Mucispirilline composition and cancer treatment method thereof

Abstract

In some embodiments, the technology described herein is directed to a composition comprising a Mucispiril (e.g., M. schaedleri). Also described herein are methods of using the Mucispirilline compositions for the treatment of cancer; and methods of using the Mucispirilline compositions for the treatment of cancer. A diet rich in sulfur-containing amino acids (SAA) or a supplement comprising SAA; and / or an XCL1 polypeptide (or an XCR1 agonist). Also described herein are methods of stratification of cancer therapy involving the detection of the level of M.schaedleri, the level of XCL1 polypeptide, the level of NKT, and / or the level of CD103 + cDC1, as well as the alteration or stratification of the corresponding therapy, and methods of stratification of cancer therapy involving the detection of the level of M.schaedleri, the level of XCL1 polypeptide, the level of NKT, and / or the level of CD103 + cDC1.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 389,382, filed July 15, 2022, pursuant to 35 U.S. SC §119(e), the contents of which are incorporated herein by reference in their entirety. Government support

[0002] This invention was completed with government support under licenses CA202704 and CA154426 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention. sequence list

[0003] This application contains a sequence list, which has been submitted in XML format via the Patent Center and is hereby incorporated in its entirety by reference. The XML copy was created on July 6, 2023, named 002806-099880WOPT_SL.xml, and has a size of 61077 bytes. Technical Field

[0004] The technologies described in this article relate to compositions including Mucispirillum and related methods for treating cancer. Background Technology

[0005] Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, and its global incidence is rising. While immunotherapy, particularly immune checkpoint inhibitors (ICIs), has been a breakthrough in the treatment of many cancers, the vast majority of CRC patients are not ICI-responsive due to intact DNA mismatch repair (MMR) and the tolerant immune tone of the colon. In addition to tumor-derived intrinsic factors such as DNA mismatch repair (MMR) status, the gut microbiota also influences responsiveness to immunotherapy and is an environmental factor in CRC development. The gut microbiota is associated with the efficacy of ICI therapy, and different bacterial species have been identified as mediators of responsiveness. See, for example, Andrews et al., Nat Med 27, 1432-1441 (2021); Frankel et al., Neopalasia 19, 848-855 (2017); Gopalakrishnan et al., Science 359, 97-103 (2018); Lee et al., Nat Microbiol 6, 277-288 (2021); Matson et al., Science 359, 104-108 (2018); Routy et al., Science 359, 91-97 (2018); their respective contents are incorporated herein by reference in their entirety. For ICI-unresponsive, immunologically cold cancers (e.g., CRC), additional treatment is required. Summary of the Invention

[0006] The techniques described herein pertain to compositions including Mucispirillum (e.g., M. schaedleri). This disclosure describes how a diet rich in sulfur-containing amino acids can increase intestinal levels of M. schaedleri, thereby increasing XCL1 secretion by NKT cells. This increased XCL1 secretion by NKT cells increases the number and / or activation of CD103+ conventional dendritic cells (cDC1) (e.g., in tumor-draining lymph nodes), thereby recruiting and activating CD8+ T cells with anti-tumor immune activity (see, for example, Figure 13 Therefore, this document describes methods for treating cancer using the Mucispirillum composition as described herein; a diet rich in sulfur-containing amino acids (SAAs) or supplements containing SAAs; and / or an XCL1 peptide (or an XCR1 agonist). This document also describes stratification methods for cancer treatment involving the detection of levels of M. schaedleri, XCL1 peptide, NKT, and / or CD103+cDC1, and corresponding changes or stratification in treatment.

[0007] Therefore, one aspect described herein is a composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria, said composition being formulated for delivery to the intestine.

[0008] In some embodiments of any aspect, the M. schaedleri bacteria are either alive or inactivated.

[0009] In some embodiments of any aspect, the M. schaedleri bacteria are in a dried, live form.

[0010] In some embodiments of any aspect, the M. schaedleri bacteria are encapsulated.

[0011] In some embodiments of any aspect, the M. schaedleri bacteria are contained in an enteric-coated capsule.

[0012] In some embodiments of any aspect, the M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

[0013] In some embodiments of any aspect, the M. schaedleri bacteria are mixed with prebiotics.

[0014] In some embodiments of any aspect, the M. schaedleri bacteria are mixed with sulfur-containing amino acids.

[0015] In some embodiments of any aspect, the sulfur-containing amino acid is methionine, cysteine, or a derivative thereof.

[0016] In some embodiments of any aspect, the M. schaedleri bacteria are formulated into a food composition.

[0017] In some embodiments of any aspect, the food composition is supplemented with sulfur-containing amino acids and / or prebiotics.

[0018] In some embodiments of any aspect, the composition further comprises 1 to 20 additional bacterial species.

[0019] In some embodiments of any aspect, the composition comprises no more than 20 bacterial strains.

[0020] One aspect described herein comprises a composition, wherein the composition is formulated for delivery to the intestine: live M. schaedleri bacteria, dead M. schaedleri bacteria, M. schaedleri conditioned medium, or an organic solvent extract of M. schaedleri conditioned medium or a fraction thereof that promotes XCL1 secretion by NKT cells.

[0021] In some embodiments of any aspect, the M. schaedleri bacteria, culture medium, or solvent extract is in a dry form.

[0022] In some embodiments of any aspect, the M. schaedleri bacteria, culture medium, or extract is encapsulated.

[0023] In some embodiments of any aspect, the M. schaedleri bacteria are contained in an enteric-coated capsule.

[0024] In some embodiments of any aspect, the M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

[0025] In some embodiments of any aspect, the M. schaedleri bacteria, culture medium, or extract is mixed with prebiotics and / or with sulfur-containing amino acids or their derivatives.

[0026] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from: Table 3 or Table 9 of U.S. Provisional Application No. 63 / 389,382, or the metabolite herein. Figure 4H , Figure 4I or Figure 16 .

[0027] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

[0028] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from the group consisting of succinic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid.

[0029] One aspect described herein is a food composition comprising the compositions as described herein.

[0030] In some embodiments of any aspect, the food composition further includes one to 20 additional bacterial strains.

[0031] One aspect described herein is a method for treating cancer or promoting anti-tumor immune activity, the method comprising administering to a subject in need the composition described herein.

[0032] In some embodiments of any aspect, the cancer is colon cancer.

[0033] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor.

[0034] One aspect described herein is a method for promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering to a subject in need the composition described herein.

[0035] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor.

[0036] In some embodiments of any aspect, the subject has colon cancer.

[0037] In some embodiments of any aspect, the subject's cancer has been identified as resistant to immune checkpoint inhibitor therapy.

[0038] In some embodiments of any aspect, the composition promotes XCL1 secretion from NKT cells.

[0039] One aspect described herein is a method for increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering to a subject in need the composition described herein.

[0040] In some embodiments of any aspect, the cDC1 is associated with a tumor.

[0041] In some embodiments of any aspect, the tumor is colon cancer.

[0042] In some embodiments of any aspect, the method further includes administering sulfur-containing amino acids.

[0043] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor.

[0044] One aspect described herein is a method for increasing XCL1 secretion by NKT cells, the method comprising administering to a subject in need the composition described herein.

[0045] In some embodiments of either aspect, the subject has cancer.

[0046] In some embodiments of any aspect, the subject has colon cancer.

[0047] One aspect described herein is a method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering to a subject in need the composition described herein.

[0048] In some embodiments of any aspect, the cDC1 is associated with a tumor.

[0049] In some embodiments of any aspect, the tumor is colon cancer.

[0050] In some embodiments of any aspect, the method further includes administering sulfur-containing amino acids.

[0051] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor.

[0052] One aspect described herein is a method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering to subjects in need a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA.

[0053] In some embodiments of any aspect, the diet rich in sulfur-containing amino acids includes more than 0.04 grams of SAA per kilogram of body weight per day.

[0054] In some embodiments of any aspect, the method further includes administering the subject the composition as described herein.

[0055] One aspect described herein is a method for establishing or maintaining a tumor-suppressive gut environment in subjects in need, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) or supplements containing SAA.

[0056] In some embodiments of any aspect, the diet rich in sulfur-containing amino acids or the supplement containing SAA includes more than 0.04 grams of SAA per kilogram of body weight per day.

[0057] In some embodiments of any aspect, the method further includes administering the subject the composition as described herein.

[0058] One aspect described herein is a method for treating cancer, which includes administering an XCL1 peptide to a subject in need.

[0059] In some embodiments of any aspect, the cancer is colon cancer.

[0060] In some embodiments of any aspect, the XCL1 polypeptide is administered to the intestine.

[0061] One aspect described herein is a method for treating cancer, which includes administering microorganisms engineered to express the XCL1 peptide to a subject in need.

[0062] One aspect described herein is a method for treating cancer, which includes administering an agonist of the XCL1 receptor, or XCR1, to a subject in need.

[0063] In some embodiments of any aspect, the XCR1 agonist comprises SEQ ID NOs: 9-11 or an amino acid sequence that is at least 95% identical to and maintains its function.

[0064] One aspect described herein is a method for treating cancer in a subject in need, the method comprising: detecting the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapy agent if the level of M. schaedleri is equal to or higher than a predetermined threshold; and administering the cancer immunotherapy agent and a composition as described herein if the level of M. schaedleri is lower than the predetermined threshold.

[0065] One aspect described herein is a method for treating cancer in a subject in need, the method comprising: obtaining a result from a assay that detects the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapy agent if the level of M. schaedleri is equal to or higher than a predetermined threshold; and administering the cancer immunotherapy agent and the composition as described herein if the level of M. schaedleri is lower than the predetermined threshold.

[0066] One aspect described herein is a method for treating cancer in a subject in need, the method comprising: detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from the subject; administering a cancer immunotherapy agent if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold; and administering a cancer immunotherapy agent and a composition as described herein if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are lower than a predetermined threshold.

[0067] One aspect described herein is a method for treating cancer in a subject in need, the method comprising: obtaining results from a assay that detects levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from the subject; administering a cancer immunotherapy agent if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold; and administering the cancer immunotherapy agent and the composition as described herein if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are lower than a predetermined threshold.

[0068] One aspect described herein is a method for stratifying subjects for cancer treatment, the method comprising: detecting the level of M. schaedleri in a sample from the subject; and classifying the subject as high-risk if the level of M. schaedleri is below a predetermined threshold; or classifying the subject as low-risk if the level of M. schaedleri is equal to or higher than the predetermined threshold.

[0069] One aspect described herein is a method for stratifying subjects for cancer treatment, the method comprising: obtaining a result from a determination of M. schaedleri levels in a sample from the subject; and classifying the subject as high-risk if the M. schaedleri level is below a predetermined threshold; or classifying the subject as low-risk if the M. schaedleri level is equal to or higher than the predetermined threshold.

[0070] One aspect described herein is a method for stratifying subjects for cancer treatment, the method comprising: detecting levels of XCL1 peptide, NKT, and / or CD103+cDC1 in samples from the subject; and classifying the subject as high-risk if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold; or classifying the subject as low-risk if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold.

[0071] One aspect described herein is a method for stratifying subjects for cancer treatment, the method comprising: obtaining results from a determination of levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from the subject; and classifying the subject as high-risk if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold; or classifying the subject as low-risk if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold.

[0072] In some embodiments of any aspect, the subject has colon cancer.

[0073] In some embodiments of any aspect, the method further includes administering a composition as described herein.

[0074] In some embodiments of any aspect, the method further includes administering sulfur-containing amino acids.

[0075] In some embodiments of any aspect, the cancer immunotherapy agent is selected from the group consisting of: immune checkpoint inhibitors, chemotherapy, dendritic cell vaccines; chimeric antigen receptor T cell (CAR-T) and NKT cell-based therapies.

[0076] In some embodiments of any aspect, the cancer immunotherapy agent includes an immune checkpoint inhibitor.

[0077] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor.

[0078] In some embodiments of any aspect, the method further includes administering a diet rich in sulfur-containing amino acids or a supplement containing SAAs.

[0079] In some embodiments of any aspect, the method produces higher therapeutic efficacy compared to treatments that do not first involve: detecting the level of M. schaedleri; obtaining results from a determination of M. schaedleri levels; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from a determination of XCL1 peptide, NKT, and / or CD103+cDC1 levels.

[0080] In some implementations of any aspect, the method produces higher therapeutic efficacy compared to treatments that do not first stratify the subjects.

[0081] In some embodiments of any aspect, the method causes fewer treatment complications compared to not first performing the following treatments: detecting the level of M. schaedleri; obtaining results from a determination of M. schaedleri levels; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from a determination of XCL1 peptide, NKT, and / or CD103+cDC1 levels.

[0082] In some implementations of any aspect, the method causes fewer treatment complications compared to treatments that do not first stratify the subjects.

[0083] One aspect described herein is an enteric delivery formulation comprising at least one metabolite selected from: Table 3 or Table 9 of U.S. Provisional Application No. 63 / 389,382, or the metabolite described herein. Figure 4H , Figure 4I or Figure 16 .

[0084] One aspect described herein is an enteric delivery formulation comprising at least one metabolite selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

[0085] One aspect described herein is an enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid.

[0086] In some embodiments of any aspect, the intestinal delivery formulation is formulated for delivery into the intestine. Attached Figure Description

[0087] Figures 1A-1J It is a series of diagrams, images and charts illustrating how dietary sulfur-containing amino acids (Saa) regulate tumor progression in a mouse CRC model. Figure 1AThe MetaCyc pathways (first four enlarged on the right) in the microbiome of immune checkpoint inhibitor (ICI) responders and non-responders for Routy's renal cell carcinoma (RCC) cohort are shown, ranked by q-values ​​from least-squares linear regression analysis; see, for example, Routy et al., Science 359, 91-97 (2018); Caspi et al., Nucleic Acids Res 42, D459-71 (2014); their respective contents are incorporated herein by reference in their entirety. Figure 1B MetaCyc pathways are shown ranked by effect size in a meta-analysis of a linear mixed-effects model from ICI responders (n=203) and non-responders (n=134). Figure 1C The left column shows the enrichment of the PWY821 superpathway for Saa biosynthesis in the fecal metagenomics of anti-PD-1 treatment responders relative to non-responders, and the right column shows the enrichment of 15 bacterial Saa biosynthesis genes from PWY-821 in 8 patient groups. Figure 1D The fecal metagenomics of PWY-821 and genes (such as...) from patients with colorectal cancer (CRC) and colonic adenoma in nine patient groups, relative to healthy controls, are shown. Figure 1C (in the middle). Figure 1E The diagram shows a schematic of the experimental setup (top subplot) and tumor volume over time in mice born in an facility fed a Saa diet and injected laterally with wild-type (WT) MC38 cells (bottom left), along with a representative image of the tumor (bottom right). Figure 1F Showing from Figure 1E The MC38 tumor weight on day 12. Figure 1G and Figure 1H A schematic diagram of the experimental setup is shown. Figure 1G The above subfigure shows the tumor volume over time in WT mice born in an institution that were fed a Saa diet, injected MC38 cells flankally, and treated intraperitoneally (ip) with α-PD-1 or allotype Abs. Figure 1G (See the sub-image below) and the MC38 tumor weight on day 13 ( Figure 1H ). Figure 1I The experimental setup is shown in the diagram above (subplot), derived from CDX2-Cre Apc fed low- or high-Saa diets. flox / + (cAPC) Representative colon photographs of mice (tumors highlighted by black circles) (neutron plot), along with data on the grade and number of developmental abnormalities and tumor lesions (bottom subplot). Each column represents data for a single mouse, and the number of each lesion type is shown in the boxes. Figure 1J Showing data from week 12 Figure 1IColon tumor weight in cAPC mice fed a Saa diet. Figure 1F , Figure 1H and Figure 1J Each symbol represents data from an individual mouse. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The error bars represent the standard error (SEM) of the mean. Figure 1E , Figure 1G and Figure 1H One-way ANOVA with FDR correction was performed. Figure 1F and Figure 1J The Mann-Whitney test was performed, and... Figure 1I Boschloo's test was performed. The data represent the... Figure 1E , Figure 1F , Figure 1G and Figure 1H Two independent experiments, and for Figure 1I and Figure 1J Four independent experiments were conducted.

[0088] Figures 2A-2H It is a series of diagrams, images and charts showing that mice fed a high-Saa diet have higher abundance of Mucispirillum schaedleri and thicker mucus. Figure 2A A volcano plot is shown, illustrating the enrichment of microbial taxa in the cecal contents of cAPC mice fed a low-Saa diet compared to a high-Saa diet, as analyzed by 16S rRNA amplicon analysis using a multivariate association (MaAsLin 2) regression model of the microbiome; see, for example, Mallick et al., PLoS Comput Biol 17, e1009442 (2021), the contents of which are incorporated herein by reference in their entirety. Figure 2B The reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of M. schaedleri 16S rRNA in cecal contents DNA from cAPC BIH mice, WT BIH mice injected with MC38, and WT ASF mice is shown. Figure 2C The relative abundance of M. schaedleri-specific reads in 16S ribosomal RNA (rRNA) amplicon sequence data from tissue biopsies of healthy controls (“normal”) or patients with colonic adenomas is shown. Figure 2DRepresentative images of Muc2 immunofluorescence staining combined with bacterial fluorescence in situ hybridization (FISH) staining in mouse colon tissue sections of altered Schaedler flora (ASF) fed with low-Saa or high-Saa diets are shown (pink fluorescence: M. schaedleri probe, blue fluorescence: universal bacterial probe, green fluorescence: Muc2; gray: DAPI). Figure 2E Measurements of the mucus layer from the image are shown (see, for example, Figures 2F-2G Each symbol represents data from an individual mouse (an average of 5 fields of view per mouse). Figures 2F-2G The results showed that in WT GF mice ( Figure 2F ) and WT ASF mice ( Figure 2G Representative images of Alcian Blue staining used to measure mucus thickness in distal colon tissue sections. Figure 2H The mucus layer thickness measurements are shown. Each symbol represents data from an individual mouse (average 23 fields of view per mouse). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent the standard error (SEM) of the mean. Figure 2B , Figure 2C , Figure 2E and Figure 2H The Mann-Whitney test was performed. The data represent the... Figure 2A and Figure 2B Three independent experiments, and for Figure 2D-Figure 2H Two independent experiments.

[0089] Figures 3A-3I This is a series of schematic diagrams, images, and charts illustrating CD8 amplification of M. schaedleri in dietary Saa and tumors. + T cells and M. schaedleri implantation alone are sufficient to increase cDC1 in tumor draining lymph nodes (TDLN). Figure 3A This shows CD8 in a representative cAPC tumor. + Flow cytometry data on the relative frequency of T cells. Figure 3B Representative immunofluorescence images of colon tumor sections from cAPC mice fed low- or high-Saa diets are shown. The image on the right is a higher magnification of the area indicated by the white rectangle on the left. Figure 3C CD3 is shown in the image. - CD8 + / CD3 - Quantification of ratios. Each symbol represents data from an individual mouse (average 4 fields of view per mouse). Figure 3DThe image shows CD8+ in tumors from cAPC mice fed the indicated diet. + Flow cytometry data, relative frequency (left), and number (right) of representative co-inhibitory receptor expression in T cells. Figure 3E The image shows IFNγ in tumors from cAPC mice fed the stated diet. + (Left) and GZMB + (Right) CD8 + The frequency of T cells. Figures 3F-3G cDC1 (CD103) in TDLN from cAPC mice fed low or high Saa diets is shown. + CD11b - CD103 - CD11b + and CD103 + CD11b + Representative data of cells; Figure 3F A representative flow cytometry illustration is shown, and, Figure 3G cDC1(CD103) is shown + CD11b - CD103 - CD11b + and CD103 + CD11b + The frequency and number of cells. Figure 3H The image shows cDC1 (CD103) in the MLN of mice fed a high-Saa diet and monoplanted with GF, M. schaedleri, or A. muciniphila. + CD11b - CD103 - CD11b + and CD103 + CD11b + Cell frequency (left) and number (right). Each symbol represents data from an individual mouse. The order of the bars in each group from left to right corresponds to the order of the legend from left to right. Figure 3I The images show cDC1 and CD103 in the mesenteric lymph nodes (MLN) of WT mice fed a low-Saa diet and intensively fed three times a week with brain heart extract (mBHI) or M. schaedleri conditioned medium (CM). - CD11b + and CD103 + CD11b +Cell frequency. Each symbol represents data from an individual mouse. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. Error bars represent the standard error (SEM) of the mean. Figure 3A , Figure 3C , Figure 3D , Figure 3E , Figure 3G and Figure 3I The Mann-Whitney test was performed, and... Figure 3H One-way ANOVA with FDR correction was performed. The data represent for... Figures 3A-3G Three independent experiments, and for Figure 3H and Figure 3I Two independent experiments.

[0090] Figures 4A-4K It is a series of diagrams and charts illustrating the high Saa diet and M. schaedleri-induced XCL1 secretion and cDC1 activation state of NKT cells associated with increased survival in CRC patients. Figures 4A-4C The experimental schematic diagram and the grades and numbers of developmental abnormalities and tumor lesions are shown below: Zbtb46-DTR cAPC mice fed a high-Saa diet and injected with PBS or diphtheria toxin per ip (PAP) Figure 4A cAPC Batf3 fed with low or high Saa diets - / - mice ( Figure 4B ) and cAPC mice fed a high-Saa diet and treated with α-XCL1 Ab or isotype control ( Figure 4C Each column represents data from an individual mouse. Figure 4D The ex vivo secretion of XCL1 from natural killer (NK) and natural killer T (NKT) cells sorted from MLNs of Saa-fed, intra-institutionally born (BIH) mice was shown. Figure 4E The frequency and quantity of NKT in TDLNs from cAPC mice fed a Saa diet are shown. Each symbol represents data from an individual mouse. Figure 4F The secretion of XCL1 in GW1 NKT cells after overnight stimulation with bacterial conditioned medium (CM) is shown. Figure 4G The secretion of XCL1 by human peripheral blood-derived NKT cells after overnight stimulation with bacterial CM is shown. Figure 4H Volcano plots of the identified metabolic signatures are shown. Thirty-six metabolic signatures enriched in M. schaedleri CM are shown. Black text: clearly identified metabolites; gray text: artificially compiled metabolites matched with a convincing library; labels: chemical formulas shown for presumptive identification. Figure 4IA heatmap is shown, which displays the abundance of 36 metabolic features enriched in M. schaedleri CM. Figure 4J The relative abundance of differentially expressed genes in cDC1 of TDLN from Saa-fed cAPC mice is shown by model-based single-cell transcriptomics analysis (MAST) (P<0.05, AvgLogFC>1.2 or <0.8). Figure 4F Survival curves for CRC patients from the Tissue Cancer Gene Mapping Dataset (TCGA-COAD / READ) of colorectal adenocarcinoma tumors are shown, stratified by tumor gene expression similarity to cDC1 from Saa-fed cAPC mice. Figure 4F In this context, "low activation" corresponds to dendritic cells with low sulfur markers (LSMD-DC), while "high activation" corresponds to dendritic cells with high sulfur markers (HSME-DC). *P value < 0.05, **P value < 0.01, ***P value < 0.001. Error bars represent the standard error (SEM) of the mean. Figure 4A , Figure 4B and Figure 4C Boschloo's test was performed. Figure 4D , Figure 4F and Figure 4G One-way ANOVA with FDR correction was performed; Figure 4E The Mann-Whitney test was performed; Figure 4J MAST analysis was performed, as well as... Figure 4K A Cox proportional hazards model was executed. The data represents [the relevant data]. Figures 4A to 4C The four independent experiments, for Figure 4D , Figure 4E and Figure 4F Three independent experiments in terms of; and for Figure 4G Two independent experiments.

[0091] Figures 5A-5G This is a series of charts showing the effects of dietary Saa on tumor growth, cecal microbiome, mucus layer thickness, and M. schaedleri detection in human fecal samples in GF cAPC mice. Figure 5A The grades of developmental abnormalities and tumor lesions are shown in GF cAPC mice fed a Saa diet. Each column represents data from an individual mouse. Figure 5B Alpha diversity analysis of cecal 16S rRNA gene amplicon samples from Saa-fed cAPC mice is shown (Chao1 and Shannon). Each symbol represents data from an individual mouse. Figure 5C and Figure 5DIt shows the use of weighted Unifrac ( Figure 5C ) or Bray-Curtis ( Figure 5D The method was used for principal coordinate analysis (PCoA) of 16S rDNA amplicon from cAPC mice fed low or high Saa diets. Each symbol represents data from an individual mouse. Figure 5E The distribution of mean bacterial phylum abundance in cAPC mouse cecal samples is shown; n=6 for each diet and 3 cages for each condition. The top-to-bottom order of the phylum bars corresponds to the top-to-bottom order of the legend. Figure 5F The abundance of 16S rRNA amplicon of M. schaedleri is shown in cecal samples from cAPC mice fed a Saa diet. Each symbol represents data from an individual mouse. Figure 5G Serum FITC-dextran levels in WTBIH mice, measured after 2 weeks of low or high Saa diets followed by 3 hours of FITC-dextran intensive feeding, are shown. Error bars represent the standard error (SEM) of the mean. *P < 0.05, ***P < 0.001. Figure 5A Boschloo's test was performed; Figure 5F and Figure 5G The Mann-Whitney test was performed. The data represent the... Figure 5A The four independent experiments, for Figures 5B to 5F Three independent experiments, and for Figure 5G Two independent experiments.

[0092] Figures 6A-6E This is a series of charts showing the effects of dietary Saa on CD8 in the colonic LP and TDLN of cAPC mice. + The influence of T cell and its immune co-suppressive receptor expression. Figures 6A-6B This shows CD8 from tumors in the TDLN of cAPC mice fed low or high Saa diets. + T cells ( Figure 6A ) and PD-1 + LAG-3 + CTLA-4 + and TIM3 + ( Figure 6B CD8 + Flow cytometry analysis of the frequency and number of T cells. Figures 6C-6D The CD8+ of cAPC mice fed with low or high Saa diets is shown. + T cells ( Figure 6C ) and PD-1 + LAG-3 + CTLA-4 +and TIM3 + ( Figure 6D Flow cytometry analysis of colonic LP frequency and number. Figure 6E Images showing CD3 in healthy colon tissue from cAPC mice + CD8 + / CD3 + Quantification of ratios. Each symbol represents data from an individual mouse. Error bars represent the standard error (SEM) of the mean. *P-value < 0.05, **P-value < 0.01. Figures 6A to 6E The Mann-Whitney test was performed. The data represent the... Figures 6A-6E Three independent experiments were conducted.

[0093] Figures 7A-7D This is a series of charts showing the CD8+ of tumors from cAPC mice fed a Saa diet. + T-cell receptor sequencing (TCR-Seq) analysis of T cells. Figure 7A It shows CD8 within the tumor + A spectrum of T cell TCRα and TCRβ clonal repertoires. Each symbol represents data from an individual mouse. The left-to-right order of the bars in each group corresponds to the top-to-bottom order of the legend. Figure 7B The tumor CD8 was shown. + tSNE plot of T cell TCRα and TCRβ diversity. Overlapping ellipses represent 95% confidence intervals. Figure 7C and Figure 7D It shows the observed clones ( Figure 7C ) or Chao1 index ( Figure 7D α-diversity of TCRα and TCRβ clonal types. Each symbol represents data from an individual mouse. Error bars represent the standard error (SEM) of the mean. Visualization and statistical tests were performed using the immnuarch R / CRAN software package workflow.

[0094] Figures 8A-8F This is a series of illustrations showing the flow cytometry gating schemes used in Figures 2, 3, 6, 9, 10, and 11. Figure 8A A single live CD45 was shown. + The cell gating scheme serves as the starting cell population for subsequent gating. Figure 8B CD8 is shown + Gating of T cell and immune checkpoint receptor expression. Figure 8C The isolated CD8 of IFNγ and GZMB is shown. + Gating of T cell expression. Figure 8D CD4 is shown + Gating schemes for T cell populations. Figure 8E A gating scheme for dendritic cells expressing CD103 / CD11b is shown. Figure 8F The gating schemes for NK and NKT cells are illustrated. Polygonal indicator gates are used.

[0095] Figures 9A-9D This is a series of charts showing CD4 in the MLN and colonic LP of WT BIH (carrying M. schaedleri) mice fed a Saa diet. + T-cell analysis. Figures 9A-9B The total CD4 count in the MLN of WT BIH mice fed a Saa diet is shown. + T cells, Th1 cells, Th2 cells, Th17 cells, and Foxp3 + The frequency of Tregs ( Figure 9A ) and quantity ( Figure 9B ). Figures 9C-9D This image shows total CD4+ T cells and Th1, Th2, Th17 cells, and Foxp3 cells in the colonic LP of WT BIH mice fed a Saa diet. + The frequency of Tregs ( Figure 9C ) and quantity ( Figure 9D Each symbol represents data from an individual mouse. Error bars represent the standard error (SEM) of the mean. *P-value < 0.05. Figures 9A-9D The Mann-Whitney test was performed. The data represent the... Figures 9A-9D Three independent experiments were conducted.

[0096] Figures 10A-10J This is a series of charts showing myeloid cell analysis in Saa-fed cAPC mice, WT BIH mice, gnotobiotic mice, and cDC1-depleted mice. Figures 10A-10B The colonic LP (low- or high-Saa) of cAPC mice fed with low- or high-Saa diets is shown. Figure 10A ) and tumors ( Figure 10B cDC1(CD103) in ) + CD11b - CD103 - CD11b + and CD103 + CD11b + The frequency and number of cells. Figures 10C-10D MLN ( ) is shown from WT BIH mice fed with low or high Saa diets. Figure 10C ) and colon LP ( Figure 10D cDC1(CD103) in ) + CD11b - CD103 -CD11b + and CD103 + CD11b + The frequency and number of cells. Figures 10E-10F MLN ( ) is shown from WT GF mice fed with low or high Saa diets. Figure 10E ) or LP ( Figure 10F cDC1 and CD103 in ) - CD11b + and CD103 + CD11b + The frequency and number of cells. Figure 10G The correlation between cDC1 frequency and M. schaedleri abundance in the cecal contents of mice is illustrated. The black line represents a linear trend line, and the gray area represents the 95% confidence interval. Figures 10H-10I MLN ( ) from WT ASF mice fed with low or high Saa diets is shown. Figure 10H ) or LP ( Figure 10I cDC1 and CD103 in ) - CD11b + and CD103 + CD11b + The frequency and number of cells. Figure 10J The images show cDC1 and CD103 in LPs from mice fed a high-Saa diet and monoplanted with GF, M. schaedleri, or Akkermansia myxophila. - CD11b + and CD103 + CD11b + Cell frequency and number. The order of the bars in each group from left to right corresponds to the order of the legend from left to right. In all plots, each point represents an individual mouse. Error bars represent the standard error (SEM) of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 10J One-way ANOVA with FDR correction was performed; Figure 10G Spearman correlation tests were performed; and the results were... Figures 10A-10F as well as Figures 10H-10I The Mann-Whitney test was performed. The data represent the... Figures 10A-10I Three independent experiments; Figure 10J This represents two independent experiments.

