FIELD OF THE INVENTION

The invention relates to the field of diagnosis and treatment of early stage breast cancer. Specifically, a method is provided for determining whether a subject has early stage breast cancer by measuring the bacterial tna oper- on in a sample from the subject. Specifically, bacterial nucleic acid coding for the TnaA peptide or the TnaA peptide is measured. A method for mitigating breast cancer promotion and/or progression in a subject is also provided.

BACKGROUND OF THE INVENTION

Dysbiotic microbiota, associated with oncological diseases, is termed the oncobiome. Oncobiosis of the distal gut is associated with breast cancer [1-11]; importantly, among the oncobiome studies common microbiome changes were identified [12, 13] suggesting a common root for the pathogenic role of the oncobiome. It remained a question whether oncobiotic transformation of the microbiome has a causative role in carcinogenesis in breast cancer or if it is an epiphenomenon. Bacterial metabolites can provide a link between the microbiome and cancer cells through the circulation [34]. Among these metabolites, short chain fatty acids, lithocholic acid and cadaverine have cytostatic properties [27, 28, 34, 39]. In breast cancer, the metabolic capacity of the microbiome is suppressed [4, 27], suggesting that the production of cytostatic metabolites decrease in breast cancer. Indolepropionic acid (IP A) and indoxyl sulphate are bacterial metabolites produced by the deamination of tryptophan [28, 40]. Approximately, 4-6% of tryptophan is metabolized to indole-derivatives through bacterial enzymes [41]. In germ-free mice, serum tryptophan levels increase, highlighting the burden of microbial degradation on tryptophan levels [42-46]. Tryptophanase (TnaA) [40] is a key enzyme in converting tryptophan into indole derivatives. Its gene can be found in the tryptophanase operon of which TnaA denotes tryptophanase enzyme. In the pathway that yields IPA, tryptophan is converted to indole lactate, then to indole acrylic acid, and subsequently to IPA [48]. Indoxyl sulphate (IS) is a metabolite of tryptophan [49]. Bacterial metabolism converts tryptophan to indole [49-51] that subsequently enters the systemic circulation. Indole is hydroxylated by Cyp2el and sulfated by SULT1 and SULT2 enzymes in the liver [49]. IS reenters circulation and is excreted through the kidneys. Indole-derivatives can activate the aryl hydrocarbon receptor [49,52].

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method for determining whether a subject has breast cancer, comprising determining the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject, wherein a decrease in the level of said nucleic acid sequence and/or gene product is indicative of the presence of breast cancer in the subject.

A method is provided for determining whether a subject is at an increased risk of developing breast cancer, comprising determining the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject, wherein a decrease in the level of said nucleic acid sequence and/or gene product is indicative of the presence of an increased risk of developing breast cancer.

Preferably the nucleic acid sequence is a nucleic acid sequence coding for tryptophanase (TnaA) or a portion thereof. Preferably the gene product is TnaA protein or a portion thereof. In another preferred embodiment the gene product is RNA.

In another aspect indolepropionic acid or a pharmaceutically acceptable salt thereof for use in preventing or mitigating initiation of breast cancer is provided. Also, indolepropionic acid or a pharmaceutically acceptable salt thereof for use in mitigating the promotion and/or progression of breast cancer in a subject is provided.

In another aspect indoxyl sulphate or a pharmaceutically acceptable salt thereof for use in preventing or mitigating initiation of breast cancer is provided. Also, indoxyl sulphate or a pharmaceutically acceptable salt thereof for use in mitigating promotion and/or progression of breast cancer in a subject is provided.

In yet another aspect the invention provides a method for mitigating promotion and/or progression of breast cancer in a subject, comprising determining the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject, administering indolepropionic acid or a pharmaceutically acceptable salt thereof and/or indoxyl sulphate or a pharmaceutically acceptable salt thereof to the subject to provide or restore physiological serum concentration of indolepropionic acid and/or indoxyl sulphate if the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in the sample is lower than the corresponding reference value, and optionally monitoring the presence of breast cancer in the subject and/or the serum level of indolepropionic acid and/or indoxyl sulphate in said subject, and if necessary, administering another anticancer therapy together or without the administration of indolepropionic acid or a pharmaceutically acceptable salt thereof and/or indoxyl sulphate or a pharmaceutically acceptable salt thereof.

In yet another aspect the invention provides a method for preventing breast cancer or mitigating initiation of breast cancer in a subject, comprising determining the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject, administering indolepropionic acid or a pharmaceutically acceptable salt thereof and/or indoxyl sulphate or a pharmaceutically acceptable salt thereof to the subject to provide or restore physiological serum concentration of indolepropionic acid and/or indoxyl sulphate if the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in the sample is lower than the corresponding reference value, and optionally monitoring the presence of breast cancer in the subject and/or the serum level of indolepropionic acid and/or indoxyl sulphate in said subject, and if necessary, administering another anticancer therapy together or without the administration of indolepropionic acid or a pharmaceutically acceptable salt thereof and/or indoxyl sulphate or a pharmaceutically acceptable salt thereof.

In yet another aspect a method is provided for promoting selection of the appropriate anticancer therapy for a patient having breast cancer, wherein a) the level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject is determined and optionally al) the number and characteristics of tumor infiltrating lymphocytes (TILs) are determined from a tumor sample obtained from the patient, b) the appropriate anticancer therapy is selected based on the value obtained in a) and optionally on the value and characteristics obtained in al), wherein the level of the nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or the level of the gene product of a gene of a bacterial tna operon correlates and/or is indicative, preferably proportional of the number of TILs.

In yet another aspect a method is provided for predicting survival for a patient having breast cancer, wherein lower expression of fecal TnaA predicts shorter survival time.

In any one of the aspects of the invention the nucleic acid sequence is preferably a nucleic acid sequence coding for tryptophanase (TnaA) or a portion thereof. In any one of the aspects of the invention the gene product is preferably TnaA protein or a portion thereof. In any one of the aspects of the invention in another preferred embodiment the gene product is RNA.

In any one of the aspects of the invention the bacterial tna operon is preferably from bacteria forming part of the gut microbiota, preferably of the healthy gut microbiota.

In any one of the aspects of the invention the bacterial tna operon is preferably from Providencia spp, Alistipes spp, Bacteroides spp, Clostridium spp, Escherichia spp.

In any one of the aspects of the invention the bacterial tna operon is preferably from Providencia rettegri, Alistipes shahii, Bacteroides xylanisolvens, Escherichia coli or any combination thereof.

In any one of the aspects of the invention the breast cancer is preferably stage 0 (in situ carcinoma) breast cancer according to the American Joint Committee on Cancer (AJCC) TNM system. Preferably, the cancer is stage 0 breast cancer and the test sample is compared to a reference value typical of the absence of stage 0 breast cancer, preferably to a reference value derived from healthy individuals (i.e. individuals not having breast cancer).

In any one of the aspects of the invention, the subject is preferably a human, preferably a female, more preferably a post-menopausal woman.

In any one of the aspects of the invention, the sample preferably comprises gut microbiota. Preferably the sample is feces.

In any one of the aspects of the invention, the level of the nucleic acid sequence from a bacterial tryptophanase (tna) operon, the level of a gene product of a gene of a bacterial tna operon, the level of IPA and the level of IS in the sample from the subject is compared to a corresponding reference value derived from the appropriate corresponding sample(s) from individuals not having the breast cancer, preferably from healthy individuals.

In another aspect a pharmaceutical composition comprising indolepropionic acid or a pharmaceutically acceptable salt thereof and/or indoxyl sulphate or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient or carrier is provided. Preferably the pharmaceutical composition is for use in preventing breast cancer, mitigating breast cancer initiation, mitigating breast cancer promotion or mitigating breast cancer progression. Preferably the pharmaceutical composition is administered to provide or restore physiological serum concentration of indolepropionic acid and/or indoxyl sulphate in a subject having in situ breast cancer or a lower level of a nucleic acid sequence from a bacterial tryptophanase (tna) operon and/or a lower level of a gene product of a gene of a bacterial tna operon. In preferred embodiments the pharmaceutical composition is used with another anticancer agent(s), preferably in combination with an estrogen receptor antagonist compound, such as tamoxifen or fulvestrant.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Supplementation of mice transplanted with 4T1 breast cancer cells with indolepropionic acid (IP A) reduces metastatic burden. Female Balb/c mice (n=10/10, 3 months of age) were grafted with 4T1 cells and were treated with IPA (1 nmol/g q.d. p.o.) or vehicle (VEH) (n=10/10) for 14 days then the mice were terminated. Upon autopsy (A) the total number and (B) total and (C) individual mass of primary tumors were determined. (D) The infiltration of the primary tumors to the surrounding tissues was scored. The (E) number of mice with metastasis were plotted and the (F) total, as well as, the (G) individual mass of the metastases were measured. In tissue sections of formalin-fixed, paraffin-embedded tissue specimens from the primary tumors (H) lymphocyte infiltration and (I) mitosis score were determined. Statistical significance was determined using Student’s t-test, except for panels D, E and I, were chi-square tests were conducted. *** indicate statistically significant difference between vehicle and treated groups at p <0.001.

