CN116407526A

CN116407526A - Breast cancer therapeutic drug, auxiliary therapeutic drug, anti-tumor immune activator and application of trimethylamine oxide and precursor product choline thereof

Info

Publication number: CN116407526A
Application number: CN202310226823.5A
Authority: CN
Inventors: 邵志敏; 王海; 赵超; 江一舟; 荣星喻; 周逸凡
Original assignee: Fudan University Shanghai Cancer Center
Current assignee: Fudan University Shanghai Cancer Center
Priority date: 2022-03-11
Filing date: 2023-03-10
Publication date: 2023-07-11

Abstract

The invention discloses a breast cancer therapeutic drug, a breast cancer auxiliary therapeutic drug, a breast cancer anti-tumor immune activation drug and application of trimethylamine oxide and choline which is a precursor product thereof. The invention discloses trimethylamine oxide and choline which is a precursor product thereof can activate anti-tumor immunity of triple negative breast cancer, which is a novel treatment method for prompting clinical dilemma of poor immune treatment effect of triple negative breast cancer, improving the curative effect of clinical immune treatment and preventing the reverse immune treatment from being effective.

Description

Breast cancer therapeutic drug, auxiliary therapeutic drug, anti-tumor immune activator and application of trimethylamine oxide and precursor product choline thereof

Technical Field

The invention belongs to the field of tumor treatment, and in particular relates to a breast cancer treatment drug, a breast cancer auxiliary treatment drug, a breast cancer anti-tumor immunity activation drug and application of trimethylamine oxide and a precursor product choline thereof.

Background

Breast cancer is the malignant tumor with the highest incidence rate of females and seriously endangers the life and health of contemporary females. Among the molecular types of breast cancer, triple negative breast cancer refers to breast cancer negative for estrogen receptor, progestogen receptor and human EGF receptor-2. Although triple negative breast cancer accounts for 10% -15% of the total breast cancer, it is the subtype with the highest malignancy degree in each molecular type of breast cancer, the recurrence and metastasis risks are higher, and the prognosis of patients is poorer. In the conventional clinical diagnosis and treatment existing at present, only chemotherapy is used as a conventional treatment scheme, and a more effective targeted treatment scheme is lacking.

In recent years, more and more clinical trials are attempting to solve the dilemma that triple negative breast cancer has poor prognosis and lacks effective treatment means from the point of view of targeted therapy and the like, wherein immunotherapy against triple negative breast cancer is regarded as a therapeutic regimen with a promising clinical application prospect. Multiple clinical triple-phase studies focused on systemic and local advanced triple-negative breast cancers, demonstrating that immunotherapy can improve survival benefits for triple-negative breast cancer patients compared to chemotherapy.

However, although immunotherapy regimens can benefit survival of some triple negative breast cancer patients, overall immunotherapy benefit is still lower in triple negative breast cancer patients compared to other cancer species such as non-small cell lung cancer or melanoma. Therefore, how to improve the therapeutic effect of immunotherapy for the whole triple negative breast cancer patient is a clinical problem to be solved urgently.

In order to further improve the curative effect of immunotherapy of patients with triple negative breast cancer, the previous research is often conducted from the perspective of the human body, and the influence of the human body or the tumor self factors on the immunotherapy and the immune tumor microenvironment is explored. However, recent studies suggest that the basal-like immunosuppressive subtype (BLIS) and the immunomodulatory subtype (IM) differ less at the genomic and expression profile levels in the two classes of subtypes that differ widely in the immune microenvironment, but differ greatly in their tumor microenvironment and responsiveness to tumor immunotherapy.

This suggests that in addition to the effects of the human body itself and the tumor itself, other factors may also have an effect on the tumor microenvironment and the therapeutic efficacy of the immunotherapy of triple negative breast cancer. As an important component of tumor microenvironment, symbiotic flora and tumor immune microenvironment are highly overlapped in spatial position, but the influence functions and mechanisms of the symbiotic flora and tumor immune microenvironment are rarely reported in the past breast cancer research.

The traditional research of flora and breast cancer mainly focuses on the intestinal flora, and the influence of the intestinal flora on the intestinal absorption and metabolism of oral medicines is studied, but the regulation and control of the flora on the tumor immunity of the breast cancer is not directly involved. In lung cancer and pancreatic cancer, the related descriptive study suggests the correlation of symbiotic flora and tumor immune microenvironment, but lacks direct causal evidence and experimental support.

Therefore, the subject plans to explore the regulation and control effect and specific mechanism of symbiotic flora on triple negative breast cancer tumor immunity, and provides a new solution for the clinical problem of poor curative effect of triple negative breast cancer tumor immunotherapy.

Based on the previous research results, the center (breast surgery team of auxiliary tumor hospitals of double denier university) constructs 360 maximum triple negative breast cancer multigroup study queues FUSCCTNBC, and the group study layer relates to genome, expression profile, metabonomics and the like. On the basis, the subject further constructs a global maximum triple negative breast cancer host-microorganism multiunit map and explores the immune correlation between symbiotic flora and host tumor.

In addition, the center first developed a refractory triple negative breast cancer FUTURE clinical trial for advanced multi-line treatment in China. The FUTURE clinical trial is an umbrella-type therapeutic clinical study in which patients with partially refractory triple negative breast cancer received immunotherapy. Based on the immune treatment queue, the subject plans to collect clinical samples related to the immune treatment effect and explores the correlation between metabolic products related to symbiotic flora and the immune treatment effect.

Disclosure of Invention

The invention discloses a breast cancer therapeutic drug, a breast cancer auxiliary therapeutic drug, a breast cancer anti-tumor immune activation drug and application of trimethylamine oxide and choline which is a precursor product thereof.

In a first aspect, the invention discloses a breast cancer therapeutic drug, wherein the active ingredient of the drug contains trimethylamine oxide and/or choline which is a precursor product of trimethylamine oxide.

Further, the breast cancer is triple negative breast cancer.

In a second aspect, the invention discloses an auxiliary breast cancer treatment drug, wherein the active ingredient of the auxiliary breast cancer treatment drug contains trimethylamine oxide and/or choline which is a precursor product of the trimethylamine oxide.

Further, the breast cancer is triple negative breast cancer.

In a third aspect, the invention discloses an anti-tumor immune activator for breast cancer, wherein the active ingredient of the anti-tumor immune activator contains trimethylamine oxide and/or choline which is a precursor product of the trimethylamine oxide.

Further, the breast cancer is triple negative breast cancer.

In a fourth aspect, the invention discloses the use of trimethylamine oxide in the treatment of breast cancer.

Further, the breast cancer is triple negative breast cancer.

In a fifth aspect, the invention discloses the use of choline, a precursor product of trimethylamine oxide, in the treatment of breast cancer.

Further, the breast cancer is triple negative breast cancer.

Further, trimethylamine oxide plays a therapeutic role by activating triple negative breast cancer anti-tumor immunity.

Further, choline, a precursor product of trimethylamine oxide, exerts a therapeutic effect by activating triple negative breast cancer anti-tumor immunity.

In a sixth aspect, the invention discloses an application of trimethylamine oxide in preparing a medicament for treating breast cancer.

Further, the breast cancer is triple negative breast cancer.

Further, the medicine is a medicine for improving the curative effect of triple negative breast cancer immunotherapy, or expressed as a medicine for activating triple negative breast cancer anti-tumor immunity to play a therapeutic role.

In a seventh aspect, the invention discloses the use of choline, a precursor product of trimethylamine oxide, in the preparation of a medicament for treating breast cancer.

Further, the breast cancer is triple negative breast cancer.

In an eighth aspect, the invention discloses a pharmaceutical composition comprising trimethylamine oxide and/or choline, a precursor product of trimethylamine oxide, and further comprising a PD-1 inhibitor.

Further, the PD-1 inhibitor is an anti-PD-1 antibody.

1. Purpose of investigation

1.1 explores the way by which symbiotic flora regulates the triple negative breast cancer tumor immune microenvironment.

1.2 provides a solution to the clinical problem of poor immune treatment effect of triple negative breast cancer from the perspective of symbiotic flora.

2. Meaning of research

2.1 clinical significance

From the perspective of symbiotic bacteria, a new treatment method is suggested for clinical dilemma of poor triple negative breast cancer immunotherapy effect, the curative effect of clinical immunotherapy is improved, and the reverse immunotherapy is ineffective.

2.2 scientific significance

The function of symbiotic flora in tumor microenvironment is explored, and the regulation and control mechanism between the symbiotic flora and the host tumor microenvironment is clear.

3. Innovative and application prospect

3.1 breakthroughs in the concept field

The prior researches lack functional researches on the breast tumor symbiotic flora, and the important functions of the flora in the tumorigenesis and development cannot be clarified. The project plan explores the important functions of symbiotic flora in triple negative breast cancer tumor immune microenvironment, and the regulation and control effects of the symbiotic flora on tumor development are clear.

The studies of most of the flora and neoplasms have been limited to correlation and descriptive analysis, but the specific causal relationship is not clear as to which bacterial structure or bacterial metabolite the flora controls the neoplasm. The subject proposes that the flora regulates tumor immunity through a specific metabolite, and the function and meaning of the flora in tumorigenesis and development are breakthroughs of the concept level.

3.2 has wide clinical application prospect

The clinical transformation of the current common flora research results mainly comprises the direct application of bacteria to human bodies, and the main methods comprise fecal bacteria transplantation and oral probiotic preparations. However, these solutions often face serious safety and effectiveness problems in clinical applications. The fecal bacteria transplanting and oral probiotics preparation has the problems of sporadic lethal risk and lower field planting transformation efficiency due to the insufficient definition of the flora type.