[0097] Figures 11A-11J This is a series of charts showing myeloid cell analysis in Saa-fed cAPC mice, WT intra-institutionally cultured (BIH) mice, gnotobiotic mice, and cDC1-depleted mice. Figure 11AThe images show cDC1 and CD103 in the TDLN of Zbtb46-DTR cAPC mice fed a high-Saa diet and injected with PBS or diphtheria toxin (DT). - CD11b + The frequency and number of cells. Figure 11B The relative abundance of M. schaedleri in the cecal contents of Zbtb46-DTR cAPC mice fed a high-Saa diet and injected with PBS or DT is shown. Figure 11C cAPCBatf3 was shown from diets with low or high Saa. - / - cDC1 and CD103 in mouse TDLN - CD11b + The frequency and number of cells. Figure 11D cAPC Batf3 was shown when fed with low or high Saa diets. - / - The relative abundance of M. schaedleri in the cecal contents of mice. Figure 11E The expression of dendritic cell activation markers CD80 and CD86 on spleen cDC1 cells after overnight in vitro incubation with sterile mBHI or non-biological bacteria CM is shown. Figure 11F The image shows OT-I CD8 after co-culturing with cDC1 loaded with OVA protein for 3 days in sterile medium or with non-biological bacteria CM. + T cell proliferation. Figure 11G The image shows OT-I CD8 after overnight incubation with DCs loaded with OVA protein or OVA peptide and treated with sterile medium or non-biological bacteria CM. + Expression of IFNg on T cells. Figure 11H Serum XCL1 concentrations in cAPC mice fed a high-Saa diet and injected with α-XCL1 or its isotype are shown. Figure 11I cDC1 and CD103 in TDLN from cAPC mice fed a high-Saa diet and injected with α-XCL1 or isotype Abs are shown. - CD11b + The frequency and number of cells. Figure 11J The frequency and number of NK cells in the TDLN of cAPC mice fed a Saa diet are shown. In all figures, each symbol represents an individual mouse. Error bars represent the standard error (SEM) of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. Figures 11A-11D as well as Figures 11H-11J The Mann-Whitney test was performed. One-way ANOVA was then applied. Figure 11E , Figure 11F and Figure 11GData represents for Figures 11A-11D The four independent experiments, for Figure 11E as well as Figures 11H-11J Three independent experiments, and for Figure 11F and Figure 11G Two independent experiments.

[0098] Figures 12A-12D This is a series of charts illustrating the identification of dendritic cell clusters in the TDLN of cAPC mice fed low or high Saa diets. Figure 12A TDLN CD11c from cAPC mice is shown. + MHCII 高 CD64 - Illustration of the Unified Manifold Approximation and Projection (UMAP) of cell clusters. Figure 12B A violin diagram showing the expression of the cDC1 marker gene is displayed. Figure 12C A heatmap of dendritic cell (DC) cluster markers in TDLN from cAPC mice fed a Saa diet is shown. Figure 12D A rapid gene set enrichment analysis (fgsea) of the cDC1 cluster in the TDLN of Saa-fed cAPC mice is shown. Only p is shown. adj Pathways <0.05.

[0099] Figure 13 A graphical model illustrating diet-microbe-host interactions in CRC is shown. Dietary saline regulates the level of colonic mucus layer, leading to the expansion of *M. schaedleri* in mice fed a high-saline diet. *M. schaedleri* exerts immunomodulatory effects on NKT cells in tumor draining lymph nodes (TDLNs), resulting in increased XCL1 expression and enhanced cDC1 recruitment. cDC1 presents and cross-presents to T cells in the TDLN, activating them and causing them to migrate to the tumor site to limit tumor growth. Tumor cell death is represented by dark brown.

[0100] Figures 14A-14B It is a series of diagrams and charts illustrating how switching to a high-Saa diet can reduce tumor growth. Figure 14A The experimental design diagram is shown (top subplot); the line graph (bottom subplot) shows the tumor volume over time in mice born in the WT facility that were fed a standard mouse diet and switched to a Saa diet 5 days after an MC38 flank injection. Figure 14B This is a box plot showing the MC38 tumor weight 12 days after injection.

[0101] Figure 15This is a schematic diagram showing a comparison between the 16S sequence of the “Lior” M. schaedleri strain used in the embodiments described herein and the 16S sequence of M. schaedleri ASF457 preserved in GENBANK (see, for example, SEQ ID NOs: 4, 41-49).

[0102] Figure 16 This is a diagram illustrating untargeted LC-MS / MS metabolomics of non-biological *M. schaedleri* CM samples (n = 4 independent samples) versus a culture medium control without *M. schaedleri* (n = 2 independent samples). Of the 4263 features detected, relatively few (in...) Figure 16 Twelve features were enriched in CM with a fold change of 2 or higher (p-value less than 0.05) using a one-way ANOVA with a post-hoc Tukey's HSD test. The identified features are described and labeled in the volcano plot (see also...). Figure 4H ).

[0103] Figure 17 This is a bar graph illustrating the use of the mouse NKT cell line GW1 to screen for identified metabolites (see, for example, Figure 16 Regarding its ability to stimulate NKT XCL1 production (detected by ELISA) in an in vitro system. Metabolites screened individually showed some activity higher than the culture medium control (indicated by solid horizontal lines).

[0104] Figure 18 This is a bar graph illustrating the use of human NKT cells to screen for identified metabolites (see, for example, Figure 16 An in vitro system was developed to assess the ability of these metabolites to stimulate NKT XCL1 production (detected by ELISA). Pools of four metabolites were generated based on their chemical similarity and solubility. XCL1 levels induced by the culture medium control are represented by solid horizontal lines. IL-12 and anti-CD3 / CD28 bound to the culture plate served as positive controls. Pools 1, 3, and 4 exhibited higher activity than the culture medium control. Pool 1 = propionic acid, and cis- and trans-aconitic acids. Pool 2 = crotonic acid, succinic acid, and itaconic acid. Pool 3 = nicotinic acid. Pool 4 = myristic acid, pentadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, and 17-hydroxyheptadecanoic acid. Detailed Implementation

[0105] Implementations of the techniques described herein pertain to compositions comprising Mucispirillum (e.g., M. schaedleri). This disclosure describes how a diet rich in sulfur-containing amino acids can increase intestinal levels of M. schaedleri, thereby increasing XCL1 secretion by NKT cells. The increased XCL1 secretion by NKT cells increases the number and / or activation of CD103+ conventional dendritic cells (cDC1) (e.g., in tumor-draining lymph nodes), thereby recruiting and activating CD8+ T cells with anti-tumor immune activity (see, for example, Figure 13 Therefore, this document describes a method for treating cancer using the Mucispirillum composition as described herein; a diet rich in sulfur-containing amino acids (SAAs) or supplements containing SAAs; and / or an XCL1 peptide (or an XCR1 agonist). This document also describes a cancer treatment stratification method involving the detection of levels of M. schaedleri, XCL1 peptide, NKT, and / or CD103+cDC1, and corresponding changes or stratification of treatment. Composition

[0106] The various aspects described herein are compositions comprising Mucispirillum bacteria. In one aspect, a composition comprising Mucispirillum bacteria is described herein, the composition being formulated for delivery to the intestine. In some embodiments of any aspect, the composition is formulated for delivery to the intestine by oral administration. In some embodiments of any aspect, the composition comprises an enteric coating or the like to remain in gastric acid and allow delivery to the small or large intestine. In one aspect, a composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria is described herein, the composition being formulated for delivery to the intestine. In some embodiments, the M. schaedleri is formulated for delivery to the small intestine, duodenum, jejunum, ileum, cecum, ileocecal junction, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, or anus.

[0107] Mucispirillum is a genus in the phylum Deferribacteres. It is represented by a single species, *Mucispirillum schaedleri*. Mucispirillum are spiral-shaped bacteria found in the mucus layer of the gastrointestinal tract of some rodents and are considered symbiotic. This species has been found in cockroaches, mice, turkeys, dogs, pigs, goats, termites, and humans. Mucispirillum is anaerobic and does not form spores. Mucispirillum is motile, flagellated, and capable of moving through mucus.

[0108] In some embodiments of any aspect, the *M. schaedleri* bacterium is *M. schaedleri* strain ASF457. In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising: SEQ ID NO: 4, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 4. In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising: SEQ ID NO: 4, or a nucleic acid sequence that is at least 95% or more identical to SEQ ID NO: 4. In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising: SEQ ID NO: 4, or a nucleic acid sequence that is at least 97% or more identical to SEQ ID NO: 4.

[0109]

[0110] In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising one of SEQ ID NOs: 4, 40-49, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to one of SEQ ID NOs: 4, 40-49 (see, for example, Figure 15 In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising one of SEQ ID NOs: 4, 40-49, or a nucleic acid sequence that is at least 95% or more identical to one of SEQ ID NOs: 4, 40-49. In some embodiments of any aspect, the *M. schaedleri* bacterium comprises a 16S sequence comprising one of SEQ ID NOs: 4, 40-49, or a nucleic acid sequence that is at least 97% or more identical to one of SEQ ID NOs: 4, 40-49.

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[0121] In some embodiments of any aspect, the composition comprises about 10 1 -10 12 M. schaedleri cells / mL, for example, about 10 1 M. schaedleri cells / mL, approximately 10 2 M. schaedleri cells / mL, approximately 10 3 M. schaedleri cells / mL, approximately 10 4 M. schaedleri cells / mL, approximately 10 5 M. schaedleri cells / mL, approximately 10 6 M. schaedleri cells / mL, approximately 10 7 M. schaedleri cells / mL, approximately 10 8 M. schaedleri cells / mL, approximately 10 9 M. schaedleri cells / mL, approximately 10 10 M. schaedleri cells / mL, approximately 10 11 M. schaedleri cells / mL, approximately 10 12 One or more *M. schaedleri* cells / mL. In some embodiments of any aspect, the composition comprises about 10 1 -10 12 The colony-forming units (CFU; e.g., as a measure of live bacterial cells) of M. schaedleri, for example, are approximately 10 1 CFU / mL, approximately 10 2 CFU / mL, approximately 10 3 CFU / mL, approximately 10 4 CFU / mL, approximately 10 5 CFU / mL, approximately 10 6 CFU / mL, approximately 10 7 CFU / mL, approximately 10 8 CFU / mL, approximately 10 9 CFU / mL, approximately 10 10 CFU / mL, approximately 10 11 CFU / mL, approximately 10 12 One CFU / mL or more.

[0122] In some embodiments of any aspect, the composition comprises about 10 1 -10 12M. schaedleri cells / g, for example, about 10 1 M. schaedleri cells / g, approximately 10 2 M. schaedleri cells / g, approximately 10 3 M. schaedleri cells / g, approximately 10 4 M. schaedleri cells / g, approximately 10 5 M. schaedleri cells / g, approximately 10 6 M. schaedleri cells / g, approximately 10 7 M. schaedleri cells / g, approximately 10 8 M. schaedleri cells / g, approximately 10 9 M. schaedleri cells / g, approximately 10 10 M. schaedleri cells / g, approximately 10 11 M. schaedleri cells / g, approximately 10 12 One M. schaedleri cell / g or more. In some embodiments of any aspect, the composition comprises about 10 1 -10 12 The colony-forming units (CFU; e.g., as a measure of live bacterial cells) of M. schaedleri, for example, are approximately 10 1 CFU / g, approximately 10 2 CFU / g, approximately 10 3 CFU / g, approximately 10 4 CFU / g, approximately 10 5 CFU / g, approximately 10 6 CFU / g, approximately 10 7 CFU / g, approximately 10 8 CFU / g, approximately 10 9 CFU / g, approximately 10 10 CFU / g, approximately 10 11 CFU / g, approximately 10 12 One CFU / g or more.

[0123] In some embodiments of any aspect, the composition comprises centrifuged Mucispirillum chaedleri culture (e.g., OD). 600nm ~0.8), and the bacterial precipitate is resuspended in a pharmaceutically acceptable carrier. In some embodiments of any aspect, the composition comprises 5 mL of centrifuged Mucispirillum chaedleri culture (e.g., OD500).600nm ~0.8), and the bacterial precipitate is resuspended in 1 mL of a pharmaceutically acceptable carrier, each unit dose of the composition comprising 100 μL of solution.

[0124] In some embodiments of any aspect, the *M. schaedleri* bacteria are alive. In some embodiments of any aspect, the *M. schaedleri* bacteria are inactivated. Non-limiting examples of bacterial inactivation methods include ethanol (e.g., 40% ethanol), ultraviolet irradiation, heating, or autoclaving. As a non-limiting example, a bacterial inactivation method includes heating the bacteria at a temperature of at least 100°C (e.g., at least 105°C, at least 110°C, at least 115°C, at least 120°C, or higher) for at least 10 minutes (e.g., at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, or longer). As another non-limiting example, a bacterial inactivation method includes heating the bacteria at a temperature of at least 70°C (e.g., at least 75°C, at least 80°C, at least 90°C, at least 95°C, or higher) for at least 40 minutes (e.g., at least 45 minutes, at least 50 minutes, at least 60 minutes, or longer). As a non-limiting example, bacterial inactivation methods include autoclaving bacteria at a temperature of at least 120°C (e.g., at least 121°C, at least 125°C, at least 115°C, at least 130°C or higher) for at least 30 minutes (e.g., at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes or longer) using saturated steam at a pressure of at least 15 psi (e.g., at least 20 psi, at least 25 psi, at least 30 psi).

[0125] In some embodiments of any aspect, the *M. schaedleri* bacteria are in a dried, live form. In some embodiments of any aspect, the *M. schaedleri* bacteria are spray-dried or freeze-dried live bacteria. In some embodiments of any aspect, the *M. schaedleri* bacteria are non-live. In some embodiments of any aspect, the composition comprises fresh *M. schaedleri* bacteria, live *M. schaedleri* bacteria, freeze-dried *M. schaedleri* bacteria, spray-dried *M. schaedleri* bacteria, non-live *M. schaedleri* bacteria, or any combination thereof.

[0126] In one aspect, this document describes a composition comprising a *M. schaedleri* conditioned medium, the composition being formulated for delivery to the intestine. In some embodiments of any aspect, the conditioned medium is prepared by incubating *M. schaedleri* bacteria in a medium for a predetermined amount of anaerobic incubation time, for example, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, or longer. In some embodiments of any aspect, the medium is a modified brain-heart extract (mBHI) medium. In some embodiments of any aspect, the mBHI medium comprises 37 g BHI, 5 g yeast extract, 2 mg vitamin K, 5 mg ferrous heme, 0.5 g L-cysteine, and 150 mL fetal bovine serum per 1 L of water. The pH of the medium is adjustable to 7.2 and filtered through a 0.2 μm filter.

[0127] In some embodiments of any aspect, the conditioned medium is prepared by removing *M. schaedleri* bacteria from the conditioned medium after a predetermined amount of anaerobic incubation time; for example, the *M. schaedleri* bacteria can be removed by centrifugation and removal of the supernatant from the bacterial precipitate. In some embodiments of any aspect, the conditioned medium is prepared by inactivating *M. schaedleri* bacteria in the conditioned medium after a predetermined amount of anaerobic incubation time; for example, the *M. schaedleri* bacteria can be inactivated by ethanol (e.g., 40% ethanol), ultraviolet irradiation, heating, or autoclaving.

[0128] As a non-limiting example, a bacterial inactivation method includes heating bacteria at a temperature of at least 100°C (e.g., at least 105°C, at least 110°C, at least 115°C, at least 120°C, or higher) for at least 10 minutes (e.g., at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, or longer). As another non-limiting example, a bacterial inactivation method includes heating bacteria at a temperature of at least 70°C (e.g., at least 75°C, at least 80°C, at least 90°C, at least 95°C, or higher) for at least 40 minutes (e.g., at least 45 minutes, at least 50 minutes, at least 60 minutes, or longer). As a non-limiting example, bacterial inactivation methods include autoclaving bacteria at a temperature of at least 120°C (e.g., at least 121°C, at least 125°C, at least 115°C, at least 130°C or higher) for at least 30 minutes (e.g., at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes or longer) using saturated steam at a pressure of at least 15 psi (e.g., at least 20 psi, at least 25 psi, at least 30 psi).

[0129] In one aspect, this document describes a composition comprising an organic solvent extract of *M. schaedleri* conditioned medium, wherein the composition is formulated for delivery into the intestine. Non-limiting examples of organic solvents used for extraction include methanol, chloroform, ethyl acetate, ethanol, acetone, or any combination thereof. In some embodiments of any aspect, the organic solvent extract of *M. schaedleri* conditioned medium is prepared by methanol:chloroform extraction (see, for example, Materials and Methods in Example 1). In some embodiments of any aspect, the organic solvent extract of *M. schaedleri* conditioned medium comprises an organic compound (e.g., organic solvent-soluble, nonpolar) secreted by *M. schaedleri* or otherwise produced. In some embodiments of any aspect, the *M. schaedleri* conditioned medium or the organic solvent extract of *M. schaedleri* conditioned medium is heat-treated, for example, incubated at a temperature of at least 100°C for at least 10 minutes.

[0130] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from: Table 3 or Table 9 of U.S. Provisional Application No. 63 / 389,382, or the metabolite herein. Figure 4H , Figure 4I or Figure 16 .

[0131] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or 12 metabolites), said metabolite being selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3), and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

[0132] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises up to 12 metabolites (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 metabolites), said metabolites being selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

[0133] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof, comprises up to seven metabolites (e.g., up to one, up to two, up to three, up to four, up to five, up to six, or up to seven metabolites), said metabolites being selected from the group consisting of succinic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid.

[0134] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises succinic acid, propionic acid, nicotinic acid, aconitic acid (cis and / or trans), pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, crotonic acid, myristic acid, and 17-hydroxyheptadecanoic acid (C 17 H 34 O3), 15-hydroxypentadecanoic acid (C 15 H 30 O3) or any combination thereof (see, for example, Equations 4-15 in Table 3).

[0135] Table 3: Exemplary Metabolites

[0136] In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof, comprises propionic acid, cis-aconitic acid, and / or trans-aconitic acid, or any combination thereof (see, for example, Figure 18Pool 1). In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises crotonic acid, succinic acid, and / or itaconic acid, or any combination thereof (see, for example, Figure 18 Pool 2). In some embodiments of any aspect, the M. schaedleri conditioned medium, or the organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof, comprises nicotinic acid (see, for example, Figure 18 Pool 3). In some embodiments of any aspect, the M. schaedleri conditioned medium, or an organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof, comprises myristic acid, pentadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid and / or 17-hydroxyheptadecanoic acid, or any combination thereof (see, for example, Pool 3). Figure 18 The pool 4).

[0137] In some embodiments of any aspect, the composition comprises the *M. schaedleri* bacteria, a culture medium, and / or a solvent extract, and the composition promotes XCL1 secretion by NKT cells. XC motif chemokine ligand 1 (XCL1) is also known as a lymphocyte chemokine, lymphotoxin, or member of the small inducible cytokine subfamily C1. XCL1 is a chemokine that plays a role in inflammatory and immune responses, inducing leukocyte migration and activation. XCL1 contributes to the chemotaxis of CD8+ T cells. NK cells release XCL1 along with IFN-γ and several other chemokines upon encountering certain bacteria, and CD8+ cells work together to cross-present antigens and deliver CD8+ T cell activation. In some embodiments of any aspect, XCL1 secretion further activates cDC1 (e.g., in tumor-draining lymph nodes), which can ultimately lead to an enhanced CD8+ T cell antitumor response. In some embodiments of any aspect, the composition comprising the *M. schaedleri* bacteria, a culture medium, and / or a solvent extract further comprises an XCL1 polypeptide (e.g., SEQ ID NO: 5-6).

[0138] In some embodiments of any aspect, the XCL1 comprises: SEQ ID NO: 5, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to and maintains its function (e.g., binding to and / or activating XCR1). In some embodiments of any aspect, the mature human XCL1 peptide comprises residues 22-114 or 22-93 of SEQ ID NO: 5.

[0139] SEQ ID NO: 5, precursor of lymphocyte chemokine (XCL1), Homo sapiens, NCBI reference sequence: NP_002986.1, 114 amino acids (aa) MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG.

[0140] In some embodiments of any aspect, the XCL1 comprises: SEQ ID NO: 6, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to and maintains its function (e.g., binding to and / or activating XCR1). In some embodiments of any aspect, the mature mouse XCL1 peptide comprises residues 22-114 or 22-93 of SEQ ID NO: 6.

[0141] SEQ ID NO: 6, Lymphocyte chemokine (XCL1) precursor, mouse, GenBank: AAA56752.1, 114 amino acids MRLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAVTLTG.

[0142] In some embodiments of any aspect, the composition comprises the *M. schaedleri* bacteria, a culture medium, and / or a solvent extract, and the composition increases XCL1 secretion from NKT cells by at least 100% (see, for example, Figures 4F-4G , Figures 17-18 In some embodiments of any aspect, the composition comprises the M. schaedleri bacteria, a culture medium, and / or a solvent extract, and the composition increases XCL1 secretion of NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to NKT cells not exposed to the composition.

[0143] In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are in a dry form (e.g., spray-dried or freeze-dried). In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are encapsulated. In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are contained in enteric-coated capsules. In some embodiments of any aspect, the composition includes an enteric coating or the like to remain in gastric acid and allow delivery to the small or large intestine. In some embodiments of any aspect, the composition is formulated for delivery as: capsules, enteric-coated capsules, tablets, pouches, pills, compressed pills, lozenges, sugar tablets, powders, granules, nutritional supplements, medical foods, sachets, liquids, suspensions, oil suspensions, gels, geltabs, semi-solids, or any combination thereof.

[0144] In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or extract are maintained in an anaerobic state in the formulation. As used herein, the term "anaerobic state" refers to an oxygen level (e.g., dissolved oxygen or gaseous oxygen) that is at or below the oxygen level normally present in the human intestinal lumen. As a non-limiting example, the anaerobic formulation of the *M. schaedleri* bacteria, culture medium, and / or solvent extract can be prepared by removing oxygen from the formulation using an inactive gas (e.g., nitrogen). In some embodiments of any aspect, the anaerobic formulation comprising the *M. schaedleri* bacteria, culture medium, and / or solvent extract does not contain detectable dissolved oxygen or gaseous oxygen, or substantially does not contain dissolved oxygen or gaseous oxygen. In some embodiments of any aspect, the anaerobic formulation comprising the *M. schaedleri* bacteria, culture medium, and / or solvent extract includes at most 0.01%, at most 0.1%, or at most 1% dissolved oxygen or gaseous oxygen.

[0145] In some embodiments of any aspect, the M. schaedleri bacteria, culture medium, and / or solvent extract are mixed with prebiotics. Non-limiting examples of prebiotics include: amino acids (e.g., arginine, glutaric acid, and ornithine), short-chain fatty acids (SCFAs; e.g., acetate, propionate, butyrate), biotin, fructooligosaccharides, galactooligosaccharides, hemicellulose (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan, inulin, chitin, lactulose, mannan oligosaccharides, inulin enriched with fructooligosaccharides, gums (e.g., guar gum, gum arabic, and carrageenan), fructooligosaccharides, dextrose oligosaccharides, tagatose, resistant maltodextrin (e.g., resistant starch), trans-galactooligosaccharides, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonic acid-I), dietary fiber (e.g., soybean fiber, beet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharides, and polyamines (e.g., but not limited to spermidine and putrescine).

[0146] In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are mixed with a sulfur-containing amino acid (SAA). In some embodiments of any aspect, the sulfur-containing amino acid is methionine, cysteine, or a derivative thereof. In some embodiments of any aspect, the sulfur-containing amino acid is methionine, cysteine, homocysteine, taurine, or a derivative thereof. In some embodiments of any aspect, the composition comprises a combination of any one of methionine, cysteine, homocysteine, and / or taurine (see, for example, Table 9). In some embodiments of any aspect, the composition comprises methionine. In some embodiments of any aspect, the composition comprises cysteine. In some embodiments of any aspect, the composition comprises homocysteine. In some embodiments of any aspect, the composition comprises taurine.

[0147] The methionine derivative or cysteine ​​derivative must contain sulfur, but may differ in structure from methionine and cysteine, and may include homocysteine ​​or taurine. In some embodiments, the methionine derivative or cysteine ​​derivative is: ribose-cysteine, ribose-methionine, N-acetylcysteine, or acetylcysteine.

[0148] As used herein, the term "methionine derivative" refers to an amino acid derivative obtained by reacting methionine with an amino or carboxyl group, or by substituting any hydrogen atom of methionine with a heteroatom; the definition generally excludes peptides containing methionine residues. Non-limiting examples of methionine derivatives include: ribo-methionine; (2S)-2-[[[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-phenylphenyl]-oxomethyl]amino]-4-(methylthio)butyric acid, 2-(1,3-benzothiazol-2-ylamino)-4-(methylthio)butyric acid, 2-[(6-bromo-4-quinazolinyl)amino]-4-(methylthio)butyric acid, methyl 2-[[(4-ethylphenyl)-oxomethyl]amino]-4-(methylthio)butyrate. 2-Amino-4-(methylthio)-N-(2-naphthyl)butyramide, N-acetylated methionine, D-methionine, L-methionine, L-methionine methyl iodide sulfonium, L-methionine, trimethylsilyl ester, methionine S-oxide, methionine sulfone, methionine sulfoxide imine, methionine isohydroxyoxime, N-(1-deoxy-1-fructosyl)methionine, N-[(2S)-2-hydroxypropionyl]methionine, N-formyl-DL-methionine, N-oleoylmethionine, or peptidylmethionine. In some embodiments, the methionine derivative is ribose-methionine.

[0149] As used herein, the term "cysteine ​​derivative" refers to an amino acid derivative obtained by reacting cysteine ​​with an amino group, carboxyl group or thiol group, or by substituting any hydrogen atom of cysteine ​​with a heteroatom; the definition generally excludes peptides containing cysteine ​​residues. Non-limiting examples of cysteine ​​derivatives include: ribose-cysteine, N-acetylcysteine, acetylcysteine, (2R;2'S)-isobutylene, 2-amino-3-(hydroxysulfonylthio)propionic acid, 2-amino-3-{[(1E)-3-(prop-2-en-1-sulfinyl)prop-1-en-1-yl]dithio}propionic acid, 2-ammonium-3-dithiopropionate, N-acetyl-S-(1Z)-propenyl-cysteine ​​sulfoxide, S-(5-acetamido-2-hydroxyphenyl)cysteine, S-2-chloroethylcysteine, S-propylcysteine, D-cysteine ​​derivatives, L-cysteine ​​derivatives, allocysteine ​​thioether, allylcysteine, sulfalanine, cysteyl amino acids, cystine (cysteine ​​dimer), γ-glutamylcysteylglutamic acid, grixazone B. Hawkinsin, L-cysteine-glycine, peptidylcysteine, isopentenylcysteine, S-(3-oxo-3-carboxy-n-propyl)cysteine, S-(allylthio)-L-cysteine, S-acetaminomethylcysteine, S-cysteine ​​succinate, or trans-S-(1-propenyl)-L-cysteine. In some embodiments, the cysteine ​​derivative is ribose-cysteine, N-acetylcysteine, or acetylcysteine.

[0150] Table 9: Exemplary SAAs in the Compositions (“x” indicates included in the composition) Methionine Cysteine homocysteine Taurine X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

[0151] In some embodiments of any aspect, the composition comprises at least 2.0 g of SAA (e.g., methionine, cysteine, homocysteine, and / or taurine or derivatives thereof). In some embodiments of any aspect, the composition comprises at least 2.4 g of SAA. In some embodiments of any aspect, the composition comprises at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some embodiments of any aspect, the composition comprises up to 5g, up to 10g, up to 20g, up to 30g, at least 40g, up to 50g, up to 60g, up to 70g, up to 80g, up to 90g, up to 100g, up to 150g, up to 200g, up to 300g, or up to 400g of SAA. In some embodiments of any aspect, the composition comprises 2.0g-400g of SAA. In some embodiments of any aspect, the composition comprises 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA.

[0152] In some embodiments of any aspect, the composition further comprises 1 to 20 additional microbial species. In some embodiments of any aspect, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 additional microbial species. In some embodiments of any aspect, the composition comprises no more than 20 microbial species. In some embodiments of any aspect, the composition comprises up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 bacterial species, including M. schaedleri bacteria.

[0153] In some embodiments of any aspect, in addition to the *M. schaedleri* bacteria, the additional bacteria in the composition are derived from sources such as: environmental isolates, commercially available isolates, at least a portion of a human microbiome sample, at least a portion of a non-human mammalian (e.g., mouse) microbiome sample, isolates from human microbiome samples, isolates from non-human mammalian microbiome samples, etc. While the benefit of a particular microbiome species depends on the specific indication, non-limiting examples of bacterial genera that may be beneficial (e.g., for the human gastrointestinal system) and included in the composition include: *Akkermansia*, *Alistipes*, *Bacillus*, *Bacteroides*; *Bifidobacterium*, *Blautia*, *Clostridium*, *Collinsella*, *Eggerthella*, *Enterococcus*, *Eubacterium*, *Faecalibacterium*. ), Fusobacterium, Gemmiger, Lactobacillus, Parabacteroides, Pararaprevotella, Phascollectobacterium, Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus, Ruthenibacterium, Streptococcus, and Subdoligranulum.

