Use of arachidonic acid for improving cytotoxic effects from chemotherapy and radiotherapy

Abstract

Disclosed herein are methods and compositions for preventing or reducing tissue damage or regenerating tissue in a subject by providing to the subject an arachidonic acid triglyceride (AA TG) or an AA precursor in the form of a triglyceride (TG). In some embodiments, the composition is provided to the subject before, during, or after a chemotherapeutic or radiotherapy process.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 482,280, filed on January 30, 2023, entitled “USE OF ARACHIDONIC ACID FOR AMELIORATION OF CYTOTOXIC EFFECTS FROM CHEMOTHERAPY ANDRADIATION THERAPY,” the entire disclosure of which is incorporated herein by reference.

[0003] Federally funded research or development

[0004] This invention was made with government support under Grant No. CA045508 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Ideally, cancer treatment should eliminate all cells that can cause cancer recurrence during the patient's lifetime. Chemotherapy and radiotherapy are two treatment modalities for several cancers. Chemotherapy includes administering a single drug or multi-drug regimen to kill cancer cells and shrink tumors, while radiotherapy uses high doses of irradiation to do this. In the case of adopting surgery as cancer treatment, combining surgery with chemotherapy or radiotherapy can improve cure rates or allow more limited surgery. Radiotherapy can be given before surgery or chemotherapy (neoadjuvant therapy) or after surgery or chemotherapy (adjuvant therapy). Although chemotherapy and radiotherapy target cancer cells, normal cells are also usually affected, resulting in adverse side effects and cytotoxic effects on normal cells and tissues, which seriously affect the quality of life of cancer patients. Summary of the Invention

[0006] The present disclosure exemplifies how the enhanced intestinal stem cell (ISC) generation or stemness-enhancing effects of oral administration or consumption of arachidonic acid triglyceride (AATG) described herein provide the basis for methods and compositions for reducing, preventing, or reversing the cytotoxic / adverse effects of chemotherapy or exposure to irradiation (e.g., by radiotherapy). In particular, treatment of intestinal organoid-derived single cells with members of the ω-6 family of fatty acids (FAs) (e.g., linoleic acid (LA); gamma-linolenic acid (γ-LA); dihomo-γ-linolenic acid (dh-γ-LA); and arachidonic acid (AA)) not only promotes the formation of spheroids with a morphology associated with an enhanced regenerative stem cell state (stemness) and reduced differentiation, but also results in a significant increase in size compared to control organoids. Furthermore, ω-6 FAs ​​converge on arachidonic acid (AA) in gastrointestinal cells, and ω-6 fatty acids converge on arachidonic acid (AA) enhance organ stemness in mice and humans. Supplementation of arachidonic acid in the form of oral administration of arachidonic acid triglycerides (AATG) / dietary elevation of AA in the intestine (e.g., by consuming an AATG-rich diet or other oral ingestion) enhances ISC regeneration.

[0007] Treatment with chemotherapy or radiotherapy often results in adverse side effects and cytotoxic effects, which is at least in part due to damage to normal cells that are not the target of treatment. Promoting cellular repair mechanisms is a potential way to prevent damage to normal cells and prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiotherapy. For example, intestinal cells may be affected by changes in the subject's diet. One of the common features of dietary interventions that promote intestinal elasticity is to increase the abundance and metabolism of fatty acids (FA) through dietary intake or release from adipose tissue (Novak et al., 2021). However, several clinical and epidemiological studies have shown that increasing total polyunsaturated fatty acids (PUFA), including ω-6 fatty acids, increases cancer risk. In summary, there is a lack of definitive evidence for the effect of ω-6 on cancer outcomes. PUFA, including ω-6 fatty acids (e.g., arachidonic acid), are important structural components of cell membranes required for the growth of rapidly proliferating cells. In addition, after tissue damage, ω-6 fatty acids are released from cell membranes to produce inflammatory bioactive lipid mediators (such as prostaglandins), which are associated with carcinogenesis (Hanson, et al. Br J Cancer (2020) 122(8): 1260-70; Sakai, et al. BMC Cancer (2012) 12: 606; Liput, et al. Int J Mol Sci (2021) 22(13): 6965; Azrad, et al. Front Oncol (2013) 3: 224).

[0008] In some embodiments, the present disclosure provides fatty acids (FA) (e.g., dietary FA) to a subject in need thereof to prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiation therapy. In some embodiments, FA (e.g., arachidonic acid (AA)), at least one AA precursor (linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA), or a combination of AA and at least one AA precursor is administered to a subject before the subject begins a course of chemotherapy or radiation therapy, during a course of chemotherapy or radiation therapy, or after the subject completes a course of chemotherapy or radiation therapy to prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiation therapy in the subject. In some embodiments, AA, at least one AA precursor, or AA and at least one AA precursor are provided to prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiation therapy in a subject. In some embodiments, AA or at least one AA precursor is in the form of a triglyceride (TG, AA TG, AA precursor TG).

[0009] In some embodiments, methods of preventing, reducing, or reversing adverse side effects due to chemotherapy or radiation therapy in a subject are disclosed.

[0010] In some embodiments, the method comprises orally administering to a subject in need thereof at least about 2 g per day (2 g / day) of arachidonic acid triglyceride (AATG) for a period of time sufficient to prevent, reduce, or reverse adverse side effects in the subject caused by chemotherapy or radiation therapy.

[0011] In some embodiments, the sufficient time is at least about 7 days; and (a) administration begins no earlier than 28 days before the subject begins a course of chemotherapy or radiation therapy; (b) administration begins no later than 28 days after the subject completes a course of chemotherapy or radiation therapy; or (c) administration begins at any time during a course of chemotherapy or radiation therapy.

[0012] In some embodiments, the sufficient period of time is at least about 14 days.

[0013] In some embodiments, the sufficient period of time is at least about 21 days.

[0014] In some embodiments, the sufficient period of time is at least about 28 days.

[0015] In some embodiments, the course of chemotherapy or radiation therapy lasts for at least about 3 months.

[0016] In some embodiments, the course of chemotherapy or radiation therapy lasts for at least about 6 months.

[0017] In some embodiments, the course of chemotherapy or radiation therapy continues for at least about 12 months.

[0018] In some embodiments, the course of chemotherapy or radiation therapy lasts from about 3 months to about 12 months.

[0019] In some embodiments, at least about 3 g AATG per day (3 g / day) is administered to the subject.

[0020] In some embodiments, at least about 20 g AA TG / day (20 g / day) is administered to the subject.

[0021] In some embodiments, at least about 30 g AA TG / day (30 g / day) is administered to the subject.

[0022] In some embodiments, at least about 60 g AA TG / day (60 g / day) is administered to the subject.

[0023] In some embodiments, at least about 90 g AA TG / day (90 g / day) is administered to the subject.

[0024] In some embodiments, at least about 100 g AATG per day (100 g / day) is administered to the subject.

[0025] In some embodiments, about 2 g AATG / day (2 g / day) to about 100 g AATG / day (100 g / day) is administered to the subject.

[0026] In some embodiments, the AATG is in the composition.

[0027] In some embodiments, the composition comprises at least about 2% by weight AATG.

[0028] In some embodiments, the composition comprises from about 20% by weight AATG to about 50% by weight AATG.

[0029] In some embodiments, the composition comprises about 40% by weight AATG.

[0030] In some embodiments, the composition comprises no more than 5% by weight of arachidonic acid (AA) esters.

[0031] In some embodiments, the composition is an oil.

[0032] In some embodiments, the oil is extracted from a fungus.

[0033] In some embodiments, the fungus is Mortierella alpina.

[0034] In some embodiments, the composition is a liquid or a powder.

[0035] In some embodiments, the composition is in food, in a capsule, or in a pill.

[0036] In some embodiments, the composition further comprises at least one AA precursor. In some embodiments, the AA precursor is in the form of a triglyceride (TG). In some embodiments, the AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

[0037] In some embodiments, the AA TG increases intestinal AA levels in a subject, which results in a beneficial effect.

[0038] In some embodiments, administration of the AATG increases plasma AA levels in the subject by at least 2-fold compared to a reference.

[0039] In some embodiments, the reference is the AA level in plasma or intestinal tissue from the subject prior to administration of the AA TG, or a predetermined AA level in plasma or intestinal tissue.

[0040] In some embodiments, the adverse side effects are gastrointestinal side effects.

[0041] In some embodiments, the adverse side effect is nausea, vomiting, diarrhea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation sickness, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation.

[0042] In some embodiments, the subject is a human.

[0043] In some embodiments, methods of preventing, reducing, or reversing the cytotoxic effects of chemotherapy or radiation therapy in a subject are disclosed.

[0044] In some embodiments, the method comprises orally administering to a subject in need thereof at least about 2 g per day (2 g / day) of arachidonic acid triglyceride (AATG) for a period of time sufficient to prevent, reduce, or reverse the cytotoxic effects caused by chemotherapy or radiation therapy in the subject.

[0045] In some embodiments, the cytotoxic effect is intestinal tissue damage.

[0046] In some embodiments, the method comprises increasing plasma arachidonic acid (AA) levels in a subject to a level indicative of intestinal AA levels that prevent, reduce, or reverse adverse side effects due to chemotherapy or radiation therapy.

[0047] In some embodiments, the method comprises (a) measuring the level of arachidonic acid (AA) in a sample from a subject in need thereof and determining whether the AA level is below a predetermined AA level sufficient to prevent, reduce, or reverse adverse side effects due to chemotherapy or radiation therapy; and (b) if the AA level is below the predetermined AA level, administering to the subject in (a) at least about 2 grams per day (2 g / day) of AATG for a period of time sufficient to increase the AA level to or above the predetermined AA level.

[0048] In some embodiments, the method further comprises (c) measuring the AA level resulting from the administration of the AATG in (b) and determining the AA level; and (d) if the AA level in (b) is not at or above a predetermined AA level, further administering to the subject a sufficient amount of AATG per day to bring the intestinal AA level to or above the predetermined AA level.

[0049] In some embodiments, the method further comprises repeating (c) through (d) to produce intestinal AA levels in the subject at or above a predetermined AA level.

[0050] In some embodiments, the sample is plasma.

[0051] In some embodiments, the sample is intestinal tissue.

[0052] In some embodiments, methods are provided for preventing, reducing, or reversing adverse side effects due to chemotherapy or radiation therapy in a subject.

[0053] In some embodiments, the AA in AATG is replaced with at least one AA precursor.

[0054] In some embodiments, the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

[0055] In some embodiments, the method comprises orally administering to a subject in need thereof at least about 2 grams per day (2 g / day) of at least one arachidonic acid (AA) precursor for a period of time sufficient to prevent, reduce, or reverse adverse side effects in the subject caused by chemotherapy or radiation therapy.

[0056] In some embodiments, the AA precursor is in the form of triglycerides (TG).

[0057] In some embodiments, the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

[0058] In some embodiments, kits are provided for preventing, reducing, or reversing adverse side effects due to chemotherapy or radiation therapy in a subject.

[0059] In some embodiments, the kit comprises (a) one or more supplement units sufficient to provide at least about 2 g per day (2 g / day) of arachidonic acid triglyceride (AATG) to a subject in need thereof for at least 7 days; and (b) instructions for preparing and consuming the one or more supplement units.

[0060] In some embodiments, one or more supplement units each contain 500 mg AATG, 1 g AATG, 2 g AATG, or 4 g AATG.

[0061] In some embodiments, the number of supplement units administered to a subject in need thereof is determined in consultation with a healthcare provider.

[0062] In some embodiments, the supplement unit is in the form of a liquid or powder.

[0063] In some embodiments, the supplement unit is in the form of a liquid or powder.

[0064] In some embodiments, the supplement unit is in the form of a pill or capsule.

[0065] In some embodiments, the supplement units are in one or more containers.