Compared with the direct application of bacteria in treatment, the bacteria related metabolites have more controllable safety, more reliable repeatability and stability. Has very strong clinical transformation value, and provides a new direction and thought for the clinical transformation of tumor treatment of the flora research result.

3.3 functional diversity of the products of the flora

In previous studies, trimethylamine oxide (TMAO) has been mainly used for causing inflammatory injury to human body, mainly including cardiovascular risk and chronic nephritis.

In the research, TMAO can improve the low reactivity of the intratumoral immunity in triple negative breast cancer tumors, thereby activating the intratumoral anti-tumor immunity and inhibiting the tumor growth, and the positive effect of TMAO on human bodies is proposed. This result suggests that bacteria and bacterial metabolites will change with different tissue types and different physiological and pathological states of the human body, revealing the diversity of functions of the flora products under different human environments.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a technical roadmap for studying a "build triple negative breast cancer tumor microenvironment flora and its metabolite profile".

Fig. 2 is a technical roadmap for a clinical transformation study to investigate the clinical relevance of two 'TMAO to substrate choline activated triple negative breast cancer anti-tumor immunity'.

FIG. 3 is a partial roadmap for studying the animal experimental phenotype of three "TMAO activating triple negative breast cancer anti-tumor immunity by inducing tumor cell apoptosis, inhibiting tumor growth".

Fig. 4 is a partial roadmap for the study of four molecular experimental mechanisms of TMAO for activating triple negative breast cancer anti-tumor immunity and inhibiting tumor growth by inducing tumor cell apoptosis.

Figure 5 is that symbiotic flora is an important component of the triple negative breast cancer tumor microenvironment.

FIG. 6 shows that the symbiotic flora compositions are different in different tumor microenvironments of triple negative breast cancer.

FIG. 7 shows enrichment of bacterial specific metabolite trimethylamine oxide in an immunocompetent tumor microenvironment.

FIG. 8 is that Clostridia is closely related to TMAO levels and that TMAO-specific metabolizing enzyme CutC is closely related to anti-tumor immune activation.

Figure 9 is a high TMAO level suggesting better therapeutic efficacy of immunotherapy.

Figure 10 is that intratumoral injection of TMAO significantly inhibited tumor growth and promoted intratumoral CD 8T cell infiltration.

FIG. 11 shows CD 8T cells and IFNγ after intratumoral injection of TMAO ⁺ CD 8T infiltration was increased.

FIG. 12 is an up-regulation of intratumoral anti-tumor cytokine levels following intratumoral injection of TMAO.

FIG. 13 is a graph of intratumoral TMAO injection that also inhibited tumor growth and activated intratumoral anti-tumor immunity in a 66cl4 cell-molded graft tumor model.

Figure 14 is RNAseq data suggesting that tumor cell apoptosis pathways and inflammatory pathways are simultaneously upregulated following TMAO treatment.

FIG. 15 shows the occurrence of scorch in tumor cells after TMAO treatment.

Figure 16 is a choline-activated triple negative breast cancer anti-tumor immunity.

Detailed Description

The invention is further described with reference to the following detailed description in order to make the technical means, the inventive features, the achieved objects and the effects of the invention easy to understand. The present invention is not limited to the following examples.

It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the practice of the invention, are not intended to be critical to the essential characteristics of the invention, but are intended to fall within the spirit and scope of the invention.

1. Study one: drawing multiple groups of microbial patterns of triple negative breast cancer, which is also called constructing triple negative breast cancer tumor microenvironment flora and metabolite patterns thereof (technical roadmap is shown in figure 1)

1.1 queue construction of triple negative breast cancer microbiome profile:

the present study was intended to complement the lack of local flora study in triple negative breast cancer. 360 samples in the natural queue of the central triple-negative breast cancer patient are utilized, the relative abundance of symbiotic flora of each sample is calculated and obtained by using a microbiome algorithm, and the differential flora of different microenvironment samples is analyzed by using Lefse analysis. 1.2 ranking and algorithm of triple negative breast cancer microbiology profile:

the 330 non-target metabonomics data in the natural queue of the central triple-negative breast cancer are utilized, related metabolites of microorganisms are defined by comprehensively referring to the past literature, and differential metabolites are screened according to different tumor microenvironments. 1.3 validation of the presence of the different Clostridia in the immunocompetent tumor microenvironment:

1.3.1 isolated culture of bacteria:

grinding fresh breast tumor tissue, culturing Clostridium by using BHI culture medium of Clostridium selective culture medium, plating in anaerobic incubator, selecting all monoclonal strains, identifying strain by mass spectrum, and determining main differential strain under Clostridium.

1.3.2 16s rDNA verification of differential species:

and (3) taking triple negative breast cancer tumor tissues, extracting bacterial DNA in the tumor tissues, and verifying that the differential bacteria are Clostridium strains through 16s rDNA full-length sequencing, wherein the results can be matched with algorithm results and culture results.

1.3.3Fish staining it was clear that the triple negative breast cancer endosymbiont flora and tumor microenvironment were highly coincident in spatial position.

1.4 in vitro bacterial culture to verify bacterial and metabolite relationship:

1.3.1 was isolated and cultured and the resulting genus Clostridia was identified, amplified in a liquid medium, choline, an essential raw material for the synthesis of trimethylamine oxide (TMAO) precursor product Trimethylamine (TMA), was administered, and the supernatant was taken after the culture, and the presence or absence of TMA in the medium was detected by targeted metabolism.

The specific experimental process is as follows:

materials and methods

1. ) Experimental materials and instrument

1.1 Clinical information of triple negative breast cancer queue of auxiliary tumor hospital at double denier university

The untreated triple negative breast cancer tumor tissue samples used in the study were from a tissue sample library of a secondary university affiliated tumor hospital and a breast surgery sample library of a secondary university affiliated tumor hospital, and the sampling scheme obtained approval and passage of the ethical committee of the secondary university affiliated tumor hospital. Each patient had a full knowledge of the use of the sample and had signed an informed consent. The invention retrospectively collects 360 samples of triple negative breast cancers. The samples are all freshly cut tissues in breast surgery in a university affiliated tumor hospital, and are strictly preserved according to the regulations of a affiliated tumor tissue library of the university, so that the requirement of sequencing is met. The samples are verified by immunohistochemical analysis of the complex denier university pathology department, and the immunohistochemical classification of the samples is determined to be triple negative breast cancer. After the samples and related clinical data were organized, they were defined as triple negative breast cancer cohort (Fudan University Shanghai Cancer Center Triple Negative Breast Cancer, FUSCC-TNBC) samples from the secondary oncology hospital at the university of double denier. The source of the queues was females with an average age of 53.+ -. 11 years. Specific queue information is found in the subject group's prior publication (DOI:

10.1016/j.ccell.2019.02.001)。

1.2 Major reagent kit

1.3 Main laboratory instrument equipment

a) Hybridization furnace

b) Anaerobic workstation

c) Anaerobic incubator

d) Polymerase chain reaction (Polymerase chain reaction, PCR) gene amplification instrument

e) Gel electrophoresis apparatus

f)Illumina Hiseq 4000platform

2. ) Experimental method

2.1 Fluorescence in situ hybridization staining (Fluorescence in situ hybridization, FISH) i. reagent preparation and formulation:

a. hybridization buffer

b. Xylene (P)

c.95% ethanol, 90% ethanol, 70% ethanol

d. Primer dissolution:

primer: EUB338 is 5' -GCTGCCTCCCGTAGGAGT-3'5' end Cy 3-labeled. Primer use

The TE solution was dissolved and stored at a concentration of 100. Mu. Mol (-20℃for light protection).

Washing buffer

ii tissue dewaxing and hydration

Taking paraffin tissue sections of a patient after triple negative breast cancer operation, sequentially placing the paraffin tissue sections in the following solutions for 10 minutes respectively, and sucking the residual redundant solution by using a paper towel every time one solution is replaced.

Hybridization staining

a. The hybridization buffer is heated in a water bath at 50-56 ℃.

b. The primers were diluted to a working concentration of 10nM with hybridization buffer.

c. The hydrated slices are placed in a wet box, and a small amount of water is added to the bottom.

d. A hydrophobic pen draws a hydrophobic ring on the slice, 150 μl of staining solution is dripped into each slice hydrophobic ring, and hybridization is carried out overnight (not more than 16 hours) in a 50 ℃ incubator.

e. The wash buffer is heated in a water bath at 50-56 ℃.

f. The sections were washed twice with washing solution.

g. Counterstaining: DAPI was dissolved in wash buffer and stained at room temperature for 10 min.

h. The sections were washed twice with washing solution.

And iv, resin sealing sheets and observing by a fluorescence microscope.

2.2 Digestion of human tumor tissue samples

The experiments used a Meitian and gentle human tumor tissue dissociation kit (Tumor Dissociation kit, human, cat # 130-095-929).

i. Preparation of digestive juice

Enzyme H in 3ml RPMI 1640 (serum free) dilution kit

b.3ml of enzyme R in RPMI 1640 (serum free) dilution kit

1ml of solution A in the kit enzyme A in the dilution kit

All diluted enzymes are preserved at-20 ℃, and are sub-packaged according to proper proportion, so that repeated freezing and thawing are prevented, and the shelf life of the diluted enzymes is 6 months.

d. Preparation of digestive working solution

Digestion of tumor tissue

a. Fresh triple negative breast cancer surgical samples were opened in an anaerobic workstation.

b. Removing fat and necrotic areas around cancerous tissue.

c. 0.2g of tumor tissue sample was weighed using an electronic balance in the bench.

d. After the sample was chopped as much as possible, 2.5ml of digestion medium was added to the petri dish

e. The anaerobic station was thermostated at 37 degrees celsius and the samples were placed in the station for digestion for 1 hour.