Figure 2. . Indolepropionic acid (IPA) reduces cell proliferation in cellular models of breast cancer. (A) 500 cells/well 4T1 cells were seeded in 6 -well plates and were then treated with IPA in the concentrations indicated for 7 days then colonies were stained according to May-Grunwald-Giemsa that were analyzed using the ImageJ software (n=3). (B) 4T1 cells were seeded in 6-well plate (75.000 cell/well) and treated with the indicated concentrations of IPA for 24 hours and stained with propidium-iodide then analyzed by flow cytometry. (C) 4T1 cells (75.000 cell/well in 6 well plates) were treated with IPA in the concentrations indicated for 24 hours the ratio of necrotic and apoptotic cells were determined with staining by propidium-iodide - FITC Annexin double staining using the V/Dead Cell Apoptosis Kit and measured by flow cytometry (n=3). (D) SKBR-3 (5.000 cell/well) were seeded in 96-well plates and were then treated with IPA in the concentrations indicated for 24 hours then total protein content was assessed in SRB assays (n=3). (E) SKBR-3 (200.000 cell/well) cells were seeded in 6-well plates and were treated with IPA in the concentrations indicated for 24 hours the ratio of necrotic and apoptotic cells were determined with staining by propidium-iodide then analyzed by flow cytometry. (F) SKBR-3 (200.000 cell/well) cells were seeded in 6-well plates and were treated with IPA in the concentrations indicated for 24 hours the ratio of necrotic and apoptotic cells were determined with staining by propidium-iodide - FITC Annexin double staining using the V/Dead Cell Apoptosis Kit and measured by flow cytometry (n=3). (G) Human fibroblast (7500 cell/well) cells were seeded in 96-well plates and were then treated with IPA in the concentrations indicated for 24 hours then total protein content was assessed in SRB assays (n=3). (H) Human fibroblast (200.000 cell/well) cells were seeded in 6-well plates and were then treated with IPA in the concentrations indicated for 24 hours then the ratio of necrotic and apoptotic cells were determined with staining by propidium-iodide then analyzed by flow cytometry (n=3). Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Tukey’s post-hoc test, except for panels D, E and F, were Dunnett’s post-hoc tests were conducted. * or *** indicate statistically significant difference between control and treated samples at p <0.05 or p<0.001, respectively.

Figure 3. Indolepropionic acid (IPA) induced oxidative stress, cellular energy stress and decreased the proportions of cancer stem cells. 500.000 cell/well 4T1 cells were treated with IPA in the concentrations indicated for 24 hours then (A) lipid peroxidation was measured by TBARS assay and (B) 4HNE expression was assessed by Western blotting (representative figure, n=3). In the same cells (C) the protein expression of NRF2 and iNOS were determined by Western blotting (n=3), while (D) the mRNA expression of catalase (cat) was determined by RT-qPCR (n=3). (E) The expression of the indicated proteins (pACC, ACC, FOXO1 and PGC-i ) were determined by Western blotting. (F) 100.000 cell/well 4T1 cells were treated with the indicated concentration of IPA for 24 hours then the proportions of aldehyde dehydrogenase-positive cells were determined in Aldefluor assays using flow cytometry (n=3). For Western blots a typical experiment was displayed. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Dunnett’s post-hoc test, except for panel F, where Student’s t-test was used. *,** and *** indicate statistically significant difference between control and treated samples at p <0.05, p <0.01 and p <0.001, respectively.

Figure 4. Indolepropionic acid (IPA) induced mesenchymal-to-epithelial transition (MET). 100.000 cell/well 4T1 cells were treated with IPA in the concentrations indicated for 24 hours then (A) cellular morphology was observed using Texas Red-X Phalloidin and DAPI staining (representative figure, n=3). Scale bar corresponds to 25 pm. Mesenchymal cells are characterized by stress filaments that are absent in epithelial cells; for details on morphology see [27] Fig. S3. (B) Normalized resistance was measured in ECIS impedance based experiments (representative figure, mean ± SD, n = 1). (C-D) In IPA-treated 4T1 cells the expression of the indicated genes were determined in (C) RT-qPCR (n=3) and (D) Western blotting (n=3). 0-actin was used as a loading control. For Western blots a typical experiment was displayed. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Dunnett’s post -hoc test, except for panel D, where chi-square test were conducted. * and ** indicate statistically significant difference between control and treated samples at p <0.05 or p <0.01, respectively. Abbreviations: Vimentin (Vim), fibroblast growth factor-binding protein 1 (FgfBpl), snail family transcriptional repressor- 1 (Snail) and P- catenin) E-cadherin, zonula occludens-1 (ZO1).

Figure 5. Indolepropionic acid (IPA) -elicited oxidative stress has central role in mediating IPA-elicited antineoplastic effects. 4T1 cells (500.000 cell/well - TBARS, 100.000 cell/well - ALDH, 1.500 cell/well - SRB) were treated with IPA in the concentrations indicated for 24 hours with or without the antioxidants, as indicated. Subsequently, (A) thiobarbituric acid-re active substances, (B) the fraction of Aldefluor -positive cells and (C) total protein content was determined. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Dunnett’s post-hoc test. *, ** and *** indicate statistically significant difference between control and treated samples at p <0.05, p<0.01 or p <0.01, respectively. Abbreviations: TBARS - Thiobarbituric acid-re active substances, ALDH - Aldehyde dehydrogenase, SRB - Sulphorhodamine B, GSH - reduced glutathione, NAC - N-acetyl-cysteine

Figure 6. AHR and PXR are responsible for the IPA-elicited antineoplastic effects. 100.000 cell/well 4T1 cells were treated with IPA in the concentrations indicated for 24 hours with or without the inhibitors, as indicated. Subsequently, (A) the actin cytoskeleton was stained using phallodin-Texas Red and morphology was assessed using confocal microscopy. On the same cells (B) lipid peroxidation was determined by TBARS assays and (C) the expression of the indicated proteins were determined by Western blotting. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Dunnett’s post-hoc test, except for panel A, where chi-square test were conducted. *, ** and *** indicate statistically significant difference between control and treated samples at p <0.05, p<0.01 or p <0.01, respectively. Abbreviations: n.s. - non-significant, PXRi - PXR inhibitor, AHRi - AHR inhibitor, ACC - acyl-CoA carboxylase (an AMPK target protein).

Figure 7. Higher expression of Pregnane X Receptor and Aryl Hydrocarbon Receptor prolongs survival in breast cancer. The effect of expression of (A) AHR or (B) PXR on survival in breast cancer was analyzed by kmplot.com, a freely accessible database. On panel (A) effect of PXR expression on survival is depicted with the data acquired from RNAseq experiments. On panel (B) effect of PXR expression on survival is depicted with the data acquired from microarray experiments and patients were stratified as a function of receptor expression. On panel (C) effect of AHR expression on survival is depicted with the data acquired from RNAseq experiments. On panel (D) effect of AHR expression on survival is depicted with the data acquired from microarray experiments and patients were stratified as a function of receptor expression. Total survival rates were assessed and all samples are represented.

Abbreviations: NR1I2 - pregnane X receptor, Aryl hydrocarbon receptor (AHR), Pregnane X receptor (PXR). The database was assessed the 19th February 2020.

Figure 8. Higher intratumoral expression of Pregnane X receptor (PXR) and nuclear Aryl hydrocarbon receptor (AHR) shows correlation with low-grade and lower-mitosis breast cancers. (A) TMA was stained with the indicated antibodies. A typical staining pattern is shown. The bar is equivalent of 50 pm. Cases in TMA were scored for receptor expression using the H-score system. (B-E) H-score of the nuclear expression of the AHR receptor were related to (B) stage of the disease, (C) mitosis score, (D) tubule formation, (E) histological subtype. (F-H) H-score of the expression of PXR receptor were related to (F) grade of the disease, (G) mitosis score and (H) mitotic index. Statistical significance on panels B and F was determined using ANOVA test followed by Dunnett’s post-hoc test, while on panels C, D, E, G and H Student’s t-test was used. Stage 0 (in situ carcinoma) and stage 1 patients, Mitosis score 2 and 3 patients and Tubule formation score 1 and 2 patients were handled together due to low number of cases. Abbreviations: Aryl hydrocarbon receptor (AHR), Pregnane X receptor (PXR), no special type (NOS).

Figure 9. The fecal expression of TnaA shows correlation with the pathological and clinical features of breast cancer. (A-B) The abundance of bacterial TnaA DNA was assessed in human fecal DNA samples from cohort study. The ct values lower than 45 are shown in Providencia rettgeri. Median values indicated by a line. On panel A all patients and controls are compared, statistical significance was calculated using Student’s t-test. On panel B patients were stratified based on the stage of the disease and statistical comparison was made using ANOVA test followed by Dunnett’s or Tukey’s post-hoc tests. Fold data were log2 transformed to achieve normal distribution.

*,** and *** indicate statistically significant difference between control and treated samples at p <0.05, p <0.01 and p <0.001, respectively.

Figure 10. IS treatment in tumor-bearing mice reduces the infiltration capacity of tumors to the surrounding tissues and reduces metastatic capacity

Female Balb/c mice were grafted with 4T1 cells and treated with IS (2 pmmol/kg q.d. p.o.) or vehicle (VEH) (n= 10/10) for 14 days before sacrifice. Upon autopsy (A) the total number of primary tumors and the (B) total and (C) individual mass of primary tumors were measured. (D) The tumor infiltration rates into the surrounding tissues were scored. (E) The number of mice with metastases, (F) the total mass of metastases, and (G) the individual mass of metastases are shown.