[0066] In some embodiments, the kit comprises (a) one or more supplement units sufficient to provide at least about 2 grams per day (2 g / day) of at least one arachidonic acid (AA) precursor to a subject in need thereof for a sufficient period of time; and (b) instructions for preparing and consuming the one or more supplement units.

[0067] In some embodiments, the AA precursor is in the form of triglycerides (TG).

[0068] In some embodiments, the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figures 1A to 1T Shown is how fatty acid (FA) screening in mouse and human organs identified arachidonic acid (AA) as a regenerative FA. Figures 1A to 1D The types of FA used in the screening are shown ( Figure 1A ), temporal dynamics analysis of organoid area ( Figure 1B ), the ratio of organoid structures with spheroid morphology to organoid structures with budding organoid morphology (spheroid ratio) in mouse intestinal organoids ( Figure 1C ) and temporal dynamics of organoid area in human intestinal patient-derived organoids (PDO) Figure 1D ). (n=4, t-test). Figures 1E to 1F Shown are the numbers of Ki67+ cells in mouse organoids treated with vehicle (V) or AA (25 μM). Figure 1E ) and representative images of Ki67 immunostaining in organoids ( Figure 1F )(n=5). Figures 1G to 1J Representative images of mouse organoids treated with V or AA are shown ( Figure 1G ), spheroid ratio in organoids treated with V or AA ( Figure 1H ), crypt domain / organoid ( Figure 1I ) and organoid area ( Figure 1J ) were quantified (n=50). Figures 1K to 1N The spheroid ratios derived from primary mouse organoids treated with V or AA are shown ( Figure 1K ), organoid area ( Figure 1L ), the number of (secondary) organoids cultured through passage ( Figure 1M ) and representative images of secondary organoids ( Figure 1N )(n=6). Figures 1O to 1Q Shown are the organoid areas in human PDO treated with V or AA (25 μM). Figure 1O ), spheroid ratio ( Figure 1P ) and representative images of human PDO ( Figure 1Q )(n=7). Figures 1R to 1T Shown are the organoid areas derived from primary PDO cultures treated with V or AA ( Figure 1R ), spheroid ratio of secondary organoids ( Figure 1S ) and representative images of secondary organoids ( Figure 1T ) (n=4). Unless otherwise indicated, the data in these figures are mean ± sem from n independent experiments. ***P < 0.001, ****P < 0.0001 (Mann-Whitney test). Scale bar represents 200 μm ( Figure 1F 、 1G , 1N, 1Q, 1T). See also Figures 8A to 8I , Table 1 and Table 2.

[0070] Figures 2A to 2S It is shown how an AA-rich diet (ARD) enhances intestinal regeneration in vivo. Figure 2A The ratios of nutrients in an isocaloric (3.8 kcal / g) control diet (Control) and an AA-enriched diet (ARD) are shown. Carbohydrate (Carb.), Protein (Prot.). Figures 2B to 2C Metabolomic analysis by liquid chromatography coupled to mass spectrometry (LC-MS) is shown, which shows the plasma of control or ARD-fed mice (n=13) ( Figure 2B ) and tissues (intestine, n=7) ( Figure 2C ) in the abundance of AA. Figures 2D to 2E Crypt lengths of small intestine from control or ARD-fed mice are shown ( Figure 2D ) and representative hematoxylin and eosin (H&E) staining images ( Figure 2E )(n=5). Figures 2F to 2G Ki67 from control or ARD-fed mice is shown + Cells / crypts ( Figure 2F ) and representative images of Ki67 immunostaining in the small intestine ( Figure 2G )(n=5). Figures 2H to 2J The spheroid ratios in organoids from control or ARD-fed mice on day 3 are shown ( Figure 2H ), crypt domain / organoid ( Figure 2I ) and representative images of organoids ( Figure 2J (n=5, Mann-Whitney test). Scale bar represents 100 μm. Figures 2K to 2L Shown is the organoid-initiating capacity of sorted Epcam+ cells derived from the crypts of control or ARD-fed mice ( Figure 2K ) and representative images of organoids ( Figure 2L ) (n=4). Scale bar represents 100 μm. Figures 2M to 2O Shown are the intestinal lengths ( Figure 2M , n = 9) and the number of viable crypts / intestinal area (N, n = 5), as well as representative H&E images of the small intestine ( Figure 2O ). The scale bar represents 50 μm. Figures 2P to 2R Shown are the EdU signal intensity per intestinal area ( Figure 2P, n=4 0Gy, n=6 15Gy), EdU+ cells / crypt ( Figure 2Q , n=6), and representative confocal microscopy images of Epcam (green), EdU (red), and DAPI (blue) staining in the intestine ( Figure 2R ). The scale bar represents 50 μm. Figure 2S Shown are intestinal lengths in control or ARD-fed mice three days after injection of 10 μM doxorubicin (Dox) or vehicle (V) (n=8 Veh, n=6 Dox). Unless otherwise indicated, data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA). See also Figures 9A to 9H and Table 3.

[0071] Figures 3A to 3L Shown is how AA induces a gene expression signature of stem cell reprogramming in mouse and human organoids. Figure 3A Depicted is a gene set enrichment analysis (GSEA) showing the enrichment of different stem cell signatures at time points (n=3 day 1 (D1) and day 3 (D3), n=4 day 6 (D6)) using bulk RNA sequencing of mouse organoids treated with AA vs. V. The scale represents the adjusted p-value for the enrichment analysis. Figure 3B Figure 2 is a heatmap showing differentially expressed (DE) genes involved in stem cell regeneration or differentiation (rows) between AA-treated vs. V-treated organoids at each time point (columns). (n = 3D1 and D3, n = 4D6). The scale represents the log2 fold change in expression between AA-treated vs. V-treated organoids. Abbreviations in the figure include "GC" for goblet cells and "EE" for enteroendocrine cells. Figure 3C Western blot of β-catenin from cytoplasmic and nuclear fractions of organoids treated with V or AA (n=5). Figures 3D to 3F Shown are the organoid areas of V- or AA-treated organoids cultured with the indicated concentrations of Wnt3a ( Figure 3D , n = 5) and organoid counts / well ( Figure 3E , n=5) quantification, and representative images of V- or AA-treated organoids with (100 ng / ml) or without (0 ng / ml) Wnt3a ( Figure 3F ). The scale bar represents 100 μm. Figure 3Gis a heatmap showing DE genes involved in Egfr signaling (receptor and ligand) between AA vs. V-treated organoids at time points (n=3D1 and D3, n=4D6). The scale represents the log2 fold change in expression between AA vs. V-treated organoids. Figures 3H to 3I Shown are the organoid areas of V- or AA-treated organoids with or without EGF supplementation (40 ng / ml) ( Figure 3H , n = 5) quantification and representative images of organoids ( Figure 3I ). The scale bar represents 100 μm. Figure 3J Shown are GSEA enrichments of different stem cell signatures using bulk RNA sequencing of human PDO treated with AA vs. (vs.) V (n=4). Scale represents adjusted p-value for enrichment analysis. Figure 3K is a heat map showing DE genes involved in stemness, differentiation, or proliferation between human PDOs treated with AA vs. V (n=4). The scale represents the log2 fold change in expression between human PDOs treated with AA vs. V. Figure 3L The relative expression of CD55, MSLN, NR4A1, NR4A2, L1CAM, and DUSP4 in human PDO treated with V or AA is shown (n=4, t-test). Unless otherwise indicated, the data in these figures are mean ± sem from n independent experiments; NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA). See also Figures 10A to 10K .

[0072] Figures 4A to 4T Shown are single-cell analyses of AA-induced stemness in vivo. Figure 4A Shown is the Uniform Manifold Approximation and Projection (UMAP) clustering of single-cell RNA sequencing (scRNA-seq) of 23,161 cells from isolated crypts of control (n=2) or ARD-fed (n=2) mice based on the expression of known marker genes (see Example 9). The scale represents the difference in density of single-cell cells between ARD vs. control on UMAP (n=2 independent experiments). Figures 4B to 4D is a split violin plot depicting the expression of S100a6 ( Figure 4B )、Lgr5( Figure 4C )、Ascl2( Figure 4D ) single-cell gene expression levels (n=2, 23,161 cells, Wilcoxon rank-sum test). Figure 4EUMAP plots of pseudo-time trajectory analysis of crypt cells from control or ARD-fed mice are shown. Arrows highlight predicted trajectories within cell clusters (n=2, 23,161 cells). Scale represents pseudo-time. Figure 4F Density plots from pseudotime trajectory analysis showing density differences along pseudotime in all cells (top), cells in Stem 1 clusters (middle), and cells in Stem 2 clusters (bottom) between control and ARD-fed mice (n=2, 23,161 cells, Fisher test). Figure 4G Included are line graphs showing the expression levels of Lgr5 (top) and Ascl2 (bottom) in control or ARD-fed mice along the indicated pseudo-time axis (n=2, 23,161 cells, Wilcoxon rank sum test). Figures 4H to 4P The expression of Lgr5 ( Figure 4H )、Ascl2( Figure 4K ) and S100a6( Figure 4N Representative confocal microscopy images of single-molecule fluorescent in situ hybridization (sm-FISH) of Lgr5 ( Figure 4I )、Ascl2( Figure 4L ) and S100a6( Figure 4O ) sm-FISH signal / crypt unit, and Lgr5 in different crypt layer positions + ( Figure 4J ), Ascl2 + ( Figure 4M ) and S100a6 + ( Figure 4P ) cells’ frequency. Figures 4Q to 4T It was shown that S100a6 is an AA-induced gene regulated by the PGE2-PTGER4-PKA-CREB / YAP pathway and is associated with regeneration. Figure 4H 、 4K The scale bars in 4N represent 10 μm. Unless otherwise indicated, the data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA). See also Figures 11A to 12T .

[0073] Figures 5A to 5M It is shown how prostaglandin E2 (PGE2) can mediate the stemness-enhancing effects of AA. Figure 5A is a heatmap showing changes in bioactive lipid mediators between organoids treated with AA vs V. Scale represents log2 fold change in metabolite abundance between organoids treated with AA vs V (n=3). Figure 5B Shown are the spheroid ratios in organoids after treatment with different AA-derived metabolites (n=5, Mann-Whitney test). Figures 5C to 5D Shown are the organoid areas of V- or AA-treated organoids cultured with the indicated concentrations of Celecoxib ( Figure 5C , n = 5) quantification, and representative images of V- or AA-treated organoids with or without celecoxib (15 μM) ( Figure 5D ). The scale bar represents 200 μm. Figures 5E to 5F Shown in mice ( Figure 5E , n=3, R 2 =0.82, P<0.001) and human PDO ( Figure 5G , n=4, R 2 =0.64, P<0.001) in the concordance between AA-induced gene expression changes and PGE2-induced gene expression as assessed by bulk RNA-seq. DE genes involved in stem cell reprogramming and differentiation are highlighted. Figure 5G Shown are UMAP clustering of single cells from organoids treated with V or PGE2. The scale represents the difference in single cell density between PGE2 vs V on the UMAP plot (n=2). Figure 5H is a density UMAP plot showing changes in gene expression characteristic of fetal spheroids between organoids treated with PGE2 vs. V. The scale represents the density difference in gene expression characteristic of fetal spheroids in single cells between PGE2 vs. V on the UMAP plot (n=2). Figures 5I to 5J is to depict the S100a6 ( Figure 5I ) and Ascl2( Figure 5J ) Split violin plots of gene expression levels in different intestinal epithelial cell clusters (n=2, Wilcoxon rank sum test). Figure 5K Shown are UMAP plots of pseudotime trajectory analysis of cells from organoids treated with V or PGE2. Arrows highlight predicted trajectories within cell clusters and the scale represents pseudotime. Figures 5L to 5MIs a line graph showing the expression levels of Ascl2 (top) and S100a6 (bottom) along the indicated pseudo-time axis in organoids treated with V or PGE2 (n=2, Wilcoxon rank sum test). Unless otherwise indicated, the data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA). See also Figures 13A to 13N .

