][69][70]. MGE significantly reduced acetic and propionic 295 acid, which are the main products of the Bacteroidetes phylum [71]. The reduction in acetic and propionic acid resulted in an increase in the relative abundance of butyric acid. Acetate, propionate, and butyrate associate with the 297 same receptors on the apical membrane of colonocytes, including monocarboxylate transporters and the G-protein 298 coupled receptors (GPR) GPR41/Ffar3 and GPR43/Ffar2. An increase in the relative abundance of butyrate would 299 increase the proportion of butyrate available to the transporters and receptors [72,73]. Consequently, in MGE-300 treated mice, butyrate may outcompete acetate and propionate for these receptors to cause a greater biological 301 impact.

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Compared to acetate and propionate, butyrate has the most potent anti-inflammatory effects and thus may 303 contribute to the reduction in serum IL-6 in MGE-treated mice [74]. Butyrate modulates gene expression through 304 inhibition of histone deacetylases and reduces cancer cell proliferation and migration [75][76][77]. Since butyrate can 305 travel through the portal vein to have direct contact with the liver, MGE-induced increases in butyrate may 306 contribute to the reduction in liver metastatic tumor burden [78]. Furthermore, butyrate concentrations in the blood For the first time, we showed that MGE inhibits TNBC metastasis in mice concomitant with MGE-induced