Numerical values are represented as average ± SEM. Statistical significance was calculated using Student’s t- test (two-tailed) except for panel D, where a Chi-square test was used.

* and *** indicate statistically significant differences between vehicle and IS groups at p <0.05 and p <0.001, respectively.

Figure 11. IS has cytostatic properties without affecting cell death

(A) 4T1 (1500 cells/well), MCF7 (4000 cells/well), SKBR-3 (5000 cells/well), MDA-MB 231 (3000 cells/well), ZR75-1 (3000 cells/well), and (E) human fibroblasts (7500 cells/well) were seeded in 96-well plates and treated with IS at the concentrations indicated for 24 hours. Total protein content was evaluated using Sulphorhodamine B assays (n=3). (B) 4T1 cells (500 cells/well) were seeded in 6-well plates and treated with IS at the indicated concentrations for 7 days. Colonies were stained according to May-Grunwald-Giemsa and counted using ImageJ software (n=3). (C) 4T1 (75,000 cells/well); MCF7 (150,000 cells/well); SKBR-3 (200,000 cells/well), and (F) human fibroblasts (200,000 cells/well) were seeded in 6-well plates and treated with the indicated concentrations of IS for 24 hours. Cells were stained with propidium-iodide and analyzed by flow cytometry (n=3). (D) Cells were treated with IS in the concentrations indicated for 24 hours. The ratios of necrotic and apoptotic cells were determined by double staining with propidium-iodide and FITC Annexin, using the V/Dead Cell Apoptosis Kit, and subjected to flow cytometry (n=3).

Numerical values are represented as average ± SEM. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined on panel A, B, and E by one-way ANOVA followed by Dunnett’s post-hoc tests; all samples were compared to controls. On panels C, D, and F ANOVAs were conducted followed by Tukey’s post-hoc tests.

*, **, and *** indicate statistically significant differences between control and treated samples at p <0.05, p<0.01, and p <0.01, respectively.

Figure 12. Indoxyl sulfate treatment induces oxidative and nitrosative stress (A) 4T1 cells (500,000 cells/well) were treated with IS at the concentrations indicated for 24 hours. Lipid peroxidation was measured using TBARS assays (n=3) and (B) 4HNE expression was determined by Western blotting (representative figure, n=3). (C) Nitrotyrosine was detected by Western blotting (representative figure, n=3). In the same cells (D) the protein levels of iNOS and NRF2 were determined by Western blotting (representative figure, n=3). (E) The mRNA expression levels of the indicated genes were determined by RT-qPCR (n=3).

Numerical values are represented as average ± SEM. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA followed by Durmett’s post-hoc test, where all values were compared to control.

* and ** indicate statistically significant differences between control and treated samples at p <0.05 and p <0.01, respectively.

Abbreviations: thiobarbituric acid reactive substances (TBARS); 4-hydroxynoneal (4HNE); nitro-tyrosine (NTyr); inducible nitric oxide synthase (iNOS); nuclear factor 2 (NRF2); glutathione peroxidase 2 (GPX2); glutathione peroxidase 3 (GPX3); superoxide dismutase 3 (SOD3); catalase (CAT).

Figure 13. IS treatment induces mesenchymal-to-epithelial transition and blocks cellular migration

(A) 4T1 cells (100,000 cells/well) were treated with IS in the concentrations indicated for 24 hours then cellular morphology was observed using Texas Red-X Phalloidin and DAPI staining (representative figure, n=3). Scale bar corresponds to 25 pm. (B) Total impedance was measured by ECIS (representative figure, mean ± SD, n = 1). (C-D) After IS treatment of 4T1 cells, the expressions of the indicated genes were determined using (C) RT- qPCR (n=3) and (D) Western blotting (representative figure, n=3). (Lactin was used as a loading control. (E) 4T1 cells (50,000 cells/well) were treated with the indicated concentration of IS for 24 hours and, subsequently, the percentages of migrated cells were determined using a Corning Matrigel invasion chamber (n=3). Cells were counted by Opera Phoenix High Content Screening System using Harmony 4.6 Software.

Numerical values are represented as average ± SEM, except for panel B, where average ± SD was plotted. Statistical significance was determined using ANOVA followed by Dunnetf s post -hoc tests, except for panel A, where a Chi-square test was conducted. For Dunnetf s tests, all comparisons were made to controls.

*, **, and *** indicate statistically significant differences between control and treated samples at p <0.05, p<0.01, and p <0.01, respectively.

Figure 14. IS treatment renders cells metabolically less flexible and reduces the proportions of cancer stem cells (A) 4T 1 cells (2000 cells/well) were treated with IS in the concentrations indicated for 24 hours then cells were subjected to a Seahorse XF96 analysis. The mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and plotted (n=3). (B) The expression levels of the indicated proteins were determined by Western blotting (n=3). (Lactin was used as a loading control. (C) 4T1 cells (100,000 cells/well) were treated with the indicated concentration of IS for 24 hours then the proportions of aldehyde dehydrogenase-positive cells were measured by Aldefluor assay using flow cytometry (n=3).

Numerical values are represented as average ± SEM. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA followed by Durmett’s post-hoc tests, except for C panel, where a Student’s t-test (two-tailed) was used. For the Dunnetf s post-hoc tests, all comparisons were made to controls.

* indicate statistically significant differences between control and treated cells at p <0.05.

Abbreviations: phospho-AMP-activated protein kinase (pAMPK); AMP-activated protein kinase (AMPK); phospho-Acetyl Co-A Carboxylase (pACC); Acetyl Co-A Carboxylase (ACC) and Forkhead box protein 01 (FOXO1).

Figure 15. Pharmacological inhibition of AHR and PXR block IS -elicited antineoplastic effects

(A) 4T1 cells (100,000 cells/well) were treated with IS in the concentrations indicated for 24 hours with or without the inhibitors, as indicated. The actin cytoskeleton and nuclei were stained using Texas Red-X Phalloidin and DAPI, then the morphology was assessed using Leica PP8 confocal system (representative figure, n=3). On the same cells, (B) lipid peroxidation (TBARS assays) (n=3) and (C) the proportions of the ALDH1 -positive 4T1 cells (n=3) were measured. (D) Expression of the indicated proteins was determined by Western blotting (representative figure, n=3). (Lactin was used as a loading control.

Numerical values are represented as average ± SEM. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA followed by Dunnetf s post-hoc tests, except for panel C, where a Student’s t-test (two-tailed) was used. For Dunnetf s tests, all comparisons were made to controls.

*, **, and *** indicate statistically significant differences between control and treated samples at p <0.05, p<0.01, and p <0.01, respectively.

Abbreviations: non-significant (ns); PXR inhibitor (PXRi); AHR inhibitor (AHRi); phospho-AMP-activated protein kinase (pAMPK); AMP-activated protein kinase (AMPK); phospho-Acetyl Co-A Carboxylase (pACC); Acetyl Co-A Carboxylase (ACC).

Figure 16. Higher expression of Cyp2El prolongs survival in breast cancer patients

The effect of expression of Cyp2El on survival in breast cancer was analyzed by kmplot.com, a freely accessible database. The data depicted stems from data acquired from microarray experiments and patients were stratified as a function of receptor expression. Total survival rates were assessed and all samples are represented. The database was assessed on March 30, 2020.

Figure 17. Higher expression of Sultl Al or Sultl A2 prolongs survival in breast cancer patients

The effect of expression of Sultl Al and Sultl A2 on survival in breast cancer was analyzed by kmplot.com, a freely accessible database. The data depicted stems from data acquired from microarray experiments and patients were stratified as a function of receptor expression. Total survival rates were assessed and all samples are represented. The database was assessed on March 30, 2020.

Figure 18. Fecal protein expression of E. coli TnaA correlates with the number of tumor infiltrating lymphocytes. The fecal protein expression of E. coli TnaA is higher in patients with higher proportions of tumor infiltrating lymphocytes. (A) E. coli total lysate was run on an SDS-PAGE gel, was transferred to nitrocellulose membrane and was probed with an anti-TnaA antibody. (B-C) Fecal samples of 36 low TIL patients (0-20% TIL) and 11 of high TIL patients (30%< TIL) were assessed by Western blotting using an anti-TnaA antibody. Protein content-normalized values were obtained. Values were tested for outliers using Grubb’s method, one value was omitted from the low TIL group. (B) Normalized TnaA expression was plotted. (C) On values from low and high TIL patients liner regression was performed.

DETAILED DESCRIPTION OF THE INVENTION

Bacterial indole biosynthesis has been found to be suppressed in breast cancer. The level of DNA from a bacterial tna operon and/or the level of a gene product of a gene from a bacterial tna operon may be used as an early indicator of the presence or risk of breast cancer. “Bacterial indole synthesis” refers to the potential of the microbiota to produce indole derivatives, such as IPA and IS from tryptophan, in which process tryptophanase is a key enzyme. The level of “bacterial indole synthesis” may depend on the number of bacteria capable of producing indole derivatives, e.g the number of bacteria comprising a coding sequence for a tryptophanase enzyme or the rate of expression of the enzyme or the level of activity of the enzyme or the level of the enzyme. The level of bacterial DNA coding for tryptophanase and the level of tryptophanase were found to be lower in fecal samples derived from patiens diagnosed with early stage breast cancer than in healthy controls. The term “human microbiota” refers to the microbes capable of living in or on the human body. The terms “gut microbiota” and “gut microbiome” refer to species of the human microbiota living in the human gastrointestinal tract. The term “microbiome” and “microbiota” as used in the description may refer to both the human microbiota and human microbiome and the gut microbiota and gut microbiome, preferably to the gut microbiota and microbiome.