No significant particles were present in the sample after 1 hour.

g. The sample was filtered into a 15ml centrifuge tube and the next experiment was performed.

2.3 Isolation and culture of anaerobic bacteria in human tumor tissue samples

i. Preparation of culture Medium

Liquid culture medium

Solid medium

* All media are sterilized at high temperature and pressure.

Amplification of anaerobes in fresh samples of triple negative breast cancer

100. Mu.l of the tumor digest was inoculated into 2ml of BHI (0.05% cysteine) medium and cultured for 24 hours in a constant temperature anaerobic incubator at 37 ℃.

Separation of anaerobes from fresh samples of triple negative breast cancer

a. 100 μl of amplified samples were smeared onto BHI (0.05% cysteine) agarose solid medium, and 6 pieces of solid medium were smeared on each amplified sample.

b. The smeared agarose culture medium is placed in a constant temperature anaerobic incubator at 37 ℃ for 5-9 days after being inverted and placed.

c. The presence or absence of newly added monoclonal antibodies on the agarose medium was observed daily from day 5.

1) Amplification of monoclonal anaerobes in fresh samples of triple negative breast cancer.

a. A new monoclonal of the agarose medium was picked up by a sterile 200. Mu.l pipette nozzle and inoculated into 3ml of BHI (0.05% cysteine) broth.

b, standing and culturing in a constant temperature anaerobic incubator at 37 ℃ for 5-14 days.

2.4 Bacterial DNA extraction

The laboratory used DNeasy PowerLyzer PowerSoil Kit (Cat # 12855-50) to extract bacterial genomic DNA.

a. The sample was transferred to a 2ml centrifuge tube, screwed down, transferred out of the anaerobic workstation and immediately placed in a centrifuge at 4 ℃,8000g for 30 minutes.

b. The supernatant was discarded, 750. Mu.l of Powerbead solution was transferred to "Powerbead Tube" provided by the kit, 60. Mu.l of solution C1 was added, and after mixing upside down, the maximum intensity was vortexed on a vortexing machine for 10 minutes.

10000g of the mixture was centrifuged at 4℃for 30 seconds, and the supernatant was transferred to a clean 2ml centrifuge tube.

d. Mu.l of solution C2 was added, vortexed for 5 seconds and incubated in a 4℃refrigerator for about 5 minutes.

e.10000g were centrifuged for 1 min and 600. Mu.l of supernatant was transferred to a new 2ml centrifuge tube. 200 μl of solution C3 was added, vortexed for 5 seconds, and incubated in a 4 degree refrigerator for about 5 minutes.

f.10000g were centrifuged for 1 min and 750. Mu.l of supernatant were transferred to a new 2ml centrifuge tube. 1200 μl of solution C4 was added and vortexed for 5 seconds.

g. Mix well and transfer 675 μl to "MB Spin Column", centrifuge 10000g for 1 min, discard waste liquid, repeat twice.

h. Mu.l of solution C5 was added and centrifuged at 10000g for 30s. And (5) discarding the waste liquid.

i. 10000g of empty tube is centrifuged for 1 minute.

j. Transfer the column to a fresh clean 1.5ml centrifuge tube and add 50 μl TE solution. Centrifuge 10000g for 1 minute.

k. The DNA concentration and purity were measured using a Nanodrop2000 spectrophotometer instrument.

2.5 Polymerase chain reaction (Polymerase Chain Reaction, PCR)

The laboratory generally uses DreamTaq DNA polymerase (Cat#EP 0703) as an amplifying enzyme for PCR systems.

PCR System (total volume 25. Mu.l)

PCR amplification procedure

Primers are used in this section

2.6 Gel electrophoresis of PCR products

i. Solution preparation

50 delta TAE solution

Gel preparation and electrophoresis

According to the target band fragment, agarose gel of suitable concentration is selected and prepared, and then electrophoresis is performed at 150V for about 30 minutes. The laboratory routinely uses a natural nucleic acid dye (170-3001). After electrophoresis, the gel is photographed and stored by a gel imager.

2.7 RNA sequencing

Firstly, an Agilent 2100 chip is used for quality control of samples of the FUSCC-TNBC queue. RNA pooling was then performed as required by the reagent manufacturer for Illumina TruSeq Stranded Total RNA LT Ribo-Zero Gold. And finishing RNA sequencing in an Illumina Hiseq platform, and outputting the result of FPKM after the obtained fastq file is matched with a human genome library for subsequent analysis. Specific sequencing procedures are found in the subject group prior published articles (DOI:

10.1016/j.ccell.2019.02.001)。

2.8 Microorganism related metabolite detection

In the triple negative breast cancer queue of the accessory tumor hospital of the university of double denier, 258 cases of samples were selected for polar metabolite detection. The 258 samples each contained data for RNA sequencing (RNA-seq) and detection of polar non-target metabolites. After freezing and homogenizing the breast tumor tissue with liquid nitrogen, the chromatographic detection was completed in 1290Infinity Series UHPLC system (Agilent Technologies) equipped with UPLC BEH Amide filter column. Subsequently, metabolite data were captured using a 6550QTOF mass spectrometer. The metabolic raw data is converted into mzXML format through Proteowizard, and the data of the abundance of the polar non-target metabolites is finally obtained through the treatment of R package XCMS (version 3.2). The specific analytical procedure is found in the prior publication of the subject group (DOI: 10.1038/s 41422-022-00614-0)

Further, bacterial related metabolites exceeding 3700 were defined based on Kyoto Encyclopedia of Genes and Genomes (KEGG), biol, gmREPO and other databases, in combination with published literature and metagenomic sequencing data. In combination with the sequencing results of the polar metabolites in this cohort, 92 microorganism-related metabolites were found in this cohort.

2.9 Immunohistochemistry)

i. Tissue dewaxing and hydration

Antigen retrieval

The laboratory routinely used Antigen Retrieval buffer (100 XCitite buffer, cat#ab 93678) from Abcam.

Diluting an antigen buffer solution by 1:100, putting the hydrated paraffin sections into an antigen retrieval liquid, boiling the paraffin sections by high fire of a microwave oven, keeping boiling by low fire, and retrieving the paraffin sections for 20 minutes.

Tap water was flushed into the vessel for 10 minutes.

A. Closure

a. The slices were placed in a wet box with a small amount of water added to the bottom.

b. The hydrophobic pen draws a hydrophobic ring on the slice, 150 μl of goat serum sealing liquid is dripped into each slice hydrophobic ring, and PBS is used for washing once after 20 minutes at room temperature.

B. Dyeing

a.1:200 primary anti-FMO3 (Abcam, ab 126711) was diluted with antigen buffer.

b. 50 μl of diluted primary antibody was added dropwise to the hydrophobic ring, and incubated overnight in a refrigerator.

c. Rewarming at room temperature for 45 minutes.

Pbs wash 3 times for 5 minutes each.

e.1:1000 dilution of the goat anti-rabit HRP secondary antibody.

f. 50 μl of the diluted secondary antibody was added dropwise to the hydrophobic ring, and incubated at room temperature for 1-2 hours.

PBS was washed 3 times for 5 minutes each.

DAB is developed for 5-10 minutes.

PBS rinse for 10 min.

j. Counterstaining with hematoxylin for 1 min.

k. And (5) hydrochloric acid alcohol differentiation.

And washing with tap water for 10-15 min.

C. And (5) performing microscopic examination after sealing.

3. ) Bioinformatics calculation method

3.1)PathSeq

The source of the RNA sequencing data was 360 patient tumor tissues of the triple negative breast cancer cohort of the secondary university affiliated hospital, and in the original RNA sequencing data, the RNA sequences of prokaryotic origin were not removed. The GATK PathSeq algorithm is used to match microbiome related sequences including bacteria, viruses, archaea and fungi in human tissues. After sequence matching is completed, the algorithm will further match sequences to different microbiomes based on different biological classification levels.

In this study, the human sequence reference dataset utilized included 5 GRCh38 (https:// software. Bromoinstall. Org/GATK/download/bundle, hg38/v 0) partially from the GATK database, virus content (chrysEBV) from IMGT and Immuno Polymorphism Database (IPD), GENCODE transcriptome (human v25;

https://www.gencodegenes.org) Human breakpoint connection sequence (GenBank accessions KY503218-KY 5808060) and NCBI UniVec cloning vector sequence https://www.ncbi.nlm.nih.gov/ tools/vecscreen/univec/)。

The relative abundance calculation method of the bacterial layer is as follows: the relative abundance of a certain bacterium in the sample = (number of unique matching regions in genome Δ1,000,000)/(total number of bacterial reads matched x bacterial genome size). And homogenizing the obtained relative abundance data according to each sample row, and finally obtaining the relative abundance of bacteria in each sample.

3.2 Species difference analysis

A linear discriminant analysis (Linear discriminant analysis Effect Size, LEfSe) algorithm was used to explore microbiome differential analysis in different group samples. To define significantly different microorganisms in immunoregulatory and non-immunoregulatory samples, the LDA (linear discriminant analysis) threshold was set to 2, and the alpha values of the Kruskal-Wallis test and the Wilcoxon test were set to 0.05.3.3 Analysis of Gene set enrichment and Gene set Difference

In the gene set enrichment analysis, R-packet GSVA was used for single sample gene set enrichment analysis, and FDR < 0.25, and nominal p < 0.05 were defined as significantly enriched. All analytical procedures were based on R (v.3.6.1,https://cran.r- project.org/) And (5) unfolding.