[0154] In some embodiments of any aspect, the composition is substantially free of pathogens. As used herein, the term "substantially" means a range or extent of complete or near-complete completeness. For example, a composition that is "substantially" free of pathogens means that the composition is completely or nearly completely free of any pathogens (e.g., free of any live pathogens). In some cases, the exact permissible deviation from absolute completeness may depend on the specific circumstances. However, in general, near-completeness will have the same overall results as those obtained with absolute and complete completeness. In some embodiments of any aspect, the pathogens in the therapeutic composition are absent or reduced to a level acceptable for human administration (e.g., as determined by the FDA).

[0155] As used herein, the term "pathogen" refers to any infectious microorganism that causes disease in an organism. In one embodiment, the pathogen includes bacteria, fungi, archaea (e.g., methanogens, halophiles, thermophiles, and psychrophiles), protozoa (e.g., Plasmodium, Entamoeba histolytica, Trypanosoma brucei, Giardia lamblia), viruses, prions (e.g., PrPres and PrPSc), microphytes (e.g., Shewanella algae, Shewanella putrefaciens, and Shewanella xiamenensis), and / or microanimals / parasites (e.g., plankton, planarians, worms, schistosomes, and trypanosomes). In some embodiments, the pathogenic virus includes, but is not limited to, RNA viruses (e.g., flaviviruses, microribonucleic acid viruses, rhabdoviruses, filamentous viruses, retroviruses (including lentiviruses)) or DNA viruses (e.g., adenoviruses, poxviruses, herpesviruses, cytomegaloviruses, hepatotropic DNA viruses) or others.

[0156] Non-limiting examples of pathogenic bacteria include spirochetes (e.g., Borrelia), actinomycetes (e.g., Actinomyces), mycoplasma, rickettsiae, Gram-negative aerobic bacilli, Gram-negative aerobic cocci, Gram-negative facultative anaerobic bacilli (e.g., Erwinia and Yersinia), Gram-negative cocci, Gram-negative cocci, Gram-positive cocci (e.g., Staphylococcus and Streptococcus), spore-forming bacilli, and spore-forming cocci. Further non-limiting examples of bacterial pathogens include certain species of the following: Bacillus, Brucella, Burkholderia, Francisella, Yersinia, Streptococcus, Haemophilus, Nisseria, Listeria, Clostridium, Klebsiella, Legionella, Escherichia (e.g., E. coli), Mycobacterium, Staphylococcus, Campylobacter, Vibrio, and Salmonella, as well as drug-resistant, multidrug-resistant, and highly virulent strains of these pathogenic bacteria. Non-limiting examples of known foodborne bacterial pathogens include some of the following species: Salmonella, Clostridium, Campylobacter, Staphylococcus, Salmonella, Escherichia (e.g., Escherichia coli), and Listeria.In some embodiments, non-limiting examples of bacterial pathogens include: Bacillus anthracis, Brucella abortus, Brucella meliensis, Brucella suis, Burkholderia mali, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, group A and B streptococci, MRSA, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Listeria monocytogenes, Clostridium difficile, Klebsiella spp., highly virulent pathogenic strains of Escherichia coli, and Mycobacterium tuberculosis. *Staphylococcus aureus*, *Campylobacter* spp., *Salmonella* spp., and *Clostridium perfringens*, as well as drug-resistant, multidrug-resistant, and highly virulent strains of these pathogenic bacteria. In some embodiments, non-limiting examples of known foodborne bacterial pathogens include: *Salmonella* spp., non-typhoidal *Clostridium perfringens*, *Campylobacter* spp., *Staphylococcus aureus*, *Salmonella* spp., non-typhoidal *Campylobacter* spp., *Escherichia coli* (STEC) O157, and *Listeria monocytogenes* spp.

[0157] In some embodiments of any aspect, the composition is substantially free of human pathogens (e.g., those described above or known in the art). In some embodiments of any aspect, the composition is substantially free of non-human mammalian pathogens. In some embodiments of any aspect, the composition is substantially free of non-human mammalian pathogens that can infect humans and / or cause disease in humans.

[0158] In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are formulated in a food composition. In some embodiments of any aspect, the food composition comprises yogurt or a yogurt beverage. In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are formulated in a medicinal food. In some embodiments of any aspect, the *M. schaedleri* bacteria, culture medium, and / or solvent extract are formulated in a supplement. In one aspect, this is described as a food composition comprising *M. schaedleri* bacteria, culture medium, and / or solvent extract as described herein. In one aspect, this is described as a medicinal food comprising *M. schaedleri* bacteria, culture medium, and / or solvent extract as described herein. In one aspect, this is described as a supplement comprising *M. schaedleri* bacteria, culture medium, and / or solvent extract as described herein. In some embodiments of any aspect, the food composition, medicinal food, or supplement is supplemented with sulfur-containing amino acids and / or prebiotics. In some embodiments of any aspect, the food composition, medical food, or supplement further includes one to 20 additional bacterial strains.

[0159] In some embodiments, the techniques described herein relate to pharmaceutical compositions comprising *M. schaedleri* bacteria, culture media, and / or solvent extracts as described herein, and optionally, pharmaceutically acceptable carriers. In some embodiments, the active ingredient of the pharmaceutical composition comprises *M. schaedleri* bacteria, culture media, and / or solvent extracts as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists substantially of *M. schaedleri* bacteria, culture media, and / or solvent extracts as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of *M. schaedleri* bacteria, culture media, and / or solvent extracts as described herein.

[0160] In some embodiments, the techniques described herein relate to pharmaceutical compositions comprising sulfur-containing amino acids (SAAs) such as those described herein, *M. schaedleri* bacteria, culture media, and / or solvent extracts. In some embodiments, the active ingredient of the pharmaceutical composition comprises an SAA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists substantially of an SAA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of an SAA as described herein.

[0161] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known to those skilled in the art. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) (11) Diols, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Pyrothermic water; (18) Isotonic saline; (19) Ringer's solution; (10) Ethanol; (11) pH buffer solution; (22) Polyesters, polycarbonates and / or polyanhydrides; (23) Fillers, such as peptides and amino acids; (24) Serum components, such as serum albumin, HDL and LDL; (25) C2-C 12 Alcohols; and (25) other non-toxic and compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, aroma enhancers, preservatives, and antioxidants may also be present in the formulation. Terms such as “excipient,” “carrier,” and “pharmaceuticalally acceptable carrier” are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent (e.g., M. schaedleri bacteria, culture media, and / or solvent extracts, and / or SAA as described herein).

[0162] Conventional dosage forms typically provide rapid or immediate release of the active ingredient from the formulation. Depending on the pharmacology and pharmacokinetics of the active ingredient, the use of conventional dosage forms can cause significant fluctuations in the concentration of the active ingredient in the patient's blood and other tissues. These fluctuations can affect many parameters, such as dosing frequency, onset time, duration of effect, maintenance of therapeutic blood levels, toxicity, side effects, etc. Advantageously, controlled-release formulations can be used to control the onset time, duration of action, levels within the therapeutic window (e.g., gastrointestinal levels), and peak levels of the active ingredient (e.g., gastrointestinal levels). In particular, controlled-release or sustained-release dosage forms or formulations can be used to ensure maximum efficacy of the active ingredient while minimizing potential side effects and safety issues that can occur due to insufficient dosage of the active ingredient (i.e., below the minimum therapeutic level) and exceeding the toxicity level of the active ingredient. In some embodiments, the *M. schaedleri* composition can be administered in a sustained-release formulation.

[0163] The common goal of controlled-release pharmaceutical products is to improve the treatment achieved by their non-controlled-release counterparts. Ideally, the use of optimally designed controlled-release formulations in medical therapy is characterized by the use of minimal active ingredient to cure or control the condition in the shortest possible time. The advantages of controlled-release formulations include: 1) prolonged activity of the active ingredient; 2) reduced dosing frequency; 3) increased patient compliance; 4) lower total dosage of the pharmaceutical composition; 5) reduced local or systemic side effects; 6) minimized accumulation of the active ingredient; 7) reduced fluctuations in levels (e.g., at the gastrointestinal level); 8) improved therapeutic efficacy; 9) reduced enhancement or loss of activity of the active ingredient; and 10) improved speed of disease or symptom control. See, for example, Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0164] Most controlled-release formulations are designed to initially release a certain amount of the active ingredient to rapidly produce the desired therapeutic effect, and then gradually and continuously release the remaining amount of the active ingredient to maintain this level of therapeutic or preventative effect over an extended period of time. To maintain this constant level of the active ingredient in the body, the active ingredient must be released from the dosage form at a controlled rate to replace the amount of active ingredient that is metabolized, excreted from the body, and / or otherwise inactivated. Controlled release of the active ingredient can be stimulated by a variety of conditions, including but not limited to: pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0165] A variety of known controlled-release or sustained-release dosage forms, formulations, and devices are suitable for the compositions described herein. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,733,566, and 6,365,185B1, each of which is incorporated herein by reference. These dosage forms can be used to provide sustained-release or controlled-release of one or more active ingredients to provide desired release profiles at different proportions, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, permeation systems (e.g., hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, permeation systems). (AlzaCorporation, Mountain View, Calif.USA) or combinations thereof. Treatment

[0166] The compositions described herein may be administered to subjects in need, for example, for: treating cancer, promoting antitumor immune activity, promoting responsiveness to immune checkpoint inhibitor cancer therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion from NKT cells, increasing CD8+ T cell infiltration in colorectal tumors, establishing or maintaining a tumor-suppressive gut environment, and / or cancer treatment stratification. Such methods may include administering one or more of the following: the Mucispirillum compositions described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAAs), and / or an XCL1 peptide (or an XCR1 agonist). In some embodiments of any aspect, the method includes administering: a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and / or an XCL1 peptide (or an XCR1 agonist); a Mucispirillum composition as described herein and a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA); a Mucispirillum composition as described herein and an XCL1 peptide (or an XCR1 agonist); a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and an XCL1 peptide (or an XCR1 agonist); or a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and an XCL1 peptide (or an XCR1 agonist).

[0167] In one aspect of any embodiment, the method described herein comprises administering a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAAs) and / or an XCL1 peptide (or an XCR1 agonist) to: treat cancer, promote antitumor immune activity, promote responsiveness to immune checkpoint inhibitor tumor therapy, increase CD103+ conventional dendritic cells (cDC1), increase XCL1 secretion from NKT cells, increase CD8+ T cell infiltration in colorectal tumors, and / or establish or maintain a tumor-suppressive intestinal environment in subjects in need.

[0168] In some embodiments, the treatment method may include prescribing a treatment as disclosed herein to a subject in lieu of administering the treatment. As used herein, the term “prescribing” means recommending and / or authorizing the use of a treatment to a subject, for example, in writing. In one aspect of any embodiment, a method described herein includes prescribing a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAAs), and / or an XCL1 peptide (or XCR1 agonist) to: treat cancer, promote antitumor immune activity, promote responsiveness to immune checkpoint inhibitor tumor therapy, increase CD103+ conventional dendritic cells (cDC1), increase XCL1 secretion from NKT cells, and / or establish or maintain a tumor-suppressive gut environment in subjects in need.

[0169] In some embodiments, the treatment method may include first diagnosing a subject or patient who may benefit from treatment with the composition described herein. In some embodiments, such diagnosis includes detecting or measuring the following aspects in a sample from the subject or patient, each being an example of an abnormal level of each analyte: for example, low levels of Mucispirillum (e.g., Mucispirillum schaedleri), low levels of sulfur-containing amino acids (SAAs), low levels of XCL1 (RNA or protein), low levels of CD103+ conventional dendritic cells (cDC1), low levels of XCL1-expressing NKT cells, low levels of antitumor immune activity, or low levels of responsiveness to immune checkpoint inhibitor tumor therapy, etc. In some embodiments, the method further includes administering to the patient the Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAAs) (or a supplement containing SAAs), and / or an XCL1 peptide (or an XCR1 agonist).

[0170] In some embodiments of any aspect, the low level of Mucispirillum (e.g., Mucispirillum schaedleri) is lower than the level measured in normal controls (e.g., in intestinal, fecal, or mucosal samples). In some embodiments of any aspect, the Mucispirillum (e.g., Mucispirillum schaedleri) level is measured using 16S rRNA abundance determined by 16S sequencing, or the level is determined using bacterial cultures (e.g., CFU / mL). As a non-limiting example, the relative 16S rRNA abundance of Mucispirillum in the feces of normal controls (e.g., healthy mouse controls) is from 1E-5 to 0.05 (e.g., 1.33E-05 to 0.0441517) or from 0.01 to 0.05 (e.g., 0.01117764 to 0.0441517). In some embodiments of any aspect, a low level of Mucispirillum in feces is a relative 16S rRNA abundance of Mucispirillum in feces of less than 0.01, less than 0.009, less than 0.008, less than 0.007, less than 0.006, less than 0.005, less than 0.004, less than 0.003, less than 0.002, less than 1E-3, less than 1E-4, less than 1E-5, or less than 1E-6. In some embodiments of any aspect, a low level of Mucispirillum is a relative 16S rRNA abundance of Mucispirillum in feces of 0 to 0.01. In some embodiments of any aspect, a low level of Mucispirillum is a relative 16S rRNA abundance of Mucispirillum in feces of 0 to 1E-5.

[0171] In some embodiments of any aspect, the level of Mucispirillum (e.g., Mucispirillum schaedleri) is measured in intestinal samples (e.g., mucosal or luminal samples) because Mucispirillum can be present at a high prevalence (e.g., an average prevalence of 42%) in human mucosal biopsy samples, but at a low prevalence (e.g., 3%) in fecal samples. In some embodiments, the intestinal sample is selected from the group consisting of: jejunal lumen, jejunal mucosa, terminal ileum lumen, terminal ileum mucosa, cecal lumen, cecal mucosa, ascending colon mucosa, transverse colon mucosa, descending colon lumen, and descending colon mucosa. In some embodiments, the intestinal sample is from the cecal lumen.

[0172] As a non-limiting example, the relative 16S rRNA abundance of *Mucispirillumschaedleri* in intestinal samples from normal controls (e.g., healthy human controls) can range from 0.02 to 1.0, depending on the sample site: for example, 0.2 (jejunum), 0.4 (jejunum mucosa), 0.1 (terminal ileum), 0.1 (terminal ileum mucosa), 1 (cecal lumen), 0.02 (cecal mucosa), 0.05 (ascending colon mucosa), 0.08 (transverse colon mucosa), 0.08 (descending colon), 0.2 (descending colon mucosa), or 0.02 (feces). In some embodiments of any aspect, a low level of Mucispirillum is a relative 16S rRNA abundance of Mucispirillum in intestinal samples of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.09, less than 0.08, less than 0.07, less than 0.06, less than 0.05, less than 0.04, less than 0.03, less than 0.02, or less than 0.01. In some embodiments of any aspect, a low level of Mucispirillum is a relative 16S rRNA abundance of Mucispirillum in intestinal samples of 0-0.01.

[0173] As another non-limiting example, the relative 16S rRNA abundance of Mucispirillum schaedleri in intestinal samples from normal controls (e.g., healthy human controls) was 0.06% (e.g., 0.057%), or 0.001%–1.4% (e.g., 0.001%–1.319%), or 0.001%–9.6% (e.g., 0.001%–9.524%). In some embodiments of any aspect, a low level of Mucispirillum schaedleri is defined as a relative 16S rRNA abundance of Mucispirillum schaedleri in intestinal samples of less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, or less than 0.001%. In some embodiments of any aspect, a low level of Mucispirillum schaedleri is defined as a relative 16S rRNA abundance of Mucispirillum schaedleri in intestinal samples of 0%–0.001%. In some embodiments of any aspect, low levels of Mucispirillum schaedleri are defined as the relative 16S rRNA abundance of Mucispirillum schaedleri in intestinal samples of 0%–0.06%. See, for example, Herp et al., Cell Host & Microbe 25(5):681–694 (2019); Zmora et al., “Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features,” Cell 174(2018):1388–1405; the contents of which are incorporated herein by reference in their entirety.

[0174] In some embodiments, the low level of sulfur-containing amino acids (SAAs) is lower than the level measured in normal controls (e.g., in plasma, intestines, or serum). As a non-limiting example, the level of said methionine in the plasma of a normal control (e.g., a normal human control) is 14-48 μmol / L (1.40-4.80 μmol / dL) methionine. In some embodiments of any aspect, the low level of methionine is less than 14 μmol / L, less than 13 μmol / L, less than 12 μmol / L, less than 11 μmol / L, less than 10 μmol / L, or less than 5 μmol / L in a plasma sample. In some embodiments of any aspect, the low level of methionine is 0-14 μmol / L methionine in a plasma sample.

[0175] As a non-limiting example, the cysteine ​​level may be measured using a cysteine ​​derivative (e.g., cystine). Cystine is the oxidized disulfide form of cysteine ​​(Cys) and is the predominant form of cysteine ​​in the blood due to its relatively high stability. Cystine is derived from dietary proteins and is formed endogenously from cysteine. In some embodiments of any aspect, the cysteine ​​level in the plasma of a normal control (e.g., a normal human control) is 0.8-27.5 μmol / L cystine. In some embodiments of any aspect, low levels of cysteine ​​(e.g., cystine) are less than 0.8 μmol / L, less than 0.7 μmol / L, less than 0.6 μmol / L, less than 0.5 μmol / L, less than 0.4 μmol / L, less than 0.3 μmol / L, less than 0.2 μmol / L, and less than 0.1 μmol / L in plasma samples. In some embodiments of any aspect, low levels of cysteine ​​(e.g., cystine) are 0-0.8 μmol / L cystine in plasma samples.

[0176] In some embodiments of any aspect, low levels of XCL1 (RNA or protein) are less than 10 pg / mL to 30 pg / mL of secreted XCL1 polypeptide (see, for example, Figure 4D , Figures 4F-4G , Figures 17-18 For example, as measured in the intestine or serum. In some embodiments of either aspect, low levels of CD103+ conventional dendritic cells (cDC1) are less than 10%-15% of MHCII+CD11c+ cells, or less than 0.5 × 10⁻⁶. 4 -1×10 4 CD103+CD11b- cells, wherein the MHCII+CD11c+ cells are CD103+CD11b- (see, for example, Figure 3G-Figure 3I(Figures 27C-27G). In some embodiments of any aspect, a low level of NKT cells (e.g., NKT cells expressing XCL1) is less than 0.25% of CD3+ cells or less than 1 × 10⁻⁶ cells. 4 NKT cells, wherein the CD3+ cells are CD1d-tetramer-bound NKT cells (see, for example, Figure 33D), as measured, for example, in the intestine, tumor draining lymph nodes, or tumors.

[0177] As used herein, the term "antitumor immune activity" refers to an immune-mediated attack on tumor cells or tissues. Antitumor immune activity (e.g., mediated by the compositions and methods described herein) may include a shift in the tumor microenvironment from an immunosuppressive immune lineage to an immune lineage that permits and / or promotes immune-mediated attack against tumor cells or tissues. An immunosuppressive immune lineage can be characterized by the presence and / or activation of regulatory T cells (Tregs), regulatory B cells (Bregs), exhausted T cells, increased expression of immune checkpoint proteins, and / or decreased activation of immune cells (e.g., cDC1, NKT, CD8+ T cells, etc.). A shift in the tumor microenvironment that permits and / or promotes immune-mediated attack against tumor cells or tissues includes: increased infiltration or activity of activated immune cells (e.g., antigen-presenting cells such as cDC1, NKT, CD8+ T cells, etc.), decreased local concentrations or activation of Tregs or Bregs, and decreased expression of immune checkpoint proteins. In some embodiments of any aspect, a low level of antitumor immune activity is a low level of antitumor immunity of CD8+ T cells, for example, fewer than 0.1 CD3+CD8+ cells per CD3+ cell per field of view, and less than 20% of CD3+CD8+ cells are IFN-γ+ (see, for example, Figure 3D ) or less than 30% of CD3+CD8+ cells are granzyme-B+ (see, for example, Figures 3C-3E ).

[0178] In some embodiments of any aspect, a low level of responsiveness to immune checkpoint inhibitor (ICI) tumor therapy is defined as the failure of tumor growth (e.g., tumor volume, tumor mass) to be slowed by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, relative to untreated ICI, despite administration of an ICI (e.g., targeting a checkpoint molecule expressed on the tumor). Alternatively or additionally, “low responsiveness” is defined as the failure of tumor-infiltrating lymphocytes (TILs) to increase after CPI administration, and / or the failure of activated CD8+ TILs (e.g., IFNγ). + and / or GZMB + It does not increase after the CPI is given.

[0179] In some embodiments of any aspect, the frequency of the CD8+ T cells may be measured in the tumor or tumor-draining lymph nodes. In some embodiments of any aspect, low levels of antitumor immune activity or low levels of responsiveness to immune checkpoint inhibitor tumor therapy may be associated with no change or increase in measurements of tumor (e.g., tumor weight or tumor volume) or with no change or increase in measurements of tumor or cancer-related symptoms or complications.

[0180] In some embodiments, the subject has been previously identified as having an abnormal level of the analyte described herein relative to a reference. In some embodiments, the reference level may be the level in samples with similar cell types, similar sample types, similar sample treatments, and / or the level in samples obtained from subjects with similar age, sex, and other demographic parameters to the sample / subject. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source, and comprise the same composition (e.g., the same number and type of cells).

[0181] As used herein, the term "sample" or "test sample" refers to a sample obtained or isolated from a biological organism, such as a blood or plasma sample from a subject. In some embodiments of any aspect, the techniques described herein cover a variety of examples of biological samples. In some embodiments of any aspect, the biological sample is a cell, or tissue, or peripheral blood or body fluid. Exemplary biological samples include, but are not limited to, the following: biopsy specimens, tumor samples, biological fluid samples, blood, serum, plasma, urine, semen, mucus, tissue biopsy specimens, organ biopsy specimens, synovium, bile, cerebrospinal fluid, mucosal secretions, effusion, sweat, saliva, and / or tissue samples. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any aspect, the test sample may include cells from a subject.

[0182] In some embodiments of any aspect, the step of determining whether the subject has an abnormal level of the analyte described herein may include: i) obtaining or having obtained a sample from the subject, and ii) performing or having performed a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether the subject has an abnormal level of the analyte described herein may include: performing or having performed a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether the subject has an abnormal level of the analyte described herein may include: requesting or requesting a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether the subject has an abnormal level of the analyte described herein may include: receiving the result of a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether the subject has an abnormal level of the analyte described herein may include: receiving a report, result, or other tool identifying the subject as having a reduced level of the analyte.

[0183] In one aspect of any embodiment, the present invention describes a method for treating cancer, promoting antitumor immune activity, promoting responsiveness to immune checkpoint inhibitor cancer therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion from NKT cells, and / or establishing or maintaining a tumor-suppressive gut environment in a subject in need, the method comprising: a) determining whether the subject has abnormal levels of the analyte described herein; and b) if the analyte level is abnormal (e.g., reduced) relative to a reference, instructing or directing the subject to receive a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAAs), and / or an XCL1 peptide. In some embodiments of any aspect, the step of instructing or directing the subject to receive a specific treatment may include providing a report of the assay results. In some embodiments of any aspect, the step of instructing or directing the subject to receive a specific treatment may include providing a report of the assay results and / or providing treatment recommendations based on the assay results. Mucispirillum method

[0184] The various aspects described herein refer to the use of the Mucispirillum composition described herein in methods including, but not limited to, methods for treating cancer, methods for promoting antitumor immune activity, methods for promoting responsiveness to immune checkpoint inhibitor tumor therapy, methods for increasing CD103+ conventional dendritic cells (cDC1), methods for increasing XCL1 secretion from NKT cells, and / or methods for increasing CD8+ T cell infiltration in colorectal tumors.

[0185] In one aspect, this document describes a method for treating cancer or for promoting antitumor immune activity, the method comprising administering a Mucispirillum composition as described herein to a subject in need. In one aspect, this document describes a method for treating colon cancer, the method comprising administering a Mucispirillum composition as described herein to a subject in need. In one aspect, this document describes a method for promoting antitumor immune activity, the method comprising administering a Mucispirillum composition as described herein to a subject in need. In one aspect, this document describes a method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering a Mucispirillum composition as described herein to a subject in need.

[0186] In one aspect, this document describes a method comprising prescribing to a subject in need a Mucispirillum composition as described herein for: treating cancer, promoting antitumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion from NKT cells, and / or increasing CD8+ T cell infiltration in colorectal tumors.

[0187] In one aspect, this document describes a method for promoting responsiveness to immune checkpoint inhibitor cancer therapy, the method comprising administering a Mucispirillum composition as described herein to a subject in need. In some embodiments of any aspect, the method further comprises administering an immune checkpoint inhibitor. In one aspect, this document describes a method for promoting responsiveness to immune checkpoint inhibitor cancer therapy, the method comprising administering an immune checkpoint inhibitor (ICI) and a Mucispirillum composition as described herein to a subject in need. In some embodiments of any aspect, the ICI is administered before administering the Mucispirillum composition. In some embodiments of any aspect, the ICI is administered after administering the Mucispirillum composition. In some embodiments of any aspect, the ICI is administered concurrently with administering the Mucispirillum composition. In some embodiments of any aspect, the subject has cancer. In some embodiments of any aspect, the subject has colon cancer. In some embodiments of any aspect, the subject has mucosal epithelial tissue cancer. In some embodiments of any aspect, the subject's cancer has been identified as resistant to immune checkpoint inhibitor therapy.

[0188] In one aspect, this document describes a method for increasing XCL1 secretion by NKT cells, the method comprising administering a Mucispirillum composition as described herein to a subject in need. In some embodiments of any aspect, the subject has cancer. In some embodiments of any aspect, the XCL1 secretion recruits cDCs and / or activates cDC1. In some embodiments of any aspect, the subject has colon cancer. In some embodiments of any aspect, the subject has mucosal epithelial tissue cancer.

[0189] In one aspect, this document describes a method for increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering a Mucispirillum composition as described herein to a subject in need. In some embodiments of any aspect, the administration increases CD103+CD11b- conventional dendritic cells (cDC1). In some embodiments of any aspect, the administration increases the number of cDCs. In some embodiments of any aspect, the administration increases the number of cDCs in tumor draining lymph nodes (TDLNs). In some embodiments of any aspect, the administration increases the activation of cDCs (e.g., increased expression of XCL1 receptor XCR1). In some embodiments of any aspect, the administration increases the activation of said cDCs in tumor draining lymph nodes (TDLNs) (e.g., increased expression of XCL1 receptor XCR1). In some embodiments of any aspect, said cDCs are recruited to tumor draining lymph nodes (TDLNs) and / or activated by XCL1. In some embodiments of any aspect, said cDC1 is tumor-associated. In some embodiments of any aspect, the cDC1 is associated with tumor draining lymph nodes (TDLNs). In some embodiments of any aspect, the tumor is colon cancer. In some embodiments of any aspect, the tumor is a mucosal epithelial tissue tumor. In some embodiments of any aspect, the tumor is an adenoma, a non-cancerous tumor originating from glandular cells of epithelial tissue. In some embodiments of any aspect, the tumor is a malignant tumor, which is cancer originating from the skin or from tissue lining or covering internal organs.

[0190] In some embodiments of any aspect, the cancer is colon cancer. In some embodiments of any aspect, the cancer is colorectal cancer (CRC). In some embodiments of any aspect, the cancer is a mucosal epithelial tissue cancer or a mucosal cancer. As used herein, the term "mucosa" refers to a mucous membrane or a membrane rich in mucous glands lining body passages and cavities (e.g., the digestive tract or respiratory tract) that is directly or indirectly connected to the outside. In some embodiments of any aspect, the cancer is a gastrointestinal cancer, including but not limited to: oral cancer, esophageal cancer, gastric cancer, small bowel cancer, colorectal cancer, or anal cancer. In some embodiments of any aspect, the cancer is a respiratory tract cancer, including but not limited to: lung cancer, laryngeal cancer, or bronchial adenoma.

[0191] In some embodiments of any aspect, the method further includes administering an immune checkpoint inhibitor. In some embodiments of any aspect, the immune checkpoint inhibitor comprises an immune checkpoint inhibitor antibody. In some embodiments of any aspect, checkpoint inhibitor immunotherapy is an inhibitor of a checkpoint molecule selected from the group consisting of: programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), adenosine A2A receptor (A2AR), CD276, CD39, CD73, B7 family immune checkpoint molecules, T-cell activation inhibitor 1 (B7H4) containing a V-set domain, B and T lymphocyte attenuation factor (BTLA), indole Amine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG-3), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase type 2 (NOX2), T cell immunoglobulin domain and mucin domain 3 (TIM-3), T cell immune receptor with Ig and ITIM domains (TIGIT), T cell activation V-domain Ig repressor (VISTA), and sialic acid-binding immunoglobulin type lectin 7 (SIGLEC7).

[0192] Non-limiting examples of immune checkpoint inhibitors (ICIs) include: pembrolizumab Nivolumab Cimipril spartalizumab, camrelizumab (AiRuiKa) TM sintilimab Tislelizumab, Toripalimab (Tuoyi TM Dotalimab (JEMPERLI), INCMGA00012, AMP-224, AMP-514 (MEDI0608), Atezolizumab Avelumab Envorimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105), durvalumab tremelimumab and ipilimumab See, for example, U.S. Patents US5811097, US5855887, US6051227, US6682736, US6984720, US7595048, US7605238, US7943743, US8008449, US8217149, US8354509, US8383796, US8728474, US8735553, US8779105, US8779108, US8907053, US8 US 900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655, US10590199, US11225522, US Patent Publication US2014341917; Storz et al., MAbs. 2016 Jan; 8(1): 10-26; their respective contents are incorporated herein by reference in their entirety.

[0193] In some embodiments of any aspect, administration of the Mucispirillum composition promotes XCL1 secretion from NKT cells. In some embodiments of any aspect, the Mucispirillum composition increases XCL1 secretion from NKT cells by at least 100% (see, for example, Figures 4F-4G , Figures 17-18 In some embodiments of any aspect, the M. schaedleri composition increases XCL1 secretion from NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more, relative to NKT cells not exposed to the composition. In some embodiments of any aspect, XCL1 secretion is measured using an XCL1-specific ELISA (e.g., R&D SYSTEMS MOUSE XCL1 / LYMPHOTACTIN DUOSET ELISA KIT, ONESTEP MOUSE XCL1 ELISA KIT (ABCAM), ONESTEP HUMAN XCL1 ELISA (ABCAM)). In some embodiments of any aspect, the NKT cells are measured and / or isolated using flow cytometry, for example, by using CD1d-tetramer-bound CD3+ cells (see, for example, Figure 8F (Gating strategy for NKT cells, Figure 30E). In some embodiments of any aspect, the NKT cells may be grown from a cell line (e.g., GW1 NKT cells).