Accordingly, the invention provides a method for diagnosing breast cancer or the risk of developing breast cancer in a subject by measuring the level of a DNA sequence from a bacterial tryptophanase (tna) operon and/or the level of a gene product of a gene of a bacterial tna operon in a sample obtained from the subject, wherein the test sample comprises microbiota of said subject.

The term “reference level” in a corresponding sample indicating the absence of breast cancer (e.g. stage 1 breast cancer) refers to a sample of the same type (e.g. blood, feces) from an individual or a group of individuals not having the breast cancer (e.g. stage 1 breast cancer).

The reference value is derived from individual(s) not having the cancer (stage) to be diagnosed. Preferably, the reference value is derived from healthy individual(s).

The “stage” of a cancer in this description is to be understood as a stage determined using the American Joint Committee on Cancer (AJCC) TNM system anatomic staging. American Joint Committee on Cancer (AJCC) (TNM system) staging system was used according to the 7th edition. Early stage breast cancer, as used herein, refers to stage 0 and/or stage 1 breast cancer according to the AJCC TNM staging system.

The methods for measuring the level of DNA, RNA or protein are well known to the skilled person and include e.g. quantitative real time PCR assays, (faecal) immunochemical tests, Western blot, turbidimetry, antibody and mass spectrometry-based assays, RT-coupled new generation sequencing. Guidance is also found in the Examples, and in e.g. Liang et al [105]; E. Nabizadeh et al. [106]; Goedert et al. [107]; Xie et al. [108].

It is also possible to search appropriate databases for bacteria known to be present in the human body, preferably in the human gastrointestinal tract, preferably in the intestine, wherein a DNA sequence coding for a tryptophanase was annoted. Accordingly, a screening method is provided, wherein bacteria suitable to indicate the presence of early stage breast cancer may be screened for. In the screening method an appropriate database is searched for bacteria wherein a DNA sequence coding for a tryptophanase has been annoted. By measuring the level of such bacteria or the DNA sequence from the tna operon from such bacteria or the level of a gene product of a gene of the tna operon in healthy individuals and early stage breast cancer patients, bacteria suitable to indicate the presence of early stage breast cancer may be identified. Such databases (e.g. the Human PanMicrobe Communities (HPMC) database, the NIH Human Microbiome Project, the Integrated reference catalog of the human gut microbiome) are well-known to the skilled person, and may be found in e.g. Turnbaugh et al. The human microbiome project: exploring the microbial part of ourselves in a changing world. Nature 2007; 449(7164): 804-810, e.g the KEGG database.

Microbial indole biosynthesis by the gut microbiome is repressed in early stages of breast cancer

To assess the link between bacterial IPA biosynthesis and breast cancer behavior, we assessed the capability of the gut microbiota to synthesize indole derivatives. To that end, we identified bacterial species in which tryptophanase operon was mapped using a database search, and designed primers to the regions coding for TnaA. We measured the abundance of the DNA coding for TnaA in fecal DNA of recently diagnosed patients vs. healthy controls (published in [3]) using qPCR.

When comparing patients against healthy individuals, the abundance of TnaA in Providencia rettgeri and Alistipes shahii was significantly reduced in patients, and there was a similar trend in Bacteroides xylanisolvens. Next, we stratified patients as a function of the stage of their disease. The decrease in TnaA abundance was accentuated in stage 0 (in situ carcinoma) patients not only for P. rettgeri, A. shahii and B. xylanisolvens, but also for a general probe on Clostridium species.

High TnaA expression is directly proportional with the number of tumor infiltrating lymphocytes.

We have assessed protein level changes to bacterial indol biosynthetic enzymes. The antibody commercially available was against the TnaA protein of E. coli origin. We stratified the patients based on the number of tumor infiltrating lymphocytes (TIL) and found that TnaA expression was trend for higher in patients with higher TIL. In good agreement with that finding, we found correlation between TIL and E. coli TnaA expression in breast cancer patients by performing linear regression.

Indolepropionic acid reduces the progression of breast cancer in vivo.

As first step, we examined the effects of IPA supplementation on tumor growth by grafting 4T1 breast cancer cells to 20 female Balb/c mice to the left and right side (200.000 cell to each site). Half of the mice received vehicle (sterilized tap water) as control, while the other half of the mice received IPA (1 nmol/g bodyweight). Per os IPA treatment did not inhibit the growth of the primary tumor, as neither the number of tumors, nor the total, nor the mass of primary tumors differed with IPA treatment. However, IPA treatment drastically reduced the level of infiltration to the surrounding tissues. IPA treatment reduced the number of mice bearing metastases and the total mass of metastases, although the mass of the individual metastases did not change. We assessed the histology of the primary tumors and found that the infiltration of lymphocytes was increased and mitosis score of the tumor cells was decreased in the IPA-treated mice.

Indoxyl-sulphate reduces the severity of breast cancer in vivo

Balb/c female mice were grafted with 4T1 breast cancer cells. Half of the mice received vehicle (sterilized tap water) as a control, while the other half received IS (2 pmol/kg bodyweight). The IS dose corresponds to a 4 pM serum concentration similar to the serum reference range of IS in humans [52]. Per os IS treatment did not inhibit tumor growth, however, IS significantly reduced the infiltration of the primary tumor to the surrounding tissues. Furthermore, IS treatment reduced the number and mass of metastases.

Indoxyl sulfate treatment inhibits the proliferation of breast cancer cells

IS reduced proliferation in multiple cell lines (SRB assays in 4T1, MCF7, SKBR-3, MDA-MB-231, ZR75-1). The anti -proliferative effects were confirmed in 4T1 cells using clonogenic assays. The proportions of apoptotic and necrotic cells in culture did not change significantly after treating with IS concentrations corresponding to the human reference concentrations. IS had no effects on non -transformed, primary human skin-derived fibroblasts.

Indolepropionic acid inhibits hallmarks of cancer

We opted for the 4T1 cells as our model for the subsequent experiments. IPA reduced the proliferation of 4T1 cells in colony forming assaysWe assessed whether IPA, in concentrations corresponding to the reference concentration and found that IPA did not increase either the proportions of propidium-iodide positive necrotic, or the Annexin-FITC - propidium-iodide double positive apoptotic cells.

We also assessed whether the effects, described above, are specific for only 4T1 cells, but can also be elicited in another breast cancer cell line. IPA, in a similar concentration range used for 4T1 cells, decreased cell proliferation as measured in SRB assays; it did not increase necrotic or apoptotic cell death in SKBR-3.

Finally, we found that IPA does not inhibit cell proliferation or exert cytotoxicity in primary, non-transformed human fibroblasts.

Next, we assessed whether IPA can modulate other cancer hallmarks. Although the role of oxidative stress in breast cancer was long debated, recent studies consistently showed that increased oxidative stress is responsible for cytostasis or cancer cell death [30, 65-69]. We assessed different oxidative stress markers to explore the redox status of IPA-treated cancer cells. IPA treatment increased lipid peroxidation, as measured by TBARS assay and by the formation of 4HNE indicative of enhanced oxidative stress. Enhanced oxidative stress was in parallel with the reduction in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor responsible for the expression of antioxidant enzymes, such as catalase. In parallel, we observed the enhanced expression of inducible nitrogen monoxide synthase (iNOS), an enzyme promoting nitrosative stress.

Finally, we assessed markers of cellular energy stress (pACC, ACC, FOXO1 and PGCip) and cancer stem cell- ness (aldehyde dehydrogenase [70]). IPA treatment reduced the proportions of cancer stem cells, while inducing the markers of cellular energy stress.

Another important feature of breast cancer is epithelial-to-mesenchymal transition (EMT). IPA treatment of 4T1 cells reverted EMT that was evidenced by dose dependent conversion of 4T1 cells to epithelial morphology. In good agreement with that, IPA treatment induced the resistance of cellular monolayer to electric current, indicative of better cell-to-cell and cell-to-surface binding. Finally, we assessed epithelial and mesenchymal markers at the protein and mRNA level. IPA treatment induced the expression of mesenchymal makers (Vimentin (Vim), fibroblast growth factor-binding proteinl (FgfBpl), snail family transcriptional repressor-1 (Snail), and P-catenin; and it upregulated the expression of epithelial markers (E-cadherin and zonula occludens-1 (ZO1) Indoxyl sulfate inhibits numerous hallmarks of cancer

We assessed whether IS can modulate cancer. First, we assessed oxidative/nitrosative stress markers, as these are major regulators of cancer hallmarks and cancer progression. Levels of thiobarbituric acid-reactive substances (TBARS) and 4-hydroxynonenal (4HNE), both markers of lipid oxidative damage, increased when cells were treated with IS (Fig. 12A-B). Next, we assessed nitro-tyrosine (NTyr) levels that indicate protein damage and nitrosative stress. NTyr levels were induced by IS treatment (Fig. 12C) suggesting increased damage to cells by reactive species and damage to cellular lipids and proteins. These changes correlated with the increased expression of inducible nitric oxide synthase (iNOS) (Fig. 12D) and the decreased expression of glutathione peroxidase 2 and 3 (GPX1 and GPX3), superoxide dismutase 3 (SOD3), and catalase (cat) (Fig. 12D-E).