[0074] Figures 6A to 6W Shown is how the Ptger4-cAMP-PKA signaling axis can regulate AA-induced stemness. Figures 6A to 6C The wild type (Ptger4 f / f ) or Ptger4 knockout (Ptger4 KO) mouse organoids ( Figure 6A , n=10), organoid area ( Figure 6B , n = 10) and representative images ( Figure 6C ). The scale bar represents 100 μm. Figure 6D Shown are the relative expressions of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in organoids of Ptger4 f / f or Ptger4 KO mice treated with V, AA, or PGE2 (n=5). Figures 6E to 6G The spheroid ratios of mouse organoids treated with V or cAMP derivative (8-bromo) (20 μM) are shown ( Figure 6E , n=7), organoid area ( Figure 6F , n = 5) and representative pictures ( Figure 6G ) (t test). Scale bar represents 100 μm. Figure 6H Shown are the relative expressions of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in mouse organoids treated with V or 8-bromopropionic acid (n=4, t-test). Figures 6I to 6K The spheroid ratios of organoids treated with V or AA with or without PKA inhibitor (H89) (20 μM) are shown ( Figure 6I , n=8), organoid area ( Figure 6J , n = 8) and representative images ( Figure 6K ). The scale bar represents 100 μm. Figure 6L Shown are the relative expressions of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in V- or AA-treated organoids with or without H89 (n=4). Figure 6MFigures 6 to 6 show the organoid area of ​​human PDO treated with V or AA with or without Ptger4 inhibitor (Ptger4i) (10 μM) ( Figure 6M , n = 8) and representative images ( Figure 6N )(n=X). Scale bar represents 100 μm. Figures 6O to 6P Shown are the organoid areas of human PDOs treated with V or AA with or without H89 ( Figure 6O , n = 5 and representative pictures ( Figure 6P ). The scale bar represents 100 μm. Figures 6Q to 6R The organoid area of ​​human PDO treated with V or 8-bromophenol is shown ( Figure 6Q , n = 5) and representative pictures ( Figure 6R ). The scale bar represents 100 μm. Figures 6S to 6T Shown are the EdU+ cells / GFP+ crypts in irradiated (15 Gy) or non-irradiated (0 Gy) control or ARD-fed Lgr5-CreERT2-IRES-GFP+;Ptger4+ / + (WT) or Lgr5-CreERT2-IRES-GFP+;Ptger4 f / f (Ptger4 iKO) mice ( Figure 6S , n=5), and representative confocal microscopy images of intestine ( Figure 6T ). Figures 6U to 6W Shows that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit AA-induced stemness. Scale bar represents 50 μm. Unless otherwise stated, data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA). See also Figures 14A to 14L .

[0075] Figures 7A to 7I We show how AA can trigger epigenetic reprogramming of stemness in a Ptger4-dependent manner. Figure 7A Shown is a pathway enrichment analysis of regions within 5 kb of the transcription start site (TSS) with increased or decreased chromatin accessibility in organoids in response to AA treatment as assessed by ATAC-seq. The scale represents the adjusted p-value and the x-axis represents the normalized enrichmentscore (NES) from GSEA (n=2 independent experiments). Figure 7BFigure 3 is a dot plot showing transcription factor (TF) motifs with regulatory potential in AA-induced chromatin accessibility changes by multivariate analysis. The scale represents the effect size of motif presence on peak accessibility changes in organoids treated with AA vs V. The dot scale represents adjusted p-values. Figure 7C Western blot of cyclic AMP-responsive element binding protein (CREB) 1, phosphorylated CREB1 (pCREB1), YAP1, and CREB-binding protein (CBP) in cytoplasmic, chromatin, and nuclear fractions from V- or AA-treated organoids (n=5). Figure 7D Figure 2 is a heatmap showing genes near regions with significant chromatin accessibility gain in response to AA (p-value < 0.01 adjusted by negative binomial analysis). The scale represents the calculated z-score of normalized ATAC-seq reads within the peak. Figure 7E Depicted is a pathway analysis showing epigenetic reprogramming around genes that are part of the regenerative stem cell signature in AA vs V-treated organoids as assessed by Cut&Run assay for the indicated histone markers. The scale represents adjusted p-values ​​and the y scale represents normalized enrichment scores by GSEA. Figure 7F Ranked list of log2 fold changes of putative enhancers defined by H3K27ac peaks around proximal and distal regulatory regions of genes in organoids treated with AA vs V, annotated by the Genomic Regions Enrichment of Annotations Tool (GREAT). Genes regulating intestinal stemness and differentiation are marked. Figure 7G Figure 3 is a scatter plot of genes that had both a significant increase in H3K4me3 signal within 10 kb of their TSS at day 3 and a significant up-regulated expression at day 6 in AA vs V-treated organoids (adjusted p-value < 0.05 by negative binomial test and log2 fold change > 0.58 for both assays). Figure 7H The ATAC-seq and Cut&Run (H3K27me3, H3K4me3, H3K4me3) tracks of the AA signature gene S100a6 locus are shown. Below the figure are the gene structure and transcription direction. Figure 7I Spectra showing the median change in H3K27ac signal of stem cell regeneration-related gene signatures in Ptger4 iKO or WT organoids treated with AA vs. vehicle (see Example 9). ***P<0.001, P value calculated by the effect of the genotype term on the linear model of H3K27ac differences according to the distance to TSS. See also Figures 15A to 15K .

[0076] Figures 8A to 8I Temporal kinetics of FA screening in mouse and human organs are shown and compared with Figures 1A to 1T Related. Figure 8A A schematic diagram showing the FA screening method using live cell imaging. Figure 8B Shown are the relative numbers of organoids (clonogenicity) in response to increasing doses of different FAs (normalized to the number of organoids in V for each FA) (n=4). Figure 8C Representative images of mouse organoids after 120 h of treatment with different FAs (25 μM) are shown. Scale bar represents 200 μm. Figure 8D Representative images of human PDOs after 120 h of treatment with different FAs (25 μM) are shown. Scale bar represents 200 μm. Figure 8E Schematic diagram showing the biosynthesis of AA from LA via desaturases and elongases. Figure 8F Shown are normalized log2-transformed (rlog) counts of gene expression for enzymes regulating AA biosynthesis (Fads1, Fads2, Elov15) using bulk RNA sequencing of mouse organoids (n=3) or human PDO (n=4). Epcam was used as a reference gene abundant in intestinal epithelial cells. Figure 8G Shown are quantifications of organoid area following pretreatment with a desaturase inhibitor (sesamin) followed by the indicated FA treatments (n=4, ANOVA). Figures 8H to 8I Shown are the microvilli lengths of organoids treated with V or AA ( Figure 8H ) and representative transmission electron microscopy images ( Figure 8I ) (n=3, Mann-Whitney test). Scale bar represents 10 μm. Unless otherwise indicated, data in these figures are mean ± sem from n independent experiments; ns, not significant, ***P < 0.001, ****P < 0.0001.

[0077] Figures 9A to 9H Characterization of the in vivo regenerative effects of an AA-rich diet (ARD) is presented and compared with Figures 2A to 2S Related. Figures 9A to 9B The weights of control or ARD-fed mice are shown ( Figure 9A ) and blood sugar levels ( Figure 9B )(n=10, t test). Figure 9C Polar metabolite analysis by LC-MS using plasma from control or ARD-fed mice is shown. The X-axis represents the log2 fold change in metabolite abundance and the Y-axis represents the -log 10 P values ​​in (n=5). Figure 9DRepresentative images of intestines from control (C) or ARD-fed mice that were irradiated (15 Gy) or non-irradiated (0 Gy) are shown (n=9). Figure 9E Shown are representative confocal images of Epcam (green), EdU (red), and DAPI (blue) staining in the intestines from control or ARD-fed mice three days after 15 Gy gamma irradiation (n=5 and scale bar represents 10 μm). Figure 9F Shown are representative images of the intestine from control (C) or ARD-fed mice three days after injection of 10 μM doxorubicin (Dox) or vehicle (V) (n=5). Figures 9G to 9H Shown are EdU+ cells / crypt ( ) in control or ARD-fed mice three days after injection of 10 μM doxorubicin (Dox) or vehicle (V). Figure 9G ), and representative confocal images of Epcam (green), EdU (red), and DAPI (blue) staining in the intestine (n=5, ANOVA). Scale bar represents 50 μm. Unless otherwise indicated, data in these figures are mean ± sem from n independent experiments; ns, not significant, ***P < 0.001, ****P < 0.0001.