3.4 Immune cell quantification

Based on RNA-seq data from the triple negative breast cancer cohort of the secondary oncology hospital at the university of double denier, with CIBERSORT (Cell-type Identification by Estimating Relative Subsets of RNA Transcripts, https://cibersort.stanford.edu/) And (3) carrying out quantitative analysis on the abundance of the immune cell subset in the tumor tissue sample according to an absolute quantitative mode in the tool.

4. ) Statistical analysis

The statistical analysis method for clinical sample queue correlation is as follows. In the statistical analysis of the comparative continuity variables, the significance of the differences was compared using Student's t test, mutation analysis (analysis of variance, ANOVA) and Mann-Whitney Wilcoxon test. In the statistical analysis of the comparative classification variables, the significance of the differences was compared using the Pearson's chi-square test and the Fisher's exact test. To calculate the correlation of the two variables, the present invention uses a paired Spearman's correlation analysis. All of the analytical procedures above were based on R (v.3.6.1,https://cran.r-project.org/) And (5) unfolding.

For statistical analysis in vitro experiments, the protocol was developed based on GraphPad Prism software (GraphPad Software) version 9.0.0. To compare differences between the two groups, a two-tailed unpaired Student's t test was used.

2. Study two: the correlation between TMAO and the therapeutic effect of triple negative breast cancer patients is explored, which is also called the clinical correlation study of TMAO and substrate choline activated triple negative breast cancer anti-tumor immunity (the technical roadmap is shown in figure 2)

2.1 study cohort and method:

patients who were immunotherapy in the C-arm of the present central FUTURE cohort (clinical three.gov identifier: NCT 03805399) were selected and analyzed for differential bacterial related metabolites based on non-target metabolic screening and targeted metabolism in patients with different therapeutic effects of immunotherapy.

2.2 validation of the correlation of differential metabolites with immune activation:

separating blood PBMC of patients with high abundance of different metabolites, sorting CD 8T cells by using magnetic beads, co-culturing the blood PBMC with human breast cancer cells in vitro, and detecting the killing function of the blood PBMC on the breast cancer cells by using an LDH release test and flow cells.

The specific experimental process is as follows:

materials and methods

1.1 Three negative breast cancer patients immunotherapy cohort

The study collected 12 patient blood samples from the FUTURE clinical trial (ClinicalTrials. Gov identifier: NCT03805399, an umbrella study of stage Ib/II based on typing and genomic biomarkers guidance). The patients in the queue are female patients with triple negative breast cancer, and the median age is 57+/-8 years after combined immunotherapy and chemotherapy. The blood sample collection protocol was agreed by the ethical committee of the affiliated oncology hospital at the university of double denier, and each patient had knowledge of the sample for scientific investigation and signed an informed consent document. The treatment information for the patients in this cohort comes from the management of the complex university affiliated oncology hospital history system.

2. ) Experimental method

2.1 Plasma targeted metabolic detection for triple negative breast cancer patients

a) According to the experimental method that ethical examination of the ethical committee of the affiliated tumor hospital of the compound denier university passes, whole blood of the patients in the group of FUTURE clinical test is collected and a viologen (an anticoagulant is EDTA) is collected and used. Avoiding violent collision and movement in the process of transporting samples so as to prevent hemolysis

b) After the whole blood sample is received by a laboratory, the whole blood sample is centrifuged for 20 minutes at the normal temperature of 1300rpm, and the whole blood sample is quickly lifted and lowered.

c) The centrifuged sample can be divided into three layers, and the upper layer is semitransparent and yellowish and is the plasma of the patient.

d) 200 microliters of the upper plasma was taken and flash frozen with liquid nitrogen. The frozen sample can be used for targeted metabolic detection.

3. ) Statistical analysis

The statistical analysis method for clinical sample queue correlation is as follows. In a statistical analysis of comparative continuity variables, the present invention compares the significance of their differences using Student's t test, ANOVA and Mann-Whitney Wilcoxon test. In the statistical analysis of the comparative classification variables, the significance of the differences was compared using the Pearson 'schi-square test and the Fisher's exact test. To calculate the correlation of the two variables, the present invention uses a paired Spearman's correlation analysis. All of the analytical procedures above were based on R (v.3.6.1, https://cran.r-project.org/) And (5) unfolding.

Statistical analysis in vivo and in vitro experiments was then based on GraphPad Prism software (GraphPad Software) version 9.0.0. To compare the differences between the two groups, the present invention uses a two-tailed unpaired Student's t test. To compare differences in tumor volume growth curves, a Two-way ANOVA with Tukey's test was used. To compare the difference in cytokine levels in tumor tissue, lactate dehydrogenase release levels and intratumoral TMAO levels in tumor infiltrating cells, significance was analyzed using One-way ANOVA with Bonferroni's assay. The other specific statistical analyses have been specifically discussed in the specific analysis section.

3. Study three: in vivo experiments prove that the microbial metabolite TMAO can inhibit tumor growth, also called TMAO can activate triple negative breast cancer anti-tumor immunity by inducing tumor cell apoptosis, and inhibit the phenotype part of tumor growth animal experiment (the technical roadmap is shown in figure 3)

Study one, two, demonstrates that TMAO high expression is closely related to activation of tumor immunity. The subsequent test shows that the microbial metabolite TMAO has tumor growth inhibiting function, and the 4T1 and 66cl4 cell lines are used in preparing mouse transplanted tumor model to simulate local TMAO high level and systemic TMAO high level via intra-tumor injection and intraperitoneal injection, and to observe whether the high TMAO content can inhibit tumor cell proliferation. The tumor, blood and spleen Th1 cells, treg cells, CD 8T cells, IFNgamma+CD8T cells, M1 type macrophages and M2 type macrophages of the mice after the molding drug are detected by flow cytometry, and the cytokine Elisa results of IFNgamma, TNF alpha, IL-2, IL-10 and the like are synthesized to evaluate the antitumor immunity level of the mice after the TMAO drug administration. Treatment groups with TMAO in combination with PD-1 inhibitors were set up simultaneously with TMAO alone, and it was observed whether upregulation of TMAO levels could reverse the immunotherapeutic inefficiency of triple negative breast cancer.

4. Study IV: verification that TMAO activates anti-tumor immunity by inducing tumor cell apoptosis, and inhibits tumor growth, also called TMAO activates triple negative breast cancer anti-tumor immunity by inducing tumor cell apoptosis, and inhibits tumor growth molecular experiment mechanism part (its technical roadmap is shown in figure 4)

The preliminary in vitro experiment result shows that the cells are in a special burst form of scorching after TMAO stimulation, and RNA-seq prompts that the relevant scorching channel of the caspase 3 of the cells is obviously up-regulated after TMAO stimulation. Taken together, TMAO presumably activates tumor immunity through pyrosis, thereby inhibiting tumor growth.

To verify this hypothesis, the present invention was designed to verify the occurrence of scorch after TMAO stimulation by cell experiments. The method comprises the main steps of comprehensively and quantitatively evaluating the coke death proportion of tumor cells from morphology and fluorescence staining proportion after PI staining, verifying whether the coke death key proteins caspase 3 and GSDME are cleaved and activated by using Western blot, and using an LDH release experiment to distinguish the coke death from the late apoptosis.

Secondly, whether the occurrence of tumor cell apoptosis caused by TMAO is a key mechanism of TMAO activation anti-tumor immunity or not is verified, a mouse transplantation tumor model is established after KO breast cancer cell GSDME genes are selected, and the infiltration activation condition and the cytokine level change condition of immune cells in the mouse tumor are observed.

In order to verify that TMAH-induced apoptosis is inhibited by activating anti-tumor immunity, the invention aims to search whether TMAH has the capability of inhibiting tumor growth when inhibiting anti-tumor immunity activation by adding a CD8 antibody inhibitor while mice are stimulated by TMAH. In summary, the present invention is to confirm whether TMAO activates anti-tumor immunity through pyrosis, and whether anti-tumor immunity activated through pyrosis is critical for inhibiting tumor proliferation.

The specific experimental procedure for study three and four was as follows:

materials and methods

1. ) Experimental materials and instrument

1.1 Cell origin

The experiment used three cell lines, 4T1, 66Cl4, MDA-MB-231 and HEK-293T, from the following sources:

1.2 Animal origin and feeding conditions

The experiment used two mice, BALB/c and NOD-SCID.

Female BALB/c with 6-8 weeks of age is bred under SPF condition, the breeding process follows 12 hours of sunlight and 12 hours of dark cycle, and the breeding room is always kept at 20-22 ℃ and 50% -70% of humidity. The mice can freely obtain feed and drinking water, and the feeding is not limited. All feeds, pads and drinking water are sterilized by radiation or high pressure steam, which meets SPF standard. The paper animal protocol was approved by the ethical committee of Shanghai laboratory animal center (Shanghai Laboratory Animal Center, SLAC).

1.3 Major reagents and kits

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1.4 Main laboratory instrument equipment

a) Carbon dioxide cell incubator

b) Super clean bench

c) Low-temperature horizontal centrifugal machine

d) Low-temperature high-speed centrifugal machine

e) Meitian gentle genetleMACS ^TM Octo Dissociator with Heaters

f) FACScan cell flow instrument

g) BD LSRFortessa cell flow instrument

h)Luminex 200System

i) Chemiluminescent imaging system

j) Full-automatic light absorption enzyme label instrument

k) Applied Biosystems real-time fluorescence quantitative PCR instrument

l) high content imaging analysis system

2. ) Experimental method

2.1 Cell culture and passage

The immortalized cell lines 4T1, 66cl4, MDA-MB-231 and HEK-293T used in this experiment were cultured under the same conditions, dulbecco's Modified Eagle Medium, DMEM, supplemented with 10% heat-shocked serum and 1% penicillin-streptomycin, and all cells were periodically tested for mycoplasma to ensure no mycoplasma infection. And (3) carrying out passage when the cell fusion degree reaches 80%, washing passage cells by PBS, digesting the passage cells by pancreatin, blowing into single cell suspension, and centrifuging the PBS, and washing the passage cells for 1:4 passage. Thin and fineThe cells are changed once every two days, the incubator is kept at 37 ℃ and CO ₂ The concentration was maintained at 5%. Isolated CD8 in lymph node of mice ⁺ The culture conditions of the cells are discussed in detail in the subsequent methodology.