The 4T1 cells reverted the endothelial-to-mesenchymal transition (EMT) after IS treatment, evidenced by dosedependent conversion of 4T1 cells to epithelial morphology (Fig. 13 A), coinciding with increased resistance (Fig. 13B). In good agreement with these findings, the mRNA and protein expression of mesenchymal markers (vimentin (Vim), fibroblast growth factor -binding protein 1 (Fgfbpl), transforming growth factor beta-3 (Tgfb3), matrix metalloproteinase 9 (MMP9), snail family transcriptional repressor- 1 (Snail) and P-catenin decreased, while the expression of epithelial markers (E-cadherin and tight junction protein- 1 (ZO-1) increased (Fig. 13C-D). In addition, cells migrated less in a Boyden-chamber experiment (Fig. 13E).

IS treatment had a profound effect on cellular metabolism. IS treatment reduced ECAR rendering cells hypometabolic and metabolically less flexible (Fig. 14A). Surprisingly, the hypometabolic switch coincided with the induction of key energy sensors, including AMPK or FOXO1 (Fig. 5B). AMPK activation upon IS treatment was marked by phosphorylation of its alpha subunit on Thrl72 and the phosphorylation of a key AMPK target protein, ACC on Ser79 (Fig. 14B). Finally, the proportions of ALDH1 -positive cancer stem cells decreased upon treatment with IS (Fig. 14C), a feature that is linked to changes in cellular metabolism.

IPA-induced cytostatic and antineoplastic effects are mediated by reactive species production

EMT, as well as, cancer stem cell characteristics are modulated by reactive species [71, 72]. With enhanced oxidative stress upon IPA treatment it was likely that the suppression of these features may be oxidative -stress driven. To get an insight on whether IPA can modify oxidative stress to exert cytostatic effects on breast cancer cells, we assessed the effects of thiol reductants glutathione (GSH) and N-acetyl-cysteine (NAC), as well as mitochondria-targeted antioxidant Mito-TEMPO, on IPA-mediated cancer hallmarks. As expected, the application of general thiol reductants (reduced glutathione (GSH) and N-acetyl-cysteine (NAC)) reduced the IPA- induced increase in thiobarbituric acid-reactive substances suggesting a reduction in IPA-induced oxidative stress. Furthermore, GSH and NAC treatment protected against the IPA-induced decrease in ALDH1 -positive cancer stem cells. More interestingly, a mitochondrial antioxidant, Mito-TEMPO, protected against IPA- induced cytostasis. These data suggest that IPA-induced reactive species production has a central role in eliciting the widespread antineoplastic effects of IPA.

IPA exerts its effects through aryl-hydrocarbon receptor (AHR) and pregnane-X-receptor (PXR)

IPA has multiple receptors [40], of which we investigated pregnane-X-receptor (PXR) and aryl-hydrocarbon receptor (AHR) in detail in our study. We used pharmacological inhibitors of AHR (CH223191) and PXR (ketoconazole [51, 52]) to interrogate the involvement of IPA receptors. Inhibition of AHR and PXR abrogated or quenched several IPA -induced features, namely, IPA-induced reversion of EMT, TBARS production, induction of NRF2 and AMPK (as marked by the phosphorylation of ACC).

We assessed whether the expression of AHR and PXR correlates with survival in human breast cancer using the kmplot.com database [63]. First, we assessed the RNAseq datasets and found that the quartile of the patient population with the highest expression has advantage in survival over the quartile with the lowest expression both in the case of PXR and AHR. Next, we assessed the microarray data as well, where we were able to stratify patients as a function of receptor expression. Expression level of AHR did not have a clear impact on breast cancer survival, nevertheless, there was a trend for better survival in the high expression quartile, similarly to the RNAseq data. Importantly, higher expression of PXR provided better survival with breast cancer, particularly during the first 4-12 years after diagnosis and for estrogen receptor-positive cancer cases. (See Table 5 in Examples.)

These in silica data were complemented by characterizing the intratumoral expression of AHR and PXR in a tissue microarray (TMA) consisting of 88 patients (the TMA was published in [30]). When AHR expression was considered in the whole cell, it did not show any consequent change, nevertheless, when we scored only the nuclear fraction of AHR that represents the active AHR [73], we found that AHR nuclear score (i.e. AHR activity) decreased with the progression of the disease. Furthermore, AHR activity was lower in highly proliferating tumors, in tumors with lower differentiation) and in not otherwise specified (NOS) tumors as compared to lobular tumors. PXR expression decreased as Nottingham grade of the patients increased and in tumors with high proliferation rate.

High extracellular tryptophan levels associate with worse survival in breast cancer (Table S8 [101]). The levels of an indole derivative, 3-indoxyl-sulphate that is a downstream metabolite of tryptophan degradation and indole-propionic acid formation, is downregulated both in estrogen receptor -positive and -negative cases ([102] Additional file 3, Table S3 Fine 44). Furthermore, in breast tumors there is a negative correlation between Ki67 positivity (a proliferation marker) and 3-indoxyl-sulphate levels ([102] Additional file 9, Table S8 line 130). These data suggest that indole derivatives support the survival of patients with breast cancer and that their levels are downregulated with progressive disease. These observations are also in good correlation with the intratumoral expression pattern and activity of AHR and PXR that are downregulated with the progression of the disease.

In breast cancer oncobiosis bacterial tryptophan metabolism is suppressed and this seems to be the most profound in early stage, in situ carcinoma cases. Decreases in IPA releases the cytostatic lockdown of breast cancer cells. Eater stage and aggressive cases are characterized by lower expression or lower activity of metabolite- elicited signaling. Apparently, oncobiosis seems to play a role in breast carcinoma progression but not in the initiation of the disease.

IS exerts its effects through the AHR and PXR receptors

As the next step, we wanted to assess which receptors are responsible for the IS -elicited effects. Indoxyl derivatives exert their effects through the aryl hydrocarbon receptor (AHR) and pregnane-X -receptor (PXR) [53]. We applied pharmacological inhibitors to interrogate the involvement of AHR and PXR in IS signaling. The AHR inhibitor, CH223191, and the PXR inhibitor, ketoconazole, were applied. Both CH223191 and ketoconazole blocked IS-elicited mesenchymal-to-epithelial transition (Fig. 15A). Inhibition of AHR and PXR also attenuated the IS-induced increases in TBARS (Fig. 15B) and suppression of ALDH1+ cells (Fig 15C). In addition, IS- induced expression of E-cadherin was blocked by CH223191, but not by ketoconazole (Fig. 15D). In contrast, the phosphorylation of ACC and AMPK was blocked by both agents (Fig. 15D).

Higher expression of the isoforms of SULT and Cyp2el correlate with better survival in breast cancer.

We assessed how the expression of IS biosynthesis enzymes affects the survival of breast cancer patients. To that end, we assessed an online database, kmplot.com. Higher expression of Cp2El and SultlAl and Sultl A2 in tumors correlated with better survival in breast cancer patients (Fig. 16-17, Table 3). Furthermore, the protective effect was lost in triple negative cases (TNBC) (Fig. 16-17, Table 6-13).

Fecal TnaA expression is lower in patients with E-cadherin negative tumors

First, we compared the TnaA expression in E-cadherin positive and negative tumors. E-cadherin is a predictive factor for metastasis formation and poorer clinical outcome. The fecal expression of E. coli TnaA was lower in E-cadherin negative cases as compared to E-cadherin positive cases.

Fecal expression of TnaA is low in lobular subtype of breast cancer

The fecal protein expression of E. coli TnaA as a function of the histological subtype. We found that the fecal expression of E. coli TnaA enzyme was lower in lobular subtype than in the ductal, not otherwise specified and mixed cases.

Low or absent E-cadherin expression was associated with reduced disease-free interval and overall survival, larger tumor size, higher histological grade, development of distant metastasis and tumors negative for estrogen receptors, in general, poor prognosis. The finding that TnaA expression was lower in E-cadherin negative cases is in good agreement with the observation that oncobiosis supports metastasis formation in breast cancer.

Indolepropionic acid (IP A) has antineoplastic features. IP A supplementation decreased the infiltration of the primary tumor to surrounding tissues, the number of metastases, cellular movement and diapedesis, while, at the same time, induced antitumor immune response, mesenchymal-to-epithelial transition, oxidative stress and influenced metabolism through two IPA receptors, AHR and PXR. IPA did not exert its cytostatic effects on non-transformed cells, exhibiting tumor cell-specific effects. IPA exerts its antineoplastic modultion through the AHR and particularly the PXR receptors.

IPA treatment increased oxidative and nitrosative stress through the inhibition of the expression of NRF2 and a subsequent reduction in cellular antioxidant defense (e.g. downregulation of caspase expression). In addition, IPA induced iNOS expression and enhanced mitochondrial reactive species production.