[0194] In some embodiments of any aspect, administration of the Mucispirillum composition increases the number and / or activation of cDC1 (e.g., in tumor-draining lymph nodes) by at least 50% (see, for example, Figure 3G-Figure 3I (Figures 27C-27F). In some embodiments of any aspect, administration of the Mucispirillum composition increases the number and / or activation of cDC1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a subject not given the Mucispirillum composition. In some embodiments of any aspect, the number of cDC1 is measured by quantifying the number of CD103+CD11b- cells from an MHCII+CDC11c+ dendritic cell population using flow cytometry (see, for example, ...). Figure 8E (Figure 30D: Gating strategy for dendritic cells expressing CD103 / CD11b). In some embodiments of any aspect, cDC1 activation can be measured by quantifying the number of cDC1 cells migrating from the intestine (e.g., lamina propria) to tumor-draining lymph nodes. In some embodiments of any aspect, cDC1 activation by XCL1 can be measured by quantifying XCR1 RNA or protein expression. Additional non-limiting examples of cDC1 activation biomarkers include Clec9a and Irf8.

[0195] In some embodiments of any aspect, administration of the Mucispirillum composition increases the number and / or activation of CD8+ T cells by at least 50%. In some embodiments of any aspect, administration of the Mucispirillum composition increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a subject who has not been given the Mucispirillum composition. In some embodiments of any aspect, administration of the Mucispirillum composition increases CD8+ T cell infiltration in colorectal tumors. In some embodiments of any aspect, flow cytometry is used to quantify the number of CD8+ T cells in tumor-draining lymph nodes or in the tumor. In some embodiments of any aspect, the activation of CD8+ T cells is quantified by measuring increased expression of effector factors (e.g., IFNγ or GZMB) or by measuring decreased expression of immune checkpoint receptors (e.g., PD-1, LAG-3, TIM-3, or CTLA-4) (see, for example, Figure 8B Figure 30B shows the gating strategy of CD8+ T cells, the expression of their immune checkpoint receptors, and the expression of IFNγ and GZMB.

[0196] In some embodiments of any aspect, the method further includes administering sulfur-containing amino acids (SAAs). In some embodiments of any aspect, the sulfur-containing amino acids (SAAs) are administered at an amount equal to or higher than the recommended daily intake. The recommended daily intake of methionine is 10.4 mg per kilogram of body weight or 4.5 mg per pound. A person weighing 70 kg (~154 lbs) should consume at least 728 mg of methionine daily. The recommended daily intake of cysteine ​​is 4.1 mg per kilogram of body weight or 1.9 mg per pound. A person weighing 70 kg (~154 lbs) should consume at least 287 mg of cysteine ​​daily. In some embodiments of any aspect, the administration of the SAA includes administering naturally occurring SAA-rich foods, SAA-supplemented foods, SAA-containing supplements, or pharmaceutical compositions comprising SAA.

[0197] In some embodiments of any aspect, the method further comprises administering a food rich in sulfur-containing amino acids, such as naturally occurring foods rich in sulfur-containing amino acids. Non-limiting examples of naturally occurring foods rich in sulfur-containing amino acids (e.g., methionine and / or cysteine) include: poultry, such as turkey (e.g., ground turkey; e.g., 931 mg methionine per 100 g of ground turkey, representing 128% of the Recommended Daily / Dietary Intake (RDI) for methionine) or chicken (e.g., 40%-195% of the RDI for methionine; e.g., lean chicken breast; e.g., 336 mg cysteine ​​per 100 g of lean chicken breast; representing 117% of the RDI for cysteine); red meat, such as beef (e.g., flank steak; e.g., 931 mg methionine per 100 g of flank steak). Methionine RDI 124%; 345 mg cysteine ​​per 100g flank steak; cysteine ​​RDI 120%); lamb, veal, or buffalo meat; fish or seafood, such as tuna (e.g., 885 mg methionine per 100g tuna; 122% methionine RDI; e.g., 321 mg cysteine ​​per 100g tuna; cysteine ​​RDI 112%), grouper, salmon, gudgeon, fish, mahi-mahi, or clams; pork, such as pork chops (e.g., 850 mg methionine per 100g pork chops; 117% methionine RDI; 350 mg cysteine ​​per 100g pork chops). methionine (mg / 100g; 122% of the RDI for methionine); pork ribs, lean ham, pork sausage, ground pork, salami, roasted ham, sausage, or bacon; soy products, such as firm tofu (e.g., 211mg methionine per 100g firm tofu; 29% of the RDI for methionine), boiled soybeans, cooked bean sprouts, or soy milk; dairy products, such as milk (e.g., 88mg methionine per 100g milk; 12% of the RDI for methionine), yogurt (e.g., 52mg methionine per 100g yogurt; 18% of the RDI for methionine) or buttermilk; cheese products, such as whey cheese (e.g., 100mg methionine per 100g whey cheese). Whey cheese (284 mg methionine per 100g; 39% of the RDI for methionine); Swiss cheese (e.g., 290 mg cysteine ​​per 100g of Swiss cheese; 133% of the RDI for cysteine); Parmesan, Gruyère, Gouda, or Fontina; nuts or seeds such as Brazil nuts (e.g., 1124 mg methionine per 100g of Brazil nuts; 154% of the RDI for methionine); sunflower seeds (e.g., 383 mg cysteine ​​per 100g of sunflower seeds; 133% of the RDI for cysteine); hemp seeds, squash seeds, pumpkin seeds, chia seeds, sesame seeds, flax seeds, cashews, pistachios, or peanuts; legumes such as white soybeans (e.g., 146 mg methionine per 100g of white soybeans).The following are included in the RDI for methionine: 20% of the total methionine intake (RDI); lentils (e.g., 118 mg cysteine ​​per 100g lentils; RDI for cysteine ​​41%); navy beans; kidney beans; black beans; long beans; pinto beans; dried peas; grains such as quinoa (e.g., 96 mg methionine per 100g quinoa; RDI for methionine 13%); rolled oats (e.g., 97 mg cysteine ​​per 100g rolled oats; RDI for cysteine ​​34%); teff; wild rice; kamut; rice or whole wheat pasta; or eggs (e.g., 292 mg cysteine ​​per 100g eggs; RDI for cysteine ​​102%).

[0198] In some embodiments of any aspect, the method further includes administering a food supplemented with at least one sulfur-containing amino acid. In some embodiments of any aspect, the food is supplemented with at least 2.0g, at least 5g, at least 10g, at least 20g, at least 30g, at least 40g, at least 50g, at least 60g, at least 70g, at least 80g, at least 90g, at least 100g, at least 150g, at least 200g, at least 300g, or at least 400g of SAA. In some embodiments of any aspect, the food has been supplemented with 2.0g-400g of SAA, 2.0g-300g of SAA, 2.0g-200g of SAA, 2.0g-100g of SAA, 10.0g-400g of SAA, 10g-100g of SAA, 50g-400g of SAA, 100g-400g of SAA, 200g-400g of SAA, or 300g-400g of SAA.

[0199] In some embodiments of any aspect, the method further includes administering a supplement comprising at least one sulfur-containing amino acid. In some embodiments of any aspect, the supplement comprises at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some embodiments of any aspect, the supplement comprises 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA. In some embodiments of any aspect, the sulfur-containing amino acid is methionine, cysteine, or a derivative thereof. In some embodiments of any aspect, the sulfur-containing amino acid is methionine, cysteine, homocysteine, taurine, or a derivative thereof. In some embodiments of any aspect, the composition comprises a combination of any one of methionine, cysteine, homocysteine, and / or taurine (see, for example, Table 9).

[0200] In some embodiments of any aspect, the method further comprises administering at least 2.0 g of a SAA (e.g., methionine, cysteine, homocysteine, and / or taurine or a derivative thereof). In some embodiments of any aspect, the method further comprises administering at least 2.4 g of a SAA. In some embodiments of any aspect, the method further comprises administering at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of a SAA. In some embodiments of any aspect, the method further includes administering up to 5g, up to 10g, up to 20g, up to 30g, at least 40g, up to 50g, up to 60g, up to 70g, up to 80g, up to 90g, up to 100g, up to 150g, up to 200g, up to 300g, or up to 400g of SAA. In some embodiments of any aspect, the method further includes administering at least 2.0g-400g of SAA. In some embodiments of any aspect, the method further includes administering 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA.

[0201] In some embodiments of any aspect, the method includes administering SAA (e.g., as a natural SAA-rich food, SAA-supplemented food, SAA-containing supplement, or SAA-containing pharmaceutical composition) once, twice, three times (e.g., with meals), four times, five times, or more times daily for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer). In some embodiments of any aspect, the administration of SAA corresponds to the duration of cancer treatment. In some embodiments of any aspect, SAA is continued after cancer treatment is discontinued.

[0202] In some embodiments of any aspect, in addition to the Mucispirillum composition, the method further includes administering at least a second composition as described herein. In some embodiments of any aspect, the Mucispirillum composition is administered co-administered with a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and / or an XCL1 peptide (or an XCR1 agonist). In some embodiments of any aspect, the Mucispirillum composition is administered co-administered with a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA). In some embodiments of any aspect, the Mucispirillum composition is administered co-administered with an XCL1 peptide (or an XCR1 agonist). In some embodiments of any aspect, the Mucispirillum composition is administered co-administered with a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and an XCL1 peptide (or an XCR1 agonist). Exemplary treatment combinations are shown in, for example, Table 10. In some embodiments of any aspect, the treatment combinations may be administered sequentially or simultaneously.

[0203] Table 10: Exemplary Treatment Combinations (“x” indicates inclusion in the treatment method) SAA diet or supplementation methods

[0204] In one aspect, this document describes a method for establishing or maintaining a tumor-suppressive gut environment in subjects in need, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA). In another aspect, this document describes a method for establishing a tumor-suppressive gut environment in subjects in need, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA). In another aspect, this document describes a method for maintaining a tumor-suppressive gut environment in subjects in need, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA). In another aspect, this document describes a method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) to subjects in need. As used herein, the term "tumor-suppressive gut environment" refers to the gut environment associated with tumor suppression (e.g., host gut cells, host immune cells, and / or associated gut microbiota); as described herein, increased gut levels of sulfur-containing amino acids (SAA), Mucispirillum, XCL1, XCL1-expressing NKT cells, and / or CD103+ conventional dendritic cells (cDC1) may be associated with tumor suppression (see, for example, Figure 13In some embodiments of any aspect, the method establishes or maintains a tumor-suppressive intestinal environment for colon cancer in subjects in need.

[0205] As used herein, the term "sulphoamino acid-rich diet" refers to a diet that includes equal to or higher than the recommended daily intake (e.g., at least 728 mg of methionine and at least 287 mg of cysteine ​​per day for a person weighing 70 kg). In some embodiments of any aspect, a sulfur-containing amino acid-rich diet includes naturally occurring SAA-rich foods or foods supplemented with SAAs as described herein. Therefore, the sulfur-containing amino acid-rich diet includes not only diets containing elevated amounts of SAAs as described herein, but also diets in which one or more SAAs are provided as supplements. In this embodiment, SAAs may be provided as supplements to a normal diet or used to enhance a diet rich in or fortified with SAAs.

[0206] In some embodiments of any aspect, the method includes administering a food supplemented with at least one sulfur-containing amino acid. In some embodiments of any aspect, the food is supplemented with at least 2.0g, at least 5g, at least 10g, at least 20g, at least 30g, at least 40g, at least 50g, at least 60g, at least 70g, at least 80g, at least 90g, at least 100g, at least 150g, at least 200g, at least 300g, or at least 400g of SAA. In some embodiments of any aspect, the food is supplemented with 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 10g-20g SAA, 20g-30g SAA, 30g-40g SAA, 40g-50g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA. In some embodiments of any aspect, the food is supplemented with about 15g of L-methionine and / or about 8g of L-cysteine. In some embodiments of any aspect, the food is supplemented with about 15g of L-methionine and / or about 8g of L-cysteine ​​(see, for example, Table 2).

[0207] In some embodiments of any aspect, the method includes administering a supplement containing at least one sulfur-containing amino acid. In some embodiments of any aspect, the supplement comprises at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some embodiments of any aspect, the supplement comprises 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 10g-20g SAA, 20g-30g SAA, 30g-40g SAA, 40g-50g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA. In some embodiments of any aspect, the supplement comprises about 15g of L-methionine and / or about 8g of L-cysteine. In some embodiments of any aspect, the supplement comprises about 15g of L-methionine and / or about 8g of L-cysteine ​​(see, for example, Table 2).

[0208] In some embodiments of any aspect, the sulfur-rich diet (or supplements including SAAs) comprises elevated levels of methionine, cysteine, or derivatives thereof, relative to a diet not rich in sulfur-containing amino acids or relative to a normal or typical diet as described herein. In some embodiments of any aspect, the sulfur-rich diet (or supplements including SAAs) comprises elevated levels of methionine, cysteine, homocysteine, taurine, or derivatives thereof, relative to a diet not rich in sulfur-containing amino acids. In some embodiments of any aspect, the sulfur-rich diet (or supplements including SAAs) comprises elevated levels of any combination of methionine, cysteine, homocysteine, and / or taurine (see, for example, Table 9).

[0209] In some embodiments of any aspect, the diet low in sulfur-containing amino acids comprises 0.01 g to 0.04 g of SAA (per kilogram (subject's) body weight) per day. In some embodiments of any aspect, the diet rich in sulfur-containing amino acids comprises more than 0.04 g of SAA (per kilogram (subject's) body weight) per day. In some embodiments of any aspect, the diet rich in sulfur-containing amino acids comprises at least 6 g of SAA (per kilogram (subject's) body weight) per day.

[0210] In some embodiments of any aspect, the diet rich in sulfur-containing amino acids comprises at least 0.04g, at least 0.05g, at least 0.06g, at least 0.07g, at least 0.08g, at least 0.09g, at least 0.1g, at least 0.2g, at least 0.3g, at least 0.4g, at least 0.5g, at least 0.6g, at least 0.7g, at least 0.8g, at least 0.9g, at least 1g, at least 2g, at least 3g, at least 4g, at least 5g, at least 6g or more of SAA (per kilogram (of the subject's) body weight per day). In some embodiments of any aspect, the diet rich in sulfur-containing amino acids comprises up to 0.05g, up to 0.06g, up to 0.07g, up to 0.08g, up to 0.09g, up to 0.1g, up to 0.2g, up to 0.3g, up to 0.4g, up to 0.5g, up to 0.6g, up to 0.7g, up to 0.8g, up to 0.9g, up to 1g, up to 2g, up to 3g, up to 4g, up to 5g, or up to 6g of SAA (per kilogram (of the subject's) body weight per day). In some embodiments of any aspect, the diet rich in sulfur-containing amino acids comprises 0.04g-0.1g, 0.1g-1.0g, 1.0g-6.0g, 0.04g-1.0g, 0.04g-6.0g, or 0.1g-6.0g of SAA (per kilogram (of the subject's) body weight per day).

[0211] Assuming an average weight of 60 kg, in some embodiments of any aspect, the SAA-rich diet comprises more than 2.4 grams of SAA per day. In some embodiments of any aspect, the SAA-rich diet comprises at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA per day. In some embodiments of any aspect, the SAA-rich diet comprises at most 5 g, at most 10 g, at most 20 g, at most 30 g, at least 40 g, at most 50 g, at most 60 g, at most 70 g, at most 80 g, at most 90 g, at most 100 g, at most 150 g, at most 200 g, at most 300 g, or at most 400 g of SAA per day. In some embodiments of any aspect, the SAA-rich diet comprises 2.0g-400g of SAA daily. In some embodiments of any aspect, the SAA-rich diet comprises 2.0g-400g SAA, 2.0g-300g SAA, 2.0g-200g SAA, 2.0g-100g SAA, 10.0g-400g SAA, 10g-100g SAA, 50g-400g SAA, 100g-400g SAA, 200g-400g SAA, or 300g-400g SAA daily. In some embodiments of any aspect, in addition to a diet rich in sulfur-containing amino acids (SAA), the method further comprises administering at least a second treatment as described herein (see, for example, Table 10).

[0212] In some embodiments of any aspect, administration of the sulfur-rich diet (or a supplement including SAA) promotes XCL1 secretion from NKT cells. In some embodiments of any aspect, the sulfur-rich diet increases XCL1 secretion from NKT cells by at least 100%. In some embodiments of any aspect, the sulfur-rich diet increases XCL1 secretion from NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more, relative to XCL1 secretion from NKT cells in subjects not given the sulfur-rich diet.

[0213] In some embodiments of any aspect, administration of the diet rich in sulfur-containing amino acids (or supplements including SAAs) increases the quantity and / or activation of cDC1 (e.g., in tumor-draining lymph nodes) by at least 25% (see, for example, Figure 3G-Figure 3I(Figures 27C-27F). In some embodiments of any aspect, administration of the sulfur-rich diet increases the number and / or activation of cDC1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to subjects not given the sulfur-rich diet. In some embodiments of any aspect, the number of cDC1 is measured by quantifying the number of CD103+CD11b- cells from an MHCII+CDC11c+ dendritic cell population using flow cytometry (see, for example, ...). Figure 8E (Figure 30D: Gating strategy for dendritic cells expressing CD103 / CD11b). In some embodiments of any aspect, cDC1 activation can be measured by quantifying the number of cDC1 cells migrating from the intestine (e.g., lamina propria) to tumor-draining lymph nodes. In some embodiments of any aspect, cDC1 activation by XCL1 can be measured by quantifying XCR1 RNA or protein expression. Additional non-limiting examples of cDC1 activation biomarkers include Clec9a and Irf8.

[0214] In some embodiments of any aspect, administration of a diet rich in sulfur-containing amino acids (or a supplement including SAAs) increases the number and / or activation of CD8+ T cells by at least 50% (see, for example, Figures 3B-3E In some embodiments of any aspect, administration of a sulfur-rich diet increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to subjects not given a diet rich in sulfur-rich amino acids. In some embodiments of any aspect, the number of CD8+ T cells in tumor-draining lymph nodes or tumors is quantified using flow cytometry. In some embodiments of any aspect, CD8+ T cell activation is quantified by measuring increased expression of effector factors (e.g., IFNγ or GZMB) or by measuring decreased expression of immune checkpoint receptors (e.g., PD-1, LAG-3, TIM-3, or CTLA-4) (see, for example, Figure 8B Figure 30B shows the gating strategy of CD8+ T cells, the expression of their immune checkpoint receptors, and the expression of IFNγ and GZMB. XCL1 peptide or XCR1 agonist method

[0215] In one aspect, this document describes a method for treating cancer, the method comprising administering an XCL1 peptide (see, for example, SEQ ID NOs: 5-6) to a subject in need. In another aspect, this document describes a method for treating cancer, the method comprising administering an XCR1 agonist to a subject in need. In another aspect, this document describes a method for treating cancer, the method comprising administering both an XCL1 peptide and an XCR1 agonist to a subject in need. In another aspect, this document describes a method for treating cancer, the method comprising administering microorganisms engineered to express the XCL1 peptide to a subject in need. In some embodiments of any aspect, the cancer is colon cancer.

[0216] XC motif chemokine receptor 1 (XCR1) is the receptor for XCL1 and XCL2 (lymphocyte chemokine-1 and lymphocyte chemokine-2, respectively). XCR1 is also known as CCXCR1 or G protein-coupled receptor (GPR5). XCR1 is a chemokine receptor belonging to the G protein-coupled receptor superfamily. Members of this family are characterized by seven transmembrane domains and a large number of conserved amino acids. XCR1 can be expressed on dendritic cells (e.g., cDC1 cells). Cross-presenting dendritic cells (DCs) in the spleen develop XCR1+DCs in the small intestine, the T-cell regions of Peyer's nodes, and the sinus and T-cell regions of the mesenteric lymph nodes. XCR1+DCs are specifically responsible for cross-presentation of orally administered antigens.

[0217] In some embodiments of any aspect, XCR1 comprises: one of SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to one of SEQ ID NO: 7 or SEQ ID NO: 8, said amino acid sequence maintaining its function (e.g., binding to XCL1 and / or XCR1-associated intracellular signaling).

[0218] SEQ ID NO: 7, Chemokine XC Receptor 1, Homo sapiens, NCBI Reference Sequence: NP_001019815.1, 333 aa MESSGNPESTTFFYYDLQSQPCENQAWVFATLATTVLYCLVFLLSLVGNSLVLWVLVKYESLESLTNIFILNLCLSDLVFACLLPVWISPYHWGWVLGDFLCKLLNMIFSISLYSSIFFLTIMTIHRYLSVVSPLSTLRVPTLRCRVLVTMAVWVASILSSILDTIFHKVLSSGCDYSELTWYLTSVYQHNLFFLLSLGIILFCYVEILRTLFRSRSKRRHRTVKLIFAIVVAYFLSWGPYNFTLFLQTLFRTQIIRSCEAKQQLEYALLICRNLAFSHCCFNPVLYVFVGVKFRTHLKHVLRQFWFCRLQAPSPASIPHSPGAFAYEGASFY

[0219] SEQ ID NO: 8, Chemokine XC Receptor 1, Mus musculus, NCBI Reference Sequence: NP_035928.2, 338 aa MDSESDALSIPASRVQMESSTAFYDYHDKLSLLCENNVIFFSTISTIVLYSLVFLLSLVGNSLVLWVLVKYENLESLTNIFILNLCLSDLMFSCLLPVLISAQWSWFLGDFFCKFFNMIFGISLYSSIFFLTIMTIHRYLSVVSPISTLGIHTLRCRVLVTSCVWAASILFSIPDAVFHKVISLNCKYSEHHGFLASVYQHNIFFLLSMGIILFCYVQILRTLFRTRSRQRHRTVRLIFTVVVAYFLSWAPYNLTLFLKTGIIQQSCESLQQLDIAMIICRHLAFSHCCFNPVLYVFVGIKFRRHLKHLFQQVWLCRKTSSTVPCSPGTFTYEGPSFY

[0220] In some embodiments of any aspect, the XCR1 agonist is a functional variant of XCL1, such as human XCL1 or mouse XCL1. In some embodiments of any aspect, the XCR1 agonist is a functional variant of XCL1 that further includes at least one additional disulfide bridge (e.g., at least two residues mutated to cysteine). In some embodiments of any aspect, the XCR1 agonist is a functional variant of XCL1 (e.g., residues 22-114 of SEQ ID NO: 5, SEQ ID NO: 6, residues 22-93 of SEQ ID NO: 5, residues 22-114 of SEQ ID NO: 6) that includes at least one of the following mutations: V21C, A59C, V59C, T10C mutation and / or the addition of an "AC" dipeptide at residue 32.

[0221] In some embodiments of any aspect, the XCR1 agonist comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to and maintains its function (e.g., binding to and / or activating XCR1) of one of the following: residues 22-114 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 5, residues 22-93 of SEQ ID NO: 5, residues 22-114 of SEQ ID NO: 6, or residues 22-93 of SEQ ID NO: 6.

[0222] In some embodiments of any aspect, the XCR1 agonist is mXCL1-V21C / A59C, which is a highly activated form of mXCL1 including mutations in V21C and A59C (see, for example, SEQ ID NO: 9). In some embodiments of any aspect, the XCR1 agonist is CC1 Ltn or CC3 Ltn, which includes at least one additional disulfide bond in hXCL to restrict XCL1 to a chemokine-like conformation having XCR1 agonist activity (see, for example, SEQ ID NOs: 10-11). In some embodiments of any aspect, the XCR1 agonist is selected from the group consisting of mXCL1-V21C / A59C, CC1 Ltn, and CC3 Ltn; see, for example, Matsuo et al., Front Immunol. 2018, 9: 2775; Tuinstra et al., Biochemistry 2007, 46(10): 2564-73; the contents of which are incorporated herein by reference in their entirety. In some embodiments of any aspect, the XCR1 agonist is selected from the group consisting of: SEQ ID NOs: 9-11.

[0223] In some embodiments of any aspect, the XCR1 agonist comprises: one of SEQ ID NOs: 9-11, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to and maintains the function (e.g., binding to and / or activating XCR1) of one of SEQ ID NOs: 9-11.

[0224] SEQ ID NO: 9, mXCL1-V21C / A59C, 93aa, the bold underlined text indicates the V21C and A59C mutations compared to residues 22-114 of mXCL1 (see, for example, SEQ ID NO: 6).

[0225] SEQ ID NO: 10, CC1 Ltn, 95aa, the bold underlined text indicates a T10C mutation compared to residues 22–114 of hXCL1 (see, for example, SEQ ID NO: 5) and the addition of the “AC” dipeptide at residue 32.

[0226] SEQ ID NO: 11, CC3 Ltn, 93aa, the bold underlined text indicates V21C and V59C mutations compared to residues 22–114 of hXCL1 (see, for example, SEQ ID NO: 5).

[0227] In some embodiments of any aspect, the XCL1 polypeptide is administered to the intestine. In some embodiments of any aspect, the XCR1 agonist is administered to the intestine. In some embodiments of any aspect, the XCL1 polypeptide is administered using bacteria engineered to express the XCL1 polypeptide and / or the XCR1 agonist (e.g., symbiotic gut bacteria; e.g., *Lactobacillus*). In some embodiments of any aspect, in addition to the XCL1 polypeptide or the XCR1 agonist, the method further includes administering at least a second composition as described herein (see, for example, Table 10).

[0228] In some embodiments of any aspect, administration of the XCL1 peptide or XCR1 agonist increases the number and / or activation of cDC1 (e.g., in tumor-draining lymph nodes) by at least 50%. In some embodiments of any aspect, administration of the XCL1 peptide or XCR1 agonist increases the number and / or activation of cDC1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a subject who has not been given the XCL1 peptide or XCR1 agonist. In some embodiments of any aspect, the number of cDC1 is measured by quantifying the number of CD103+CD11b- cells from an MHCII+CDC11c+ dendritic cell population using flow cytometry (see, for example, Figure 8E (Figure 30D: Gating strategy for dendritic cells expressing CD103 / CD11b). In some embodiments of any aspect, cDC1 activation can be measured by quantifying the number of cDC1 cells migrating from the intestine (e.g., lamina propria) to tumor-draining lymph nodes. In some embodiments of any aspect, cDC1 activation by XCL1 can be measured by quantifying XCR1 RNA or protein expression. Additional non-limiting examples of cDC1 activation biomarkers include Clec9a and Irf8.

[0229] In some embodiments of any aspect, administration of the XCL1 peptide or XCR1 agonist increases the number and / or activation of CD8+ T cells by at least 50%. In some embodiments of any aspect, administration of the XCL1 peptide or XCR1 agonist increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a subject who has not been given the XCL1 peptide or XCR1 agonist. In some embodiments of any aspect, flow cytometry is used to quantify the number of CD8+ T cells in tumor-draining lymph nodes or the tumor. In some embodiments of any aspect, the activation of CD8+ T cells is quantified by measuring increased expression of effector factors (e.g., IFNγ or GZMB) or by measuring decreased expression of immune checkpoint receptors (e.g., PD-1, LAG-3, TIM-3, or CTLA-4) (see, for example, Figure 8B Figure 30B shows the gating strategy of CD8+ T cells, the expression of their immune checkpoint receptors, and the expression of IFNγ and GZMB. Treatment stratification method

[0230] In several respects, this document describes a method for treatment stratification related to the detection of M. schaedleri. In one respect, this document describes a method for treating cancer in a subject in need, the method comprising: detecting the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapy agent if the level of M. schaedleri is equal to or higher than a predetermined threshold; and administering a cancer immunotherapy agent and a Mucispirillum composition as described herein if the level of M. schaedleri is lower than the predetermined threshold.

[0231] In one aspect, this document describes a method for treating cancer in a subject in need, the method comprising: obtaining a result from a determination of M. schaedleri levels in a sample from the subject; administering a cancer immunotherapy agent if the M. schaedleri level is equal to or higher than a predetermined threshold; and administering the cancer immunotherapy agent and a Mucispirillum composition as described herein if the M. schaedleri level is lower than the predetermined threshold.

[0232] In one aspect, this document describes a method for treating cancer in a subject in need, the method comprising: detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from the subject; administering a cancer immunotherapy agent if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold; and administering a cancer immunotherapy agent and a Mucispirillum composition as described herein if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are lower than a predetermined threshold.

[0233] In one aspect, this document describes a method for treating cancer in a subject in need, the method comprising: obtaining results from a determination of levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from said subject; administering a cancer immunotherapy agent if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold; and administering a cancer immunotherapy agent and a Mucispirillum composition as described herein if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are lower than a predetermined threshold.

[0234] In one aspect, this document describes a method for stratifying subjects for cancer treatment, the method comprising: detecting the level of M. schaedleri in a sample from the subject; and classifying the subject as high-risk (e.g., high risk of having colorectal cancer symptoms or complications) if the level of M. schaedleri is below a predetermined threshold; or classifying the subject as low-risk (e.g., low risk of having colorectal cancer symptoms or complications) if the level of M. schaedleri is equal to or higher than the predetermined threshold.

[0235] In one aspect, this document describes a method for stratifying subjects for cancer treatment, the method comprising: obtaining a result from a determination of M. schaedleri levels in a sample from the subject; and classifying the subject as high-risk (e.g., high risk of having colorectal cancer symptoms or complications) if the M. schaedleri level is below a predetermined threshold; or classifying the subject as low-risk (e.g., low risk of having colorectal cancer symptoms or complications) if the M. schaedleri level is equal to or higher than the predetermined threshold.

[0236] In one aspect, this document describes a method for stratifying subjects for cancer treatment, the method comprising: detecting levels of XCL1 peptide, NKT, and / or CD103+cDC1 in samples from the subject; and classifying the subject as high-risk (e.g., high risk of having colorectal cancer symptoms or complications) if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold; or classifying the subject as low-risk (e.g., low risk of having colorectal cancer symptoms or complications) if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold.