2.2 In situ injection of mouse breast cancer cells

i. Treatment of tumor cells

4T1, 66cl4 or MDA-MB-231 is passaged for 1-2 days in advance, so that the cell fusion degree on the day of tumor implantation is ensured to be about 70% -80%, and meanwhile, mycoplasma PCR detection is carried out, so that the cell mycoplasma is ensured to be negative.

Cells were washed twice with pbs and then digested with pancreatin for about 3 minutes, 2 volumes with serum medium to terminate digestion.

c. Cells were washed twice with PBS and resuspended with the appropriate volume of PBS after centrifugation.

d. Cell counts, cells adjust cell concentration to 0.5Δ10 per 25 μl ⁶ Individual cells.

e. Adding equal volume matrigel (without cell factor) into cells, mixing uniformly, placing on ice, and injecting as soon as possible.

in situ injection of breast cancer cells

a. The mice were fixed in supine position after qi-anaesthesia, with the midline opening in the lower abdomen, without shearing the peritoneum.

b. The left inguinal region was blunt-separated and inguinal mammary fat pad was found.

c. A50. Mu.l volume of the homogeneous cell suspension was injected with an insulin needle.

d. The abdominal opening is sutured.

e. The anesthesia is revived.

Observing wound healing condition on 1-5 days after operation, and preventing infection and off-line condition.

2.3 Monitoring of tumor growth curve

After generally successful modeling on day 6 (4T 1 cell line) and day 9 (66 cl4 cell line), the tumor grows to a size that can be visually observed and measured. Tumor measurements tumor length (L) and width (W) were measured and recorded using vernier calipers. Tumors were measured every 3 days and tumor size was calculated as follows:

Tumor volume = 0.5 delta long diameter (L) delta wide diameter squared (W) ² )。

2.4 Intratumoral injection and intraperitoneal injection of mice

The experiment adopts the medicine, the administration method and the concentration

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Intratumoral injection is a multi-point injection with a volume of 20 μl per injection and intraperitoneal injection with a volume of 100 μl per injection. In the present invention, the terms "anti-PD-1 antibody" and "PD-1 antibody" both refer to the same meaning, and the English terms are anti-PD-1 anti; the "CD8 antibody" is also the same substance as the "anti-CD 8 antibody".

2.5 Digestion of mouse tumor tissue

The experiment uses a Meitian gentle mouse tumor tissue dissociation kit (Tumor Dissociation kit, mouse, cat#130-096-730)

i. Preparation of digestive juice

3ml RPMI-1640 medium (serum free) diluted enzyme H in the kit.

2.7ml RPMI-1640 medium (serum free) was used to dilute the enzyme R in the kit.

1ml of solution A in the kit dilutes the enzyme A in the kit.

d. Preparation of digestive working solution

Digestion of tumor tissue

a) The tumors were removed intact after mice were sacrificed.

b) Removing fat and necrotic areas around cancerous tissue.

c) A maximum of 1g of tumor tissue sample was weighed using an electronic balance in the bench.

d) After the samples were chopped as much as possible, they were transferred to C-tubes (Meitian Tsaoko gentleMACS C Tube) and 2.5ml of digestion working fluid (working fluid preheated at 37 ℃).

e) Screw the tube port and transfer the sample to genemacs ^TM Octo Dissociator with Heaters.

f) The 37c_m_tdk_2 program is selected and run.

g) And taking down the C pipe after the program operation is finished. PBS was added for rinsing and the samples transferred onto a cell filter, and the digested tumor tissue was filtered into a 50ml centrifuge tube from which PBS was added to the nozzle.

h) Cells were centrifuged at 300g for 7 min and washed twice with PBS.

i) Cell counts, appropriate volumes of PBS resuspended cells and placed on ice.

2.6 Isolation of immune cells in mouse tumor microenvironment

The laboratory adopts a density gradient centrifugation method to separate immune cells in tumors, and adopts a separation system of 40% of Percoll and 80% of Percoll aiming at the tumors formed by 4T 1.

i. Solution configuration

100％Percoll(100ml)

40％Percoll(100ml)

80％Percoll(100ml)

The Percoll is equilibrated to room temperature prior to use.

Density gradient centrifugation

a) The resulting tumor cell suspension after centrifugation was resuspended in appropriate volume of 40% percoll.

b) The pipette was adjusted to minimum speed, the pipette was inserted into the bottom of the tube, and an equal volume of 80% percoll was slowly added. Ensuring that the interface between the two is not disturbed as much as possible.

c) And the centrifugation is carried out at room temperature for 23 minutes at 2300 rpm.

d) The interface cells were pipetted into a new 15ml centrifuge tube, filled with room temperature PBS, and resuspended.

e) Cells were centrifuged at 300g for 10 min at normal temperature.

f) The supernatant was discarded, the cells resuspended in 15ml PBS and washed twice by centrifugation.

g) The cells were resuspended in the appropriate volume of ice MACS buffer and counted. And entering the subsequent steps.

2.7 Flow cytometry

i. Solution configuration

MACS buffer

Closure of

a. Taking 2X 10 ⁶ Tumor infiltrating lymphocytes (tumor infiltration lymphocytes, TIL) isolated in 2.6 were diluted with 50. Mu.l of ice MACS buffer.

100 volumes of CD16/CD32 blocking antibody were added.

c. Incubate on ice for 10 minutes.

d. All the following procedures are strictly protected from light.

Dead living staining

a. The laboratory was stained routinely with U-bottom 96-well plates, resuspended in 200 μl ice PBS, centrifuged at 300g for 5 min, and washed twice.

b.1:20000 LIVE/DEAD Fixable DEAD Cell Stain was diluted in PBS (without serum).

50 μl of dead live staining solution resuspended cells and stained on ice for 10 min.

d. Surface antigen staining

200 μl ice PBS was resuspended, centrifuged at 300g for 5 min and washed twice.

f. The surface-stained antibodies were diluted with MACS buffer in the optimal ratio as suggested by the results of the antibody titration experiments, and the surface-stained antibodies for each stained Panel were mixed in 1 tube.

The cells were resuspended in 50. Mu.l of surface staining solution and stained for 30 minutes at room temperature.

200 μl ice MACs buffer was resuspended, centrifuged at 300g for 5 min and washed twice.

iv fixing and rupture of membranes

a. The laboratory routinely uses the eBioscience Foxp 3/transcription factor staining buffer kit (Sieimer fly, cat#00-5523-00) for fixed rupture.

b. According to 1 (Fixation/Permeabilization Concentrate): 3

(Fixation/Permeabilization Diluent) preparing the fixative solution.

c. Cells were resuspended with 200 μl fixative. Incubate for 60 minutes in refrigerator or 30 minutes at room temperature.

d. (if there is no staining with intracellular antigen, the immobilized cells can be detected on-line after centrifugation by resuspension with 500. Mu.l MACs buffer.)

e. Centrifuging and discarding the supernatant.

f. ddH at a ratio of 1:10 ₂ Membrane rupture liquid was prepared by O dilution Permeabilization Buffer (10×).

200 μl of the membrane-rupture liquid was used to wash the cells twice, and the cells were centrifuged at 600g for 4 minutes at room temperature.

Intracellular antigen staining

a. The intracellular staining antibodies were diluted with MACS buffer at the optimal ratio indicated by the results of the antibody titration experiments, and the intracellular staining antibodies for each staining Panel were mixed in 1 tube.

50 μl of intracellular staining solution resuspended cells and stained for 30 min at room temperature.

200 μl membrane rupture solution was resuspended, centrifuged at 600g for 5 min, and washed twice.

Cells after intracellular staining were resuspended in 500. Mu.l MACs buffer and were ready for on-machine detection.

Flow instrument detection

a. Single-stained tubes were prepared with anti-CD19 antibodies of the corresponding colors and spleen single-cell suspensions.

b. The appropriate voltage is set and the compensation is adjusted by means of a single dye tube.

c. Sample detection is carried out, a facs file is stored, and result analysis is carried out.

2.8 Immunohistochemistry)

i. Tissue dewaxing and hydration

Taking paraffin tissue sections of a patient after triple negative breast cancer operation, sequentially placing the paraffin tissue sections in the following solutions for 10 minutes respectively, and sucking residual redundant solution by using a paper towel after replacing one solution

Antigen retrieval

The laboratory routinely used Abcam's Antigen Retrieval buffer (100 XCitrate buffer, cat#ab 93678).

a) Diluting an antigen buffer solution by 1:100, putting the hydrated paraffin sections into an antigen retrieval liquid, boiling the paraffin sections by high fire of a microwave oven, keeping boiling by low fire, and retrieving the paraffin sections for 20 minutes.

b) Tap water was flushed into the vessel for 10 minutes.

Sealing off

a) The slices were placed in a wet box with a small amount of water added to the bottom.

b) The hydrophobic pen draws a hydrophobic ring on the slice, 150 μl of goat serum sealing liquid is dripped into each slice hydrophobic ring, and PBS is used for washing once after 20 minutes at room temperature.