NRF2 and iNOS have profound roles in setting the redox balance in cancer cells [66, 93]. Increased oxidative and nitrosative stress is cytostatic in breast cancer [30, 65, 68, 69]. Increase in reactive species production is vital to exert the cytostatic properties of IPA. Furthermore, oxidative stress is a key regulator of cancer cell stem-ness [94], and higher levels of oxidants switch cancer stem cells to lose their stem properties [95-97]. IPA treatment reduced the proportions of cancer stem cells, an effect that was reverted by the addition of thiol reductants. IPA induced mesenchymal-to-epithelial transition (MET). Inducing MET slows down cell movement, diapedesis and metastasis formation. In our models we observed that IPA supplementation reduced metastasis formation.

In addition to these findings, we showed that IPA induces AMPK, FOXO1, and PGCip, enzymes inducing mitochondrial biogenesis, in an AHR/PXR-dependent fashion. The activation and overexpression of these enzymes were shown to be associated with better survival in breast cancer [27, 53, 99, 100]

High TnaA expression, indicative of higher IPA production, is directly proportional with the number of tumor infiltrating lymphocytes.

High extracellular tryptophan levels associate with worse survival in breast cancer (Table S8 [101]). The levels of an indole derivative, 3-indoxyl-sulphate that is a downstream metabolite of tryptophan degradation and indole-propionic acid formation, are downregulated both in estrogen receptor-positive and -negative cases ([102] Additional file 3, Table S3 Line 44). Furthermore, in breast tumors there is a negative correlation between Ki67 positivity (a proliferation marker) and 3-indoxyl-sulphate levels ([102] Additional file 9, Table S8 line 130). These data suggest that indole derivatives support the survival of patients with breast cancer and that their levels are downregulated with progressive disease. In addition to these, we showed that the IPA biosynthetic capacity of the microbiome is reduced in women newly diagnosed with breast cancer, especially for women with in situ carcinoma. These observations are also in good correlation with the intratumoral expression pattern and activity of AHR and PXR that are downregulated with the progression of the disease, or were lower in aggressive, highly proliferative and undifferentiated breast cancer cases. Decreases in IPA releases the cytostatic lockdown of breast cancer cells. Later stage and aggressive cases are characterized by lower expression or lower activity of metabolite-elicited signaling.

EXAMPLES

Chemicals

Chemicals, among them, indolepropionic acid (IPA), indoxyl-sulphate (IS), glutathione (GSH), N-acetyl- cysteine (NAC), Mito-TEMPO and ketoconazole were from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. IPA was used 0.4 pM and 0.8 pM. [40, 49, 50]. IS was used at concentrations of 2 pM and 4 pM, which correspond to the normal human serum concentration of IS [53,60,61]. GSH and NAC antioxidants were used at final concentration of 5 mM. The mitochondria -targeted antioxidant Mito-TEMPO was used at concentration of 5 pM. The aryl hydrocarbon receptor (AHR) inhibitor, CH223191, was obtained from MedChemExpress (MCE, Monmouth Junction, USA) and was applied at concentration of 10 pM. Pregnane X receptor (PXR) downstream signaling was inhibited using ketoconazole at final concentration of 25 pM [51, 52], The Silencer Select siRNAs targeting AHR (AHR— siRNA ID: si 198) and PXR (NR1I2— siRNA ID: sl6910) and the negative control siRNA #1 (cat.no. 4390843) were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and each was used at a final concentration of 30 nM.

Cell culture

4T1 murine breast cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, R5886) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 1 % pyruvate at 37 °C with 5 % CO ₂ . MCF7 human breast cancer cells were maintained in MEM (Sigma-Aldrich, M8042) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine at 37 °C with 5 % CO ₂ .

SKBR-3 human breast cancer cells were maintained in DMEM (Sigma- Aldrich, 1000 mg/1 glucose, D5546) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine at 37 °C with 5 % CO ₂ .

Human primary fibroblasts cells were maintained in DMEM (Sigma- Aldrich, 1000 mg/1 glucose, D5546) medium containing 20 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine at 37 °C with 5 % CO ₂ .

In vitro cell proliferation assays

Cellular proliferation was assessed using Sulphorhodamine B assay and colony forming assay as described in [53]. Cells were seeded in a 96-well plates (4T1- 1500 cell/well; MDA-MB-231 - 3000 cell/well; SKBR-3 - 5000 cell/well; MCF7 - 4000 cell/well; human fibroblast - 7500 cell/well) in complete medium and were treated with the indicated concentrations of IS, IPA or Mito-TEMPO mitochondria-targeted antioxidant (5 pM) at the presence of IPA (0,8 pM) for 24 hours. Then cells were fixed in 50% trichloroacetic acid (TCA - final concentration: 10 %) and the plates were incubated for 1 hour at 4°C. Plates were washed 5 times with water and stained with 0.4 % (w/v) sulphorhodamine B solution in 1 % acetic acid. Unbound dye was removed by washing 5 times with 1 % acetic acid. Bound stain was solubilized with 10 mM Tris base and the absorbance was measured on an automated plate reader (Thermo Labsystems Multiskan MS, Walthman, Massachusetts, USA) at 540 nm.

For colony forming assays, cells were seeded in a 6-well plate (4T1 - 500 cell/well; MCF7 - 1000 cell/well; SKBR-3 - 1500 cell/well) in complete medium and were cultured with the indicated concentrations of IPA for 7 days. After treatment, the plates were washed twice with PBS. Colonies were fixed in methanol for 15 minutes, dried and stained with May-Grunwald-Giemsa solution for 20 minutes. Cells were washed with water and the colonies were determined using Image J software [54] .

Detection of cell death

IPA or ISinduced cytotoxicity was assessed by propidium iodide (PI; Biotium, Fremont, CA, 40016) uptake assays as in [55]. Cells were seeded in 6-well plate (4T1 - 75.000 cell/well; MCF7 - 150.000 cell/well; SKBR-3 - 200.000 cell/well; human fibroblast - 200.000 cell/well) and treated with the indicated concentrations of IPA or IS for 24 hours, then stained with 100 pg/mL PI for 30 minutes at 37°C, washed once in PBS, and analyzed by flow cytometry (FACS Calibur, BD Biosciences).

To assess changes in apoptotic and necrotic cell death we used an Annexin V+PI double staining assay kit (Invi- trogen, Oregon, USA, V13242). Cells were seeded in 6-well plate (4T1 - 75.000 cell/well; MCF7 - 150.000 cell/well; SKBR-3 - 200.000 cell/well; human fibroblast - 200.000 cell/well) treated with the indicated IPA or IS concentrations for 24 hours. Then cells were stained with 100 pg/mL PI solution and 5 pl FITC Annexin V according to the manufacturer’s instructions. The number of apoptotic and necrotic cells were counted using FacsCalibur flow cytometer.

2.5 Electric Cell-substrate Impedance Sensing (ECIS)

ECIS (Electric cell-substrate impedance sensing) measurements (ECIS model Z0, Applied BioPhysics Inc. Troy, NY, USA) were used to monitor cell-to-cell and cell-to -surface connections. 4T1 cells were seeded (40.000 cell/well) on type 8W10E arrays. Cells were treated with vehicle , 0.4 pM or 0.8 pM IPA or 2 pM or 4 pM IS for 20 hours and total impedance values were measured for 24 hours. Multifrequency measurements were taken at 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, 16000, 32000 and 64000 Hz. The reference well was set to a no-cell control with complete medium. ECIS assays were performed similarly to [27].

Immunocytochemisry

Immunocytochemistry was performed similarly to [27]. 4T1 cells were grown on glass coverslips for 1 day, and treated with the indicated concentrations of IPA, IS and the AHR inhibitor, CH223191 (10 pM), or the PXR inhibitor, ketoconazole (25 pM), for 24 hours. Then the cells were washed in PBS, then fixed with 4% paraformaldehyde for 15 minutes and permeabilized using 1 % Triton X-100 in PBS for 5 minutes. Cells were then blocked with 1% BSA in PBS for 1 hour and incubated with TexasRed-X Phalloidin (T7471, 1:150, Invitrogen, Oregon, USA) for 1 hour at 4°C. Cells nuclei were visualized with DAPI (R37606, 1:10, Thermo Fischer Scientific Inc., Rockford, IL, USA) and rinsed in PBS twice for 10 minutes. Coverslips mounted in Mowiol/Dabco solution. Confocal images were acquired with a Leica TCS SP8 confocal microscope and were processed using LAS AFv3.1.3 software. Typical mesenchymal-like and epithelial-like morphology of 4T1 cells are shown in [27], mRNA preparation and quantitation

Reverse transcription-coupled PCR (RT-qPCR) was performed similarly to [56]. Total RNA from cells were isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen Corporation, Carlsbad, CA). For the assessment of the expression of indicated genes 2 pg of RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The qPCR reactions were performed with the qPCRBIO syGreen Lo-ROX Supermix (PCR Biosystems Ltd., London, UK) on Light- Cycler 480 Detection System (Roche Applied Science). Gene expression was normalized to the geometric mean of human 36B4 and cyclophyllin values. Primers are listed in Table 1.

Table 1. Murine and human primers used in RT-qPCR reactions

Bacterial TnaA quantitation

The human fecal DNA library from breast cancer patients and matched control women was published in [3]. For the assessment of the abundance of bacterial Tryptophanase (TnaA) coding DNA in human fecal, DNA samples 10 ng of DNA was used for qPCR reactions. Primers are listed in Table 2.