[0237] In one aspect, this document describes a method for stratifying subjects for cancer treatment, the method comprising: obtaining results from a determination of levels of XCL1 peptide, NKT, and / or CD103+cDC1 in a sample from the subject; and classifying the subject as high-risk (e.g., high risk of having colorectal cancer symptoms or complications) if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold; or classifying the subject as low-risk (e.g., low risk of having colorectal cancer symptoms or complications) if the levels of XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold.

[0238] In some embodiments of any aspect, the subject has colon cancer. In some embodiments of any aspect, the stratification method further includes administering a Mucispirillum composition as described herein. In some embodiments of any aspect, the stratification method further includes administering sulfur-containing amino acids. In some embodiments of any aspect, the stratification method further includes administering a diet rich in sulfur-containing amino acids. In some embodiments of any aspect, the stratification method further includes administering a Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or an XCL1 peptide (or XCR1 agonist), or any combination thereof (see, for example, Table 10).

[0239] In some embodiments of any aspect, the cancer immunotherapy agent includes an immune checkpoint inhibitor. In some embodiments of any aspect, the stratification method further includes administering an immune checkpoint inhibitor, as exemplified herein by non-limiting examples. In some embodiments of any aspect, the cancer immunotherapy agent is selected from the group consisting of: immune checkpoint inhibitors, chemotherapy, dendritic cell vaccines; chimeric antigen receptor T-cell (CAR-T) and NKT-based therapies. See, for example, Nelson et al., Cancers vol. 13, 20 5174. October 15, 2021, the contents of which are incorporated herein by reference in their entirety.

[0240] In some embodiments of any aspect, the treatments described herein (e.g., the Mucispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or an XCL1 peptide (or XCR1 agonist)) are administered as a monotherapy, for example, without administering another treatment for cancer to the subject. In some embodiments of any aspect, the methods described herein may further include administering a second agent and / or treatment to the subject (e.g., as part of a combination therapy).

[0241] Non-limiting examples of second-agent and / or treatments may include cancer therapies selected from the group consisting of: radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents (e.g., thiotepa and...). Cyclophosphamides; alkyl sulfonates (e.g., busulfan, indomethacin, and piperosulfan); aziridines (e.g., benzodopa, carboquinone, meturedopa, and uredopa); ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trihydroxymethylmelamine; polyacetyls (especially anomericin and bulbatacinone); camptothecin (including its synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin). Cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; pyruvicin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, naphthalenemustine, cholophosphamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trofosfamide, uracil). mustard); nitrosoureas (e.g., carmustine, chloramphenicol, fomomustine, lomustine, nimustine, and ramustine); antibiotics, such as ethynylene antibiotics (e.g., chachiin, especially chachiin γ1I and chachiin ΩI1 (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates (e.g., clophosphonates); esperamicin; and new carcinogen chromophores and related chromoprotein ethynylene antibiotic chromophores), aclarubicin, actinomycin, autramycin, azoserine, bleomycin, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, Doxorubicins (including doxorubicin morpholino, doxorubicin cyanomorpholino, doxorubicin 2-pyrrolidone, and doxorubicin deoxyribonucleic acid), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C, mycophenolic acid, nogalamycin, olipomycin, pepromycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, fenestrated statin, and zorubicin); antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thia Miprine, thioguanine; pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine); androgens (e.g., carotestosterone, drotalbutone propionate, cyclothiosterol, mepitiostane, and testolactone); antiadrenergics (e.g., aminoglutethimide, mitotan, trilostane); folic acid supplements (e.g., frolinicacid); aceglatone; aldehyde phosphoramide glycoside; aminolevulinic acid; eniluracil; acridine; bestrabucil; edatraxate; defofamine; colchicine; diaziquone; elformithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine (e.g., maytansine and anserine); mitoguazone; mitoantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; Polysaccharide complexes (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; Alternaria alternata ketone; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); carbamates; vinblastine; dacarbazine; mannomustine; dibromomannitol; dibromoeusine; pipebroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes (e.g., Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) Paclitaxel-based albumin-engineered nanoparticle formulations without polyoxyethylene castor oil (American Pharmaceutical Partners, Schaumberg, Ill.) and Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chloranbucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (e.g., cisplatin, oxaliplatin, and carboplatin); vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; Novantrone; Teniposide; Edatarexate; Daunorubicin; Aminopterin; Xeloda; Ibandronic Acid; Irinotecan (Camptosar, CPT-11) (including irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids (e.g., retinoic acid); Capecitabine; Cobbutatin; Leucovorin (LV); Oxaliplatin, including oxaliplatin regimens (FOLFOX); Lapatinib Inhibitors of PKC-α, Raf, H-Ras, and EGFR that reduce cell proliferation (e.g., erlotinib) Inhibitors of VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0242] Those skilled in the art can readily identify the chemotherapeutic agents used (see, for example, Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS, ed.: The Cancer Chemotherapy Handbook, 4th edition. St. Louis, Mosby-Year Book, 2003).

[0243] Furthermore, the treatment method may further include the use of radiation or radiotherapy. Additionally, the treatment method may further include the use of surgical treatment.

[0244] In some embodiments of any aspect, the level of *M. schaedleri* is quantified using standard detection methods for bacteria, including but not limited to quantitative 16S sequencing, serial dilution plate assays, direct counting via an optical microscope, etc. In some embodiments of any aspect, the level of the XCL1 peptide is quantified using standard detection methods for peptides, including but not limited to ELISA (enzyme-linked immunosorbent assay), Western blotting, immunoprecipitation, or immunofluorescence using detection reagents (e.g., antibodies or protein binding reagents). In some embodiments of any aspect, the level of NKT and / or CD103+cDC1 is quantified using standard detection methods for immune cells, including but not limited to flow cytometry for blood or tissue samples, or laser capture microdissection, immunohistochemistry, or immunofluorescence for tissue samples.

[0245] In some embodiments of any aspect, the stratification method produces higher therapeutic efficacy compared to not first performing the following methods: detecting the level of M. schaedleri; obtaining results from a determination of M. schaedleri level; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from a determination of XCL1 peptide, NKT, and / or CD103+cDC1 level.

[0246] In some embodiments of any aspect, the stratification method results in an increase in therapeutic efficacy of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to treatments that do not first involve: detecting the level of M. schaedleri; obtaining results from a determination of the level of M. schaedleri; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from a determination of the levels of XCL1 peptide, NKT, and / or CD103+cDC1.

[0247] In some embodiments of any aspect, the stratification method produces higher therapeutic efficacy compared to a treatment method that does not first stratify the subjects. In some embodiments of any aspect, the stratification method causes an increase in therapeutic efficacy of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a treatment method that does not first stratify the subjects.

[0248] In some embodiments of any aspect, the stratification method results in fewer treatment complications compared to treatments that do not first involve: detecting the level of M. schaedleri; obtaining results from a assay that detects the level of M. schaedleri; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from an assay that detects the levels of XCL1 peptide, NKT, and / or CD103+cDC1.

[0249] In some embodiments of any aspect, the stratification method reduces treatment complications by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to treatments that do not first involve: detecting the level of M. schaedleri; obtaining results from a determination of M. schaedleri levels; detecting the levels of XCL1 peptide, NKT, and / or CD103+cDC1; or obtaining results from a determination of XCL1 peptide, NKT, and / or CD103+cDC1 levels.

[0250] In some embodiments of any aspect, the stratification method causes fewer treatment complications compared to a treatment method that does not first stratify the subjects. In some embodiments of any aspect, the stratification method causes a reduction in treatment complications of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400% or more compared to a treatment method that does not first stratify the subjects.

[0251] Non-limiting examples of complications from cancer treatments (e.g., chemotherapy, radiation therapy) that can be reduced using the treatments or stratification methods described herein include: anemia, loss of appetite, bleeding and bruising (e.g., thrombocytopenia), constipation, delirium, diarrhea, edema, fatigue, fertility problems in boys and men, fertility problems in girls and women, flu-like symptoms, hair loss (e.g., alopecia areata), infections and neutropenia, lymphedema, memory or attention problems, oral and throat problems, nausea and vomiting, neurological problems (e.g., peripheral neuropathy), immunotherapy and organ-related inflammation, pain, sexual health problems in men, sexual health problems in women, skin and nail changes, sleep problems, or urinary and bladder problems. give

[0252] In some embodiments, the methods described herein involve treating a subject with or diagnosed with cancer with the Mucispirillum composition described herein, a diet rich in sulfur-containing amino acids (SAAs), and / or an XCL1 peptide (or an XCR1 agonist). Subjects with cancer may be identified by a physician using current methods for diagnosing cancer.

[0253] Symptoms and / or complications of cancer that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to: extreme fatigue or tiredness that does not improve with rest; unexplained weight loss or gain of more than 10 pounds; eating problems (e.g., not feeling hungry); difficulty swallowing, abdominal pain, or nausea and vomiting; swelling or lumps in any part of the body; thickening or lumps in the breast or other parts of the body; pain, especially new or unexplained pain that does not go away or worsens; skin changes (e.g., bleeding or scaly lumps), new moles or changes in moles, non-healing sores, or yellowing of the skin or eyes (e.g., jaundice); persistent cough or hoarseness; unexplained abnormal bleeding or bruising; changes in bowel habits, such as persistent constipation or diarrhea, or changes in the appearance of stool; bladder changes (e.g., painful urination, blood in urine, or need to urinate more frequently or less); fever or night sweats; headache; vision or hearing problems; oral changes (e.g., sores, bleeding, pain, or numbness).

[0254] Symptoms and / or complications of colorectal cancer that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to: persistent changes in bowel habits, including diarrhea or constipation or changes in stool consistency; rectal bleeding or blood in the stool; persistent abdominal discomfort (e.g., abdominal cramps, bloating, or pain); a feeling that the bowels are not completely empty; weakness or fatigue; or unexplained weight loss.

[0255] Tests that can aid in the diagnosis of, for example, colorectal cancer include, but are not limited to: colonoscopy, proctoscopy, colon or rectal biopsy, and stool tests (e.g., Genetic testing (e.g., changes in KRAS, NRAS, or BRAF genes; microsatellite instability (MSI); changes in any mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2); changes in the EPCAM gene), CT-guided needle biopsy, ultrasound, MRI, and PET scans are also helpful. A family history of colorectal cancer or exposure to risk factors for colorectal cancer (e.g., lack of regular physical activity; a diet low in fruits and vegetables; a diet low in fiber and high in fat or rich in processed meats; overweight and obesity; alcohol consumption; or tobacco use) can also help determine whether a subject is likely to have colorectal cancer or aid in the diagnosis of colorectal cancer.

[0256] The compositions and methods described herein may be administered to subjects who have or have been diagnosed with cancer (e.g., colorectal cancer, also referred to herein as colon cancer). In some embodiments, the methods described herein comprise administering to a subject an effective amount of the compositions described herein (e.g., Mucispirillum compositions as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or XCL1 peptides (or XCR1 agonists)) to alleviate symptoms of cancer (e.g., colorectal cancer). As used herein, “alleviate symptoms of cancer” means to improve any condition or symptom associated with cancer (e.g., colorectal cancer). This reduction is, as measured by any standard technique, at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, compared to an equivalent untreated control. Various methods of administering the compositions described herein to subjects are known to those skilled in the art. Such methods may include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, skin, local, injection, or intratumoral administration. Administration can be local or systemic.

[0257] As used herein, the term "effective amount" refers to the amount of a Mucispirillum composition, sulfur-containing amino acid (SAA)-rich diet, and / or XCL1 peptide (or XCR1 agonist) as described herein required to alleviate at least one or more symptoms of a disease or disorder, and relates to an amount of pharmacological composition sufficient to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to an amount of a Mucispirillum composition, sulfur-containing amino acid (SAA)-rich diet, and / or XCL1 peptide (or XCR1 agonist) as described herein, sufficient to provide a specific anticancer effect when administered to a typical subject. Effective amounts as used herein also include, in various cases, an amount sufficient to delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. Therefore, specifying an exact "effective amount" is generally not feasible. However, for any given situation, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experiments.

[0258] Effective doses, toxicity, and therapeutic efficacy can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as procedures for determining the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population). The doses can vary depending on the dosage form and route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Additionally, doses can be tailored in animal models to achieve circulating plasma concentration ranges including the IC50 (i.e., concentrations of the Mucispirillum composition, a diet rich in sulfur-containing amino acids (SAAs), and / or an XCL1 peptide (or XCR1 agonist) as determined in cell cultures or in suitable animal models, achieving half-maximal inhibition of symptoms) for example, by high-performance liquid chromatography (HPLC) to measure plasma or intestinal levels. The effect of any particular dose can be monitored by appropriate bioassays (e.g., measurement of levels of Mucispirillum bacteria, XCL1 peptide, NKT, and / or CD103+cDC1, etc.). The dose may be determined by a physician and adjusted as needed to accommodate observed therapeutic effects.

[0259] Pharmaceutical compositions comprising the Mucispirillum composition as described herein, sulfur-containing amino acids (SAAs), and / or XCL1 peptides (or XCR1 agonists) can also be formulated for oral administration, for example, in discrete dosage forms, such as, but not limited to, tablets (including, but not limited to, scored or coated tablets), pills, capsules, chewable tablets, powders, packets, flat capsules, lozenges, rice paper capsules, aerosols, or liquids, such as, but not limited to, syrups, elixirs, solutions, or suspensions (in aqueous, non-aqueous, oil-in-water, or water-in-oil emulsions). Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of a disclosed compound and can be prepared by pharmaceutical methods well known to those skilled in the art. Generally see, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0260] In some embodiments, a single effective dose of the composition as described herein may be administered to a patient, the composition comprising the Mucispirillum composition as described herein, a sulfur-containing amino acid (SAA)-rich diet, and / or an XCL1 peptide (or XCR1 agonist). In some embodiments, an effective dose of the composition comprising the Mucispirillum composition as described herein, a sulfur-containing amino acid (SAA)-rich diet, and / or an XCL1 peptide (or XCR1 agonist) may be administered to a patient repeatedly. For systemic administration, a therapeutic amount of the composition comprising the Mucispirillum composition as described herein, a sulfur-containing amino acid (SAA), and / or an XCL1 peptide (or XCR1 agonist) may be administered to the subject, for example, such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0261] In some implementations, treatment may be administered at a lower frequency after the initial treatment regimen. For example, after three months of treatment every two weeks, treatment may be repeated monthly for six months, a year, or longer. Treatment according to the methods described herein may reduce the level of symptoms or markers of a condition (e.g., cancer, such as colorectal cancer) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0262] The dosage of the compositions described herein may be determined by a physician and adjusted as needed to accommodate observed therapeutic effects. For the duration and frequency of treatment, a skilled clinician typically monitors the subject to determine when treatment provides therapeutic benefit and to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosing schedule may vary from once weekly to once daily, depending on various clinical factors, such as the subject's sensitivity to the Mucispirillum compositions described herein, a diet rich in sulfur-containing amino acids (SAAs), and / or XCL1 peptides (or XCR1 agonists). The desired dose or amount may be administered once or divided into sub-dose, such as 2-4 sub-dose, and administered over a period of time, such as at appropriate intervals throughout the day or other suitable schedules. In some embodiments, administration may be long-term, such as once or multiple times daily for weeks or months. Examples of dosing and / or treatment schedules include administration once daily, twice daily, three times daily, or four or more times daily for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or longer. Compositions including Mucispirillum compositions as described herein, sulfur-containing amino acid (SAA)-rich diets, and / or XCL1 peptides (or XCR1 agonists) may be administered over a period of time, such as within 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0263] According to the methods described herein, the dosage range for administration of the compositions described herein depends, for example, on the form of the Mucispirillum composition described herein, the diet rich in sulfur-containing amino acids (SAA) and / or XCL1 peptides (or XCR1 agonists), their potency, and the degree to which symptoms, markers, or indicators of the condition described herein are expected to be reduced, such as the expected percentage reduction in tumor and / or cancer symptoms, or the expected degree of increase in levels of, for example, Mucispirillum bacteria, XCL1 peptides, NKT, and / or CD103+cDC1. The dosage should not be too high to avoid adverse side effects such as sepsis or autoimmune diseases. Generally, the dosage will vary with the patient's age, condition, and sex and can be determined by those skilled in the art. The dosage may also be adjusted by an individual physician in the event of any complications.

[0264] The efficacy of the Mucispirillum compositions, sulfur-containing amino acid (SAA)-rich diets, and / or XCL1 peptides (or XCR1 agonists) in the treatment of conditions described herein, or in inducing responses as described herein, can be determined by a skilled clinician. However, treatment is considered “effective treatment” as used herein if one or more signs or symptoms of the condition described herein change in a beneficial manner, other clinically accepted symptoms improve or even lessen, or a desired response is induced (e.g., at least 10% after treatment according to the methods described herein). Efficacy can be assessed, for example, by measuring biomarkers, indicators, symptoms, and / or the incidence of conditions treated according to the methods described herein, or any other suitable measurable parameter, such as the levels of Mucispirillum bacteria, XCL1 peptides, NKT, and / or CD103+cDC1, for example, cancer indicators such as tumor size, tumor growth, and / or tumor metastatic activity. Efficacy can also be measured by hospitalization assessment that the individual no longer deteriorates or no longer requires medical intervention (i.e., disease progression stops). Methods for measuring these indicators are well known to those skilled in the art or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) suppressing the disease, such as preventing the worsening of symptoms (e.g., pain or organ damage; for example, symptoms and / or complications of cancer, as further described herein; for example, symptoms and / or complications of colorectal cancer, as further described herein); or (2) alleviating the severity of the disease, such as causing symptom resolution. An effective amount of disease treatment means an amount sufficient, when administered to a subject in need, to produce an effective treatment for the disease as defined herein. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response, such as increased levels of Mucispirillum bacteria, XCL1 peptide, NKT, and / or CD103+cDC1; for example, a decrease in cancer indicators (such as tumor size, tumor growth, and / or tumor metastatic activity). It is entirely within the capabilities of those skilled in the art to monitor the efficacy of the administered and / or treatment by measuring any such parameter or combination of parameters. Efficacy can be assessed in animal models of the conditions described herein, such as the treatment of cancer (e.g., colorectal cancer). When using experimental animal models, the efficacy of treatment is demonstrated when statistically significant changes in biomarkers are observed, such as increased levels of Mucispirillum bacteria, XCL1 peptide, NKT and / or CD103+cDC1, or decreased levels of cancer indicators (e.g., tumor size, tumor growth and / or tumor metastatic activity).

[0265] This document provides in vitro and animal model assays that allow for the evaluation of given doses of the Mucispirillum composition as described herein, a sulfur-containing amino acid (SAA)-rich diet, and / or an XCL1 peptide (or XCR1 agonist). As a non-limiting example, the effects of doses of the Mucispirillum composition as described herein, a sulfur-containing amino acid (SAA)-rich diet, and / or an XCL1 peptide (or XCR1 agonist) can be evaluated through clinical trials in human volunteers. The efficacy of a given dose combination can also be evaluated in animal models, including but not limited to the mouse models further described herein (see, for example, Example 1). cancer

[0266] As used in this article, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body via the blood and lymphatic system. There are several major types of cancer. Malignant tumors are cancers that begin in the skin or in tissues lining or covering internal organs. Sarcomas are cancers that begin in bones, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is a cancer that begins in hematopoietic tissues (such as bone marrow) and causes the production of large numbers of abnormal blood cells that enter the bloodstream. Lymphoma and multiple myeloma are cancers that begin in cells of the immune system. Cancers of the central nervous system are cancers that begin in tissues of the brain and spinal cord.

[0267] In some embodiments of any aspect, the cancer is primary cancer. In some embodiments of any aspect, the cancer is malignant cancer. As used herein, the term "malignant" refers to a group of tumor cells in which one or more uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other sites in the body via the lymphatic or bloodstream). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. A tumor formed by spreading cells is called a "metastatic tumor" or "metastatic tumor." Metastatic tumors contain cells similar to those in the primary (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to a tumor that may grow but does not spread to other parts of the body. Benign tumors are self-limiting and generally do not invade or metastasize.

[0268] "Cancer cells" or "tumor cells" refer to individual cells of cancerous growths or tissues. A tumor generally refers to a swelling or lesion formed by abnormal cell growth, which may be benign, premalignant, or malignant. Most cancer cells form tumors, but some (such as leukemia) do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cells) and tumor (cells) are used interchangeably.

[0269] As used herein, the term "vesicle" refers to any new and abnormal tissue growth, such as an abnormal mass of tissue that grows beyond and out of sync with the growth of normal tissue. Therefore, a growth can be benign, precancerous, or malignant.

[0270] A subject with cancer or a tumor is one in whom there are objectively measurable cancer cells present. This definition includes malignant, actively proliferating cancers, as well as potentially dormant tumors or micrometastases. The cancer has migrated from its initial location and seeded other vital organs, potentially leading to the subject's death through functional deterioration of the affected organs.

[0271] Examples of cancers include, but are not limited to, malignant tumors, lymphomas, germ cell tumors, sarcomas, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancers; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatocellular carcinoma; hepatocellular carcinoma; intraepithelial neoplasia; kidney cancer or renal cell carcinoma; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma); lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma. Cellular tumors; oral cancers (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancers; salivary gland malignancies; sarcomas; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancers; vulvar cancer; and other malignancies and sarcomas; as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, and large masses). Diseases including NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with nevus hamartomatosis, edema (e.g., edema associated with brain tumors), and Meigs syndrome.

[0272] Cancer cells are cancerous, precancerous, or transformed cells in vivo, in vitro, or in tissue cultures that exhibit spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. While transformation can be caused by infection with transforming viruses and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, resulting in mutations in endogenous genes. Transformation / cancer is associated with, for example, morphological changes, cell immortalization, abnormal growth control, lesion formation, anchorage independence, malignancy, loss of contact inhibition and growth density limitation, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts (e.g., nude mice). definition

[0273] For convenience, the meanings of some terms and phrases used in the specification, embodiments, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. If there is a significant difference between the usage of terms in the art and their definitions provided herein, the definitions provided in the specification shall prevail.

[0274] As used herein, the terms “colonized” or “colonized” broadly refer to the presence of a microbiome in the body (e.g., in the gastrointestinal tract or skin of a mammalian organism) without any noticeable significant alterations other than the presence of bacteria. In contrast to transient passage through the gastrointestinal tract, a colonized microbiome is established and / or proliferated in the gastrointestinal tract non-transiently (e.g., semi-permanently) (e.g., in the GI lumen or associated with the GI epithelium or mucus layer). “Colonized” or “colonized” can also refer to the presence of a microbiome on the surface of food or the environment. The terms “colonized” and “colonized” contrast with the terms “infectious” or “infectious,” which are generally understood to require noticeable harmful alterations as part of their definition. “Colonized” and “colonized” can also refer to the presence of bacteria in or on a human or animal without noticeable damage, alteration, or disease. “Colonized” and “colonized” may be associated with human or animal benefits.

[0275] As used herein, the term "isolated" means that the bacteria or other entity or substance has been (1) isolated from at least some of the components with which it was originally associated (whether in nature (e.g., human feces) or in a laboratory facility (e.g., a Petit's dish composed of artificial growth media)) and / or (2) produced, prepared, purified, and / or manufactured artificially. The isolated bacteria, proteins, metabolites, or combinations thereof may be isolated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, the isolated bacteria, proteins, metabolites, or combinations thereof have a purity greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. As used herein, a substance is “pure” if it is substantially free of other components (e.g., other bacterial species). As recognized by those skilled in the art of bacterial culture or related techniques (e.g., chemistry), the terms “purified,” “purified,” and “refined” mean that bacteria or other material have been isolated from at least some of the components associated with them at the time of their initial production or generation (e.g., whether in nature or in a laboratory setting) or at any time after their initial generation. If bacteria or bacterial communities are isolated during or after production (e.g., from materials or environments containing said bacteria or bacterial communities), they can be considered purified, and purified bacteria or bacterial communities may contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% of other material, and are still considered “isolated.” In some embodiments, the purified bacteria and bacterial communities have a purity greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. In the case of the bacterial compositions provided herein, one or more bacterial types present in the composition can be independently purified from materials or environments containing said bacterial types and / or from one or more other bacteria present. In some embodiments, if the bacteria or bacterial community comprises a single bacterial strain, it is "isolated". In some embodiments, such isolated bacteria can be mixed with or given with other isolated bacteria (e.g., in a defined isolated bacterial community).

[0276] As used in this article, "probiotics" is understood to refer to live microorganisms that, when given in appropriate amounts, confer health benefits to the host.

[0277] As used herein, “prebiotic” is understood to mean an ingredient that allows or promotes specific changes in the composition and / or activity of the microbiome (e.g., the gut microbiome), which may or may not confer a benefit to the host.

[0278] As used in this article, “medicinal food” is understood to mean food that is formulated for consumption or enteral administration under the supervision of a physician and is intended for the specific dietary management of a disease or condition, based on recognized scientific principles and medical evaluation to establish the unique nutritional requirements of the disease or condition.

[0279] As used herein, “supplement” (also referred to as dietary supplement) is understood to mean an oral product that includes one or more ingredients intended to supplement a diet and not considered as food (e.g., vitamins, minerals, amino acids, isolated microorganisms or their products as described herein). As a non-limiting example, supplements may be in the form of capsules, enteric-coated capsules, tablets, pouches, pills, lozenges, sugar tablets, powders, or granules.

[0280] The term "intestinal tract" is understood to refer to the human gastrointestinal tract, also known as the digestive tract. The intestinal tract includes the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum and colon), and rectum.

[0281] As used in this article, “bacteria” is understood to be a single bacterial cell of a given species.

[0282] As used herein, the terms “reduction,” “lowering,” “reduction,” or “inhibition” refer to a statistically significant reduction. In some implementations, “reduction,” “lowering,” “reduction,” or “inhibition” generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “lowering” or “inhibition” compared to a reference level does not encompass complete inhibition or reduction. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction may preferably be reduced to an acceptable level within the normal range, for example, for an individual without a given disorder.

[0283] As used herein, the terms “increased,” “enhanced,” “strengthened,” or “activated” all refer to an increase of a statistically significant amount. In some implementations, the terms “increased,” “enhanced,” or “activated” may mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level. In the context of a biomarker or symptom, “increased” means that the level is statistically significant.

[0284] As used herein, “subject” refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0285] Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer (e.g., colorectal cancer). Subjects can be male or female.

[0286] Subjects may be those who have been previously diagnosed with or identified as suffering from or suffering from a condition (e.g., cancer) or one or more complications associated with such a condition, and optionally, have already undergone treatment for cancer or one or more cancer-related complications. Alternatively, subjects may be those who have not previously been diagnosed with cancer or one or more cancer-related complications. For example, a subject may be one who exhibits one or more risk factors for cancer or one or more cancer-related complications, or a subject who does not exhibit any risk factors.

[0287] "Subjects in need" for treatment of a specific condition can be those who have the condition, have been diagnosed with the condition, or are at risk of developing the condition.

[0288] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues linked together by peptide bonds between adjacent α-amino and carboxyl groups. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “polypeptide” are generally used to refer to larger polypeptides, while the term “peptide” is generally used to refer to smaller polypeptides; however, the use of these terms overlaps in the art. When referring to gene products and fragments thereof, the terms “protein” and “polypeptide” are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, the aforementioned fragments and analogs.

[0289] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conserved modified variants, and / or conserved substitution variants of any of the specific polypeptides are also considered. Regarding amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence (which alter a single amino acid or a small percentage of amino acids in the coding sequence) constitute "conserved modified variants," wherein the changes result in the substitution of an amino acid with a chemically similar amino acid while maintaining the desired activity of the polypeptide. Such conserved modified variants are complementary to, but not excluded from, polymorphic variants, interspecies homologs, and alleles consistent with this disclosure.

[0290] A given amino acid can be substituted with residues having similar physicochemical characteristics, for example, replacing one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala substituted for each other), or replacing one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conserved substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. Peptides including conserved amino acid substitutions can be tested to confirm the desired activity, for example, maintaining the activity and specificity of the native or reference peptide (e.g., XCL1).

[0291] Amino acids can be grouped according to the similarity of their side chain properties (see Allhninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side-chain properties: (1) hydrophobic: ortholeucine, Met, Ala, Val, Leu, Ile; (2) neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would require exchanging one member of these classes for another. Specific conservative substitutions include, for example: Ala substituted with Gly or Ser; Arg substituted with Lys; Asn substituted with Gln or His; Asp substituted with Glu; Cys substituted with Ser; Gln substituted with Asn; Glu substituted with Asp; Gly substituted with Ala or Pro; His substituted with Asn or Gln; Ile substituted with Leu or Val; Leu substituted with Ile or Val; Lys substituted with Arg, Gln, or Glu; Met substituted with Leu, Tyr, or Ile; Phe substituted with Met, Leu, or Tyr; Ser substituted with Thr; Thr substituted with Ser; Trp substituted with Tyr; Tyr substituted with Trp; and / or Phe substituted with Val, Ile, or Leu.

[0292] In some embodiments, the polypeptide (or nucleic acid encoding such polypeptide) described herein may be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a segment or segment of a polypeptide that retains at least 50% of the activity of a wild-type reference polypeptide. The functional fragment may include conserved substitutions of the sequences disclosed herein.

[0293] In some embodiments, the polypeptide described herein may be a variant of the polypeptide sequence described herein. In some embodiments, the variant is a conserved modified variant. For example, a conserved substitution variant can be obtained by mutating a natural nucleotide sequence. As described herein, a “variant” is a polypeptide that is substantially homologous to a natural or reference polypeptide, but whose amino acid sequence differs from that of a natural or reference polypeptide due to one or more deletions, insertions, or substitutions. When compared to a natural or reference DNA sequence, the DNA sequence encoding the variant polypeptide encompasses sequences including the addition, deletion, or substitution of one or more nucleotides, but the protein or fragment thereof it encodes retains the activity of the natural or reference polypeptide. A wide variety of site-specific mutagenesis methods, such as PCR-based methods, are known in the art and can be applied by those skilled in the art to generate and test artificial variants.

[0294] The variant amino acid or DNA sequence may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identical to the natural or reference sequence. The degree of homology (percentage of identity) between the natural and mutant sequences may be determined, for example, by comparing the two sequences using computer programs (e.g., BLASTp or BLASTn, using default settings) commonly used on the Internet for this purpose and freely available.