Dyeing process

a) 1:200 primary anti-CD8 or anti-GSDME was diluted with antigen buffer.

b) 50 μl of diluted primary antibody was added dropwise to the hydrophobic ring, and incubated overnight in a refrigerator.

c) Rewarming at room temperature for 45 minutes.

d) The PBS was washed 3 times for 5 minutes each.

e) Diluting the goat anti-rabit HRP secondary antibody at 1:1000.

f) 50 μl of the diluted secondary antibody was added dropwise to the hydrophobic ring, and incubated at room temperature for 1-2 hours.

g) The PBS was washed 3 times for 5 minutes each.

h) DAB was developed for 5-10 minutes.

i) The PBS was washed for 10 minutes.

j) Counterstaining with hematoxylin for 1 min.

k) And (5) hydrochloric acid alcohol differentiation.

l) washing with tap water for 10-15 minutes.

v, microscopic examination after sealing.

2.9 Detection of cytokine levels in tumor tissue

Cytokine level detection in tumor tissues in this paper was detected on the Luminex 200System (R & D System) platform using ProcartaPlex Mouse Cyotokine Panel (eBioscience, cat#epx 360-26092-901). Before tumor tissue was examined, about 20mg was weighed by an electronic balance, cell lysis buffer (Invitrogen, cat#EPX-999999-000) was added thereto, homogenized at-20℃for 10 minutes in a homogenizer, and the supernatant was centrifuged to obtain a supernatant for examination.

2.10 Intra-lymph node CD8 ⁺ Sorting of T cells

The experiment uses CD8a ⁺ T cell Isolation Kit mouse kit (Methaemal and gentle, cat # 130-104-075) for CD8 in tumor drainage region ⁺ Sorting of T cells. The kit is a negative selection kit, and the first laboratory needs to use the flow detection separation efficiency to ensure the correct experimental operation.

i. Preparing a suspension of lymph node single cells,

a. tumor-bearing mice were sacrificed and the lymph nodes in the tumor drainage area were removed and ground on a cell strainer.

b. The milled cell filter was rinsed with PBS.

c. The cells were centrifuged, resuspended with an appropriate volume of ice MACS buffer, and counted.

Sorting CD8 ⁺ T cell

a. Taking 1×10 ⁷ Cells were resuspended by centrifugation, using 40. Mu.l MACS buffer.

b. Mu.l Biotin-Antibody Cocktail was added. Mix well and incubate in the chill room for 5 minutes. No bubbles are generated in the mixing process.

c. Add 30. Mu.l MACS buffer and mix well.

d. Mu.l of Anti-Biotin Microbeads was added. Mix well and incubate in the chill room for 10 minutes. No bubbles are generated in the mixing process.

e. LS columns were placed on MACS separators and 15ml centrifuge tubes were placed in the corresponding positions below.

3ml MACS buffer rinse LS column.

g. 1ml MACS buffer was added to the sample and resuspended and the sample was applied to the LS column.

The LS column was washed twice with 3ml MACS buffer.

i.15ml of liquid collected in a centrifuge tube, 100g was centrifuged for 10 minutes.

j. The supernatant was discarded, resuspended in the appropriate volume of MACS buffer and counted.

2.11 Tumor cells and CD 8) ⁺ Co-culture of T cells

a) Configuration of culture Medium

b) 66cl4 scramble or 66cl4-shGsdme1 cells were seeded 24 hours in 96 well plates at 5000 cells per well.

c) Collecting 20000 sorted CD8 ⁺ T cells were resuspended in formulated medium after centrifugation.

d) Co-cultivation was performed for 24 hours.

e) Shooting under the light mirror.

f) Taking the culture medium supernatant, centrifuging, and detecting the content of lactate dehydrogenase (Lactate dehydrogenase, LDH) in the supernatant.

2.12 Cell supernatant lactate dehydrogenase (Lactate dehydrogenase, LDH) assay

a) The present laboratory uses the Cytotox 96Non-Radioactive Cytotoxicity Assay kit (Promega Cat#G1780) to detect lactate dehydrogenase content in the culture supernatant.

b) All experiments required positive control wells that did not require drug treatment but were reserved. Negative control wells were set and the corresponding medium was directly added for the same time without seeding the cells.

c) The detection time was 45 minutes earlier and cells in positive control wells were lysed with Lysis Solution (10×).

d) Cell culture plates were centrifuged at 250g for 3 min and the supernatant was taken.

e) Mu.l of the supernatant was placed in a flat bottom 96-well plate and 50. Mu.l of Cytotox 96Reagent was added as soon as possible using a multichannel pipette.

f) Incubate for 30 min at room temperature in the dark.

g) The color was observed to prevent excessive reaction.

h) Mu.l of Stop solution was added. The shorter the time, the better the multichannel pipettes are added.

i) The plate was read with 490nm laser.

j) The drug or CD8 was calculated according to the following formula ⁺ T cell toxicity.

k) % cytoxity = (assay well reading-negative control well reading)/(positive control well reading-negative control well reading)

2.13 Propidium iodide staining

All three stains in this laboratory were performed using commercially available kits.

After cells are treated in 6-well plates, staining is performed at an appropriate time, and the staining and testing steps strictly follow the instructions provided by the commercial products.

2.14 Fecal bacterial DNA extraction

a. The experiment was performed using DNeasy PowerLyzer PowerSoil Kit (Qiagen, cat#)

12855-50) extracting human and mouse genome DNA.

b. About 0.25g of feces was added to 750. Mu.l of Powerbead solution, transferred to "Powerbead Tube" provided by the kit, added with 60. Mu.l of solution C1, and mixed upside down and vortexed for 10 minutes at maximum intensity on a vortexing instrument.

i. 10000g of empty tube is centrifuged for 1 minute.

k. The DNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer.

2.15 Fecal 16S rDNA sequencing

All 16S rDNA sequencing was done after DNA extraction. After 16S rDNA quality inspection, the sequencing fragment is a variable segment of V3+V4, and the amplification primers are as follows

Sample-specific barcode of about 7bp is added before the primer of each sample, amplified for 25 cycles, and the amplicon is purified and then inspected. Samples passing quality inspection were sequenced on a llumina MiSeq platform with MiSeq Reagent Kit v3 platform. The data obtained by sequencing is processed by QIIME2 2019.4%https://docs.qiime2.org/ 2019.4/tutorials/) Processing to obtain OTU with cut off value of 97%. Matching of different OTUs to generic information using a classify-sklear of feature-classifer plug-inn naive Bayes taxonomy classifier. The reference database is SILVA Release 132.

2.16 Administration method of mouse choline

The choline is added into the feed of the mice. The control group feed is AIN93G standard feed, 1% choline is added into the feed to synthesize high choline feed, and SPF is completed subsequently.

2.17 Treatment of mouse plasma samples

a) Mice were bled into Eppendorf tubes after deep anesthesia without anticoagulants.

b) Standing at room temperature for 30 minutes.

c) And the mixture is centrifuged at 1300rpm at normal temperature for 20 minutes, and the mixture rises and falls rapidly.

d) The centrifuged sample is taken from the upper layer of translucent yellowish person's plasma.

e) Mu.l of the upper plasma was taken and flash frozen with liquid nitrogen. The frozen sample can be used for targeted metabolic detection.

3) Statistical analysis

Results:

study one: construction of triple negative breast cancer tumor microenvironment flora and its metabolite profile triple negative breast cancer symbiotic flora is an important component of the tumor microenvironment (FIGS. 5 and 6)

FISH staining found that the symbiotic flora and tumor microenvironment were highly coincident in spatial position, suggesting that the symbiotic flora is an important component of the triple negative breast cancer tumor microenvironment. By using 360 cases of triple negative breast cancer multiunit data in the center, a triple negative breast cancer host-microorganism multiunit map is drawn, and the fact that individual differences exist in the composition of breast cancer symbiotic flora is found, so that different breast symbiotic flora structures can play different functions in the occurrence and development of breast cancer of different individuals.

Based on previous triple negative breast cancer typing studies, a particular subtype, the IM subtype, was found to exist in triple negative breast cancer. Compared to other subtypes, it is characterized by enrichment of immune cells and cytokines in the microenvironment and a higher probability of benefiting from immunotherapy. Based on microbiome data, the present invention found that clostridium was enriched in IM subtypes that are immunologically relatively active, suggesting that clostridium is immunologically associated with the activated tumor. In addition, the high abundance of clostridium also suggests better prognosis for survival of triple negative breast cancer patients, suggesting that clostridium is associated with activated triple negative breast cancer tumor microenvironment and good prognosis.

The flora-associated specific metabolite TMAO was associated with an activated tumor microenvironment (FIGS. 7 and 8)

Flora metabolism is one of the important ways for flora to influence immunity in humans. Based on non-target metabolic data of tumor tissue in retrospective large cohorts, 249 flora-related metabolites were defined. Through volcanic image analysis, it was found that bacterial-related specific metabolite trimethylamine oxide (TMAO) was significantly enriched in the IM subtype, and the concentration in the IM subtype was significantly higher than in the non-IM subtype group. Analysis of the expression profile pathway suggests that in samples with high TMAO abundance, T cell-related pathways are significantly activated, and also suggests that TMAO is associated with triple negative breast cancer-activated tumor immune microenvironment.

In order to further verify the association of the metabolite TMAO with the flora, the invention combines the microbiology data and the metabonomics data, discovers that the high abundance of the genus Clostridium is also related to the high TMAO concentration, and the expression of the specific flora metabolism gene CutC related to TMAO in triple negative breast cancer tissues is also positively related to the infiltration of CD 8T cells, which suggests the correlation of the metabolic profile data and the microbiology data.

The specific results are as follows:

1. colonization of tumor symbiotic flora in triple negative breast cancer

In order to understand the function and role of the tumor-associated flora in triple negative breast cancer, the invention first determines the spatial positional association of the tumor-associated flora with triple negative breast cancer by fluorescent in situ hybridization staining (Fluorescence in situ hybridization, FISH) of 16S rDNA. From FISH staining results of triple negative breast cancer patient origin, it was found that tumor symbiotic bacteria were present in triple negative breast cancer tissues (fig. 5A).