Table 2. Primers for the determination of abundance of TnaA using RT-qPCR reactions

Quantitation of fecal E. coli TnaA protein

Fecal proteins were isolated as described in [28] and [108]. Fecal samples (100 mg) were lysed in 500 pl RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 0.5 % sodium deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1 mM NaF, protease inhibitor cocktail) and sonicated (Qsonica Q125 Sonicator,

Newtown, Connecticut) 3 times for 30 seconds with 50% amplitude. After centrifugation, 8 pl - mercaptoethanol and 25 pl 5 X SDS sample buffer (50 % glycerol, 10 % SDS, 310 m Tris HC1, pH 6.8, 100 m DTT, 0.01 % bromophenol blue) were added to each 100 pl extract. Then, fecal protein samples were heat- ed for 10 minutes at 96 °C and held on ice until loading. Protein extract (40 pl) was loaded on 8 % SDS-PAGE gels and proteins were separated and transferred onto nitrocellulose membrane. Ponceau-red staining took place after transfer, but before blocking. Ponceau-stained membranes were photographed and used in subsequent analyses. Membranes were cut at the 70 kDa standard and the top part was blotted for TnaA (E. coli TnaA, 1:2000, Assaypro (33517-05111)). Membranes were blocked in TBST containing 5 % BSA for 1 hour and incubated with anti-LdcC primary antibody overnight at 4°C. After washing with lx TBS-TWEEN solution, the membranes were probed with IgG HRP-conjugated peroxidase secondary antibodies (1:2000, Cell Signaling Technology, Inc, Beverly, MA, USA). Bands were visualized by enhanced chemiluminescence reaction (SuperSignal West Pico Solutions, Thermo Fisher Scientific Inc., Rockford, IL, USA). Blots were evaluated by densitometry using Image J software and antibody signals were normalized to total protein stained by Ponceau - red (Sigma-Aldrich).

Aldefluor assay

Aldehyde dehydrogenase (ALDH) activity was measured using Aldefluor Stem Cell kit (StemCell Technologies, Vancouver, Canada). 4T1 cells were seeded on 6 well plates (4T1- 100.000 cell/well) and treated with the indicated concentration of IPA or IS for 24 hours. Cells then were collected and prepared according to the manufacturer’s instructions. For positive control samples SKBR-3 cell line was used based on the manufacturers’ instructions. Changes in the level of ALDH was determined by flow cytometry and the results were analyzed by using Flowing Software 2.5.1. Aldefluor assay for assessing sternness was performed similarly to [28, 57, 58].

Seahorse metabolic flux analysis

Changes in oxygen consumption rate (OCR, reflecting mitochondrial oxidative capacity) and pH, termed extracellular acidification rate (ECAR, reflecting glycolysis) were measured using an XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA). The 4T1 cells were seeded in 96-well Seahorse assay plates (4T1, 2000 cells/well) and treated with vehicle or the indicated IS concentrations for 24 hours. The OCR and ECAR values were recorded every 30 minutes to monitor the effects of IS treatment. Data were normalized to protein content and normalized readings were used for calculations.

SDS-PAGE and Western blotting

SDS PAGE and Western blotting was performed as in [59]. Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% TritonX 100, 0.5% sodium deoxycolate, ImM EDTA, ImM Na ₃ VO ₄ , 1 mM PSMF, 1 mM NaF, protease inhibitor cocktail). Protein extracts (20-50 pg) were separated on 10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Then membranes were blocked in TBST containing 5 % BSA for 1 hour, and incubated with primary antibodies overnight at 4°C. After washing with lx TBST solution, the membranes were probed with IgG HRP-conjugated peroxidase secondary antibodies (1:2000, Cell Signaling Technology, Inc., Beverly, MA, USA). Bands were visualized by enhanced chemiluminescence reaction (SuperSignal West Pico Solutions, Thermo Fisher Scientific Inc., Rockford, IL, USA). Blots were quantified by densitometry using the Image J software. The primary and secondary antibodies are listed in Table 3.

Table 3. List of antibodies used for Western blot

Determination of lipid peroxidation

Lipid peroxidation was evaluated by determining the production rate of thiobarbituric acid-reactive substrate using the thiobarbituric acid-reactive substances (TBARS) assay as described in [60]. The 4T1 cells were seeded in T75 flasks and exposed to to AHR (10 pM) and PXR (25 pM) inhibitors together with IS (2 p or 4 pM), or different concentration of IPA or Mito-TEMPO mitochondria-targeted antioxidant (5 pM) /GSH and NAC antioxidants (5 mM) / AHR (10 pM) and PXR (25 pM) inhibitors together with IPA (0,4 pM or 0,8 pM) for 24 hours. Cells were washed in PBS and scraped, then collected by centrifugation. After adding 8.1% SDS, 20 % acetic acid, 0.8 % thiobarbituric acid (TBA) and distilled water to the pellet, the samples were heated at 96°C for 1 hour. Samples were cooled down on ice and centrifuged. The absorbance of the supernatants were identified at 540 nm. The levels of 4-hydroxynonenal (4HNE) -modified proteins, as marker for lipid peroxidation were also assessed using western blotting.

Invasion

Matrigel invasion assays were performed with 4T1 cells using Coming BioCoat Matrigel Invasion Chambers (Corning, NY, USA). Cells were seeded in the chambers (50,000 cells/well) in serum free medium and grown overnight. Then, the cells were exposed to the indicated concentrations of IS for 24 hours. The lower chamber was filled with 4T1 medium with lOOng/ml SDFl-alpha (Sigma-Aldrich, SRP4388) as a chemoattractant. Cells were prepared and stained with Hematoxylin-Eosin (VWR, PA, USA, 340374T 341972Q) dye according to the manufacturer’s instructions. Cells were then analyzed on the Opera Phoenix High Content Screening System using Harmony 4.6 Software. Migration was calculated from the percentage of migrated cells through the Matrigel control membranes.

Animal study

Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Debrecen and the National Board for Animal Experimentation (1/2015/DEMAB) and were performed according to the NIH guidelines (Guide for the care and use of laboratory animals) and applicable national laws. Animal studies are reported in compliance with the ARRIVE guidelines.

Experimental animals were BALB/c female mice (14-16 weeks of age, 20-25 g). Animals were randomized for all experiments. Mice were bred in the “specific pathogen free” zone of the Animal Facility at the University of Debrecen, and kept in the “minimal disease” zone during the experiments. No more than 5 mice were housed in each cage (standard block shape 365 x 207 x 140 mm, surface 530 cm ² ; 1284 L Eurostandard Type II. L from Techniplast). Cages were changed once a week, on the same day. Animals had paper tubes to enrich their environment. Dark/light cycle was 12 hours, and temperature was 22 ± 1°C. Mice had ad libitum access to food and water (sterilized tap water). The animal facility was overseen by a veterinarian. A total of 20 female mice were used in the study, 10 randomly selected control and 10 IP A fed mice.

4T1 cells were suspended (2xlO ⁶ /mL) in ice cold PBS-Matrigel (1:1, Sigma-Aldrich) at 1:1 ratio. 20 female BALB/c mice received 50 pL injections to their second inguinal fat pads on both sides (10 ⁵ cells/inj ection site). Tumor growth and animal wellbeing was controlled daily.

IPA treatment was administered by oral gavage in the dose of 1 nmol/g body weight once a day on each day of the experiment. The dose was planned not to exceed the serum reference concentration of IPA [40, 49, 50]. Animals received single daily oral IPA treatment. IPA stock was prepared in sterilized tap water at lOOx concentration (15 mM) for storage at -20°C. IPA stock was diluted each day to a working concentration of 150 pM in sterile tap water before treatment. Animals received a daily oral dose of 100 pl/30 g bodyweight from the IPA solution (10 mice) or vehicle (sterilized tap water, 10 mice). Researchers involved in IPA and vehicle solution administration were blinded. Treatment was administered every day at the same time between 9 a.m. and 11 a.m. Animals were sacrificed on day 14 post grafting by cervical dislocation, then primary tumors and metastases were collected for subsequent analysis.

IS was administered by oral gavage at a dose of 2 pmol/kg as a bolus once a day. The dose correlated with the serum reference concentration of IS [53,60,61]. IS stock (60 mM) was prepared in sterilize tap water and stored at -20 °C. The IS stock solution was diluted on the day of treatment. Animals were randomized into two groups, 10 mice were treated with IS and 10 mice were treated with vehicle (sterilized tap water). The researchers administering IS and vehicle solutions were blinded. Treatment was carried out every day at the same time between 9 a.m. and 11 a.m. Animals were sacrificed on day 14 post grafting by cervical dislocation, and primary tumors and metastases were harvested for subsequent analysis Upon autopsy primary tumors were visually evaluated and scored based on their infiltration rate into surrounding tissues and macroscopic appearance of the tumor [27, 28]. Tumors were classified as a “low infiltration” class if the primary tumor remained in the mammary fat pads without any attachment to muscle. “Medium infiltration” tumor means that the tumor mass attached to the muscle tissue, but did not penetrate to the abdominal wall. In the case the tumor grew into the muscle tissue and totally penetrated the abdominal wall, it was scored as a “high infiltration” tumor. For the assessment of primary tumor infiltration rate the researchers were blinded. Both primary and metastatic tumor masses were removed from the animals and were measured on analytical balance in preweighed Eppendorf tubes. From the sections of HE-stained, formalin- fixed, paraffin embedded tumor tissues tumor infiltrating lymphocyte (TIL) content was determined as the number of TILs per 100 tumor cells.