[0295] Variant amino acid sequences may be at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher similar to native or reference sequences. As used herein, “similarity” means the same amino acid or a conservedly substituted amino acid as described herein. Therefore, the percentage of “sequence similarity” is the percentage of the same or conservedly modified amino acids; for example, “sequence similarity” = (sequence identity %) + (conservative modification %). It should be understood that a sequence having a specific percentage of similarity to a reference sequence necessarily encompasses sequences having the same specific percentage of identity to that reference sequence. Those skilled in the art will understand various computer programs using different mathematical algorithms that can be used to determine the identity or similarity between two sequences. For example, one can use the computer program employing the Needleman and Wunsch algorithm (Needleman et al., (1970)), the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego USA), the algorithm of E. Meyers and W. Miller (Meyers et al., (1989)), which has been incorporated into the ALIGN program (version 2.0), or more preferably, BLAST (a basic local alignment tool using default parameters), see, for example, U.S. Patent 10,023,890, the contents of which are incorporated herein by reference in their entirety.

[0296] In some embodiments, sequencing includes 16S rRNA gene sequencing, which may also be referred to as “16S ribosomal RNA sequencing,” “16S rDNA sequencing,” or “16S rRNA sequencing.” Sequencing of the 16S rRNA gene can be used for genetic research because it is highly conserved across different bacterial species, but it is not present in eukaryotic species. In addition to the highly conserved regions, the 16S rRNA gene also includes nine species-dependent hypervariable regions (V1–V9). 16S rRNA gene sequencing typically involves using multiple universal primers that bind to the conserved regions of the 16S rRNA gene, PCR amplification of the bacterial 16S rRNA gene region (including the hypervariable regions), and sequencing the amplified 16S rRNA gene using next-generation sequencing technologies as described herein (see also, for example, U.S. Patents 5,654,418, 6,344,316, and 8,889,358, and U.S. Patent Applications US2013 / 0157265 and US2018 / 0195111, which are incorporated herein by reference in their entirety).

[0297] Alterations to natural amino acid sequences can be achieved using any of a variety of techniques known to those skilled in the art. For example, mutations can be introduced at a specific locus by synthesizing an oligonucleotide containing a mutated sequence flanked by a restriction site that allows ligation to a fragment of the natural sequence. After ligation, the resulting reconstructed sequence encodes an analogue with the desired amino acid insertion, substitution, or deletion. Alternatively, site-specific mutagenesis procedures directed by oligonucleotides can be used to provide altered nucleotide sequences having specific codons changed according to the desired substitution, deletion, or insertion. Various site-specific mutagenesis methods are known in the art and can be applied by those skilled in the art to introduce mutations into specific nucleic acid loci, such as the Kunkel method, cassette mutagenesis, site-directed PCR mutagenesis (e.g., conventional PCR, primer extension, or reverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, and CRISPR / Cas-guided mutagenesis. The techniques used to make such changes are well-established and include those disclosed, for example, those published as follows: Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd edition); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014) Cell 157(6): 1262-78; Cerchione et al. (2020) PLOS ONE 15(4):e0231716 and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine ​​residues that do not participate in maintaining the proper conformation of the polypeptide can often also be replaced with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bonds can be added to the polypeptide to improve its stability or promote oligomerization.

[0298] In some embodiments, the methods described herein involve measuring, detecting, or determining the level of at least one marker. As used herein, the terms “detection” or “measurement” refer to observing a signal from, for example, a probe, marker, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a specific labeled portion can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescence, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some embodiments of any aspect, the measurement may be a quantitative observation.

[0299] In some embodiments of any aspect, the peptide, nucleic acid, or cell described herein may be engineered. As used herein, “engineered” means an aspect that has been artificially manipulated. For example, a peptide is considered “engineered” when at least one aspect of it (e.g., its sequence) has been artificially manipulated to differ from its aspect present in nature. As understood and commonly practiced by those skilled in the art, progeny of engineered cells are generally still referred to as “engineered” even if the actual manipulation was performed on a prior entity.

[0300] As used herein, the terms “treatment” (treatment, treat, treating) or “improvement” refer to therapeutic interventions aimed at reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of a condition associated with a disease or disorder (e.g., cancer, such as colorectal cancer). The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with cancer (e.g., colorectal cancer). Treatment is generally “effective” if it reduces one or more symptoms or clinical markers. Alternatively, treatment is “effective” if the progression of the disease is reduced or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also the cessation or at least slowing of the progression or worsening of symptoms compared to what would have been expected without treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delaying or slowing disease progression, improving or alleviating the disease state, mitigating (whether partially or completely) and / or reducing mortality, whether detectable or undetectable. The term “treatment” for a disease also includes providing relief (including palliative care) for the symptoms or side effects of the disease.

[0301] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues within a reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a synthetic or engineered carrier (e.g., a carrier in which the active ingredient is not found to exist in nature or within the body).

[0302] As used herein, the term "administration" means placing a compound disclosed herein into a subject by a method or route that produces at least partial delivery of the pharmaceutical agent at a desired site. Pharmaceutical compositions comprising compounds disclosed herein may be administered by any appropriate route that induces effective treatment in a subject. In some embodiments, administration includes physical activities such as injection, ingestion, application, and / or manipulation of a delivery device or machine. Such activities may be performed, for example, by a medical professional and / or the subject being treated.

[0303] The terms “statistically significant” or “significantly” refer to statistical significance and typically mean a difference of two standard deviations (2SD) or greater.

[0304] Except as provided in the operational examples or otherwise indicated, all figures used herein to indicate amounts of components or reaction conditions shall be understood to be modified by the term “about” in all cases. When used in conjunction with percentages, the term “about” may mean ±1%.

[0305] As used herein, the term "includes" means that other elements may exist in addition to the elements defined therein. The use of "includes" indicates inclusion rather than limitation.

[0306] The term "composed of" means any composition, method and its respective components as described herein, excluding any elements not listed in the description of the embodiments.

[0307] As used herein, the term "consistently of" refers to those elements required for a given implementation. This term allows for the presence of additional elements that do not materially affect the basic and novel or functional features of the implementation of the invention.

[0308] As used herein, the term "corresponding to" refers to an amino acid or nucleotide at an enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide equivalent to an amino acid or nucleotide enumerated in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by aligning candidate sequences using a procedure known in the art (e.g., BLAST).

[0309] Unless the context clearly indicates otherwise, the singular terms “a, an, and the” include plural references. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and”. Although methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. The abbreviation “eg” originates from the Latin *exempli gratia* and is used in the text to indicate a non-limiting example. Therefore, the abbreviation “eg” is synonymous with the term “for example”.

[0310] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members or other elements of the group visible herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to include the modified group thereby satisfying the written description of all Markush groups used in the appended claims.

[0311] Unless otherwise defined herein, the scientific and technical terms used in connection with this application should have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, and therefore is subject to variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention (which is defined only by the claims). Definitions of commonly used terms in cell biology, immunology, and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al., eds., The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers, Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan... Mowat, edited by Casey Weaver, WW Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, edited by Jon Lorsch, Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), edited by Frederick M.Ausubel, John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), edited by John E.Coligan, John Wiley and Sons Sons, Inc., 2005; and Current Protocols The contents of *Immunology* (CPI) (John E. Coligan, ADA M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, eds., John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737) are incorporated herein by reference in their entirety.

[0312] Other terms are defined herein within the description of various aspects of the invention.

[0313] All patents and other publications (including references, granted patents, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies, such as those described in such publications, that may be used in conjunction with the techniques described herein. These publications are provided solely because their publications predate the filing date of this application. In this respect, nothing should be construed as an admission that the inventor was not entitled to prior disclosure due to a prior invention or any other reason. All statements regarding dates or representations regarding the content of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or content of these documents.

[0314] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of this disclosure, as will be recognized by those skilled in the art. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially simultaneously. The teachings of this disclosure provided herein can be suitably used in other procedures or methods. The various embodiments described herein may be combined to provide further embodiments. If desired, aspects of this disclosure may be modified to employ the compositions, functions, and concepts of the foregoing references and applications to provide further embodiments of this disclosure. Furthermore, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the biological or chemical action in terms of type or amount. These and other changes may be made to this disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0315] Specific elements of any of the foregoing embodiments may be combined with or substitute for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of this invention.

[0316] Some implementations of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria, the composition being formulated for delivery to the intestine. 2. The composition according to paragraph 1, wherein the M. schaedleri bacteria is alive or inactivated. 3. The composition according to paragraph 1, wherein the M. schaedleri bacteria are in a dried, live form. 4. The composition according to any one of paragraphs 1-3, wherein the M. schaedleri bacteria is encapsulated. 5. The composition according to any one of paragraphs 1-4, wherein the M. schaedleri bacteria is contained in an enteric-coated capsule. 6. The composition according to any one of paragraphs 1-5, wherein the M. schaedleri bacteria are maintained in an anaerobic state in the formulation. 7. The composition according to any one of paragraphs 1-6, wherein the M. schaedleri bacteria is mixed with a prebiotic. 8. The composition according to any one of paragraphs 1-7, wherein the M. schaedleri bacteria is mixed with sulfur-containing amino acids. 9. The composition according to paragraph 8, wherein the sulfur-containing amino acid is methionine, cysteine, or a derivative thereof. 10. The composition according to any one of paragraphs 1-9, wherein the M. schaedleri bacterium is formulated in the food composition. 11. The composition according to paragraph 10, wherein the food composition is supplemented with sulfur-containing amino acids and / or prebiotics. 12. The composition according to any one of paragraphs 1-11, wherein the composition further comprises 1 to 20 additional microbial species. 13. The composition according to any one of paragraphs 1-11, wherein the composition comprises no more than 20 microbial strains. 14. A composition comprising live M. schaedleri bacteria, dead M. schaedleri bacteria, M. schaedleri conditioned medium, or an organic solvent extract of M. schaedleri conditioned medium or a fraction thereof that promotes XCL1 secretion by NKT cells, wherein the composition is formulated for delivery to the intestine. 15. The composition according to paragraph 14, wherein the M. schaedleri bacteria, culture medium or solvent extract is in a dry form. 16. The composition according to paragraph 14 or paragraph 15, wherein the M. schaedleri bacteria, culture medium or extract is encapsulated. 17. The composition according to any one of paragraphs 14-16, wherein the M. schaedleri bacteria is contained in an enteric-coated capsule. 18. The composition according to any one of paragraphs 14-17, wherein the M. schaedleri bacteria are maintained in an anaerobic state in the formulation. 19. The composition according to any one of paragraphs 14-18, wherein the M. schaedleri bacteria, culture medium or extract is mixed with prebiotics and / or sulfur-containing amino acids or their derivatives. 20. The composition according to any one of paragraphs 14-19, wherein the M. schaedleri conditioned medium, or the organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises a fraction selected from... Figure 4H , Figure 4I or Figure 16 At least one metabolite of [the substance]. 21. The composition according to any one of paragraphs 14-20, wherein the M. schaedleri conditioned medium, or the organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3). 22. The composition according to any one of paragraphs 14-21, wherein the M. schaedleri conditioned medium, or the organic solvent extract or fraction thereof of the M. schaedleri conditioned medium, comprises at least one metabolite selected from the group consisting of: succinic acid, nicotinic acid, cis and / or trans aconitine, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid. 23. A food composition comprising any one of the compositions according to paragraphs 14-22. 24. The food composition according to paragraph 23, wherein the food composition further comprises 1 to 20 additional microbial strains. 25. A method for treating cancer or promoting antitumor immune activity, the method comprising administering to a subject in need the composition according to any one of paragraphs 1-24. 26. The method described in paragraph 25, wherein the cancer is colon cancer. 27. The method according to paragraph 25 or 26, the method further comprising administering an immune checkpoint inhibitor. 28. A method for promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering to a subject in need the composition according to any one of paragraphs 1-24. 29. The method according to paragraph 28, the method further comprising administering an immune checkpoint inhibitor. 30. The method according to paragraph 28 or 29, wherein the subject has colon cancer. 31. The method according to any one of paragraphs 28-30, wherein the subject's cancer has been determined to be resistant to immune checkpoint inhibitor therapy. 32. The method according to any one of paragraphs 28-31, wherein the composition promotes XCL1 secretion by NKT cells. 33. A method for increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering to a subject in need the composition according to any one of paragraphs 1-24. 34. The method according to paragraph 33, wherein the cDC1 is associated with a tumor. 35. The method described in paragraph 34, wherein the tumor is colon cancer. 36. The method according to any one of paragraphs 33-35, wherein the method further comprises administering a sulfur-containing amino acid. 37. The method according to any one of paragraphs 33-36, the method further comprising administering an immune checkpoint inhibitor. 38. A method for increasing XCL1 secretion by NKT cells, the method comprising administering to a subject in need the composition according to any one of paragraphs 1-24. 39. The method according to paragraph 38, wherein the subject has cancer. 40. The method according to paragraph 38 or 39, wherein the subject has colon cancer. 41. A method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering to a subject in need the composition according to any one of paragraphs 1-24. 42. The method according to paragraph 41, wherein the cDC1 is associated with a tumor. 43. The method described in paragraph 42, wherein the tumor is colon cancer. 44. The method according to any one of paragraphs 41-43, the method further comprising administering a sulfur-containing amino acid. 45. The method according to any one of paragraphs 41-44, the method further comprising administering an immune checkpoint inhibitor. 46. ​​A method for increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering to a subject in need a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA. 47. The method according to paragraph 46, wherein the diet rich in sulfur-containing amino acids comprises more than 0.04 grams of SAA per kilogram of body weight per day. 48. The method according to paragraph 46 or 47, the method further comprising administering to a subject the composition according to any one of paragraphs 13 to 20. 49. A method for establishing or maintaining a tumor-suppressive gut environment in subjects in need, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA. 50. The method according to paragraph 49, wherein the diet rich in sulfur-containing amino acids or the supplement containing SAA comprises more than 0.04 grams of SAA per kilogram of body weight per day. 51. The method according to paragraph 49 or 50, the method further comprising administering to a subject the composition according to any one of paragraphs 14 to 21. 52. A method for treating cancer, the method comprising administering an XCL1 peptide to a subject in need. 53. The method described in paragraph 52, wherein the cancer is colon cancer. 54. The method according to paragraph 52 or 53, wherein the XCL1 polypeptide is administered to the intestine. 55. A method for treating cancer, the method comprising administering to a subject in need a microorganism engineered to express an XCL1 peptide. 56. A method of treating cancer, the method comprising administering an agonist of the XCL1 receptor, i.e., XCR1, to a subject in need. 57. The method according to paragraph 56, wherein the XCR1 agonist comprises SEQ ID NOs: 9-11 or an amino acid sequence that is at least 95% identical thereto and maintains its function. 58. A method for treating cancer in a subject in need, the method comprising: The level of M. schaedleri in samples from the subjects was detected; If the level of M. schaedleri is equal to or higher than a predetermined threshold, a cancer immunotherapy agent is administered; and If the level of M. schaedleri is below a predetermined threshold, a cancer immunotherapy agent and the composition according to any one of paragraphs 1-24 are administered. 59. A method for treating cancer in a subject in need, the method comprising: Results were obtained from the determination of the level of M. schaedleri in samples from the subjects; If the level of M. schaedleri is equal to or higher than a predetermined threshold, a cancer immunotherapy agent is administered; and If the level of M. schaedleri is below a predetermined threshold, a cancer immunotherapy agent and the composition according to any one of paragraphs 1-24 are administered. 60. A method for treating cancer in a subject of need, the method comprising: The levels of XCL1 peptide, NKT and / or CD103+cDC1 in samples from the subjects were detected; If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold, a cancer immunotherapy agent is administered; and If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold, a cancer immunotherapy agent and the composition according to any one of paragraphs 1-24 are administered. 61. A method for treating cancer in a subject of need, the method comprising: Results were obtained from the determination of levels of XCL1 peptide, NKT and / or CD103+cDC1 in samples from the subjects. If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold, a cancer immunotherapy agent is administered; and If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold, a cancer immunotherapy agent and the composition according to any one of paragraphs 1-24 are administered. 62. A method for stratifying subjects for cancer treatment, the method comprising: The levels of M. schaedleri were detected in samples from the subjects; and If the level of M. schaedleri is below a predetermined threshold, the subject is classified as high-risk; or If the level of M. schaedleri is equal to or higher than a predetermined threshold, the subject is classified as low risk. 63. A method for stratifying subjects for cancer treatment, the method comprising: Results were obtained from measurements of M. schaedleri levels in samples from the subjects; and If the level of M. schaedleri is below a predetermined threshold, the subject is classified as high-risk; or If the level of M. schaedleri is equal to or higher than a predetermined threshold, the subject is classified as low risk. 64. A method for stratifying subjects for cancer treatment, the method comprising: The levels of XCL1 peptide, NKT, and / or CD103+cDC1 in samples from the subjects were detected; and If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold, the subject is classified as high-risk; or If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold, the subject is classified as low risk. 65. A method for stratifying subjects for cancer treatment, the method comprising: Results were obtained from the determination of levels of XCL1 peptide, NKT, and / or CD103+cDC1 in samples from the subjects; and If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are below a predetermined threshold, the subject is classified as high-risk; or If the levels of the XCL1 peptide, NKT, and / or CD103+cDC1 are equal to or higher than a predetermined threshold, the subject is classified as low risk. 66. The method according to any one of paragraphs 58-65, wherein the subject has colon cancer. 67. The method according to any one of paragraphs 62-65, the method further comprising administering the composition according to any one of paragraphs 1-24. 68. The method according to any one of paragraphs 58-67, the method further comprising administering a sulfur-containing amino acid. 69. The method according to any one of paragraphs 58-61, wherein the cancer immunotherapy agent is selected from the group consisting of: immune checkpoint inhibitors, chemotherapy, dendritic cell vaccines, chimeric antigen receptor T cells (CAR-T), and NKT cell-based therapies. 70. The method according to any one of paragraphs 58-61, wherein the cancer immunotherapy agent comprises an immune checkpoint inhibitor. 71. The method according to any one of paragraphs 62-65, the method further comprising administering an immune checkpoint inhibitor. 72. The method according to any one of paragraphs 58-71, the method further comprising administering a diet rich in sulfur-containing amino acids or a supplement containing SAAs. 73. The method according to any one of paragraphs 58-61 or 66-69, wherein the method produces higher therapeutic efficacy compared to a treatment method that does not first perform: detecting the level of M. schaedleri; obtaining results from a determination of the level of M. schaedleri; detecting the levels of the XCL1 peptide, NKT and / or CD103+cDC1; or obtaining results from a determination of the levels of the XCL1 peptide, NKT and / or CD103+cDC1. 74. The method according to any one of paragraphs 62-72, wherein the method produces higher therapeutic efficacy compared to a treatment method that does not first stratify the subject. 75. The method according to any one of paragraphs 58-61 or 66-72, wherein the method causes fewer treatment complications compared to treatments that do not first involve: detecting the level of M. schaedleri, obtaining results from a determination of the level of M. schaedleri, detecting the levels of the XCL1 peptide, NKT and / or CD103+cDC1, or obtaining results from a determination of the levels of the XCL1 peptide, NKT and / or CD103+cDC1. 76. The method according to any one of paragraphs 62-72, wherein the method causes fewer treatment complications compared to treatment methods that do not first stratify the subjects. 77. An enteral delivery formulation, said enteral delivery formulation comprising a group selected from... Figure 4H , Figure 4I or Figure 16 At least one metabolite of [the substance]. 78. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of: succinic acid, propionic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3), and 15-hydroxypentadecanoic acid (C 15 H 30 O3). 79. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid. 80. An intestinal delivery formulation according to any one of paragraphs 77-79, wherein the intestinal delivery formulation is formulated for delivery into the intestine.

[0317] The techniques described herein are further illustrated by the following examples, but should not be construed as further limitations. Example Example 1: Diet-microbe interactions enhance anti-tumor immunity in colon cancer

[0318] Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, but responses to ICIs are not universal, particularly for colorectal cancer (CRC). While certain gut microbiota members have been associated with ICI treatment response in patients, the mechanisms controlling microbial regulation of antitumor immunity remain unclear. This study considers that microbial metabolic capacity (rather than specific taxonomic constructs) may better distinguish ICI responders from ICI non-responders. Since the pathogenesis of CRC is associated with both the microbiota and dietary factors, the biochemistry influenced by dietary factors was investigated. Microbial metabolic profiles were examined in ICI-responsive patients. As described in this paper, sulfur-containing amino acid (Saa) metabolic pathways and gene abundance in the gut microbiota were positively correlated with ICI responsiveness, and a Saa-rich diet slowed tumor growth to a degree comparable to that of α-PD-1 therapy in an ectopic mismatch repair-deficient CRC model. In genetically engineered mouse models of mismatch repair intact and ICI-resistant CRC, a high-Saa diet delayed the progression of vegetations and promoted the expansion of a niche of *Mucispirillum schaedleri*, a mucus-dwelling immunomodulatory species present in the colons of humans and mice. The high-Saa diet (studied in mice in this study and achievable in humans via dietary supplementation) induced massive proliferation of *M. schaedleri*, converting “cold” CRC tumors to “warm” ones by increasing XCL1 secretion from NKT cells, amplifying and activating cDC1 in tumor-draining lymph nodes, ultimately resulting in enhanced tumor CD8. + This study describes diet-microbiome-host interactions and demonstrates microbiome-targeted diet-based cancer therapy. It also describes, in several aspects, how diet-based microbiome analysis of immunotherapy-responsive patients enhances anti-tumor immunity in a CRC model.

[0319] As further described in this paper, the abundance of sulfur-containing amino acid (Saa) metabolism genes in the gut microbiome is associated with improved antitumor immunity and ICI response, facilitating the testing of the effects of dietary Saa in a CRC mouse model. A high-Saa diet slowed tumor growth in an ectopic CRC model, and this diet enhanced CD8+ in a gene-driven CRC model.+ T-cell infiltration and reduced vegetation progression. A high-Saa diet increased the abundance of Mucispirillum schaedleri, which increased CD103 in tumor draining lymph nodes (TDLNs). + The abundance and activation status of conventional dendritic cells (cDC1) enhanced antitumor immune responses. *M. schaedleri* conditioned medium (CDM) induced XCL1 production in natural killer T (NKT) cells in vitro, indicating a direct causal effect of *M. schaedleri* on this cDC1 activation pathway. Therefore, this article describes the characteristics of diet-microbiota-host interactions and the modulation of antitumor immunity, providing a framework for identifying and evaluating microbiota-targeted, diet-based adjuvant cancer therapies.

[0320] The Saa biosynthesis gene is enriched in ICI-responsive patients.

[0321] To quantify microbial pathways enriched in the microbiome of ICI responders, grouped analyses were performed on the microbial metabolic potential of fecal metagenomic datasets from ICI responders vs. non-responders in eight ICI microbiome studies; see, for example, Andrews et al., 2021, ibid.; Frankel et al., 2017, ibid.; Gopalakrishnan et al., 2018, ibid.; Lee et al., 2021, ibid.; Matson et al., 2018, ibid.; Routy et al., 2018, ibid.; Baruch et al., Science 371, 602-609 (2021); Davar et al., Science 371, 595-602 (2021); their respective contents are incorporated herein by reference in their entirety. The initial testing focused on a cohort of patients with renal cell carcinoma (RCC) treated with intracellular immunotherapy (ICI) because this patient population reported less antibiotic (Abx) use compared to patients with non-small cell lung cancer (NSCLC) and melanoma, and Abx could serve as an additional modulator of the microbiome and ICI response. Functional profiles from HUMAnN 2 (see, for example, Franzosa et al., Nat Methods 15, 962-968 (2018), the contents of which are incorporated herein by reference in their entirety; see, for example, Supplementary Methods) were analyzed against fecal metagenomics from the RCC cohort (see, for example, Routy et al., 2018, ibid.) to select those who met the Responsive Evaluation of Solid Tumors (RECIST) criteria for partial or complete response and to exclude those with stable disease. The profiles from these samples (n=16) were compared with those from ICI non-responders (n=32) of RCC patients by a least-squares linear model-based differential abundance analysis.

[0322] The MetaCyc superpathway for Saa biosynthesis (PWY-821) is the most enriched metabolic pathway in the ICI responder vs. non-responder microbiome, followed by three other sulfur-related pathways: sulfate reduction 1, the superpathway for L-methionine biosynthesis (PWY-5347), and the superpathway for sulfate assimilation (see, for example, Figure 1A Based on these results, an unbiased meta-analysis was performed on the microbiomes of responders (R) vs. non-responders (NR) from eight ICI-microbiome studies (R, n = 203, NR, n = 134) with melanoma, RCC, or non-small cell lung cancer (NSCLC). Six Saa pathways were among the top 10% of the pathways enriched in ICI responders vs. non-responders (ranked by effect size, Cohen's D test), including the super-pathway for Saa biosynthesis PWY-821 (see, for example, Figure 1B (Table 1). Targeted analysis of superpathways for Saa biosynthetic abundance in each study independently revealed that it was enriched in responder vs. non-responder microbiomes in 7 out of 8 studies (see, for example, Figure 1C All analyzed ICI microbiome cohorts controlled for common covariates such as age, sex, and TNM stage (tumor (T), nodule (N), and metastasis (M)), but not all had available survival or dietary pattern data. To support this approach, inosine-related microbial metabolic pathways produced by lactic acid bacteria and reported to enhance responses to ICI therapy in preclinical cancer models were also enriched in the top 10% of pathways in ICI R vs. NR (see, for example, Table 1; see, for example, Mager et al., Science 369, 1481 (2020), the contents of which are incorporated herein by reference in their entirety).

[0323] To expand the MetaCyc pathway analysis, a group of 15 bacterial gene homologs were examined based on E. coli Saa biosynthesis genes, covering a superpathway for Saa biosynthesis (n = 11 genes), and also including genes from the superpathway for methionine biosynthesis enriched in ICI-responders (see, for example, Figure 1B Saa is ubiquitous in the metagenomics of the human gut. A bacterial pan-genome database containing 15 Saa metabolic gene homologs was generated (standard: e-value 10). -9 (80% coverage), and the fecal metagenomic sequence was aligned with this reference sequence. Consistent with the HUMANN MetCyc analysis, Saa metabolic genes were enriched in ICI responders vs. non-responders (see, for example, in...). Figure 1C (The log2 multiple change is shown in the figure).

[0324] Enrichment of microbiome Saa metabolism gene libraries (carriages) from ICI responders with different cancer types indicates the role of microbial Saa metabolism in antitumor immunity. This provides an opportunity to enhance antitumor immunity in immunologically “cold” tumors, such as mismatch repair intact CRC. Fecal metagenomics were examined from nine CRC cohorts (containing a total of 812 patients (610 CRC patients and 202 adenoma patients) and 639 healthy controls) to analyze Saa metabolism-related gene libraries in CRC patients vs. controls. See, for example, Feng et al., Nat Commun 6, 6528 (2015); Gupta et al., mSystems 4, e00438-19 (2019); Hannigan et al., mBio 9, e02248-18 (2018); Vogtmann et al., PLoSOne 11, e0155362 (2016); Wirbel et al., Nat Med 25, 679-689 (2019); Yachida et al., Nat Med 25, 968-976 (2019); Yu et al., Gut 66, 70-78 (2017); Zeller et al., Mol Syst Biol 10, 766 (2014); Thomas et al., Nat Med 25, 667-678 (2019); their respective contents are incorporated herein by reference in their entirety. Common covariates such as age, sex, and TNM stage were controlled for in all CRC cohorts selected for analysis, but survival data were lacking. To date, no publicly available CRC ICI microbiome profiling studies have been performed. In these histopathologically, genetically, and geographically diverse CRC cohorts, the Saa superpathway and 15 Saa metabolic gene homologs were present and enriched compared to controls in some cohorts (see, for example,...). Figure 1D The shared abundance of these Saa genes in some CRC cohorts is similar to the pattern observed in many ICI responders, leading to the hypothesis that some CRC patients may respond to targeting the Saa pathway.

[0325] Dietary Saa and microbiome regulation of vegetation progression in CRC mouse model.

[0326] Because Saa metabolic genes are enriched in the microbiome of ICI responders (see, for example, Figure 1AFurthermore, diet can influence the microbiome by altering nutrient niche availability, thus increasing interest in microbiome-guided foods for modulating immune system function in cancer patients. As described here, the Saa pathway can be targeted via dietary Saa supplementation, which can then influence antitumor immunity in CRC. Two isocaloric mouse diets were formulated to represent the boundary cases of human Saa consumption: diets with low vs. high methionine and cysteine ​​levels (see, for example, Table 2). The low-Saa diet contained sufficient methionine to avoid methionine restriction, and the high-Saa diet was well below the threshold for hyperhomocysteinemia, thus representing physiologically relevant human Saa consumption that can be achieved through dietary adjustments or supplementation.

[0327] A mouse model of mismatch repair deficient (dMMR) ectopic colonic adenocarcinoma (MC38 cells) that responded to anti-PD-1ICI therapy was used, and the results were examined in a microbiome meta-analysis (see, for example, Figures 1A-1C Wild-type (WT) C57BL / 6 mice housed in animal enclosures were fed either a high- or low-Saa diet for two weeks prior to lateral injection into MC38 cells, and were monitored for 12 days post-injection. Mice fed a high-Saa diet showed slower tumor growth, approximately a 50% reduction in tumor volume, and a 30% reduction in tumor weight (see example,). Figures 1E-1F Since MC38 tumors respond to anti-PD-1 antibody (Ab) therapy, it was necessary to test whether a diet could exert an additive or synergistic effect by binding to anti-PD-1Abs. While a high-Saa diet did not additively or synergistically interact with anti-PD-1Abs, the reduction in terminal tumor volume and weight was similar to that observed with anti-PD-1Ab treatment, indicating that a high-Saa diet has efficacy comparable to ICI therapy in this model (see, for example...). Figures 1G-1H ).