[0078] Figures 10A to 10K The induction of AA-mediated stem cell reprogramming gene expression in mouse and human organs is shown and is consistent with Figures 3A to 3L Related. Figure 10A Schematic diagram showing temporal dynamics of bulk RNA-seq experiments in organoids. Figure 10B Shown are principal component analysis (PCA) of temporal kinetic bulk RNA-seq data from mouse organoids after one day (D1, n=3), three days (D3, n=3), or six days (D6, n=4) of AA treatment. Figure 10C Included are upset plots (right) showing overlapping gene sets between AA-induced genes and different stem cell signatures (see Example 9). Boxplots (left) show the log2 fold changes of the gene sets indicated in each row at each time point in organoids treated with AA vs. V. Figure 10D Depicted is an assessment of the statistical significance of AA-induced upregulation of genes that are part of different stem cell signatures based on a permutation test using 100,000 permutations to calculate the distribution of the differences between the two mean profiles. The solid line depicts the empirical null model and the dashed line indicates the observed values ​​of the measured profiles. Figure 10EShown are the relative expressions of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in organoids treated with V or AA for one day (D1) or three days (D3) (n=4, ANOVA). Figure 10F Shown is how GSEA can enrich pathways at time points (D1, D3, D6) using bulk RNA sequencing of mouse organoids treated with AA vs. V. The color scale represents the adjusted p-value for the enrichment analysis. Figure 10G Figure 2 is a heatmap showing DE genes that are Wnt / β-catenin targets between AA vs. V treated organoids at time points (n=3D1 and D3, n=4D6). The scale represents the log2 fold change in expression between AA vs. V treated organoids. Figures 10H to 10I The relative number of organoids (clonogenicity) in cultures set up with Egf or other Egfr ligands Areg and Ereg (normalized to the number of organoids in Wnt3a alone) is shown ( Figure 10H ), and representative images of organoids (n=5, ANOVA). Scale bar represents 100 μm. Figure 10J Shown are PCA analyses of bulk RNA-seq data from human PDOs treated with V or AA (n=4). Figure 10K This figure shows how GSEA can enrich pathways using bulk RNA sequencing of human PDO treated with AA vs. V. The scale represents the adjusted p-value for the enrichment analysis. Unless otherwise noted, data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0079] Figures 11A to 11I Single-cell analysis of AA-induced stemness in vivo is shown, and Figures 4A to 4P Related. Figure 11A is a bubble plot showing marker gene expression levels for cluster identification from scRNAseq analysis of 23,161 epithelial cells in intestinal crypts (n=2 independent experiments). Clusters were identified based on the expression of known marker genes. Figure 11B Depicted are stacked bar graphs showing the proportions of different epithelial cell clusters identified by scRNAseq according to crypts of C (n=2) or ARD-fed (n=2) mice. Figure 11C Depicted are histograms showing the fraction of cells in different epithelial cell clusters identified by scRNAseq in crypts of C (n=2) or ARD-fed (n=2) mice. Figure 11DShown are split violin plots depicting single-cell gene expression levels of Ly6a, Clu, and Msil in different intestinal epithelial cell clusters in control or ARD-fed mice (Wilcoxon rank sum test). Figure 11E Depicted are line graphs showing the expression levels of S100a6 (top) and Ly6a (bottom) of control or ARD-fed mice along the indicated pseudo-time axis (Wilcoxon rank sum test). Figure 11F is a density UMAP plot showing changes in gene expression of different stem cell signatures in clusters between ARD-fed mice vs. control mice (see Example 9). The scale represents the density difference in gene expression of the specified signature in single cells between ARD-fed mice vs. controls on the UMAP plot (n=2). Figures 11G to 11I The expression of Lgr5 ( Figure 11G )、Ascl2( Figure 11H ) and S100a6( Figure 11I Lower magnification representative confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) of ). Figures 11G to 11I Showing the location of Lgr5 in different crypt layers + ( Figure 11G ), Ascl2 + ( Figure 11H ) The frequency of cells, and the scale bar represents 50 μm. Unless otherwise stated, the data in these figures are the mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0080] Figures 12A to 12T Single-cell analysis of AA-induced stemness in vitro is shown, and Figures 4A to 4P Related. Figure 12A Is a bubble plot showing the expression levels of marker genes for cluster identification from scRNAseq analysis of 23,599 single epithelial cells from organoids treated with V (n=2) or AA (n=2). Clusters were identified based on the expression of known marker genes (see Example 9). Figure 12B Presented are stacked bar graphs showing the proportions of different epithelial cell clusters identified by scRNAseq based on organoids treated with V (n=2) or AA (n=2). Figure 12C Bar graphs showing the fraction of cells in different epithelial cell clusters identified by scRNAseq according to crypts of organoids treated with V (n=2) or AA (n=2) are conveyed. Figure 12DUMAP clustering of 23,599 single cells from V- or AA-treated organoids is presented, and the scale represents the difference in density of single cell clusters between AA vs V treatment on the UMAP plot (n=2). Figures 12E to 12J is to depict S100a6 ( Figure 12E )、Ly6a( Figure 12F )、Clu( Figure 12G )、Ascl2( Figure 12H )、Lgr5( Figure 12I ) and Msi1( Figure 12J ) Split violin plots of gene expression levels in different intestinal epithelial cell clusters (n=2, Wilcoxon rank sum test). Figure 12K Density UMAP plots showing changes in gene expression of different stem cell signatures in clusters between AA vs. V treated organoids. The scale represents the difference in density of gene expression of the indicated signature in single cells between AA vs. V on the UMAP plot (n=2). Figure 12L Presented are UMAP plots of pseudotime trajectory analysis of cells from organoids treated with V or AA. Arrows highlight predicted trajectories within cell clusters and the scale represents pseudotime. Figures 12M to 12P is a density plot from pseudo-time trajectory analysis showing the differences in all cells between AA vs. V treated organoids ( Figure 12M ), cells in the Stem1 cluster ( Figure 12N ), cells in the Stem2 cluster ( Figure 12O ) and cells in the Stem3 cluster ( Figure 12P ) along pseudotime (Fisher test). Figures 12Q to 12T Figure 3 shows that S100a6 ( Figure 12Q )、Ly6a( Figure 12R )、Ascl2( Figure 12S ) and Lgr5( Figure 12T ) Line graph of expression levels along the indicated pseudo-time axis (Wilcoxon rank sum test). Unless otherwise noted, the data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0081] Figures 13A to 13N showed that AA metabolism to PGE2 leads to stem cell reprogramming and Figures 5A to 5M Related. Figure 13AFigure 2 is a heat map showing the DE genes regulating AA metabolism to bioactive lipid mediators at time points (n=3D1 and D3, n=4D6) between organoids treated with AA vs. V. The color scale represents the log2 fold change in expression between organoids treated with AA vs. V. Figure 13B Shown are the abundances of bioactive lipid mediators from the intestines of control or ARD-fed mice as assessed by LC-MS (n=4, ANOVA). Figure 13C Representative images of organoids treated with the indicated bioactive lipid mediators are presented, and the scale bar represents 200 μm. Figures 13D to 13E Shown are the organoid areas of V- or AA-treated organoids cultured with the indicated concentrations of indomethacin ( Figure 13D ) Quantification, representative images of organoids treated with V or AA with or without indomethacin (15 μM) ( Figure 13E ) (n=5, ANOVA), and the scale bar represents 200 μm. Figure 13F Shown are PGE2 levels in sorted Epcam+ intestinal epithelial cells from crypts after treatment with V or AA as assessed by ELISA (n=3, ANOVA). Figure 13G Shown are the relative expression of enzymes regulating prostaglandin production (Ptges, Ptgs1, Ptgs2) in organoids treated with V, AA, or PGE2 (n=5, ANOVA). Figure 13H Split violin plots depicting single-cell gene expression levels of Ptges in different intestinal epithelial cell clusters in V- or AA-treated organoids are presented (Wilcoxon rank-sum test). Figure 13I Shown are bubble plots showing marker gene expression levels for cluster identification from scRNAseq analysis of 23,599 single epithelial cells from organoids treated with V (n=2) or PGE2 (n=2). Clusters were identified based on expression of known marker genes (see Example 9). Figure 13J Delivered are stacked bar graphs showing the proportions of different epithelial cell clusters identified by scRNAseq in organoids treated with V (n=2) or PGE2 (n=2). Figure 13K The concordance between AA-induced gene expression changes and PGE2-induced gene expression as assessed by scRNAseq is presented. Figure 13L Density UMAP plots showing changes in gene expression of different stem cell signatures in clusters between organoids treated with PGE2 vs. V are shown (see Example 9). The color scale represents the difference in density of gene expression of the indicated signature in single cells between PGE2 vs. V on the UMAP plots (n=2). Figure 13MSplit violin plots depicting single-cell gene expression levels of Lgr5, Ly6a, and Clu in different intestinal epithelial cell clusters in V- or AA-treated organoids are conveyed (Wilcoxon rank-sum test). Figure 13N Presented are line graphs showing the expression levels of Lgr5 and Ly6a along the indicated pseudo-time axis in organoids treated with V or PGE2 (Wilcoxon rank sum test). Unless otherwise noted, the data in these figures are means ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0082] Figures 14A to 14L Shows how AA can promote stem cell reprogramming through PGE2-Ptger4 signaling and Figures 6A to 6T Related. Figures 14A to 14B Quantification of organoid area of ​​V- or AA-treated organoids cultured with the indicated PGE2 receptor inhibitors (Ptger1i, Ptger2i, Ptger3, Ptger4i) is presented ( Figure 14A ), Representative images of organoids treated with V or AA with or without inhibitors (10 μM) ( Figure 14B ) (n=5), and the scale bar indicates 200 μm. Figures 14C to 14E Organoid area of ​​mouse organoids treated with V, AA or PGE2 with or without Ptger4 inhibitor (Ptger4i) (10 μM) is presented. Figure 14C , n = 10), spheroid ratio ( Figure 14D , n = 10) and representative images ( Figure 14E ). The scale bar represents 200 μm. Figure 14F Shown are the relative expressions of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in V-, AA-, or PGE2-treated organoids with or without Ptger4i (n=4). Figures 14G to 14H shows the use of mouse organoids ( Figure 14G , n=3) or human PDO ( Figure 14H rlog counts of gene expression of PGE2 receptors (Ptger1, Ptger2, Ptger3, Ptger4) from bulk RNA sequencing of 100 μg / mL PE2 receptors (n=4). Figures 14I to 14L Figure 3 is presented depicting Ptger1 in organoids treated with V or AA. Figure 14I )、Ptger2( Figure 14J )、Ptger3( Figure 14K ) and Ptger4( Figure 14L) Split violin plots of single-cell gene expression levels in different intestinal epithelial cell clusters (Wilcoxon rank sum test). Unless otherwise noted, the data in these figures are mean ± sem from n independent experiments; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0083] Figures 15A to 15K showed that PTGER4-dependent epigenetic reprogramming of stemness induced by AA was associated with Figures 7A to 7I Related. Figure 15A The numbers of open and closed chromatin peaks annotated by genic regions, FANTOM enhancers, and CpG islands are presented for ATACseq of AA vs. vehicle-treated organoids (adjusted p<0.01 by negative binomial test and log2 fold change >0.58). Figure 15B We report differential gene expression differences in differentially accessible peaks by integrating ATACseq and RNAseq of cells treated with AA vs. V (adjusted p-value < 0.01 and log2 fold change > 0.58 by negative binomial test for ATACseq). Genes were associated with peaks within 2.5 kb of their TSS. (Off vs. Stable: p = 4.9e -6 , open vs. stable: p<2.2e -16 , closed vs. open: p = 6.4e -9 , Wilcoxon rank sum test). Figure 15C The number of differential peaks for the indicated histone marks in AA vs. V-treated organoids is presented (adjusted p-value < 0.01 by negative binomial test and log2 fold change > 0.58). Figures 15D to 15F H3K4me3 near the TSS of genes differentially up-regulated or down-regulated in organoids treated with V or AA is presented. Figure 15D )、H3K27ac( Figure 15E ) and H3K27me3( Figure 15F ) Spectrum of median reads per genome coverage (RPGC) of histone marks. P values ​​were calculated by finding enrichment of significantly up-regulated and down-regulated gene signatures within all genes ranked by changes in Cut&Run signal within 2.5 kb of the TSS using GSEA. Figure 15G Shown are scatter plots of genes that had both a significant increase in H3K4me3 signal within 10 kb of their TSS at day 3 and significantly upregulated expression at day 3 in AA vs. V-treated organoids (adjusted p-value < 0.05 by negative binomial test and log2 fold change > 0.58 for both assays). Figure 15HShown are scatter plots of genes that had both a significant increase in H327ac signal within 25 kb of their TSS at day 3 (left) and a significant up-regulated expression at day 3 (left) or day 6 (right) in AAvs.V-treated organoids (adjusted p-value < 0.05 by negative binomial test and log2 fold change > 0.58 for both assays). Figure 15I ATAC-seq and Cut&Run (H3K27me3, H3K4me3, H3K4me3) tracks of regeneration-related AA signature genes Msln, Anxa10, Ly6a, and Ascl2 are shown. Gene structure and transcription direction are shown below the figure. Figure 15J Presented are profiles of the median per genome coverage reads (RPGC) of H3K27ac enrichment in cells treated with AA and vehicle near the TSS (± 5.0 kb) of a list of differentially upregulated or downregulated genes from AA vs. V treatment RNAseq. Two conditions are shown: WT (top) and Ptger4 iKO (bottom). P values ​​were calculated by finding enrichment of significantly upregulated and downregulated gene signatures within all genes ranked by changes in Cut & Run signals within 2.5 kb of the TSS using GSEA. Figure 15K Shown are the median changes in H3K27ac signal in Ptger4 iKO or WT organoids treated with AA vs. vehicle for upregulated or downregulated genes. ***P < 0.001, P values ​​calculated using a linear model of the effect of genotype on H3K27ac differences according to distance to TSS.

[0084] Figures 16A to 16E Shown is a schematic diagram detailing the potential mechanisms of stem cell reprogramming in vitro and in vivo in response to AA.

[0085] Figures 17A to 17F It was shown that YAP, CREB1, and CBP are required for the regenerative effects of dietary AAs.

[0086] Figures 18A to 18C It was shown that loss of S100a6 blunts stem cell regeneration.

[0087] Figures 19A to 19C showed that a diet rich in AA (ARD, also known as FA1) does not increase small intestinal ( Figure 19B ),colon( Figure 19B ) or the whole ( Figure 19C ) tumor burden. Figure 19A An overview of the experimental procedure is shown.

[0088] Figures 20A to 20CShows that in a mouse model of metastatic colon cancer, an AA-enriched diet (ARD) does not reduce survival compared to a control diet (C) Figure 20B ) or increase the transfer rate ( Figure 20C ). Figure 20A An overview of the experimental procedure is shown.

[0089] Figures 21A to 21B Shown are the kinetics of AA plasma levels in mice fed a control diet for four weeks, an AA-enriched diet (ARD) for four weeks (Arasco), or an ARD for two weeks followed by a control diet for two weeks (ArascoRev). Figure 21A Shown are plasma lipid levels on days 3, 7, or 14 of mice fed a control diet or an AA-enriched diet. Figure 21B Plasma lipid levels after 4 weeks are shown for all three groups.