2. Drawing a triple negative breast cancer tumor symbiotic flora map

After demonstrating the presence of active tumor-associated flora in triple negative breast cancer, the present invention further analyzes the characteristics of triple negative breast cancer tumor-associated flora. The invention utilizes PathSeq algorithm, and calculates and obtains the microorganism abundance of each phylum, class, order, family and genus layer of the tumor symbiotic flora based on RNA sequencing (RNA-seq) data of 360 cases of triple negative breast cancer queues of the auxiliary tumor hospital of the double denier university. Among them, the most abundant bacterial species at the genus level are Bacteroides (bacterioides), pseudomonas (Pseudomonas), acinetobacter (Acinetobacter), etc. (FIG. 5B).

3. Enrichment of Clostridium in immunomodulatory triple negative breast cancers

In order to further study the association of triple negative breast cancer intratumoral bacteria with tumor immunity, the present invention, in combination with the tumor symbiotic flora data obtained by the PathSeq algorithm, found based on linear discriminant analysis that the bacteria significantly enriched in immunoregulatory triple negative breast cancer include rhodobacter mucilaginosus (Blautia), ruminococcus (Ruminococcus), etc., all belonging to the genus clostridium (fig. 6A) compared to non-immunoregulatory type. In addition, high abundance of clostridia was also associated with better prognosis for survival for triple negative breast cancer patients (fig. 6B).

This result suggests, on the one hand, that clostridium is associated with an immunomodulatory triple negative breast cancer and a more active triple negative breast cancer tumor microenvironment; on the other hand, most of the bacteria enriched in the active immune microenvironment belong to clostridium and also suggest that triple negative breast cancer tumor symbiotic bacteria can play a regulatory role by taking clostridium as a unified bacterium, so that the physiological and pathological states of the human body are influenced.

4. Enrichment of flora-related metabolites TMAO in immunoregulatory triple negative breast cancer

As part of the immune system, studies of the association of anti-tumor immunity with tumor-associated bacteria are in the initiation phase, and the way and mechanism how tumor-associated bacteria regulate tumor immunity is not yet clear. Therefore, the invention further explores the way by which the tumor symbiotic bacteria influence the anti-tumor immunity of the triple negative breast cancer.

Based on the data of the polar non-target metabolome of 258 triple negative breast cancer patient source samples, 92 flora-related metabolites were detected. Subsequently, the differences in the flora-related metabolites of immunoregulated and non-immunoregulated triple negative breast cancers were further compared, and it was found that the flora-related metabolites including trimethylamine oxide (TMAO) were enriched in the IM subtype (fig. 7A). After the triple negative breast cancers are grouped according to the height of TMAO in tumors, the invention discovers that the TMAO in high tumors is activated by the passage related to anti-tumor immunity compared with the TMAO in low tumors, which indicates that the TMAO is related to anti-tumor immunity.

The key regulatory process of TMAO is regulated by the bacterial metabolizing enzyme choline trimethylamine lyase (Choline trimethylamine Lyase, cutC), and most of the genus bacteria expressing the cutC gene are enriched under clostridia. Studies demonstrated that the higher the clostridia abundance, the higher the abundance of TMAO in the tumor, and the higher the relative abundance of the cutC gene in immunomodulatory triple negative breast cancer (fig. 8). These results suggest that TMAO may be associated with immunomodulatory triple negative breast cancer.

TMAO is a class of metabolites that are primarily regulated by bacterial metabolism. The bacteria take choline, carnitine and the like as substrates, the substrates are metabolized into Trimethylamine (TMA) by metabolism enzymes cutC carried by the bacteria (coded by bacterial cutC genes), and further TMAO is produced mainly by flavin monooxygenase 3 (Flavin Containing Dimethylaniline Monoxygenase 3, FMO 3) in human cells. The rate-limiting step of its metabolism is the metabolism of choline to TMA by cutC, which occurs in bacteria and is expressed only by bacteria, eukaryotic cells are not expressed and therefore have been defined by previous studies as bacterial related metabolites.

Study two: clinical relevance research of TMAO and substrate choline activated triple negative breast cancer anti-tumor immunity

TMAO high levels suggested better clinical tumor immunotherapy efficacy (fig. 9)

Based on correlation analysis against retrospective large cohorts, the present invention found that TMAO was associated with an activated tumor immune microenvironment. In order to further find the correlation between TMAO and immunotherapy effect, the invention verifies in the FUTURE immunotherapy queue, and finds that compared with the patients with poor immunotherapy effect, the patients with better immunotherapy effect have higher TMAO concentration. A higher concentration of TMAO suggests a higher rate of regression of the target lesion after immunotherapy and also suggests that the patient is able to benefit from immunotherapy more permanently.

The specific results are as follows:

1. high plasma TMAO concentration correlates with better immunotherapeutic efficacy for triple negative breast cancer patients

In order to realize the transformation from research results to clinical application, the invention further explores clinically accessible transformation and application treatment strategies. The invention selects an advanced triple negative breast cancer immunotherapy queue of the central lead, each patient collects peripheral blood samples before starting treatment, and completes the targeted metabolic detection of peripheral blood TMAO concentration. Patients were divided into disease progression group (PD) and partial remission group (PR) based on efficacy assessment of patients after five immunotherapy cycles (fig. 9A). The peripheral blood TMAO concentration was higher in the partially buffered group of patients with better efficacy compared to the disease progression group with worse efficacy (fig. 9A). The patients were classified into high peripheral blood TMAO group and low peripheral blood TMAO group according to the median of TMAO concentration in peripheral blood of the patients, and the correlation analysis result suggested that the proportion of tumor retraction of the patients was proportional to the peripheral blood plasma TMAO concentration (fig. 9B). Based on the research of clinical advanced triple negative breast cancer immunotherapy queues, the high peripheral blood plasma TMAO concentration is related to better immunotherapy curative effect of patients with triple negative breast cancer.

2. High plasma TMAO concentrations correlate with longer immunotherapy life cycles in triple negative breast cancer patients

In addition to the ratio of immunotherapy effect to tumor regression, the impact of immunotherapy on patient survival is also critical. The invention is based on the advanced triple negative breast cancer immunotherapy queue, and divides patients into a high peripheral blood TMAO group and a low peripheral blood TMAO group according to the median of the peripheral blood TMAO concentration, and is based on the change condition of the tumor tissue size of the patients. Patients in the high peripheral blood TMAO group had longer progression free survival than those in the low peripheral blood TMAO group, and the patients benefited from immunotherapy for longer periods of time (fig. 9C).

Study three and four: TMAO and its substrate choline activate triple negative breast cancer anti-tumor immunity by inducing tumor cell apoptosis, and inhibit tumor growth

In vivo experiments prove that TMAO can improve the curative effect of triple negative breast cancer tumor immunotherapy (figures 10-13)

By combining the previous data analysis, the TMAH is found to be related to tumor immune activation and immune therapy curative effect, so the subsequent invention explores the functions and mechanisms of the TMAH based on an in-vivo and in-vitro model. Previous studies suggest that the mouse triple negative breast cancer 4T1 cell transplantation tumor model alone has poor therapeutic effect of immunotherapy, so that the animal model is selected for in vivo phenotype verification later. According to the tumor growth curve, TMAO in combination with PD-1 inhibitors was found to significantly inhibit tumor growth compared to the PD-1 inhibitor alone group. The result of immunohistochemistry of tumor tissues also suggests that TMAO combined with PD-1 inhibitor can improve CD 8T cell infiltration in tumors compared with the PD-1 inhibitor alone.

Through flow data verification, TMAO is found to promote intratumoral killer T cells, i.e. IFNgamma ⁺ CD 8T cell infiltration.

The invention not only can observe the activation condition of anti-tumor immunity in tumor through immune cell infiltration, but also can observe the level of inflammatory factors in tumor. The rise of the levels of cytokines such as IFNgamma, TNF alpha and the like is found, which indicates that the anti-tumor function of CD 8T is enhanced, and the TMAO combined with the PD-1 inhibitor can activate the immune microenvironment of the triple negative breast cancer tumor and improve the curative effect of immunotherapy compared with the single PD-1 inhibitor treatment group.

In addition, the invention also uses 66cl4 triple negative breast cancer cell line modeling to get similar conclusions, and also verifies that TMAO can inhibit tumor proliferation and increase the intratumoral duty cycle of anti-tumor immune cells, up-regulate the intratumoral level of anti-tumor cytokines, as evidenced by the findings in the 4t1 cell line.

TMAO activates tumor immunity through the pyro-death pathway (FIGS. 14 and 15)

Previous animal experiments suggest that the flora-specific metabolite TMAO is capable of activating tumor immunity in mice. In order to further explore the mechanism by which TMAO activates tumor immunity, the invention discovers that after TMAO stimulation, tumor cells apoptosis and inflammatory pathways are activated based on RNA-SEQ data, and speculates that TMAO activates tumor immunity by inducing inflammatory apoptosis, i.e., pyrosis.

Firstly, the validation is carried out from the aspect of morphology, and the 4T1 cells after TMAO treatment are found to have typical pyro-death morphology, cell membranes are typically ruptured, and the uptake rate of PI dye is obviously up-regulated. LDH release experiments also suggest that TMAO can increase the proportion of cell apoptosis. In addition, the invention also verifies through Western Blot that TMAO can increase the amounts of key proteins caspase 3 and GSDME protein of the activated scorch pathway after cleavage, and suggests that the scorch pathway of tumor cells is activated after TMAO treatment.