Human studies

We assessed the abundance of the bacterial TnaA coding DNA in human fecal DNA samples. We used two cohorts in the study.

Cohort 1.

Cohort 1 consisted of fecal DNA samples and were used for the quantitation of the TnaA coding DNA. The cohort involved 48 controls and 46 breast cancer patients.

The human feces samples were collected from healthy volunteers and breast cancer patients by collaborators at the National Cancer Institute (NCI), Kaiser Permanente Colorado (KPCO), the Institute for Genome Sciences at the University of Maryland School of Medicine, and RTI International. The study protocol and all study materials were confirmed by the Institutional Review Boards at KPCO, NCI, and RTI International (IRB number 11CN235). The study results were published in [3]. Informed consent was obtained from the study participants. That cohort was designed and powered to compare healthy controls versus breast cancer patients, but we also stratified patients using the available clinical data.

Cohort 2.

This cohort was used in assessing the Intratumoral expression of IPA receptors. The TMA study was carried out using archived tissue blocks of 88 breast cancer patients.

Formalin-fixed, paraffin embedded tissues from breast cancer surgeries were collected and tissue microarray (TMA) blocks were built [62]. The cohort is described in [30]. The study was approved by the local ethical committee at the University of Debrecen. That cohort was designed and powered to compare healthy controls versus breast cancer patients, but we also stratified patients using the available clinical data.

Cohort 3.

Fecal protein expression was assessed in a cohort of female breast cancer patients of 35 participants with a mean age of 57 years. Samples from stage I-III and Nottingham grade 1-3 patients were used in the study. The average stage and grade were not statistically different amongst the groups. The cohort was published in [ 108].

The collection and biobanking of feces were authorized by the Hungarian national authority (ETT). Patients and healthy volunteers meeting the following criteria were excluded from the study: 1) has a previous history of breast cancer or had been operated due to neoplasia, 2) has a disease of unknown origin, 3) has a chronic contagious disease, 4) had contagious diarrhea 6 months prior to enrollment, 5) taken antibiotics within the 6 months prior to enrollment, 6) had chemotherapy, biological therapy, or immunosuppressive therapy 6 months prior to enrollment, 7) used intravenous drugs 12 months prior to enrollment, 8) had piercing, tattooing, acupuncture, or other endangering behavior or action 12 months prior to enrollment, 9) exposure to an allergen to which the enrolled individual had been sensitized to, or 10) underwent colonoscopy 12 months prior to enrollment. The first morning feces was sampled; samples were frozen and deposited in the biobank within two hours after defecation. Samples were stored at -70 oC until subsequent use. We obtained informed consent from study participants. The patient’s routine pathological data were assessed in the study and were compared to the fecal expression of E. coli TnaA.

Tissue Microarray, immunohistochemistry and analysis

TMA in combination with immunohistochemistry was performed as in [61]. From every block there were three replicates and the staining was assessed applying H-score system [62]. In immunohistochemical studies, Leica Bond Max™ protocol was used. The antibodies and conditions are listed in Table 4.

Table 4. List of antibodies and conditions used in tissue microarray (TMA) analysis

Database screening

The kmplot.com database [63] was used to examine the connection between gene expression levels (AHR and PXR) and breast cancer survival in humans. The case numbers are listed in Table 5 (below). The sequence of the TnaA ORFs was retrieved from the KEGG (www.genome.jp/kegg/), the PATRIC (www.patricbrc.org/) and the Uniprot (www.uniprot.org) databases.

Statistical analysis

For the comparison of two groups, we used two tailed Student’s t-test, unless stated otherwise. Fold data were log ₂ transformed to achieve normal distribution. Statistical significance was determined for multiple comparisons with one-way analysis of variance test (ANOVA) followed by Tukey’s or Dunnetf s honest significance difference (HSD) post-hoc test. All data are presented as average ± SEM unless stated otherwise. Texas Red-X Phalloidin-labelled fluorescent pictures were analyzed using Cell Profiler 2.0 followed by Advanced Cell Classifier 3.0. FACS results were analyzed using Flowing Software 2.0. Statistical analysis was done using GraphPad Prism 7 software unless stated otherwise.

Statistical analysis

All data are represented as average ± SEM, and n denotes the number of patients in a group. Statistical tests are mentioned in figure captions. For statistical analysis Graphpad 8.1 software was used.

TABLE 5. LINK BETWEEN AHR OR PXR EXPRESSION AND BREAST CANCER PATIENT SURVIVAL The effect of expression of AHR or PXR, Cyp2El or Sult isoform on survival in breast cancer was analyzed by kmplot.com, a freely accessible database. Total survival rates were assessed and all samples are represented in different subpopulations of breast cancer. Numbers in bold represent statistically significant results.

Table 6. Breast cancer survival and SULT1A1 (203615_x_at)

Numbers in bold represent statistically significant results.

Table 7.

Breast cancer survival and SULT1 Al (215299_x_at) Numbers in bold represent statistically significant results.

Table 8.

Breast cancer survival and SULT1A2 (207122_x_at) Numbers in bold represent statistically significant results.

Table 9.

Breast cancer survival and SULT1 A2 (211385_x_at) Numbers in bold represent statistically significant results.

Table 10.

Breast cancer survival and CYP2E1 (1431_at) Numbers in bold represent statistically significant results.

Table 11.

Breast cancer survival and CYP2E1 (209975_at) Numbers in bold represent statistically significant results.

Table 12.

Breast cancer survival and CYP2E1 (209976_s_at) Numbers in bold represent statistically significant results.

Table 13.

Breast cancer survival and CYP2E1 (222100_at) Numbers in bold represent statistically significant results.

SEQUENCE LISTING

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<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223 > CAT Murine reverse primer

<400> 2 caagtttttg atgccctggt 20

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > VIM Murine forward primer

<400> 3 ctccagagag aggaagccga aag 23

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence <220>

<223 > VIM Murine reverse primer

<400> 4 cctggatctc ttcatcgtgc agt 23

<210> 5

<211> 23

<212> DNA

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<220>

<223 > FgfBpl Murine forward primer

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<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > FgfBpl Murine reverse primer

<400> 6 agctccaaga ttccccacag aac 23

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Tgfb3 Murine forward primer

<400> 7 ggcgtctcaa gaagcaaaag gat 23

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Tgfb3 Murine reverse primer

<400> 8 ccttaggttc gtggacccat ttc 23

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223 > MMP9 Murine forward primer

<400> 9 cattcgcgtg gataaggagt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223 > MMP9 Murine reverse primer

<400> 10 acctggttca cctcatggtc 20

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223 > GPX2 Murine forward primer

<400> 11 gttctcggct tcccttgc 18

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223 > GPX2 Murine reverse primer

<400> 12 ttcaggatct cctcgttctg a 21

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223 > GPX3 Murine forward primer

<400> 13 ggcttccctt ccaaccaa 18

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223 > GPX3 Murine reverse primer

<400> 14 cccacctggt cgaacatact 20

<210> 15

<211> 19

<212> DNA <213> Artificial Sequence

<220>

<223 > S0D3 Murine forward primer

<400> 15 ctcttgggag agcctgaca 19

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223 > SOD3 Murine reverse primer

<400> 16 gccagtagca agccgtagaa 20

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223 > Cyclophilin A Murine forward primer

<400> 17 tggagagcac caagacagac a 21

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223 > Cyclophilin A Murine reverse primer

<400> 18 tgccggagtc gacaatgat 19

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 36B4 Murine forward primer

<400> 19 agattcggga tatgctgttg g 21

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223 > 36B4 Murine reverse primer <400> 20 aaagcctgga agaaggaggt c 21

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence

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<223 > AHR Human forward primer

<400> 21 ttgaaccatc cccatacccc ac 22

<210> 22

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223 > AHR Human reverse primer

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<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223 > PXR Human forward primer

<400> 23 agtgaaggtt cccgaggaca tg 22

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223 > PXR Human reverse primer

<400> 24 ttgtcacaga gcatacccag ca 22

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223 > Cyclophilin A Human forward primer

<400> 25 gtctcctttg agctgtttgc agac 24

<210> 26

<211> 21 <212> DNA

<213> Artificial Sequence

<220>

<223 > Cyclophilin A Human reverse primer

<400> 26 cttgccacca gtgccattat g 21

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> 36B4 Human forward primer

<400> 27 ccattgaaat cctgagtgat gtg 23

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223 > 36B4 Human reverse primer

<400> 28 gtcgaacacc tgctggatga c 21

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > Alistipes shahii Forward primer

<400> 29 gcctcgaaga cgatcaagga gat 23

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > Alistipes shahii Reverse primer

<400> 30 tcgaacatga tgctgaacga cat 23

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > Providencia rettegri Forward primer <400> 31 cgtttacgtg agggagggat ttc 23

<210> 32

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223 > Providencia rettegri Reverse primer

<400> 32 accgcacgaa caccagattc taa 23

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Bacteriodes xylanisolvens Forward primer

<400> 33 aactggaaat cccgttcaaa gga 23

<210> 34

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Bacteriodes xylanisolvens Reverse primer

<400> 34 gtacgggttt gccgtatttg tea 23

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Clostridium sp. Forward primer

<400> 35 gatggaaegt ccaaacactt teg 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Clostridium sp. Reverse primer

<400> 36 atattttccg ccttccggaa ett 23 REFERENCES

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