[0090] Figures 22A to 22C Shown are the effects of diet on AA levels observed in a mouse model ( Figure 22B ) and intestinal cell proliferation after irradiation ( Figure 22C to 22D). Figure 22A An overview of the experimental protocol is shown.

[0091] Figures 23A to 23B Shown are changes in gene expression in mice fed a control diet for four weeks, an AA-enriched diet (ARD) for four weeks, or an ARD for two weeks followed by a control diet for two weeks. Figure 23A An overview of the experimental protocol is shown. Figure 23B The results of several comparisons are shown.

[0092] Figures 24A to 24B Shown is the in vivo regenerative effect of an AA-enriched diet (ARD) in mice treated with different doses of 5-fluorouracil (5-FU). Figure 24A A schematic diagram of the experimental design is shown. Figure 24B Shown are the % body weight loss of control or ARD-fed mice treated with 250 mg / kg 5-FU on days 1 to 3 (D1, D2, D3) after 5-FU treatment.

[0093] Figures 25A to 25B In vivo regenerative effects of an AA-enriched diet (ARD) in mice treated with a multiple-dose regimen of 5-fluorouracil (5-FU) and oxaliplatin. Figure 25A A schematic diagram showing the experimental procedure. Figure 25BShown are the % weight changes of control or ARD-fed mice treated with 100 mg / kg 5-FU and 6 mg / kg oxaliplatin once weekly for two weeks, allowed to recover for two weeks, and then repeated weekly for two weeks. DETAILED DESCRIPTION

[0094] Adverse side effects of chemotherapy or radiation therapy often include gastrointestinal discomfort due to intestinal tissue damage. In some embodiments, the present disclosure relates to preventing or reducing intestinal tissue damage by providing a certain amount (beneficial dose) of arachidonic acid (AA) or its precursor in the form of a triglyceride to a subject who will undergo or be exposed to a chemotherapy or radiation therapy course before starting the chemotherapy or radiation therapy course. In some embodiments, AA or its precursor in the form of a triglyceride (TG) is administered during the chemotherapy or radiation therapy course. In some embodiments, AA or its precursor in the form of a TG is administered before and during the chemotherapy or radiation therapy course. In some embodiments, the present disclosure relates to administering or supplementing AA or its precursor in the form of a triglyceride for promoting tissue regeneration in a subject who has been exposed to a chemotherapy or radiation therapy course.

[0095] Accumulating evidence indicates that nutrients and metabolic pathways not only influence cell growth and proliferation but also affect cell function and fate by signaling to transcription factors and altering the epigenetic landscape (Beyaz et al., 2016; Beyaz et al., 2021b; Beyaz and Yilmaz, 2016; Chandel et al., 2016; Chen et al., 2020; Cimmino et al., 2018; Lu and Thompson, 2012). Recent studies have explored the metabolic regulation of intestinal stem cell (ISC) activity through fatty acid (FA) oxidation (Chen et al., 2020; Mihaylova et al., 2018; Stine et al., 2019), ketone body signaling (Cheng et al., 2019), mitochondrial pyruvate metabolism (Rodriguez-Colman et al., 2017; Schell et al., 2017), vitamins (Jijon et al., 2018; Lukonin et al., 2020; Peregrina et al., 2015), and microbiome-derived metabolites (Kaiko et al., 2016; Lee et al., 2018).

[0096] The present disclosure provides the following: Dietary AA and AA precursors affect stemness and epigenetic regulation of gene expression, for example in the intestinal epithelium. These findings provide a basis for using AA and AA precursors (which can be in the form of triglycerides (TG)) to prevent, reduce or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiation therapy in subjects in need thereof. Arachidonic acid (AA) and AA precursors (precursor AA)

[0097] In some embodiments, a method of preventing, reducing, or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject is disclosed. In some embodiments, the method comprises administering arachidonic acid (AA) in the form of a triglyceride (AATG) to a subject in need thereof for a sufficient time to prevent, reduce, or reverse adverse side effects caused by chemotherapy or radiation therapy in the subject. In some embodiments, the method comprises orally administering to a subject in need thereof at least one precursor of arachidonic acid (precursor AA) for a sufficient time to prevent, reduce, or reverse adverse side effects caused by chemotherapy or radiation therapy in the subject.

[0098] Arachidonic acid (AA) is a 20-carbon chain fatty acid with four methylene-intercalated cis-double bonds. In some embodiments, AA is in the form of a glyceride. In some embodiments, AA is in the form of a triglyceride (AA triglyceride or AATG). In some embodiments, AA is in the form of a free fatty acid (AA). In some embodiments, the free fatty acid AA is bound to a carrier protein (e.g., albumin). In some embodiments, AA is in the form of a phospholipid (AA phospholipid or AAPL). In some embodiments, AA PL is used in the compositions, methods, and kits disclosed herein. In some embodiments, AA is not associated with triglycerides (TG) or phospholipids (PL). In some embodiments, AA precursors are used in the compositions, methods, and kits disclosed herein. In some embodiments, the precursor AA is linoleic acid (LA), α-linoleic acid (ALA), γ-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA); LA and ALA; LA and γ-LA; LA and dh-γ-LA; ALA and γ-LA; ALA and dh-γ-LA; γ-LA and dh-γ-LA; LA, ALA, γ-LA; LA, ALA, γ-LA and dh-γ-LA; ALA, γ-LA and dh-γ-LA; LA, γ-LA, dh-γ-LA; γ-LA, ALA and γ-LA; LA, γ-LA and dh-γ-LA; or LA, ALA, γ-LA and dh-γ-LA. In some embodiments, the AA precursor is in the form of a triglyceride (TG). In some embodiments, AA or an AA precursor is in the form of a phospholipid (PL, AA PL, precursor AAPL).

[0099] In some embodiments, the composition comprises AATG. In some embodiments, the composition is an oil. In some embodiments, the oil is extracted from an organism (e.g., a plant, a fungus, etc.). In some embodiments, the organism is a microorganism (see, e.g., U.S. Patent No. 8,389,808, the contents of which are incorporated by reference in their entirety). In some embodiments, the microorganism belongs to the genus Mortierella, Entomophthora, Pythium, or Porphyridium. In some embodiments, the microorganism belongs to the genus Pythium. In some embodiments, the microorganism is Pythium insidiuosum. In some embodiments, the organism is a fungus. In some embodiments, the fungus belongs to the genus Mortierella. In some embodiments, the fungus is Mortierella alpina.

[0100] In some embodiments, the oil comprises about 10% or at least about 10% AA TG, about or at least about 15% AATG, about 20% or at least about 20% AA TG, about 25% or at least about 25% AATG, about 30% or at least about 30% AA TG, about 35% or at least about 35% AA TG, about 40% or at least about 40% AA TG, about 45% or at least about 45% AA TG, about 50% or at least about 50% AA TG, about 55% or at least about 55% AA TG, about 60% or at least about 60% AA TG. In some embodiments, the oil comprises about 20% AA TG to about 60% AA TG. In some embodiments, the oil comprises about 20% AA TG to about 50% AA TG. In some embodiments, the oil comprises about 30% AA TG to about 50% AA TG. In some embodiments, the oil comprises at least 40% AA TG or about 40% AA TG. In some embodiments, the percent AA TG is calculated as a volume / volume percentage. In some embodiments, the percent AA TG is calculated as a weight / volume percentage. In some embodiments, the percent AA TG is calculated as a weight / weight percentage.

[0101] In some embodiments, disclosed are methods for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiotherapy. In some embodiments, the method includes orally administering a composition to a subject in need thereof, the composition comprising (a) an oil comprising arachidonic acid triglycerides (AATG); and (b) an oil other than the oil in (a), wherein the ratio of the oil of (a) to the oil of (b) is about 3:4, wherein the composition is administered at least 7 days before the subject begins a course of chemotherapy or radiotherapy, thereby preventing, reducing or reversing adverse side effects caused in the subject by chemotherapy or radiotherapy in the subject.

[0102] Application

[0103] In some embodiments, AATG is administered to a subject in need thereof to prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiation therapy in the subject. In some embodiments, a beneficial dose of AATG is administered. In some embodiments, the beneficial dose is a therapeutic dose, an effective dose, or a therapeutically effective dose. In some embodiments, the beneficial dose is a clinically effective dose. In some embodiments, administration is a supplement or includes a supplement. In some embodiments, administration is a supplementing act or includes a supplementing act.

[0104] In some embodiments, an amount of AATG / day is administered to a subject in need thereof. In some embodiments, about or at least about 2 g of AATG / day is administered to a subject. In some embodiments, about 2.5 g or at least about 2.5 g of AATG / day is administered to a subject. In some embodiments, about 3 g or at least about 3 g of AATG / day is administered to a subject. In some embodiments, about 4 g or at least about 4 g of AATG / day is administered to a subject. In some embodiments, about 5 g or at least about 5 g of AATG / day is administered to a subject. In some embodiments, about 6 g or at least about 6 g of AATG / day is administered to a subject. In some embodiments, about 7 g or at least about 7 g of AATG / day is administered to a subject. In some embodiments, about 8 g or at least about 8 g of AATG / day is administered to a subject. In some embodiments, about 9 g or at least about 9 g of AATG / day is administered to a subject. In some embodiments, about 10 g or at least about 10 g of AATG / day is administered to a subject. In some embodiments, about 15 g or at least about 15 g of AATG / day is administered to a subject. In some embodiments, about 20 g or at least about 20 g of AATG / day is administered to a subject. In some embodiments, about 25 g or at least about 25 g of AATG / day is administered to a subject. In some embodiments, about 30 g or at least about 30 g of AATG / day is administered to a subject. In some embodiments, about 40 g or at least about 40 g of AATG / day is administered to a subject. In some embodiments, about 50 g or at least about 50 g of AATG / day is administered to a subject. In some embodiments, about 60 g or at least about 60 g of AATG / day is administered to a subject. In some embodiments, about 70 g or at least about 70 g of AATG / day is administered to a subject. In some embodiments, about 80 g or at least about 80 g of AATG / day is administered to a subject. In some embodiments, about 90 g or at least about 90 g of AATG / day is administered to a subject. In some embodiments, about 100 g or at least about 100 g of AA TG per day is administered to the subject.

[0105] In some embodiments, about 2 g to about 100 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 90 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 80 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 70 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 60 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 50 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 40 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 30 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 20 g of AATG / day is administered to a subject. In some embodiments, about 2 g to about 10 g of AATG / day is administered to a subject.

[0106] In some embodiments, about 5 g to about 100 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 90 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 80 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 70 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 60 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 50 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 40 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 30 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 20 g of AATG / day is administered to a subject. In some embodiments, about 5 g to about 10 g of AATG / day is administered to a subject.

[0107] In some embodiments, AATG is administered to a subject in need thereof based on the subject's weight. In some embodiments, about 50 mg or at least about 50 mg AATG / kg body weight, about 100 mg or at least about 100 mg AA TG / kg body weight, about 150 mg or at least about 150 mg AA TG / kg body weight, about 200 mg or at least about 200 mg AATG / kg body weight, about 300 mg or at least about 300 mg AA TG / kg body weight, about 400 mg or at least about 400 mg AA TG / kg body weight, about 500 mg or at least about 500 mg AATG / kg body weight, about 600 mg or at least about 600 mg AA TG / kg body weight, about 700 mg or at least about 700 mg AA TG / kg body weight, about 800 mg or at least about 800 mg AATG / kg body weight, about 900 mg or at least about 900 mg AATG / kg body weight, about 1 g or at least about 1 g AA TG / kg body weight, about 1.5 g or at least about 1.5 g AA TG / kg body weight, about 2 g or at least about 2 g AATG / kg body weight, or any range or combination thereof.

