Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

Abstract Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.

(A) Schematic design for treatment regimen.Balb/c mice were injected with 3x10 4 4T1 cells at the fourth mammary fat pads of mice (n=6 for each group).Mice were treated with BFT-1 (1mg/kg) or PBS (CTRL) by intratumoral injection (IT) every two days for eight times in total.Docetaxel (DTX, 15 mg/kg) was given by intraperitoneal injection (IP) every other day starting at day 14 (n=6 for each group).

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Figure.S2 The beta diversity analysis of T and PT from four CR or five NR patients to TNC.
(B) The tumor sizes of 4T1 cell allograft were measured every three days (n=6) and the tumor growth curve was shown (All values were presented as mean ± SEM, * p< 0.05, *** p< 0.001, ns no significance).(C) The tumor weights of 4T1 cell allograft at the end of experiments were analyzed and graphed.The bar graph was presented as mean ± SEM, * p< 0.05, **** p< 0.0001, ns no significance.(D) The percentage of ALDH + BCSCs was detected by ALDEFLUOR assay in tumor cells at the end of experiments.The bar graph was presented as mean ± SEM, * p< 0.05, **** p< 0.0001, ns no significance.

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Figure.S4 ETBF promotes the tumor growth of 4T1 cells in Balb/c mice.(A) Schematic design for treatment regimen.Balb/c mice were treated with ATBs via DW, and infected with ETBF (1x10 9 CFU) or NTBF (1x10 9 CFU) by IG every other day for six times.Mice were injected with 3x10 4 4T1 cells at the fourth mammary fat pads of mice (n=6 for each group).(B) The sizes of 4T1 cell allograft tumors treated with ETBF or NTBF were measured every other day in Balb/c mice and the tumor growth curve was shown.Mice were infected with ETBF (1x10 9 CFU), NTBF (1x10 9 CFU) or water (CTRL) by intragastric gavage (IG) every two days for six times in total (All values were presented as mean ± SEM, * p< 0.05, ns no significance vs CTRL).(C) The weights of 4T1 allograft tumors in Balb/c mice treated with ETBF or NTBF were shown.The bar graph was presented as mean ± SEM, ** p< 0.01, ns no significance.(D) The percentage of ALDH + cells were detected by ALDEFLUOR assay in tumor cells from 4T1 cell allograft tumors treated with ETBF or NTBF.The bar graph was presented as mean ± SEM, ** p< 0.01, ns no significance.

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Figure.S5 Pathway enrichment signatures are assessed by gene set enrichment analysis (GSEA) in T compared to PT. Pathway enrichment signatures were assessed by gene set enrichment analysis (GSEA) in T compared to PT from both NR and CR to TNC.The rows stated to canonical pathways from the Reactome Pathway Database and the columns stated to tissue samples.The blue or red color of each cell referred to the -log10 (p-value).

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Figure.S6 The efficiency of NOD1 overexpressing or knockdown in breast cancer cells.(A) The protein level of NOD1 was detected by western blotting in EM or NOD1overexpressing breast cancer cells.(B) The mRNA expression level of NOD1 was detected by qRT-PCR in EM or NOD1-overexpressing breast cancer cells.The bar graph was presented as mean ± SEM of three biological independent experiments, **** p< 0.0001.(C) The protein level of NOD1 was detected by western blotting in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) breast cancer cells.(D) The mRNA expression level of NOD1 was detected by qRT-PCR in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) breast cancer cells.The bar graph was presented as mean ± SEM of three biological independent experiments, **** p<

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Figure.S9 Non-toxigenic Bacteroides fragilis (NTBF) has no effects on breast cancer cell stemness.(A) The percentage of ALDH + cells was detected by ALDEFLUOR assay in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) breast cancer cells treated with or without NTBF.The bar graph was presented as mean ± SEM of three biological independent experiments, ns no significance.(B) The mRNA expression levels of stemness genes SOX2, OCT4 and NANOG were detected by qRT-PCR in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) breast cancer cells treated with or without NTBF.The bar graph was presented as mean ± SEM, * p< 0.05, ** p< 0.01, ns no significance.