Choline activated triple negative breast cancer anti-tumor immunity (FIG. 16)

Choline is a precursor metabolite of TMAO. The study team increased plasma TMAO and intratumoral levels by feeding mice with the TMAO precursor metabolite choline and demonstrated that supplementation with choline could activate triple negative breast cancer anti-tumor immunity, enhancing the efficacy of immunotherapy.

The specific experimental results are as follows:

1. in vivo experiments prove that TMAO inhibits the growth of triple negative breast cancer

In study one, the invention is based on analysis of a plurality of groups of chemical queues of clinical samples of triple negative breast cancer, and found that clostridium genus and clostridium related metabolite TMAO are related to triple negative breast cancer activated antitumor immune microenvironment. Thus, the present invention proposes the assumption based on the correlation obtained by analysis of clinical sample data: the bacterial related metabolite TMAO can inhibit the growth of triple negative breast cancer by activating the anti-tumor immunity of the triple negative breast cancer, and improve the immune treatment effect of the triple negative breast cancer.

The invention selects a murine triple negative breast cancer cell line 4T1 cell line, uses BALB/c mice to make a model, injects tumor cells into the mammary fat pad of the mice, and constructs a transplantation tumor model. Mice were divided into 4 groups and received the following different treatments: 1) Physiological saline in combination with IgG; 2) Normal saline in combination with a mouse anti-PD-1 antibody (anti-PD-1); 3) TMAO in combination with IgG; 4) TMAO was combined with mouse anti-PD-1 antibodies. Wherein physiological saline is a TMAO control, and the two are administered by intratumoral injection; igG is a control for the mouse PD-1 antibody, both of which were administered by intraperitoneal injection (fig. 10).

The results of the tumor growth curve of the mice indicate that compared with a control group, the anti-PD-1 alone can not significantly inhibit the growth of a triple negative breast cancer in-vivo model, which is proved by the situation that the clinical triple negative breast cancer immunotherapy effect is poor. In the case of no anti-PD-1 antibody, TMAO was used to significantly inhibit the growth rate of the triple negative breast cancer in vivo model compared to physiological saline. And the combination of TMAO and anti-PD-1 can significantly inhibit the growth of tumor compared with the single anti-PD-1 (figure 10A). Therefore, the triple negative breast cancer cell transplantation tumor model suggests that TMAO can effectively inhibit the growth of triple negative breast cancer and improve the immune treatment effect of triple negative breast cancer.

TMAO treatment activates anti-tumor immunity of mice triple negative breast cancer

One result of the study suggests that TMAO is associated with an activated anti-tumor immune microenvironment associated with an activated immune pathway of triple negative breast cancer, thus speculating that TMAO further inhibits growth of triple negative breast cancer by activating anti-tumor immunity of triple negative breast cancer.

The invention first verifies whether TMAO treatment can activate intratumoral anti-tumor immunity. Immunohistochemical staining and flow cytometry were used to assess the differences in intratumoral immune microenvironment of the above 4 groups of mice after receiving different treatments. The results of immunohistochemical and flow cytometry analysis show that anti-PD-1 alone cannot effectively improve CD8 in tumor ⁺ Infiltration of T cells, while intratumoral injection of TMAO increased compared to controlCD8 ⁺ Infiltration ratio of T cells in tumor. The simultaneous combination of TMAO and anti-PD-1 can effectively increase CD8 in tumor compared with single anti-PD-1 ⁺ T infiltration (FIG. 10B, FIG. 11).

Meanwhile, the invention utilizes ELISA technology to detect the cytokine levels of interferon gamma, tumor necrosis factor alpha and interleukin 2 in the tumor tissues of 4 different treatment methods, and the result shows that compared with a control group, the content of interferon gamma, tumor necrosis factor alpha and interleukin 2 in the tumor tissues of a TMAO treatment group is obviously increased. Meanwhile, the anti-PD-1 alone did not affect the levels of interferon gamma and tumor necrosis factor alpha in tumor tissues, but the combined use of TMAO and anti-PD-1 also significantly up-regulated the levels of interferon gamma and tumor necrosis factor alpha in tissues as compared to the anti-PD-1 alone (FIG. 12). In summary, the invention verifies that TMAO treatment can activate anti-tumor immunity in mice tumors.

To explore the conclusions more fully, the invention selects a second murine triple negative breast cancer cell line 66cl4, uses BALB/c mice for modeling, injects tumor cells into the mammary fat pad of the mice, and constructs a transplanted tumor model. Mice were divided into 4 groups and received the following different treatments: 1) Physiological saline in combination with IgG; 2) Normal saline in combination with a mouse anti-PD-1 antibody (anti-PD-1); 3) TMAO in combination with IgG; 4) TMAO was combined with mouse anti-PD-1 antibodies. Wherein physiological saline is a TMAO control, and the two are administered by intratumoral injection; igG is a control for mouse anti-PD-1 antibodies, both of which were administered by intraperitoneal injection (fig. 13). Similar to the results in the 4T1 cell line, TMAO was also found to inhibit the growth of triple negative breast cancer cells modeled on the 66cl4 cell line, enhancing the anti-tumor immunotherapeutic response (fig. 13).

TMAO activation of tumor cell focal death pathway

To further explore the molecular mechanism of TMAO activation of triple negative breast cancer anti-tumor immunity, the invention sequenced the RNA of tumor cells before and after TMAO treatment, and found that apoptosis and interferon-related anti-tumor immune pathway activation in tumor cells (fig. 14) based on gene enrichment analysis (Gene Set Enrichment Analysis), which suggested that TMAO might be related to pyroapoptosis (also known as inflammatory apoptosis).

Upon treatment with TMAO at 66cl4 and 4T1 triple negative breast cancer cell lines, the cells were observed to undergo a "balloon-like" morphological change, and the balloon-like change in cell membrane permeability was verified by Propidium Iodide (PI) staining experiments (fig. 15a, b). It was also found that lactate dehydrogenase (Lactate dehydrogenase, LDH) content was increased in tumor cell supernatants after TMAO treatment (fig. 15C). The results based on western blot analysis also suggest that TMAO is able to activate GSDME protein-mediated focal death pathways.

4. Oral choline can inhibit growth of triple negative breast cancer and activate anti-tumor immunity

The intestinal flora can convert the metabolic substrates such as choline, L-carnitine and betaine into TMA through the metabolic function. TMA is further metabolized in the liver to produce TMAO. Thus, oral administration of TMAO-associated metabolic substrates in the hope of up-regulating TMAO concentrations is a viable clinical conversion tool.

It is speculated that oral choline activates triple negative breast cancer anti-tumor immunity. Based on this, the present invention devised an animal experiment, inoculated a triple negative breast cancer cell line transplantation tumor model in a mouse mammary fat pad, and divided the mice into 6 groups, with normal feed as a control for high choline feed, normal drinking water as a control for 3,3 dimethyl-1-butanol (DMB) drinking water added, and IgG as a feed for anti-PD-1 (fig. 16A). Compared with a control group, the high choline feed can obviously improve the concentration of TMAO in tumors and inhibit the growth of a triple negative breast cancer transplantation tumor model in a mouse. While DMB can significantly reduce TMAO concentration in tumors, significantly reduce tumor suppression of high choline feeds (fig. 16b, c).

To further explore the change of immune microenvironment in the mouse engraftment tumor model, the present invention digests mouse tumor tissue into single cells and performs flow cytometry fluorescent staining. Compared with the control group, the high choline feed can obviously improve the CD8 in the tumor of the mice ⁺ Infiltration of T cells and Interferon gamma ⁺ CD8 ⁺ T cell ratio. Compared with the high choline feed group, DMB drinking water and high choline feed can obviously reduce CD8 in mice tumor ⁺ T cellIs (1) infiltration and interferon gamma ⁺ CD8 ⁺ T cell ratio (fig. 16d, e). In addition, TMAO concentration was positively correlated with clostridia abundance suggesting that the antitumor immune function of choline was flora-regulated (fig. 16F). The in vivo experiments of the mice indicate that oral choline can obviously inhibit the growth of triple negative breast cancer and activate CD8 ⁺ Is a clinically feasible treatment means.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. A breast cancer therapeutic agent, which is characterized in that the active ingredient contains trimethylamine oxide and/or choline which is a precursor product of trimethylamine oxide.

2. An auxiliary therapeutic drug for breast cancer is characterized in that the active ingredient of the auxiliary therapeutic drug contains trimethylamine oxide and/or choline which is a precursor product of trimethylamine oxide.

3. An antitumor immune activator for breast cancer is characterized in that the active ingredient contains trimethylamine oxide and/or choline which is a precursor product of trimethylamine oxide.

4. Use of trimethylamine oxide in the treatment of breast cancer.

5. The use of choline, a precursor product of trimethylamine oxide, in the treatment of breast cancer.

6. Application of trimethylamine oxide in preparing medicine for treating breast cancer is provided.

7. The use of claim 6, wherein the breast cancer is a triple negative breast cancer.

8. The use of claim 6, wherein the medicament is a medicament for improving the efficacy of triple negative breast cancer immunotherapy.

9. The application of choline, a precursor product of trimethylamine oxide, in the preparation of medicaments for treating breast cancer.

10. The use of claim 9, wherein the breast cancer is a triple negative breast cancer.

11. The use of claim 9, wherein the medicament is a medicament for improving the efficacy of triple negative breast cancer immunotherapy.

12. A pharmaceutical composition comprising trimethylamine oxide and/or choline, a precursor product of trimethylamine oxide, and further comprising a PD-1 inhibitor.

13. The pharmaceutical composition of claim 12, wherein the PD-1 inhibitor is an anti-PD-1 antibody.