[0108] In some embodiments, the amount of AATG to be administered to a subject in need thereof takes into account one or more of the subject's age, sex, height, concomitant medications, and pre-existing conditions. In some embodiments, when the subject is a pediatric patient, the amount of AATG to be administered is adjusted based on the pediatric patient's age and the amount of AATG to be administered to an adult as disclosed herein (as amount / day or amount / kg body weight). A non-limiting example of determining a pediatric dose is Young's Rule, according to the equation: [age / (age+12)]×recommended adult dose=pediatric dose. (See, e.g., ncbi.nlm.nih.gov / books / NBK554603, which is readily available to one of ordinary skill in the art). If the age of the pediatric patient is unknown, Young's Rule can be used. If the age of the pediatric patient is known, Clark's Rule or the body surface area rule can be implemented. (See, e.g., ncbi.nlm.nih.gov / books / NBK541104 / , which is readily available to one of ordinary skill in the art).

[0109] In some embodiments, administration of an AATG increases AA levels in the subject's blood or blood components (e.g., plasma) relative to a reference. In some embodiments, AA levels are increased in the subject's intestine relative to a reference. In some embodiments, AA levels are increased in the subject's plasma and intestine relative to a reference.

[0110] In some embodiments, AA levels are measured in the subject's blood or blood components (e.g., plasma). In some embodiments, AA levels are measured in the subject's intestine. In some embodiments, AA levels are measured in the subject's intestine and in the subject's blood or blood components (e.g., plasma).

[0111] In some embodiments, administration of AATG to a subject in need thereof increases expression of stemness markers, such as increased expression of genes associated with stemness, relative to a reference. In some embodiments, the gene associated with stemness is at least one of: leucine rich repeat containing g protein-coupled receptor 5 (Lgr5), achaete-scute family BHLH transcription factor 2 (Ascl2), lymphocyte antigen 6 complex locus A (Ly6a) or S100 calcium binding protein A6 (S100a6), LY6 / PLAUR domain-containing 6 (Lypd6), connective tissue growth factor (CTGF), or IL-64 receptor agonist. factor, Ctgf), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (cromer blood type) (Cd55), epiregulin (Ereg), myoferlin (Myof), and mesothelin (Msln).

[0112] In some embodiments, administration of AATG increases expression of genes associated with stemness by about 75% or at least about 75%; about 100% or at least about 100%; about 125% or at least about 125%; about 150% or at least about 150%; about 175% or at least about 175%; about 200% or at least about 200%; about 300% or at least about 300%; about 400% or at least about 400%, about 500% or at least about 500%, about 600% or at least about 600%; about 700% or at least about 700%, about 800% or at least about 800%, or any range or combination thereof, relative to a reference. In some embodiments, expression of genes associated with stemness is increased in cells obtained from a subject (e.g., epithelial cells, intestinal cells, etc.). In some embodiments, expression of a gene associated with stemness is increased in a sample obtained from a subject (eg, blood, a component of blood (eg, plasma, serum, etc.), or a tissue (eg, intestinal tissue)).

[0113] In some embodiments, administration of an AATG to a subject in need thereof increases AA levels in the subject (e.g., a sample from the subject) relative to a reference. In some embodiments, administration of an AATG to a subject in need thereof increases AA levels by about 2-fold or at least about 2-fold relative to a reference. In some embodiments, administration of an AATG to a subject in need thereof increases AA levels by about 2-fold or at least about 2-fold, about 3-fold or at least about 3-fold, about 4-fold or at least about 4-fold, about 5-fold or at least about 5-fold, about 6-fold or at least about 6-fold, about 7-fold or at least about 7-fold, about 8-fold or at least about 8-fold, about 9-fold or at least about 9-fold, about 10-fold or at least about 10-fold, about 11-fold or at least about 11-fold, about 12-fold or at least about 12-fold, about 13-fold or at least about 13-fold, about 14-fold or at least about 14-fold, about 15-fold or at least about 15-fold, or any range or combination thereof relative to a reference. In some embodiments, administration of AA TG to a subject in need thereof increases AA levels by about 3-fold to about 20-fold relative to a reference. In some embodiments, administration of AA TG to a subject in need thereof increases AA levels by about 3-fold to about 15-fold relative to a reference. In some embodiments, administration of AA TG to a subject in need thereof increases AA levels by about 3-fold to about 10-fold relative to a reference. In some embodiments, administration of AA TG to a subject in need thereof increases AA levels by about 1.5-fold to about 3-fold relative to a reference.

[0114] In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG is administered prior to a course of chemotherapy, radiation therapy, or a combination of chemotherapy and radiation therapy. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered for a total time of 7 days, about 7 days, or at least 7 days, 10 days, about 10 days, or at least 10 days, 2 weeks or about 2 weeks, or at least 2 weeks, 3 weeks, about 3 weeks, or at least 3 weeks, 4 weeks, about 4 weeks, or at least 4 weeks, 5 weeks, about 5 weeks, or at least 5 weeks, 6 weeks, about 6 weeks, or at least 6 weeks, 7 weeks, about 7 weeks, or at least 7 weeks, 8 weeks, about 8 weeks, or at least 8 weeks, 9 weeks, about 9 weeks, or at least 9 weeks, 10 weeks, about 10 weeks, or at least 10 weeks, 11 weeks, about 11 weeks, or at least 11 weeks, 12 weeks, about 12 weeks, or at least 12 weeks prior to a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered 2 to 4 weeks prior to a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered 1 to 3 weeks, 3 to 5 weeks, 4 to 6 weeks, or 5 to 7 weeks prior to a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0115] A subject can complete one course of chemotherapy or one course of radiation therapy or more than one course of chemotherapy or one course of radiation therapy. The number of courses of chemotherapy or the number of courses of radiation therapy can depend on the needs of the subject.

[0116] In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof for a sufficient time to prevent, reduce, or reverse adverse side effects or cytotoxic effects caused by chemotherapy or radiotherapy in the subject. In some embodiments, the sufficient time is at least about 3 days; and (a) administration begins before the subject starts a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy. In some embodiments, the sufficient time is at least about 5 days; and (a) administration begins before the subject starts a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy. In some embodiments, the sufficient time is at least about 7 days; and (a) administration begins before the subject starts a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy. In some embodiments, the sufficient time is at least about 14 days; and (a) administration begins before the subject begins a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy. In some embodiments, the sufficient time is at least about 21 days; and (a) administration begins before the subject begins a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy. In some embodiments, the sufficient time is at least about 28 days; and (a) administration begins before the subject begins a course of chemotherapy or radiotherapy; (b) administration begins after the subject completes a course of chemotherapy or radiotherapy; or (c) administration begins at any time during a course of chemotherapy or radiotherapy.

[0117] In some embodiments, the sufficient period of time is about 1 month, at least 1 month, about 2 months, at least 2 months, about 3 months, at least 3 months, about 4 months, at least 4 months, about 5 months, at least 5 months, about 6 months, at least 6 months, about 7 months, at least 7 months, about 8 months, at least 8 months, about 9 months, at least 9 months, about 10 months, at least 10 months, about 11 months, at least 11 months, about 1 year, or at least 1 year; and (a) the administration begins before the subject begins a course of chemotherapy or radiation therapy; (b) the administration begins after the subject completes a course of chemotherapy or radiation therapy; or (c) the administration begins at any time during a course of chemotherapy or radiation therapy.

[0118] In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered for a cycle that is or includes administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about 1 week or at least 1 week, and then not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about 1 week or at least 1 week; administering for a cycle that is or includes administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about 2 weeks or at least 2 weeks, and then not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about 2 weeks or at least 2 weeks; administering for a cycle that is or includes administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about 3 weeks or at least 3 weeks, and then not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG. TG for about 3 weeks or at least 3 weeks; administer a cycle that is or comprises administering AATG, at least one precursor AATG, or AATG, and at least one precursor AATG for about 4 weeks or at least 4 weeks, and then not administering AATG, at least one precursor AATG, or AATG, and at least one precursor AATG for about 4 weeks or at least 4 weeks. In some embodiments, AATG, at least one precursor AATG, or AATG, and at least one precursor AATG are administered for about 1 week or at least 1 week, about 2 weeks or at least about 2 weeks, about 3 weeks or at least about 3 weeks, about 4 weeks or at least 4 weeks, and then not administering AATG, at least one precursor AATG, or AATG, and at least one precursor AATG for about 1 week or at least 1 week, about 2 weeks or at least about 2 weeks, about 3 weeks or at least about 3 weeks, about 4 weeks or at least 4 weeks.

[0119] In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof every day. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are not administered to a subject in need thereof every day. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof every other day. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof at least once a day. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof every other day. In some embodiments, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are administered to a subject in need thereof two, three, or four times a day.

[0120] In some embodiments, the AA level in a sample from a subject in need thereof is below a predetermined AA level in the absence of administration of an amount of AATG that increases the AA level in the subject in need thereof to or above the predetermined AA level. In some embodiments, the predetermined AA level is a two-fold increase in the AA level measured in the sample from the subject relative to the AA level measured in the sample from the subject before administration of the AATG. In some embodiments, the predetermined AA level is a 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in the AA level measured in the sample from the subject relative to the AA level measured in the sample from the subject before administration of the AATG. In some embodiments, the predetermined AA level is a clinically relevant AA level. In some embodiments, the predetermined AA level is a clinically relevant plasma AA level or intestinal AA level. In some embodiments, the predetermined AA level is an AA level sufficient to prevent, reduce, or reverse adverse side effects caused by chemotherapy or radiation therapy. In some embodiments, the predetermined AA level is the lowest AA level at which a beneficial effect is observed in the subject.

[0121] In some embodiments, the beneficial effect is increased expression of a stemness marker relative to a reference, such as increased expression of a stemness-associated gene relative to a reference. In some embodiments, the stemness-associated gene is at least one of the following: leucine-rich repeat-containing g-protein coupled receptor 5 (Lgr5), hairless-scale family BHLH transcription factor 2 (Ascl2), lymphocyte antigen 6 complex locus A (Ly6a), or S100 calcium binding protein A6 (S100a6), LY6 / PLAUR domain-containing 6 (Lypd6), connective tissue growth factor (Ctgf), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (cromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln).

[0122] In some embodiments, a beneficial effect is observed when the expression of a stemness-related gene in a subject in need thereof is increased by about 10% or at least about 10%; about 25% or at least about 25%; about 50% or at least about 50%; about 75% or at least about 75%; about 100% or at least about 100%; about 125% or at least about 125%; about 150% or at least about 150%; about 175% or at least about 175%; about 200% or at least about 200%; about 300% or at least about 300%; about 400% or at least about 400%, about 500% or at least about 500%; about 600% or at least about 600%; about 700% or at least about 700%, about 800% or at least about 800%; or any range or combination thereof relative to a reference. In some embodiments, the expression of a stemness-related gene is increased in cells obtained from a subject (e.g., epithelial cells, intestinal cells).

[0123] In some embodiments, the beneficial effect is the prevention, reduction, or reversal of an adverse side effect caused by chemotherapy or radiation therapy in a subject relative to a reference, as determined by a healthcare provider (e.g., a medical doctor). For example, a healthcare provider can determine that one or more symptomatic measures are reduced relative to a reference, including, but not limited to, frequency, volume, amount, or occurrence of diarrhea, blood in the stool, calprotectin in the stool, vomiting, nausea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation sickness, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation. In some embodiments, the reference is the frequency, volume, amount or occurrence of diarrhea, blood in the stool, calprotectin in the stool, vomiting, nausea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation sickness, radiation enteritis, abdominal pain, rectal bleeding, bloating or constipation caused by chemotherapy or radiation therapy experienced by a subject in need thereof prior to administration of AATG, at least one precursor AATG, or AATG and at least one precursor AATG to the subject.