[0328] Next, we will test whether the Saa dietary intervention can affect tumor progression in a gene-driven colon cancer model that is unresponsive to ICI and has intact mismatch repair (pMMR). flox / + Mice were crossbred with CDX2-Cre mice for colon-specific loss of heterozygosity of the tumor suppressor Apc (cAPC mice), as their pMMR status and Apc inactivation reflected two characteristics observed in 85% of CRC patients; see, for example, Hinoi et al., Cancer Research 67, 9721 (2007), the contents of which are incorporated herein by reference in their entirety. At 6–8 weeks of age, cAPC mice were randomly assigned to either a low- or high-Saa diet intervention for 12 weeks. Consistent with findings in the MC38 CRC model, the incidence of advanced vegetation lesions (i.e., adenocarcinoma) was reduced fourfold in cAPC mice fed a high-Saa diet (see, for example, Figure 1I Furthermore, the average weight of these tumors was half that of cAPC mice fed a low-Saa diet (see, for example, Figure 1J These data support the view that dietary Saa supplementation is a regulator of tumor progression in MMR-deficient (dMMR) and pMMR mouse CRC models.

[0329] Dietary Saa regulates the abundance and mucus layer thickness of Mucispirillum schaedleri.

[0330] To determine whether the effects on tumor growth and progression depend on the gut microbiota, cAPC mice were germically reintroduced, and dietary interventions were repeated in germ-free (GF) cAPC mice starting at two months of age. During our experimental period of over 4 months (>4 months), no colonic tumors were observed in either Saa-fed GF mice (see, for example, Figure 5A This supports the microbiome-dependent nature of CRC. In summary, the data point to dietary Saa supplementation as a regulator of tumor progression in dMMR and pMMR mouse CRC models.

[0331] Since tumor development in cAPC mice is microbiome-dependent (see, for example, Figure 5A This study investigated whether the gut microbiota composition was affected by dietary Saa by conducting a 16S rRNA amplicon survey of cecal contents from conventionally bred cAPC mice fed a low-Saa diet versus a high-Saa diet. As expected with such subtle dietary changes, no statistically significant differences in α-diversity were found (see, for example,...). Figure 5B ) or β-diversity separation (see, for example, Figures 5C-5D The abundance of only one taxa changed significantly; Mucispirillum schaedleri was enriched in the cecal contents of mice fed a high-Saa diet (see, for example, Figure 2A and Figures 5E-5F To analyze variations within a specific operational taxonomic unit (OTU) that are independent of feeding conditions, MaAsLin 2 can be used. Although *Ackermania myxophilus* appeared to be increased in the cecal contents of mice on a low-Saa diet (see, for example, Figure 26A), this was not statistically significant and could be attributed to a cage effect (see, for example, 28F).

[0332] *M. schaedleri* is a member of the family Gram-negative anaerobic deferrobacteria, which inhabits the outer mucus layer of the mouse colon; *M. schaedleri* has been detected in up to 42% of human colonic mucosal biopsies; see, for example, Herp et al., Cell Host Microbe 25, 681-694.e8 (2019); Loy et al., mSystem 2, e00171-16 (2017); Robertson et al., Int J Syst Evol Microbiol 55, 1199-1204 (2005); Zmora et al., Cell 174, 1388-1405.e21 (2018); the contents of which are incorporated herein by reference in their entirety. Quantitative (q)-PCR analysis of cecal DNA from cAPC mice confirmed that *M. schaedleri* was increased in mice fed a high-Saa diet versus a low-Saa diet (see, for example, Figure 2B (Left subfigure). The effects were observed in WT mice from animal feeding boxes (see, for example, Figure 2B It is detectable in the intermediate subgraph and observed in altered Schaedler colony (ASF) mice, which contain M. schaedleri as part of its 8-member community (see, for example, Figure 2B (Right sub-figure).

[0333] To assess the human relevance of *M. schaedleri*, *M. schaedleri*-specific reads were retrieved from human microbiome datasets (see, for example, Methods). The analysis did not detect a reliable signal of *M. schaedleri* in human fecal metagenomics. Since *M. schaedleri* is associated with mucus, it is considered that searching for its presence in human mucosal tissue microbiome samples may be more effective than in fecal metagenomic samples. Tissue-related levels of *M. schaedleri* are readily detectable in colon biopsy samples compared to adenomas, and are enriched in normal colon biopsy samples (see, for example, Methods). Figure 2C Such samples and analyses are not available to ICI-responsive patients who have never experienced ICI-induced colitis.

[0334] Given these human mucosal data (see, for example, Figure 2CThe localization of *M. schaedleri* in the mouse colon was examined using bacterial directed fluorescence in situ hybridization (FISH). Since *M. schaedleri* localizes to the mucus layer, it is considered that dietary amino acids (SAA) could influence the abundance of *M. schaedleri* by modulating mucus layer thickness, creating a more favorable ecological niche for *M. schaedleri*. Dietary amino acid supplementation (including L-cysteine) increases colonic mucin synthesis in rats; see, for example, Faure et al., J Nutr 136, 1558-1564 (2006), the contents of which are incorporated herein by reference in their entirety. *M. schaedleri* proliferates extensively in the colonic mucus of mice fed a high-SAA diet (see, for example, Figure 2D ), and the mucus layer also appears thicker (see, for example, Figure 2E ).

[0335] To assess the effects of dietary saa and its microbiota dependence on mucus layer thickness, GF mice were first fed low-Saa and high-Saa diets, and the distal colonic mucus layer was measured by alcine blue staining. In the absence of a microbiota, the high-Saa diet increased the internal mucus layer thickness by 20% (see, for example, Figure 2F , Figure 2H Next, this analysis was extended to examine mice with altered Schaedler colony (ASF) microbiota, including aggregates of the eight defined species, including *M. schaedleri*; see, for example, Wymore et al., ILAR J56, 169-178 (2015), the contents of which are incorporated herein by reference in their entirety. A high Saa diet induced an increase in mucus layer thickness of approximately 50% (see, for example, Figures 2G-2H This indicates that the microbiome amplifies the effect of the high-Saa diet on mucus thickness.

[0336] In these mice, as assessed by qPCR (see, for example, Figure 28E), increased abundance of *M. schaedleri* in the mucus layer was observed in response to a high-Saa diet. qPCR was also performed to quantify *M. schaedleri* levels in the background of the microbiota of more fully cultured mice in the aforementioned dMMR ectopic CRC mice, and these results further confirmed the increased abundance of *M. schaedleri* in mice fed a high-Saa diet (see, for example, Figure 28F).

[0337] The study also investigated whether this difference in mucus thickness altered colonic barrier integrity, since reduced colonic barrier integrity is involved in CRC-promoted smoldering inflammation. However, the FITC-dextran intestinal permeability test did not reveal any difference between low vs. high Saa diet-fed WT mice (see, for example, Figure 5G In summary, these data indicate that dietary Saa levels regulate the abundance of M. schaedleri (a member of the mouse and human microbiome) by influencing the colonic mucus layer and its habitability as an ecological niche for this species.

[0338] A high-Saa diet produces an increased number of CD8+ T cells in cAPC tumors.

[0339] To identify the mechanistic link between combined dietary saa, microbial activity, and antitumor immunity, intratumoral cytotoxic CD8, a key taxonomy of "hot" tumors, was evaluated in tumors from cAPC mice fed high- or low-Saa diets. + T cells. As used herein, the term “hot tumor” refers to a tumor exhibiting a “T cell inflammatory” phenotype, and conversely, the term “cold tumor” refers to a tumor lacking or absent tumor T cell infiltration; see, for example, Bonaventura et al., FrontImmunol 10, 168 (2019), the contents of which are incorporated herein by reference in their entirety. CD8+ in tumors from high-Saa-fed cAPC mice compared to tumors from low-Saa-fed mice. + The frequency and number of T cells are higher (see, for example, Figure 3A CD8 + T cell frequency and number remain relatively constant in adjacent normal lamina propria (LP) and tumor draining lymph nodes (TDLN) (see, for example, Figures 6A-6B CD8 was also observed in tumors from cAPC mice fed a high-Saa diet using immunofluorescence microscopy. + The ratio of T cells increased twofold (see example) Figures 3B-3C CD3 in the surrounding colon LP + CD8 + / CD3 + No difference was observed in cell ratio (see, for example, Figure 6C To gain a deeper understanding of the function of tumor-infiltrating CD8+ T cells enriched during Saa feeding, the expression of multiple immunosuppressive receptors IFNγ and granzyme B (GZMB) was analyzed. IFNγ + and GZMB + CD8 + The proportion of T cells was significantly increased in tumors from mice fed a high-Saa diet (see, for example, Figure 3E ), while PD-1 + and LAG-3 + CD8 + The proportion of T cells was significantly reduced (see, for example, Figure 3D ), indicating these CD8 +T cells were more capable of initiating anti-tumor immune responses. The expression of immune checkpoint markers TIM-3 and CTLA-4 did not differ between the two groups. No differences were observed in the expression of immune checkpoint markers in either LP or TDLN (see, for example, Figures 6D-6E In summary, the tumor microenvironment in mice fed a high-Saa diet was immunologically warmer than that in mice fed a low-Saa diet.

[0340] Because tumor neoantigen diversity can contribute to the frequency and number of tumor-infiltrating T cells, it can affect the intratumoral CD8+ cells from cAPC mice. + T cells were analyzed using TCR-Seq. TCR-Seq analysis did not reveal CD8+ in tumors from cAPC mice fed a low-Saa diet versus a high-Saa diet. + Any difference in the composition or abundance of T cell clonal types (see, for example, Figure 7). Therefore, dietary Saa does not affect TCR diversity in cAPC colon tumors, thus prompting the examination of antigen-presenting cells as a mechanism by which diet influences tumor invasion and CD8. + T cell activation.

[0341] Mucispirillum schaedleri abundance and CD103 in tumor draining lymph nodes (TDLN) + CD11b - Dendritic cell (cDC1) frequency is correlated.

[0342] *M. schaedleri* is considered a mucus-resident bacterium that enhances its interaction with host cells, similar to other mucosa-associated microorganisms (e.g., segmented filamentous bacteria (SFBs)). However, while the ability of SFBs to induce Th17 immune responses has been extensively studied, the immunomodulatory role and underlying mechanisms of *M. schaedleri* remain poorly characterized. *M. schaedleri* in Nod2... - / - x Cybb - / - T induction in mice HType 1 colitis and plays a role in the development of peripheral regulatory T cells (Tregs); see, for example, Caruso et al., Sci Immunol 4, eaaw4341 (2019); Campbell et al., Immunity 48, 1245-1257.e9 (2018); their respective contents are incorporated herein by reference in their entirety. To address what immunomodulatory effects can be induced by M. schaedleri in a diet-CRC model, immune cell populations in the colonic LP and TDLN of cAPC mice fed a Saa diet and in the colonic LP and MLN of mice cultured in an in-house WT facility (BIH) were analyzed (see, for example, gating strategy in Figure 8). CD4 + Analysis of the major subsets of T cells (including Th1 cells, Th2 cells, Th17 cells, and Tregs) did not reveal any differences in colonic LP or MLN between mice fed a low or high Saa diet (see, for example, Figure 9).

[0343] Conversely, analysis of myeloid cells revealed CD103 in the TDLN of cAPC mice fed a high-Saa diet. + CD11b - The frequency and number of dendritic cells (called cDC1) were significantly increased, while other CD11c cells were significantly reduced. + The cell population remains unchanged (see, for example, Figure 3G ). LP and tumor CD11c + Cell frequency and number remained unchanged between low- and high-Saa-fed cAPC mice (see, for example, Figures 10A-10B cDC1 cells are initiators of antitumor immunity because they cross-present tumor-derived antigens in TDLNs to elicit an antitumor response in CD8+ T cells. Similar results were obtained for myeloid cell populations in the LP and MLNs of WT BIH mice fed a Saa diet (see, for example, Figures 10C-10D To determine whether the increase in cDC1 was dependent on microbial factors, germ-free (GF) mice were fed low- and high-Saa diets, and their immune cell populations were analyzed. In GF mice fed a high-Saa diet, cDC1 was not increased in either the MLN or LP (see, for example, Figures 10E-10F Using data from all conventionally bred mice (WT and cAPC), a significant positive correlation was found between M. schaedleri cecal abundance and MLN / TDLN cDC1 group frequency (Spearman p = 0.585, p = 0.0002) (see, for example, Figure 10G ).

[0344] Next, three methods were used to assess whether *M. schaedleri* contributed to the increase of cDC1. First, ASF mice containing *M. schaedleri* were fed either a low- or high-Saa diet, and an increase in cDC1 was observed only in the high-Saa diet-fed MLN group (see example). Figures 10H-10I These data suggest that eight ASF conglomerates, including *M. schaedleri*, are sufficient to increase cDC1 in the presence of a high-Saa diet. Next, GF mice were individually colonized with either *M. schaedleri* or *Ackermania myxophilus*, and both groups were fed a high-Saa diet. *Ackermania myxophilus* was chosen because it is also a mucus-resident bacterium with immunomodulatory properties associated with ICI responses. The frequency and quantity of cDC1 increased only in the MLN of mice colonized with *M. schaedleri*, but not in those colonized with *Ackermania myxophilus*, suggesting that not only the presence of intestinal colonization itself is required for this observed cDC1 effect (see, for example,...). Figure 3H and Figure 10J Third, since many bacteria secrete bioactive molecules, the effects of abiotic M. schaedleri conditioned medium (“CDM” or “CM”) on cDC1 frequency in the MLN of C57BL / 6BIH mice were tested. Aggressive feeding of M. schaedleri CDM over a two-week period increased cDC1 frequency in the MLN, while the abiotic medium control had no effect (see, for example, Figure 3I These results indicate that M. schaedleri and the specific factors it secretes are sufficient to increase the frequency and quantity of MLN / TDLN cDC1.

[0345] Depletion of cDC1 or XCL1 eliminates the antitumor effects of a high-Saa diet.

[0346] Testing cDC1 cells in CD8 against the background of CRC +The role of T cell-mediated antitumor immunity. To determine whether cDC1 cells mediate the aforementioned high-Saa diet-dependent and M. schaedleri-dependent effects on antitumor immunity and tumor growth as described above, three methods were used to deplete cDC1. First, bone marrow chimeric mice were generated using hematopoietic cells from Zbtb46-DTR mice that express diphtheria toxin receptor (DTR) under the regulation of a cDC1-specific promoter upstream of Zbtb46 and allow diphtheria toxin-mediated cDC1 depletion; see, for example, Meredith et al., J Exp Med 209, 1153-1165 (2012), the contents of which are incorporated herein by reference in their entirety. These cAPC Zbtb46-DTR mice were fed a high-Saa diet (which drives M. schaedleri expansion and subsequent MLN-TDLN cDC1 increase) and began receiving diphtheria toxin (DT) injections (or PBS injections as a control) 4 weeks after irradiation and engraftment to deplete cDC1. The expected reduction in TDLNcDC1 levels was observed in DT-treated mice, while other CD11c levels were lower. + The frequency and number of cells remain unchanged (see, for example, Figure 11A There was no difference in the abundance of *M. schaedleri* in the cecum between the DT and PBS treatment groups (see, for example, Figure 11B cDC1 depletion increases the frequency of vegetation lesions, even in the presence of a high Saa diet (see, for example, Figure 4A This finding further supports the role of cDC1 as an antitumor immunomodulator and a mediator of vegetation progression, and confirms the function of cDC1 in mediating the antitumor effect of high-Saa diet-induced proliferation of M. schaedleri.

[0347] As a second method for depleting cDC1 in cAPC mice, the cAPC line is crossed with Batf3 knockout (KO) mice to produce cAPC Batf3. - / - Mice. Like Zbtb46, Batf3 is a key transcription factor for the development of cDC1. Batf3 for cAPC - / - Mice were fed either a high- or low-Saa diet, and as expected, the frequency and number of cDC1, rather than cDC2, in the TDLN were reduced in both groups (see, for example, Figure 11C Higher abundance of *M. schaedleri* was also detected in the cecal contents of mice fed a high-Saa diet compared to mice on a low-Saa diet (see, for example, Figure 11D ). Figure 4ASupporting findings include that Batf3 deficiency leads to loss of the protective effect of a high-Saa diet, since there was no difference in tumor number or vegetation progression between the two dietary groups (see, for example, Figure 4B Since the data supports that cDC1 is a key link connecting the aforementioned Saa diet, M. schaedleri, and antitumor immunity (see, for example, Figures 3F-3I and Figures 4A-4B The study examined whether *M. schaedleri* directly acts on cDC1. In vitro assays of non-biological *M. schaedleri* CM against cDC1 do not support its ability to directly enhance CD8 by increasing the expression or cross-presentation of co-stimulatory molecules. + T cell cDC1 activation (see, for example, Figures 11E-11G ).

[0348] To determine what drives the observed effects against cDC1, further testing focused on factors known to activate and recruit cDC1, namely lymphocyte chemokine (XCL1). In a third approach to interfering with cDC1, treatment with an α-XCL1 antibody (Ab) was used to deplete this cDC1-specific chemokine and cytokines in a facility with a high Saa diet; see, for example, Matsumoto et al., J Immunol 199, 82-90 (2017); Lei et al., Microbes Infect 14, 262-267 (2012); their respective contents are incorporated herein by reference in their entirety. cAPC mice treated with α-XCL1 Ab had reduced serum XCL1 levels and lower cDC1 levels in their TDLN (see, for example, Figures 11H-11I Decreased XCL1 levels led to a significant increase in adenocarcinoma in cAPC mice fed a high-Saa diet (see, for example, Figure 4C Even though α-XCL1Ab only reduces serum levels by an average of two-fold (see example, Figure 11H In summary, these experiments confirm the immunomodulatory role of cDC1 in tumor growth in cAPC mice and link diet-microbe interactions with antitumor immunity.

[0349] Dietary Saa and M. schaedleri induce XCL1 secretion from natural killer T (NKT) cells.

[0350] Increased MLN / TDLN cDC1 levels were observed in M. schaedleri mice fed a high-Saa diet, prompting investigation into factors driving cDC1 accumulation and activation. Testing focused on XCL1, as it plays a role in the protective effect of a high-Saa diet in cAPC mice (see, for example, Figure 4CFurthermore, cDC1 selectively expresses the XCL1 receptor XCR1. Both NK cells and NKT cells can secrete XCL1, thereby directing cDC1 expression on tissue CD8+. + The role of T cell in antitumor immunity; see, for example, Barry et al., Nat Med 24, 1178-1191 (2018); et al., Cell 172, 1022-1037.e14 (2018); their respective contents are incorporated herein by reference in their entirety. To determine whether XCL1 secretion in these cell types is affected by dietary Saa, NK and NKT cells from the MLN of BIH C57BL6 WT mice fed two Saa diets were sorted; NKT cells from mice fed a high Saa diet secreted higher levels of XCL1 in vitro compared to NKT cells from mice fed a low Saa diet (see, for example, Figure 4D Regardless of diet, NK cells secrete less XCL1 than NKT cells (see, for example, Figure 4D The frequency of TDLN NKT cells, rather than NK cells, was increased in cAPC mice fed a high-Saa diet versus a low-Saa diet (see, for example, Figure 4E , Figure 11J This indicates the frequency and amount of XCL1 secreted by NKT cells induced by a high-Saa diet and its increased presence in TDLNs.

[0351] To test whether *M. schaedleri* could directly affect XCL1 production in NKT cells, an in vitro system using the NKT cell line GW1 (see, for example, Donado et al., Cell Rep 31, 107466 (2020); the contents of which are incorporated herein by reference in their entirety) and non-biological bacterial conditioned media (CDM) was utilized. While CDM derived from two immunomodulatory gut bacteria (*Lactobacillus plantarum* and *Akermansia myxotroph*) had no effect, *M. schaedleri* CDM significantly stimulated XCL1 production to a degree comparable to that of IL-12 treatment (see, for example,...). Figure 4F The XCL1-stimulatory activity of *M. schaedleri* CDM is thermally unstable and is primarily found in the organic phase of the supernatant (see, for example, Figure 4F Since GW1 cells are a mouse cell line, the findings were then extended to human NKT cells. NKT cells were expanded from peripheral blood mononuclear cells from healthy human donors; these NKT cells were then treated with *M. schaedleri* CDM. Compared to untreated NKT cells, the treated cells showed an approximately 4-fold increase in XCL1 (see, for example, Figure 4GIn summary, these data indicate that XCL1 production derived from NKT cells contributes to the accumulation of activated cDC1 in MLN / TDLN, and that dietary and microbial factors trigger this NKT XCL1 production in mouse and human NKT cells.

[0352] To gain a deeper understanding of the *M. schaedleri* activity driving NKT XCL1 secretion, untargeted LC-MS / MS metabolomics were performed on non-biological *M. schaedleri* CM samples (n = 4 independent samples) and culture medium controls without *M. schaedleri* (n = 2 independent samples). Of the 4263 features examined, relatively few features were enriched in CM (n = 36), with fold changes of 2 or higher and p-values ​​below 0.05. A one-way ANOVA using Tukey's HSD test (see, for example, in this paper) was performed post-hoc. Figure 4H and Figure 4I (and Table 3 of U.S. Provisional Application No. 63 / 389,382). Consistent with the extraction results (see, for example, Figure 4F Many known or predicted metabolic features enriched in abiotic CM of M. schaedleri are lipids and lipid components (e.g., fatty acids) (see, for example, in this article). Figure 4H and Figure 4I (and Table 3 of U.S. Provisional Application No. 63 / 389,382). Not wishing to be bound by theory, this paper considers the synergistic effect of multiple *M. schaedleri* metabolites to enhance NKT XCL1 secretion, with XCL1 acting on cDC1 via CD8. + The T-cell compartment promotes its anti-tumor immunity.

[0353] Activated transcriptional signatures in TDLN cDC1 of cAPC mice fed a high-Saa diet were associated with better survival in CRC patients.

[0354] In the ICI-responsive microbiome, high-Saa diet, and *M. schaedleri* studies described in this article, cDC1 emerged as a convergence point for understanding diet-microbiome-immune stimulation interactions in CRC. Therefore, to clarify the effects of dietary Saa-*M. schaedleri* on cDC1, single-cell RNA sequencing (scRNA-Seq) was performed on FACS-sorted DCs from the TDLN of cAPC mice fed low- and high-Saa diets. The TDLN is a key immunomodulatory site, where events related to antitumor CD8... +For T cells, cDC1 cross-presentation is a key event. Furthermore, these events are readily available for study in mice and have not yet been analyzed in human CRC scRNA-seq studies, thus representing an underutilized cell population with potential prognostic uses.

[0355] For ~12,500 CD11c + MHCII + CD64 - Cell sequencing revealed substantial cellular heterogeneity with six DC clusters, as defined by an artificially designed examination of known DC markers (see, for example, Figure 12A (Table 4). The largest cluster includes CD11b. + Cells, representing cDC2, macrophages, and monocytes, followed by clusters of migrating DCs (expressing CCR7), and DC-SIGN. + (CD209)DC clusters, and cDC1 clusters identified by expression of the Xcr1, Clec9a, and Irf8 genes (see, for example, Figures 12B-12C Rapid gene set enrichment analysis of Hallmark gene set pathways (fgsea) revealed that the inflammatory response pathway (M5932) was enriched in cDC1 in TDLN isolated from mice with a high-Saa diet-M. schaedleri (Ms) amplification (see, for example, Figure 12D Cell cycle-related pathways were enriched in cDC1 from mice fed a low-Saa diet (see, for example, Figure 12D ).

[0356] Differentially expressed genes were identified in cDC1 from mice on low-Saa diets versus high-Saa diets (see, for example, Table 5). cDC1 from high-Saa diet-Ms amplification (HSME) mice expressed higher levels of Cxcl9 and Ccl4 transcripts (see, for example, Table 5). Figure 4J Both play a crucial role in antitumor immune responses. Other transcripts expressed at high levels in cDC1 from HSME mice include genes encoding calcium-binding proteins S100A6 and S100A4, which function in efficient DC antigen presentation, co-stimulatory molecule expression, and T cell activation (see, for example, Figure 4J Genes that are more highly expressed in cDC1 from mice fed a low-Saa diet (or reduced with a high-Saa diet) include the thioesterase Ppt1, which balances viral resistance vs. T cell cross-activation (see, for example...). Figure 4J(Table 5). Reduced Ppt1 expression enhanced tumor clearance in mice. Overall, scRNA-Seq analysis showed that cDC1 from HSME mice exhibited higher activation and transcription of immunostimulatory genes, consistent with the finding that a high-Saa diet limited tumor growth and reduced tumor growth progression.

[0357] Using differentially abundant cDC1 genes, cDC1 gene expression signatures associated with beneficial antitumor effects dependent on a high-Saa diet in M. schaedleri were generated (see, for example, Table 5). The association of these signatures with survival data from the Tissue Cancer Genome Atlas (TCGA) dataset of large-scale RNA-Seq transcriptomes of colorectal adenocarcinoma tumors (TCGA COAD-READ, N=359) was examined. Patients with transcriptome scores more similar to HSME mouse cDC1 transcriptional status (HSME-DC; “high activation”) had statistically significantly longer survival compared to patients with transcriptional scores similar to those of low-Saa-M. schaedleri depletion (LSMD; “low activation”) mice (Cox model, univariate analysis P=0.005, disease stage-adjusted multivariate analysis P=0.017) (see, for example, Table 5). Figure 4K For patients with tumor gene expression more similar to LSMD-DC, univariate analysis showed an increased hazard ratio of 1.611 (1.152–2.253, 95% confidence interval (CI)), and a hazard ratio of 1.521 (1.077–2.147, 95% CI) in multivariate analysis including stage. Therefore, gene expression signatures detected in cDC1 from HSME cAPC mice are associated with improved survival in CRC patients. discuss

[0358] In the search for microbial bioactivity implicit in antitumor immunity, the enrichment of Saa pathways and genes in the gut microbiome has been identified as a shared feature of ICI responsiveness across cohorts. Targeting these pathways through dietary Saa supplementation slows tumor progression in both MMR-deficient and MMR-intact CRC models. Methionine dependence has been reported in CRC tumor cells, and methionine restriction has limited tumor growth in mouse models. Such results raise the question: why was reduced tumor growth observed in the high-Saa diets discussed in this paper? This paper considers that very low or high Saa diets may be tumor-repressive for different underlying reasons, with low Saa diets limiting cancer cell growth due to metabolic dependence, while high Saa diets enhance antitumor immunity, as described in this paper. Low Saa diets are insufficient to limit methionine to affect tumor growth in this paper. Dietary sulfur takes many different forms in organic and inorganic compounds; for example, total dietary sulfur intake has shown a correlation with human colonic adenomas and CRC. Dietary Saa regulation also has pleiotropic effects on the host and microbiome, ranging from altering redox potential to post-translational protein modifications, all within a dose range that is non-toxic to the host. Given the antitumor immune effects observed in this study, clinical studies of Saa supplementation in a cohort of CRC patients are warranted. Furthermore, given the enrichment of this pathway in the microbiome of ICI-resistant melanoma patients who have received fecal microbiota transplantation and become ICI-responsive, further investigations could be conducted in ICI-refractory patients. See, for example, Gao et al., Nature 572, 397-401 (2019); Hoffman (Humana Press, 2019); Komninou et al., Nutr Cancer 54, 202-208 (2006); Tan et al., Clin Cancer Res 5, 2157-2163 (1999); Nguyen et al., Gastroenterology 158, 1313-1325 (2020); Nguyen et al., Gastroenterology 161, 1423-1432.e4 (2021); Yan et al., Journal of Biological Chemistry 285, 41525-41532 (2010); Lobel et al., Science 369, 1518-1524 (2020); Baruch et al., 2021, ibid.; their respective contents are incorporated herein by reference in their entirety.

[0359] This article describes the identification of common functional features across multiple gut microbiota taxa associated with ICI responses in patients, which can be targeted to improve antitumor immunity. This article also describes a dietary intervention targeting the Saa pathway, which attenuates tumor growth in malignant CRC, which is often resistant to ICI therapy and considered immunologically cold. The dietary intervention initiates the mucin-M. schaedleri-antitumor immune cascade (in... Figure 13 (Simplified Chinese) This indicates that functionalized foods can improve antitumor immunity in CRC. M. schaedleri data suggest that mucus is a link between diet, microbiota, and immune function, and that the colonic mucus layer is a microbial active site involved in antitumor immunity and immune function. Furthermore, the immunomodulatory effects of M. schaedleri focus on NKT cells as a link between diet, gut microbiota factors, and CD8 aggregation. + The link between enhanced cDC1 cell function on T cells and other pathways. These findings confirm the role of cDC1 in antitumor immunity. The cDC1 depletion data presented in this paper, combined with data on XCL1 depletion (which is a cDC1-specific chemokine and activator), demonstrate the role of cDC1 in linking enhanced immunity against Saa, M. schaedleri, and CRC.

[0360] In summary, the data presented in this paper highlight the utility of mouse models in exploring diet-microbe interactions, not only for identifying biogeography such as the mucus layer or tumor-draining lymph nodes, but also for identifying prognostic features of CRC located at the intersection of diet, microbiome, and host immunity. In conclusion, the experiments presented in this paper elucidate how diet-microbiome interactions enhance antitumor immunity in a multi-step manner, thereby modulating innate immune cell responses to control tumor progression in CRC. Overall, the data presented in this paper provide an examination of the fecal metagenomic profiles of ICI-responsive patients to identify the mechanisms by which they enhance antitumor immunity against immunologically cold tumors through microbiome-dietary interactions. Materials and methods

[0361] Meta-analysis of microbial metabolic pathways in the microbiomes of antiPD-1 therapy responders vs. non-responders.

[0362] Shotgun reads of fecal metagenomics were obtained from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) for the ICI and CRC datasets. Any metagenomic samples known to have used antibiotics (as described in the available clinical meta-analyses) were excluded from the meta-analysis. Raw reads were quality-trimmed using TRIMMOATIC (v0.39), configured to perform a sliding window scan with the following parameters: “ILLUMINACLIP:${adapter_library_FASTA}::2:36:7:1:keepBothReads LEADING:3TRAILING:3SLIDINGWINDOW:4:15MINLEN:36.”. To construct an expanded set of reference genes for bacterial Saa biosynthesis from the MetaCyc pathway database, 72,380 complete and draft-level bacterial GENBANK genome assemblies with sufficient taxonomic coverage across representative human gut microbiome genera were accessed. A priority list of 15 Saa protein sequences was integrated for a custom BLAST+ search (options: e-value 1e-09, coverage 80%), and the protein-coding regions of each assembly were iteratively annotated using PROKKA (a fast hierarchical genome annotation pipeline), resulting in the detection of 484,023 bacterial Saa gene homologs. See, for example, Bolger et al., Bioinformatics 30, 2114-2120 (2014); Xiao et al., Nature Biotechnology 33, 1103-1108 (2015); Seemann, Bioinformatics 30, 2068-2069 (2014); their respective contents are incorporated herein by reference in their entirety.