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Figure.S10 The mRNA expression level of NOD1 was not affected by BFT-1 in breast cancer cells.The mRNA expression levels of NOD1 were detected by qRT-PCR in MDA-MB-231 cells treated with or without 10 nM BFT-1.The bar graph was presented as mean ± SEM of three biological independent experiments, ns no significance.

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Figure.S11 NOD1 promotes cancer cell stemness and chemoresistance.(A) The cell proliferation was analyzed by MTT assay in EM or NOD1overexpressing breast cancer cells All values between both groups were presented as mean ± SEM, ** p< 0.01.(B) The percentage of ALDH + cells was detected by ALDEFLUOR assay in EM or NOD1-overexpressing breast cancer cells.The bar graph was presented as mean ± SEM of three biological independent experiments, ** p< 0.01.(C) The self-renewal ability was determined by primary mammosphere formation assay (sphere number and diameter) in EM or NOD1-overexpressing breast cancer cells.The bar graph was presented as mean ± SEM of three biological independent experiments, ** p< 0.01, *** p< 0.001, **** p< 0.0001.(D) The self-renewal ability was determined by secondary mammosphere formation assay (sphere formation efficiency) in EM or NOD1-overexpressing breast cancer cells.The bar graph was presented as mean ± SEM of three biological independent

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Figure.S14 NUMB expression level is shown in ALDH -or ALDH + cells.(A) Representative images of NUMB -or NUMB + cells were shown by immunofluorescence staining.(B) The percentages of NUMB + and NUMB -cells in ALDH -or ALDH + cells were shown.Sorted ALDH + or ALDH -cells from EM or NOD1-overexpressing SUM159cells were plated at one cell per well in 96-well plate.After 18 hours, single cells were stained for DAPI (blue), Tubulin (green) and NUMB (red).One representative result of three independent experiments with cells was shown in the bar graph.

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Figure.S15 The mRNA expression level of NUMB was not affected by NOD1 in breast cancer cells.(A) The mRNA expression level of NUMB was detected by qRT-PCR in EM or NOD1-overexpressing SUM159 cells.The bar graph was presented as mean ± SEM of three biological independent experiments, ns no significance.(B) The mRNA expression level of NUMB was detected by qRT-PCR in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells.The bar graph was presented as mean ± SEM of three biological independent experiments, ** p< 0.01.

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Figure.S16 Identification of NOD1-binding proteins.(A) A representative image of silver staining after Co-Immunoprecipitation was shown in FLAG-tagged NOD1-overexpressing SUM159 cells.(B) Candidate proteins interacting with NOD1 were identified by a liquid chromatography-tandem mass spectrometry (LC/MS) analysis.(C) GAK peptides were identified in the Co-Immunoprecipitation samples of FLAGtagged NOD1-overexpressing SUM159 cells by a LC/MS analysis.

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Figure.S19 MNZ efficiently eliminates ETBF in mice.ETBF clearance efficiency was analyzed by qRT-PCR in ETBF-pretreated Balb/c mice after being treated with Metronidazole (MNZ, 25 mg/kg) by IG for consecutive four days.

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Figure.S20 Simultaneous NOD1 inhibition and ETBF clearance improve the chemosensitivity in breast cancer.(A) ETBF abundances and NOD1 expression in PDX Lsl17 were measured by FISH and IHC respectively.(B) Schematic design for treatment regimen.PDX Lsl17 mice were treated with MNZ (25 mg/kg) by IG every two days for four times in total and treated with MNZ (0.5 g/L) via DW throughout the experiment.ETBF (1x10 9 CFU) was infected via IG for six times at day 12 (n=5 for each group).(C) The tumor size was measured every three days and the tumor growth curve were shown for PDX Lsl17 tumors in nude mice treated with ETBF (n=5).All values were presented as mean ± SEM, **** p< 0.0001.(D) The Hematoxylin and Eosin (H&E) histology images were shown for different organs.