[0124] In some embodiments, the beneficial effect is the prevention, reduction or reversal of the cytotoxic effect caused by chemotherapy or radiotherapy relative to a reference, as determined by a health care provider (e.g., a doctor). For example, a health care provider can determine a beneficial effect relative to a reference. In some embodiments, after administering AATG, at least one precursor AATG or AATG and at least one precursor AATG, relative to a reference, intestinal tissue damage is prevented, reduced or reversed in an object in need thereof. In some embodiments, the reference is to the extent of intestinal tissue damage before administering AATG, at least one precursor AATG or AATG and at least one precursor AATG to an object in need thereof. Intestinal damage caused by chemotherapy is discussed in Sougiannis, et al. Am J Physiol Gastrointest Liver Physiol (2021) 320 (5): G712-G719, which can be obtained by one of ordinary skill in the art and is incorporated herein by reference in its entirety. In some embodiments, the cytotoxic effect is intestinal tissue damage.

[0125] In some embodiments, preventing or a method of prevention refers to the clinical absence of observation of one or more adverse side effects that would be expected in a subject undergoing a similar course of chemotherapy or radiation therapy.

[0126] In some embodiments, the beneficial effect is assessed in a sample obtained from a subject in need thereof. In some embodiments, the sample is a cell (e.g., epithelial cell, intestinal cell, etc.), blood, a blood component (e.g., serum, plasma), or feces obtained from a subject in need thereof.

[0127] In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof relative to a reference. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof by about 2-fold or at least about 2-fold relative to a reference. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold; at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 11-fold, at least or about 12-fold, at least or about 13-fold, at least or about 14-fold, or at least or about 15-fold, or any range or combination thereof, relative to a reference. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof by about 3-fold to about 15-fold relative to a reference. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof by about 3-fold to about 10-fold relative to a reference. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof by about 1.5-fold to about 3-fold relative to a reference.

[0128] In some embodiments, the reference is the level of AA in the subject before exposure to chemotherapy, radiation, or a course of both chemotherapy and radiation. In some embodiments, the reference is the level of an AA population in a sample from the subject before chemotherapy, radiation, or a course of both chemotherapy or radiation. In some embodiments, the reference is the level of AA in the subject before administration of an AATG to the subject. In some embodiments, the reference is the level of AA in the subject without exposure to chemotherapy, radiation, or a course of both chemotherapy and radiation. In some embodiments, the reference is the level of AA in a subject with the same condition as the subject to be exposed to the course of chemotherapy or radiation, but the subject with the same condition has not been exposed to the course of chemotherapy or radiation. In some embodiments, the condition is a condition to be treated with chemotherapy, radiation, or a course of chemotherapy and radiation. In some embodiments, the condition is cancer. In some embodiments, the reference is the level of AA in a cell before exposure to the course of chemotherapy or radiation.

[0129] In some embodiments, AATG is administered orally to a subject in need thereof. In some embodiments, AATG is administered orally. In some embodiments, AATG is administered by nasogastric tube. In some embodiments, AATG is administered into the stomach, for example, by a gastric tube (G tube) or injection. In some embodiments, AATG is administered by nasoduodenal tube or nasojejunal tube. In some embodiments, AATG is administered to the small intestine by jejunostomy (J tube). Administration can be by a variety of non-parenteral routes. In some embodiments, AATG is not administered by intragastric injection. In some embodiments, AATG is in a composition, wherein the composition is in the form of a liquid or powder. In some embodiments, the composition is in the form of a pill or capsule. In some embodiments, AA is administered rectally as a free fatty acid bound to a carrier protein (e.g., albumin), for example, using a suppository.

[0130] The effect of administering AATG can be assessed by comparing the extent of one or more adverse side effects, the extent of cytotoxicity, or both after administering AATG to the extent of the same one or more side effects, the extent of cytotoxicity, or both in a subject in need thereof before administering AATG. Similarly, the effect of administering at least one precursor AATG (precursor of the TG form of AA) or administering AATG and at least one precursor AATG (precursor of the TG form of AA) can be assessed by comparing the extent of one or more adverse side effects, the extent of cytotoxicity, or both after administering at least one precursor AATG (precursor of the TG form of AA) or administering AATG and at least one precursor AATG (precursor of the TG form of AA) to the extent of the same one or more side effects, the extent of cytotoxicity, or both in a subject in need thereof before administering at least one precursor AATG (precursor of the TG form of AA) or administering AATG and at least one precursor AATG (precursor of the TG form of AA).

[0131] Object

[0132] In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a mouse. In some embodiments, the subject is a domesticated animal (e.g., a dog, a cat, etc.). In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0133] In some embodiments, the subject in need thereof is a human in need thereof. In some embodiments, the subject in need thereof is a subject before the subject begins a course of chemotherapy or radiation therapy. In some embodiments, the subject in need thereof is a subject that has been exposed to a course of chemotherapy or radiation therapy. In some embodiments, the subject in need thereof is a subject with cancer who will be exposed to or treated with the following process: chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AATG before the course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AATG during the course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AATG before and during the course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0134] Many polymorphisms in the fatty acid (FA) desaturase gene cluster are strongly associated with metabolic traits and diseases including IBD (Sabatti et al. 2009; Dupuis et al., 2010; Costea et al., 2014). In some embodiments, such polymorphisms can be used to select a subject population for treatment described herein.

[0135] In some embodiments, the method comprises increasing plasma arachidonic acid (AA) levels in a subject to a level indicative of intestinal AA levels that prevent, reduce, or reverse adverse side effects due to chemotherapy or radiation therapy.

[0136] In some embodiments, the method comprises (a) measuring the level of arachidonic acid (AA) in a sample from a subject in need thereof and determining whether the AA level is below a predetermined AA level sufficient to prevent, reduce, or reverse adverse side effects due to chemotherapy or radiation therapy; and (b) if the AA level is below the predetermined AA level, administering to the subject in (a) at least about 2 grams per day (2 g / day) of AATG for a period of time sufficient to increase the AA level to or above the predetermined AA level.

[0137] In some embodiments, the method further comprises (c) measuring the AA level resulting from administering the AATG in (b) and determining the AA level; and (d) if the AA level in (b) is not at or above a predetermined AA level, further administering to the subject daily a sufficient amount of AATG to bring the intestinal AA level to or above the predetermined AA level.

[0138] In some embodiments, the method further comprises repeating (c) through (d) to produce an intestinal AA level in the subject at or above a predetermined AA level.

[0139] In some embodiments, methods of increasing AA levels in a subject to prevent or reduce tissue damage due to chemotherapy or radiotherapy are disclosed. In some embodiments, the method comprises (a) measuring the AA level in a sample obtained from the subject, (b) determining whether the AA level in the subject in (a) is below a predetermined AA level; (c) administering an amount of AATG sufficient to increase the AA level to or above a predetermined AA level, wherein the AA level increased in (c) prevents or reduces tissue damage caused by chemotherapy, radiotherapy, or chemotherapy and radiotherapy in the subject. In some embodiments, the methods disclosed herein further comprise (d) after administering AATG in (c), measuring the AA level in a sample obtained from the subject and determining whether the AA level in the sample in (d) is at or above a predetermined AA level. In some embodiments, if the AA level is below the predetermined AA level described above, the amount of AATG to be administered to the subject is adjusted to an amount sufficient to increase the AA level in the subject to an amount above the predetermined AA level.

[0140] In some embodiments, measuring AA levels comprises collecting a sample from a subject in need thereof and measuring the AA level in the sample. In some embodiments, the sample is blood. In some embodiments, the sample is serum. In some embodiments, the sample is plasma. In some embodiments, the sample is or comprises tissue. In some embodiments, the sample is or comprises feces. In some embodiments, the tissue is intestinal tissue. In some embodiments, the AA in the AA levels is free AA fatty acids. In some embodiments, AA fatty acids are associated with a carrier protein (e.g., albumin). In some embodiments, the AA in the AA levels is AA PL.

[0141] In some embodiments, AA in a sample obtained from a subject is measured by detecting AA in the sample. In some embodiments, methods for measuring AA levels in a sample obtained from a subject include, but are not limited to, mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry (LC-MS), gas chromatography, thin layer chromatography, size exclusion chromatography, enzyme-linked immunosorbent assay (ELISA), and nuclear magnetic resonance (NMR).

[0142] Prevent or reduce tissue damage

[0143] In some embodiments, methods of preventing or reducing tissue damage are disclosed. In some embodiments, the methods comprise administering to a subject a form of AA disclosed herein prior to exposure to chemotherapy, radiation therapy, or chemotherapy and radiation therapy to prevent or reduce tissue damage caused by the chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0144] In some embodiments, preventing or reducing tissue damage comprises predicting tissue damage in a subject and prophylactically administering an AATG to the subject prior to exposure to chemotherapy, radiation therapy, or a course of chemotherapy and radiation therapy. In some embodiments, preventing or reducing tissue damage further comprises observing less tissue damage in the subject relative to the degree of tissue damage that occurred or existed prior to administration of the AATG or relative to a reference.

[0145] In some embodiments, preventing or reducing tissue damage comprises observing about 10% less or at least 10% less tissue damage, about 15% less or at least 15% less tissue damage, about 20% less or at least 20% less tissue damage, about 25% less or at least 25% less tissue damage, about 30% less or at least 30% less tissue damage, about 35% less or at least 35% less tissue damage, about 40% less or at least 40% less tissue damage, about 45% less or at least 45% less tissue damage, relative to the level of tissue damage that occurred or existed prior to administration of the AATG or relative to a reference. In some embodiments, the prevention or reduction of tissue damage comprises preventing all tissue damage relative to a reference. In some embodiments, the reference is tissue that has been damaged due to chemotherapy, radiation, or chemotherapy and radiation treatment but to which AATG has not been administered (e.g., prophylactically). In some embodiments, the prevention or reduction of tissue damage is determined by a health care provider (e.g., a physician). For example, a health care provider can determine a symptomatic measure that includes, but is not limited to, diarrhea, blood in the stool, or a decrease in calprotectin in the stool relative to a reference.

[0146] In some embodiments, tissue damage is damage expected based on chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, tissue damage is measured histologically. In some embodiments, tissue damage is measured or assessed as understood by one of ordinary skill in the art. In some embodiments, tissue damage is inferred based on the subject's clinical symptoms. In some embodiments, tissue damage is inferred based on digestive tract symptoms, such as nausea, vomiting, diarrhea, weight loss, etc. In some embodiments, prevention or reduction of tissue damage is represented by a decrease in clinical symptoms in a subject who has received chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0147] Tissue regeneration

[0148] In some embodiments, a method of promoting tissue regeneration is disclosed, comprising administering an AATG to a subject having tissue damage caused by chemotherapy, radiation therapy, or both to promote tissue regeneration in the subject, wherein the AATG increases the AA level in the subject by at least 2-fold relative to a reference. Tissue regeneration includes the regrowth of tissue that has undergone tissue damage. Tissue damage includes, but is not limited to, damage to a subject's tissue from a chemotherapy or radiation treatment process. In some embodiments, the damage results from a combination of chemotherapy and radiation therapy, or from a process that includes both chemotherapy and radiation therapy.

[0149] In some embodiments, promoting the regeneration of damaged tissue from chemotherapy, radiation therapy, or chemotherapy and radiation therapy processes includes observing damage to the tissue, administering AATG, and observing less damage to the tissue. In some embodiments, in each case, tissue regeneration includes 100% recovery, about 95% recovery, about 90% recovery, about 80% recovery, about 70% recovery, about 60% recovery, about 50% recovery, about 40% recovery, or about 30% recovery relative to a reference from tissue damage. In some embodiments, the reference is tissue that is damaged due to chemotherapy, radiation, or chemotherapy and radiation processes but is not administered (e.g., prophylactically administered) AATG. In some embodiments, the reference is tissue that is damaged due to chemotherapy, radiation, or chemotherapy and radiation processes and before administering AATG. In some embodiments, the regeneration of damaged tissue is determined by a health care provider (e.g., a doctor). For example, a health care provider can determine a symptomatic measure that includes, but is not limited to, diarrhea, blood in the stool, or calprotectin in the stool is reduced in the subject relative to a reference.