[0363] When readings were mapped using BWA-MEM v0.7.17 with default parameters and quantified using htseq-count (the default co-occurrence method) from HTSeq, these Saa were found to utilize 120,861 non-zero abundances from gene homologs. The gene read count matrix was constructed by aggregating total gene abundance (reads per kilobase; RPK) across the bacterial genome. These data, along with pathway relative abundance distributions generated using HUMAnN2, were used to fit a linear mixed-effects model (LMM) with the study effect as a random covariate, as implemented in the lmerTest R / CRAN package. Effect sizes were derived from fitted LMMs of pathways present in at least 20% of the samples using the emmeansR / CRAN package. Rank-based combined mean fold changes guided by quartile ranges were calculated to correlate with the biomes of interest. See, for example, Li, arXiv1303.3997v2 (2013); Anders et al., Bioinformatics 31, 166-169 (2015); Kuznetsova et al., Journal of Statistical Software 82, (2017); Russell, emmeans: Estimated Marginal Means, aka Least-Squares Means (2021); Xiao et al., Bioinformatics 30, 801-807 (2014); their respective contents are incorporated herein by reference in their entirety.

[0364] Mice and dietary intervention.

[0365] Mice (WT C57BL / 6, CDX2-Cre APC) were used flox / + (cAPC), Zbtb46-DTR, cAPC Batf3 - / - and Ptprc a(Ly5.1, CD45.1) mice were housed in a barrier facility with a constant ambient temperature of 24°C and a 12-hour day / night cycle. Institutionally born (BIH) mice were routinely housed, specific pathogen-free (SPF) C57BL6 / J mice bred in barrier facility animal housings. All mouse strains were purchased from Jackson Laboratory and then bred in-house. For gnotobiotic experiments, mice were housed in semi-rigid isolators (PLASTIC CONCEPTS INC.) and experiments were conducted in a separate, ventilated ISOCAGEP system (TECNIPLAST). Fecal samples and cage swabs were routinely monitored, including 16S rRNA gene amplicon sequencing and qPCR analysis (using universal 16S rDNA primers) and Sanger sequencing, to validate gnotobiotic status (sterile, monotrophic, or ASF). To regenerate cAPC mice, pregnant female mice were euthanized and their uteruses were removed under aseptic conditions using a chemical sterilizer (MB-10, QUIP LABS) in a semi-rigid isolator. Germ-free pups were introduced into germ-free foster mothers. After 3 weeks, the germ-free status and genotype of the mice were assessed using PCR.

[0366] Sulfur amino acid (Saa) diets are formulated to represent the boundary conditions of Saa consumption, taking the following into consideration. Human dietary cysteine ​​and methionine consumption is typically between 0.03 and 0.06 g / kg body weight / day. In human diets within the protein consumption range (44 g–140 g / day), low cysteine ​​levels are between 0.01 and 0.04 g / kg / day. Very high levels of methionine or cysteine ​​are ≥6 g / kg / day; such levels raise concerns about hyperhomocysteinemia in humans. See, for example, Elshorbagy et al., J Nutr Biochem 23, 332-340 (2012); Paul et al., Nature 509, 96-100 (2014); O'Keefe et al., J Nutr 137, 175S-182S (2007); O'Keefe et al., Nat Commun 6, 6342 (2015); Nimni et al., Nutr Metab (Lond) 4, 24 (2007); David et al., Nature 505, 559-563 (2014); the contents of each of these are incorporated herein by reference in their entirety. Based on these data and with veterinary approval, the two isocaloric diets used in this paper were formulated and manufactured by RESEARCH DIETS, INC. (see, for example, Table 2, for dietary formulations). For the gnotobiotic experiments, the same irradiated formulations were ordered from TEST DIET.

[0367] At 6-8 weeks of age, administer WT, cAPC, and cAPC Batf3. - / - Alternatively, cAPC Zbtb46-DTR mice were transitioned to a Saa diet. After 12 weeks on the Saa diet, mice were sacrificed and tissues (normal, vegetation, and adjacent normal) were collected for histology, flow cytometry, or immunofluorescence, and cecal contents were frozen for microbiological analysis. For gnotobiotic experiments, mice monoplanted with GF, ASF, M. schaedleri, or Akkermansia myxophila were transitioned to a Saa diet at 6–8 weeks of age. After 4 weeks, mice were sacrificed and tissues were analyzed by flow cytometry. GF cAPC mice were transitioned to a Saa diet at weaning and maintained for 12 weeks. In bacterial CDM feeding experiments, WT-cultured mice on a low-Saa diet were force-fed every two days with 100 μL of filtered culture supernatant of 5–7-day-old M. schaedleri or sterile mBHI medium.

[0368] To generate cAPC Zbtb46-DTR mice, cAPC mice were irradiated with a single dose of 1000 rad at 10 weeks of age, followed by injection of 10 rads of [unclear - possibly a specific drug or treatment] from Zbtb46-DTR mice. 6 Bone marrow cells. To deplete Zbtb46-expressing cells in cAPC Zbtb46-DTR bone chimera mice, mice were injected with 400 ng (~20 ng / g body weight) of diphtheria toxin (DT) 4 weeks after irradiation, followed by injections of 100 ng of DT (~4 ng / g body weight) twice weekly to maintain Zbtb46-expressing cell depletion for the remainder of the experiments. For XCL1 depletion, cAPC mice were fed a high-Saa diet for 8 weeks, followed by intraperitoneal injections of 300 μg of anti-XCL1 antibody or InVivoMAb rat IgG1 isotype control, or anti-trinitrophenol (BIOXCELL) every 2–3 days for 4 weeks. Animal studies and experiments have been approved and conducted in accordance with animal use and care guidelines.

[0369] MC38 colon cancer lateral abdominal tumor model.

[0370] MC38 mouse colon cancer cells (KERAFAST) were grown in RPMI GLUTAMAX medium supplemented with 10% fetal bovine serum (FBS), 500 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol. Cells were harvested at ~80% confluence, washed in PBS, and then resuspended on ice at a concentration of 3 × 10⁶ cells / mL in a 1:1 solution of CULTREX growth-reducing basement membrane extract (R&D SYSTEMS) and PBS. Two weeks prior to MC38 engraftment, C57BL / 6J mice bred at the WT facility were placed on a low- or high-Saa diet. On the day of engraftment, the left flank of the mice was shaved, and 150 μL (0.5 × 10⁶ cells / mL) was subcutaneously injected. 6 MC38 cell solution (number of cells). Mice were monitored every other day, using a volume = (width) 2 The tumor volume was calculated as (×length) / 2 (Equation 1). Twelve days post-implantation, mice were sacrificed, and the tumors were excised and weighed. For the anti-PD-1 experiment, mice were treated as described above and intraperitoneally injected on days 6, 9, and 12 with either INVVOMAB anti-mouse PD-1 (BIOXCELL) or INVVOMAB rat IgG2a isotype control anti-trinitrophenol (BIOXCELL), 250 μg / mouse. Mice treated with isotype or anti-PD-1 were sacrificed on the thirteenth day post-implantation, and tumor weight was measured. See, for example, Corbett et al., Cancer Res 35, 2434-2439 (1975); Faustino-Rocha et al., Lab Anim (NY) 42, 217-224 (2013); their respective contents are incorporated herein by reference in their entirety.

[0371] Histopathology.

[0372] After euthanasia, the colon was opened with blunt scissors and its contents were removed. The cecal contents were rapidly frozen in liquid N2. The tissue was fixed in 4% paraformaldehyde, processed using standard protocols through the rodent histopathology core, and paraffin-embedded. Five hematoxylin and eosin (H&E) stained slides (five horizontal sections obtained at 50 μm intervals) from each sample were blind-assessed by a committee-certified GI pathologist for vegetation lesions—abnormal crypt lesions (ACF), adenomas, or adenocarcinomas (adenoCA).

[0373] DNA was extracted from the cecum and analyzed by real-time quantitative PCR (RT-qPCR).

[0374] Mouse cecal contents were collected into 1.5 mL tubes and immediately frozen in liquid N2. The thawed cecal contents were resuspended in 300 μL of Tris-EDTA solution (100 mM Tris and 15 mM EDTA) in a 2 mL tube with ~300 μL zirconium beads (20 μm), and 500 μL of TE-saturated phenol (SIGMA-ALDRICH) was added. The tube was placed in a bead grinder for 2 minutes and centrifuged at maximum speed for 15 minutes. The aqueous phase was transferred to a clean tube and phenol:chloroform:isoamyl alcohol (25:24:1) was added in a 1:1 volume ratio. The tube was vortexed for 1 minute and then centrifuged at maximum speed for 2 minutes. This process was repeated 3 times. The aqueous phase was transferred to a clean tube and 2 volumes of 100% ethanol (EtOH) and 1 / 10 volume of sodium acetate (NaOAc) (pH 5.2) were added. After 1 hour at -20°C, the tubes were centrifuged at 14,000 rpm for 20 minutes at 4°C, the liquid was discarded, and 1 mL of cold 70% EtOH was added to wash the precipitate. The tubes were then centrifuged again at 14,000 rpm for 20 minutes at 4°C. Finally, the liquid was aspirated, and the precipitate was air-dried at room temperature for 10 minutes, then resuspended in 100 μL of sterile molecular biology grade H2O. DNA concentration was measured spectrophotometrically at 260 nm. For RT-PCR analysis, 50 ng of cecal DNA was mixed with 10 μL of 2X SYBR Green (KAPPA SYBR FAST) and 0.29 μM of each forward / reverse primer set (see, for example, Table 6) in 20 μL of reaction mixture. Real-time PCR was performed on an APPLIEDBIOSYSTEMS STRATAGENE MX3005P instrument. 2 -((XaCt-XbCt)-(YaCt-YbCt)) (Equation 2) Calculate ΔΔCt, where X and Y are genes, and a and b are biological samples, with b being the mean of the reference sample or biological replicate. Raw data were extracted and analyzed using the LIBREOFFICE Calc program and RStudio.

[0375] Generation and sequencing of bacterial 16S rRNA gene amplicon library.

[0376] The procedures in this section were performed in a biosafety cabinet to minimize potential contamination and were based on the EARTHMICROBIOME PROJECT protocol. 50 ng of cecal DNA was used in the PCR reaction using THERMO FISHER PLATINUM HOT START PCRMASTER MIX (catalog number 13000014) according to the reagent protocol. Forward (10 μM) and reverse (1.3 μM) primers (see, for example, Table 6) were used to amplify the V4 region of the 16S rRNA gene. For each sample, the reverse primer contained a unique 12 bp GOLAY barcode. Each sample was amplified in triplicate in a 25 μL reaction volume in a 96-well plate. Sterile molecular biology grade water and E. coli genomic DNA served as negative and positive controls, respectively. The PCR reaction was initiated at 94 °C for 3 min, followed by 35 cycles of 45 sec at 94 °C, 60 sec at 50 °C, and 90 sec at 72 °C, and then at 72 °C for 10 min. After amplification, triplet reactions were combined, and amplicones were purified using AMPURE magnetic beads. DNA concentration was determined using a QUBIT machine via a dsDNA wide-range assay kit (THERMO FISHER), and samples from multiple libraries were run on agarose gels to visualize specific amplicones. Libraries were merged to achieve a final DNA concentration of 50 ng / μL, with each library exhibiting equal abundance. The merged amplicon libraries were analyzed on an AGILENT 4200TAPESTATION system. DNA sequencing was performed at the biopolymer core on an ILLUMINA MISEQ machine using the MISEQ V2 kit with 250 bp end-paired reads. See, for example, Thompson et al., Nature 551, 457-463 (2017); Walters et al., mSystems 1, e00009-15 (2016); their respective contents are incorporated herein by reference in their entirety.

[0377] Analysis of bacterial 16S rRNA gene amplicon survey in mice.

[0378] 16S rRNA gene amplicon survey analysis was performed using the microbiome-helper software package according to the standard operating procedure (SOP) of Comeau et al., mSystems 2, e00127-16 (2017). Briefly, fastq files were obtained for each library (median end-paired read count: 79584 250 bps), and read quality was assessed using FastQC (v0.11.5). Based on the sequence quality report, reads were trimmed using the fastx-toolkit to retain only high-confidence base calls and spliced ​​using PEAR. Chimeric reads were filtered using VSEARCH. Operational taxonomic units (OTUs) were selected using QIIME (v1.9) with SortMeRNA filtering, and OTUs with less than 0.1% reads were excluded as low-confidence. Finally, the OTU read count data were reduced to the minimum library size. Alpha- and β-diversity (weighted UnifracPCoA) analyses were performed and visualized using the phyloseq R / Bioconductor (v1.30) and metaencoder R / CRAN software packages. Differential OTU abundance between the two diets was analyzed using MaAsLin 2 with cages as random covariates. To measure the abundance of *M. schaedleri* in human mucosa-associated bacteria amplicon samples, 16S rRNA gene sequences were analyzed using the mothur software suite following 454 and the ILLUMINA MISEQ SOP. See, for example, Comeau et al., mSystems 2, e00127-16 (2017); Andrews, FastQC, available online: bioinformatics.babraham.ac.uk / projects / fastqc / (2010); Zhang et al., Bioinformatics 30, 614-620 (2014); Rognes et al., PeerJ 4, e2584 (2016); Caporaso et al., Nat Methods 7, 335-336 (2010); Kopylova et al., Bioinformatics 28, 3211-3217 (2012); McMurdie et al., PLoS One 8, e61217 (2013); Schloss et al., Appl Environ Microbiol 75, 7537-7541 (2009); the contents of each of these are incorporated herein by reference in their entirety.

[0379] Alixin Blue staining.

[0380] Unopened colon sections were fixed overnight in a methanol-Carnoy's solution (6:3:1 methanol:chloroform:glacial acetic acid), followed by routine paraffin embedding and sectioning. Sections were dewaxed, hydrated, and stained with 1% Alcian blue solution to visualize the mucus layer, then counterstained with nuclear solid red (AMRESCO 1B1369). HISTOMOUNT (NATIONALDIAGNOSTIC HS-103) was used as the mounting medium, and images were acquired using a NIKON ECLIPSE NI-U microscope.

[0381] Immunofluorescence staining and FISH.

[0382] To detect CD3 and CD8 positive T cells in mouse colonoma tissue, immunofluorescence staining was performed on formalin-fixed paraffin-embedded 5-micron sections of mouse colon tissue. The colon sections were dewaxed, pretreated with 3% hydrogen peroxide in methanol, and then subjected to antigen retrieval with 1 mM EDTA pH 8.0 (at >90°C for 15 minutes). After blocking in Tris-buffered saline (TBS) with 10% donkey serum and 1% BSA, the sections were stained overnight (O / N) at 4°C with anti-CD3 (1:3400, ABCAMab5690) and anti-CD8 (1:2500, EBIOSCIENCE 14-0808-80). After washing, donkey anti-rat ALEXA FLUOR 594 IgG (JACKSON IMMUNORESEARCH 712-585-153) was administered to detect CD8, followed by donkey anti-rabbit HRP IgG (JACKSON IMMUNORESEARCH 711-035-152), and then TSA fluorescein reagent (1:2200, NEL741E001KT, PERKINELMER) to reveal CD3-positive cells. 4′,6-diamidinyl-2-phenylindole (DAPI) was used as the nuclear counterstain, and PROLONG GOLD anti-fading agent was used as the mounting medium (P36934, LIFE TECHNOLOGIES). Images were acquired on a NIKON ECLIPSE TI laser scanning microscope with 20X and 60X objectives.

[0383] For the detection of Mucispirillum spp. and visualization of the mucus layer and goblet cells, unopened terminal colon sections were fixed overnight in methanol-Carnos fixative, followed by routine paraffin embedding and sectioning. Colon sections were dewaxed, pretreated with 3% hydrogen peroxide in methanol, and blocked with TRUEBLACK LIPOFUSCINAUTOFLUORESCENCE QUENCHER (BIOTIUM 23007) as recommended by the manufacturer. In 5% formamide-0.1% SDS-TBS buffer, fluorescence in situ hybridization was performed at 50°C for 90 min using the following probes: 2.5 ng / μL of each Mucispirillum genus-specific probe MCS487 (5'-Cy5-GCCGGGGCTGCTTATACAGGT-3', SEQ ID NO: 1) and MCS547 (5'-Cy5-CAGTCACTCCGAACAACGCT-3', SEQ ID NO: 2), and 5 ng / μL of the eubacterial 16S RNA sequence-specific probe EUB338 (5'-Cy3-GCTGCCTCCCGTAGGAGT-3', SEQ ID NO: 3). After washing, all subsequent steps were performed at 4°C. Tissue sections were blocked with 3% donkey serum in 1% BSA-TBS for 1 hour and stained for O / N using anti-Muc2 antiserum and donkey anti-rabbit Alexa Fluor 488 IgG (INVITROGEN A21206). DAPI was used as the nuclear counterstain, and PROLONGGOLD anti-fading agent was used as the mounting medium (P36934, LIFE TECHNOLOGIES). Images were acquired on a NIKONE CLIPSE NI-U equipped with a 40X objective lens. See, for example, Berry et al., ISME J 6, 2091-2106 (2012); Johansson et al., PNAS 105, 15064-15069 (2008); the contents of their respective articles are incorporated herein by reference in their entirety.

[0384] Intestinal barrier leakage measurement.

[0385] Mice were force-fed 10 mg of FITC-dextran (SIGMA 46944) in 100 μL PBS, with free access to food and water. Serum was collected 3 hours later. The serum was diluted 1:1 in PBS, and fluorescence levels were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Standard curves were generated by diluting FITC-dextran at various concentrations in a 1:1 mixture of control serum (from mice force-fed alone in PBS) and PBS.

[0386] Cell suspension preparation and flow cytometry analysis.

[0387] The colon was removed and placed in a 50 mL conical tube in 10 mL of phosphate-buffered saline (PBS) containing 1 mM dithiothreitol (DTT) and incubated on ice for 10 min. Subsequently, the colon was transferred to a new tube containing 10 mL of PBS (5 mM EDTA and 3% FBS) and placed on a rotating wheel at 37°C for 15 min. Next, the epithelial cell suspension was passed through a 100 μm cell filter to a new tube, and the colon tissue was collected and transferred to a new 50 mL tube containing 10 mL of PBS (5 mM EDTA and 3% FBS), and this procedure was repeated. The final filtrate was stored as an epithelial cell fraction. The tissue was transferred to a new 50 mL conical tube containing 20 mL of PBS and centrifuged at 1500 rpm for 5 min at 4°C to wash away the EDTA and DTT from the colon. Next, the tissue was transferred to a 60 mm Petit's dish and manually minced for 1 minute in 2 mL of digestion medium (containing GLUTAMAX, 10% FBS, penicillin-streptomycin, 0.5 mg / mL dispersin (STEM CELL TECHNOLOGY), 1 mg / mL collagenase D (ROCHE), and 50 μg / mL DNase I in RPMI). 8 mL of digestion medium was added, and the tissue suspension was transferred to a 50 mL conical tube and incubated on a rotating wheel at 37°C for 30 minutes. The lamina propria (LP) cell suspensi...

Claims

1. A composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria, the composition being formulated for delivery to the intestine.

2. The composition according to claim 1, wherein The M. schaedleri bacteria are alive or inactivated.

3. The composition according to claim 1, wherein The M. schaedleri bacteria are in a dried, live form.

4. The composition according to claim 1, wherein The M. schaedleri bacteria are encapsulated.

5. The composition according to claim 1, wherein The M. schaedleri bacteria are contained in enteric-coated capsules.

6. The composition according to claim 1, wherein The M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

7. The composition according to claim 1, wherein The M. schaedleri bacteria are mixed with prebiotics.

8. The composition according to claim 1, wherein The M. schaedleri bacteria are mixed with sulfur-containing amino acids.

9. The composition according to claim 8, wherein The sulfur-containing amino acid is methionine, cysteine ​​or a derivative thereof.

10. The composition according to claim 1, wherein The M. schaedleri bacteria are formulated in a food composition.

11. The composition according to claim 10, wherein The food composition is supplemented with sulfur-containing amino acids and / or prebiotics.

12. The composition of claim 1, further comprising 1 to 20 additional bacterial species.

13. The composition of claim 1, comprising no more than 20 bacterial species.

14. A composition comprising live M. schaedleri bacteria, dead M. schaedleri bacteria, M. schaedleri conditioned medium, or an organic solvent extract of M. schaedleri conditioned medium or a fraction thereof that promotes NKT cells to secrete XCL1, wherein: The composition is formulated for delivery to the intestine.

15. The composition according to claim 14, wherein The M. schaedleri bacteria, culture medium or solvent extract is in a dried form.

16. The composition according to claim 14, wherein The M. schaedleri bacteria, culture medium or extract is encapsulated.

17. The composition according to claim 14, wherein The M. schaedleri bacteria are contained in enteric-coated capsules.

18. The composition according to claim 14, wherein The M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

19. The composition according to claim 14, wherein The M. schaedleri bacteria, culture medium or extract is mixed with prebiotics and / or sulfur-containing amino acids or derivatives thereof.

20. The composition according to claim 14, wherein The M. schaedleri conditioned medium, or the organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof comprises at least one metabolite selected from FIG. 4H , FIG. 4I , or FIG. 16 .

21. The composition according to claim 14, wherein The M. schaedleri conditioned medium, or the organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof comprises at least one metabolite selected from the group consisting of succinic acid, propionic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

22. The composition according to claim 14, wherein The M. schaedleri conditioned medium, or an organic solvent extract of the M. schaedleri conditioned medium or a fraction thereof comprises at least one metabolite selected from the group consisting of succinic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid and crotonic acid.

23. A food composition comprising the composition according to claim 14.

24. The food composition of claim 23, further comprising 1 to 20 additional bacterial species.

25. A method for treating cancer or promoting anti-tumor immune activity, the method comprising administering the composition according to claim 1 to a subject in need thereof.

26. The method according to claim 25, wherein: The cancer is colon cancer.

27. The method according to paragraph 25, further comprising administering an immune checkpoint inhibitor.

28. A method of promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering the composition of claim 1 to a subject in need thereof.

29. The method of claim 28, further comprising administering an immune checkpoint inhibitor.

30. The method of claim 28, wherein: The subject suffers from colon cancer.

31. The method of claim 28, wherein: The subject's cancer has been determined to be resistant to immune checkpoint inhibitor therapy.

32. The method of claim 28, wherein: The composition promotes XCL1 secretion by NKT cells.

33. A method for increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering the composition of claim 1 to a subject in need thereof.

34. The method of claim 33, wherein: The cDC1 is associated with tumors.

35. The method of claim 34, wherein: The tumor is colon cancer.

36. The method of claim 33, further comprising administering a sulfur-containing amino acid.

37. The method of claim 33, further comprising administering an immune checkpoint inhibitor.

38. A method for increasing XCL1 secretion by NKT cells, the method comprising administering the composition according to claim 1 to a subject in need thereof.

39. The method of claim 38, wherein: The subject suffers from cancer.

40. The method of claim 38, wherein: The subject suffers from colon cancer.

41. A method of increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering the composition of claim 1 to a subject in need thereof.

42. The method according to claim 41, wherein: The cDC1 is associated with tumors.

43. The method of claim 42, wherein: The tumor is colon cancer.

44. The method of claim 41, further comprising administering a sulfur-containing amino acid.

45. The method of claim 41, further comprising administering an immune checkpoint inhibitor.

46. ​​A method of increasing CD8+ T cell infiltration in colorectal tumors, the method comprising administering to a subject in need thereof a diet rich in sulfur-containing amino acids (SAA) or a supplement comprising SAA.

47. The method of claim 46, wherein: The sulfur-containing amino acid-rich diet includes greater than 0.04 grams of SAA per kilogram of body weight per day.

48. The method of claim 46, further comprising administering to the subject a composition according to any one of claims 13 to 20.

49. A method of establishing or maintaining a tumor suppressive intestinal environment in a subject in need thereof, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) or a supplement comprising SAA.

50. The method of claim 49, wherein: The sulfur-containing amino acid-rich diet or supplement comprising SAA comprises greater than 0.04 grams of SAA per kilogram of body weight per day.

51. The method of claim 49, further comprising administering to a subject a composition according to any one of claims 14 to 21.

52. A method of treating cancer, the method comprising administering an XCL1 polypeptide to a subject in need thereof.

53. The method of claim 52, wherein: The cancer is colon cancer.

54. The method of claim 52, wherein: The XCL1 polypeptide is administered to the intestine.

55. A method of treating cancer, the method comprising administering to a subject in need thereof a microorganism engineered to express an XCL1 polypeptide.

56. A method of treating cancer, the method comprising administering to a subject in need thereof an agonist of the XCL1 receptor, ie, XCR1.

57. The method of claim 56, wherein: The XCR1 agonist comprises SEQ ID NO: 9 to SEQ ID NO: 11 or an amino acid sequence that is at least 95% identical thereto and maintains its function.

58. A method of treating cancer in a subject in need thereof, the method comprising: detecting the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapeutic agent if the level of M. schaedleri is equal to or above a predetermined threshold; and If the level of M. schaedleri is below a predetermined threshold, a cancer immunotherapeutic agent and the composition of claim 1 are administered.

59. A method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay to detect the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapeutic agent if the level of M. schaedleri is equal to or above a predetermined threshold; and If the level of M. schaedleri is below a predetermined threshold, a cancer immunotherapeutic agent and the composition of claim 1 are administered.

60. A method of treating cancer in a subject in need thereof, the method comprising: detecting the level of XCL1 polypeptide, NKT and / or CD103+cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent if the level of the XCL1 polypeptide, NKT and / or CD103+cDC1 is equal to or above a predetermined threshold; as well as If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is below a predetermined threshold, a cancer immunotherapeutic agent and the composition according to claim 1 are administered.

61. A method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay that detects the level of XCL1 polypeptide, NKT, and / or CD103+cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent if the level of the XCL1 polypeptide, NKT and / or CD103+cDC1 is equal to or above a predetermined threshold; as well as If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is below a predetermined threshold, a cancer immunotherapeutic agent and the composition according to claim 1 are administered.

62. A method of stratifying subjects for cancer treatment, the method comprising: detecting the level of M. schaedleri in a sample from the subject; as well as If the level of M. schaedleri is below a predetermined threshold, classifying the subject as high risk; or If the level of M. schaedleri is equal to or above a predetermined threshold, the subject is classified as low risk.

63. A method of stratifying subjects for cancer treatment, the method comprising: obtaining a result from an assay to detect the level of M. schaedleri in a sample from the subject; as well as If the level of M. schaedleri is below a predetermined threshold, classifying the subject as high risk; or If the level of M. schaedleri is equal to or above a predetermined threshold, the subject is classified as low risk.

64. A method of stratifying subjects for cancer treatment, the method comprising: detecting the level of XCL1 polypeptide, NKT and / or CD103+cDC1 in a sample from the subject; as well as If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is below a predetermined threshold, classifying the subject as high risk; or If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is equal to or above a predetermined threshold, the subject is classified as low risk.

65. A method of stratifying subjects for cancer treatment, the method comprising: obtaining a result from an assay that detects the level of XCL1 polypeptide, NKT, and / or CD103+cDC1 in a sample from the subject; as well as If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is below a predetermined threshold, classifying the subject as high risk; or If the level of XCL1 polypeptide, NKT and / or CD103+cDC1 is equal to or above a predetermined threshold, the subject is classified as low risk.

66. The method according to any one of claims 58 to 65, wherein: The subject suffers from colon cancer.

67. The method of any one of claims 62-65, further comprising administering a composition of any one of claims 1-24.

68. The method of any one of claims 58-65, further comprising administering a sulfur-containing amino acid.

69. The method of any one of claims 58-61, wherein the cancer immunotherapeutic agent is selected from the group consisting of: immune checkpoint inhibitors, chemotherapy, dendritic cell vaccines; chimeric antigen receptor T cells (CAR-T) and NKT cell-based therapies.

70. The method according to any one of claims 58 to 61, wherein: The cancer immunotherapeutics include immune checkpoint inhibitors.

71. The method of any one of claims 62-65, further comprising administering an immune checkpoint inhibitor.

72. The method of any one of claims 58-65, further comprising administering a diet rich in sulfur-containing amino acids or a supplement comprising SAA.

73. The method according to any one of claims 58-61 or 66-65, wherein: The method produces a higher therapeutic efficacy compared to a treatment method that does not first: detect the level of M. schaedleri; obtain a result from an assay that detects the level of M. schaedleri; detect the level of XCL1 polypeptide, NKT and / or CD103+cDC1; or obtain a result from an assay that detects the level of XCL1 polypeptide, NKT and / or CD103+cDC1.

74. The method according to any one of claims 62-65, wherein: The method results in a higher therapeutic efficacy compared to a treatment approach that does not first stratify the subjects.

75. The method according to any one of claims 58 to 61, wherein: The method causes fewer treatment complications compared to treatment methods that do not first: detect the level of M. schaedleri; obtain results from an assay that detects the level of M. schaedleri; detect the level of XCL1 polypeptide, NKT and / or CD103+cDC1; or obtain results from an assay that detects the level of XCL1 polypeptide, NKT and / or CD103+cDC1.

76. The method of any one of claims 62-65, wherein: The method results in fewer treatment complications compared to treatment approaches that do not first stratify the subjects.

77. An enteric delivery formulation comprising at least one metabolite selected from Figure 4H, Figure 4I or Figure 16.

78. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid, propionic acid, nicotinic acid, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, C8H 15 NO3S, crotonic acid, myristic acid, 17-hydroxyheptadecanoic acid (C 17 H 34 O3) and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

79. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid, niacin, cis- and / or trans-aconitic acid, pentadecanoic acid, itaconic acid, 16-hydroxyhexadecanoic acid, and crotonic acid.

80. The enteric delivery formulation of any one of claims 77-79, formulated for delivery to the intestine.

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