[0150] In some embodiments, tissue damage is damage expected based on chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, tissue damage is measured histologically. In some embodiments, tissue damage is measured or assessed as understood by one of ordinary skill in the art. In some embodiments, tissue damage is inferred from clinical symptoms of the subject. In some embodiments, tissue damage is inferred from digestive tract symptoms, such as nausea, vomiting, diarrhea, weight loss, etc. In some embodiments, regeneration of damaged tissue is represented by a reduction in clinical symptoms in a subject who has received chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0151] In some embodiments, regeneration of damaged tissue comprises increased expression of stemness markers relative to a reference, such as increased expression of stemness-related genes relative to a reference. In some embodiments, the stemness-related genes are at least one of the following: leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), hairless-scale family BHLH transcription factor 2 (Ascl2), lymphocyte antigen 6 complex locus A (Ly6a), or S100 calcium binding protein A6 (S100a6), LY6 / PLAUR domain-containing 6 (Lypd6), connective tissue growth factor (Ctgf), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (cromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln). In some embodiments, the expression of stemness-related genes is increased relative to a reference by at least or about 75%; at least or about 100%; at least or about 125%; at least or about 150%; at least or about 175%; at least or about 200%; at least or about 300%; at least or about 400%, at least or about 500%, at least or about 600%; at least or about 700%, at least or about 800%, or any range or combination thereof. In some embodiments, the reference is the expression level of stemness-related genes in tissue that was damaged due to chemotherapy, radiation, or a chemotherapy and radiation process but was not administered (e.g., prophylactically). In some embodiments, the reference is the expression level of stemness-related genes in tissue that was damaged due to chemotherapy, radiation, or a chemotherapy and radiation process and prior to administration of AATG. In some embodiments, regeneration of damaged tissue is determined by a healthcare provider (e.g., a physician). For example, a healthcare provider can determine a symptomatic measure that includes, but is not limited to, diarrhea, blood in stool, or a decrease in calprotectin in stool relative to a reference in a subject.

[0152] In some embodiments, the present disclosure relates to a method for promoting the regeneration of damaged or injured cells, comprising contacting damaged or injured cells with AATG that increases the level of AA in the damaged or injured cells or the environment surrounding the damaged or injured cells by at least 2-fold relative to a reference, to promote the regeneration of damaged or injured cells. In some embodiments, the damaged or injured cells are isolated from damaged tissue caused by chemotherapy, radiation, or chemotherapy and radiation.

[0153] In some embodiments, promoting the regeneration of damaged or injured cells comprises observing damage in the cell, administering AATG, and observing less damage in the cell. In some embodiments, regeneration comprises 100% recovery, about 95% recovery, about 90% recovery, about 80% recovery, about 70% recovery, about 60% recovery, about 50% recovery, about 40% recovery, or about 30% recovery from cell damage relative to a reference. In some embodiments, the reference is a damaged or injured cell caused by chemotherapy, radiation, or chemotherapy and radiation processes but without the administration (e.g., prophylactic administration) of AATG. In some embodiments, the prevention or reduction of cell damage or cell injury is determined by a health care provider.

[0154] In some embodiments, regeneration of damaged cells comprises increased expression of stemness markers relative to a reference, such as increased expression of stemness-related genes relative to a reference. In some embodiments, the stemness-related genes are at least one of the following: leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), hairless-scale family BHLH transcription factor 2 (Ascl2), lymphocyte antigen 6 complex locus A (Ly6a), or S100 calcium binding protein A6 (S100a6), LY6 / PLAUR domain-containing 6 (Lypd6), connective tissue growth factor (Ctgf), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (cromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln). In some embodiments, the expression of stemness-related genes is increased by at least or about 75%; at least or about 100%; at least or about 125%; at least or about 150%; at least or about 175%; at least or about 200%; at least or about 300%; at least or about 400%, at least or about 500%, at least or about 600%; at least or about 700%, at least or about 800%, or any range or combination thereof relative to a reference. In some embodiments, the reference is the expression level of stemness-related genes in tissue that has been damaged due to chemotherapy, radiation, or a chemotherapy and radiation process but has not been administered (e.g., prophylactically). In some embodiments, the reference is the expression level of stemness-related genes in tissue that has been damaged due to chemotherapy, radiation, or a chemotherapy and radiation process and prior to administration of AATG.

[0155] In some embodiments, the damaged or injured cells are or comprise damaged epithelial cells or injured epithelial cells. In some embodiments, the damaged or injured cells are or comprise damaged intestinal cells or injured intestinal cells. In some embodiments, the damaged or injured cells are or comprise damaged oral cells or injured oral cells. In some embodiments, the damaged or injured cells are or comprise damaged skin cells or injured skin cells.

[0156] cell

[0157] In some embodiments, the present disclosure relates to methods of preventing or reducing cell damage or cell injury comprising contacting a cell with an AATG prior to exposure to chemotherapy, radiation therapy, or a combination of chemotherapy and radiation therapy.

[0158] In some embodiments, the cell is or comprises an epithelial cell. In some embodiments, the cell is or comprises an oral cell, a skin cell, or an intestinal cell. In some embodiments, the cell is or comprises an intestinal cell. In some embodiments, the cell is or comprises a cultured cell. In some embodiments, the cell is or comprises a component of an organoid. In some embodiments, the cell is or comprises a human cell. In some embodiments, the cell is or comprises an animal cell. In some embodiments, the cell is or comprises a mammalian cell. In some embodiments, the cell is or comprises a portion of a tissue. In some embodiments, the tissue is or comprises epithelial tissue. In some embodiments, the tissue is or comprises intestinal tissue. In some embodiments, the cell is or comprises a cell of a living multicellular organism. In some embodiments, the cell is or comprises a cell obtained from a subject.

[0159] In some embodiments, AA levels are at least or about 75%, at least or about 100%, at least or about 125%, at least or about 150%, at least or about 175%, at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500%, at least or about 600%, at least or about 700%, at least or about 800%, or any range or combination thereof relative to a reference. In some embodiments, AA levels are measured inside cells. In some embodiments, AA levels are assessed in the environment surrounding the cells.

[0160] In some embodiments, the level of AA in a cell is increased by 1.5-fold, about 1.5-fold, or at least 1.5-fold; 2-fold, about 2-fold, or at least 2-fold; 3-fold, about 3-fold, or at least 3-fold; 4-fold, about 4-fold, or at least 4-fold; 5-fold, about 5-fold, or at least 5-fold; 6-fold, about 6-fold, or at least 6-fold; 7-fold, about 7-fold, or at least 7-fold; 8-fold, about 8-fold, or at least 8-fold; 9-fold, about 9-fold, or at least 9-fold; 10-fold, about 10-fold, or at least 10-fold; 11-fold, about 11-fold, or at least 11-fold; 12-fold, about 12-fold, or at least 12-fold; 13-fold, about 13-fold, or at least 13-fold; 14-fold, about 14-fold, or at least 14-fold; 15-fold, about 15-fold, or at least 15-fold relative to a reference. In some embodiments, the reference is the level of AA in the cell or an identical cell before administration of the AA TG. In some embodiments, the reference is the level of AA in a control sample of cells.

[0161] cancer

[0162] Cancer (malignant neoplasm) is a type of disease in which a group of cells exhibits the properties of uncontrolled growth (growth and division beyond the normal range), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spreading to other locations in the body through the lymph or blood). Most cancers form tumors, but some cancers (e.g., leukemias) do not. In some embodiments, the cancer is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelia sarcoma, synovialoma, mesothelioma, Ewing's sarcoma ...ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, sarcoma), leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioblastoma, neuronoma, craniopharyngioma, schwannoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma neuroama), oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia and lymphoma, acute lymphocytic leukemia and acute myelocytic polycythemia vera, multiple myeloma, Waldenstrom macroglobulinemia and heavy chain disease, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, colorectal cancer, urinary tract cancer, uterine cancer, oral cancer, skin cancer, stomach cancer, brain cancer, liver cancer, laryngeal cancer, esophageal cancer, breast tumors, childhood naked acute lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute myeloid leukemia, myelomonocytoid leukemia, acute megakaryocytoid leukemia, Burkitt's lymphoma lymphoma), acute myeloid leukemia, chronic myeloid leukemia and T-cell leukemia, small and large non-small cell lung cancer, acute myeloid leukemia, germ cell tumors, endometrial cancer, gastric cancer, head and neck cancer, chronic lymphocytic leukemia, hairy cell leukemia, or thyroid cancer.

[0163] Cancer treatment

[0164] In some embodiments, the AATG is administered to a subject receiving or intending to receive chemotherapy or radiation therapy. In some embodiments, chemotherapy comprises administering one or more pharmaceutical compositions including, but not limited to, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids. In some embodiments, the chemotherapeutic agent is one or more agents selected from the group consisting of altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, dapoxetine ... halan), oxaliplatin, temozolomide, thiotepa, trabectedin, nitrosoureas, azacytidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine abine), gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine and tipiracil combination, daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, actinomycin D, mitomycin-C, mitoxantrone one), irinotecan, irinotecan liposome, topotecan, etoposide (VP-16), teniposide, taxanes, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinca alkaloids, vinblastine, vincristine, vincristine liposome, vinorelbine, prednisone, methylprednisone, dexamethasone, all-trans retinoic acid, arsenic trioxide,Asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, or vorinostat.

[0165] In some embodiments, the subject receives or is intended to receive radiation therapy. In some embodiments, radiation therapy comprises external beam radiation therapy. In some embodiments, radiation therapy comprises three-dimensional conformal radiation therapy (3D-CRT). In some embodiments, radiation therapy comprises intensity modulated radiation therapy (IMRT). In some embodiments, radiation therapy comprises proton beam therapy. In some embodiments, radiation therapy comprises image-guided radiation therapy (IGRT). In some embodiments, radiation therapy comprises stereotactic radiation therapy (SRT). In some embodiments, radiation therapy comprises internal radiation therapy. In some embodiments, internal radiation therapy comprises permanent implants. In some embodiments, internal radiation therapy comprises temporary internal radiation therapy. In some embodiments, radiation therapy comprises intraoperative radiation therapy (IORT). In some embodiments, radiation therapy comprises systemic radiation therapy. In some embodiments, radiation therapy comprises radioimmunotherapy. In some embodiments, radiation therapy comprises a radiosensitizer and a radioprotectant. In some embodiments, radiation therapy comprises neoadjuvant radiation therapy. In some embodiments, radiation therapy comprises adjuvant radiation therapy. In some embodiments, radiation therapy comprises palliative radiation therapy.

[0166] medicine box

[0167] In some embodiments, a kit for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject is disclosed. In some embodiments, (a) one or more supplement units are sufficient to provide at least about 2g (2g / day) of arachidonic acid triglycerides (AA TG) to a subject in need thereof for at least 7 days; and (b) instructions for the preparation and consumption of one or more supplement units. In some embodiments, (a) one or more supplement units are sufficient to provide at least about 2g (2g / day) of at least one precursor of arachidonic acid (AA) to a subject in need thereof for a sufficient period of time; and (b) instructions for the preparation and consumption of one or more supplement units.

[0168] In some embodiments, the number of supplement units administered to a subject in need thereof is determined in consultation with a healthcare provider.

[0169] In some embodiments of the present invention, the medicine box may include a formulation vial, a formulation diluent vial, AATG, at least one precursor AATG, or AATG and at least one precursor AATG, and another pharmaceutical agent. The diluent vial contains a diluent, for example, for diluting an edible composition that can be a solution or powder (e.g., a concentrated solution or lyophilized powder) of AATG, at least one precursor AATG, or AATG and at least one precursor AATG. In some embodiments, the edible composition is a fruit or vegetable puree. In some embodiments, the edible composition is a nutritional milkshake, etc.

[0170] In some embodiments, the instructions include instructions for mixing a specific amount of diluent with a specific amount of concentrated solution or lyophilized powder to prepare a final formulation for administration. In some embodiments, the instructions include instructions for a syringe or other administration device. In some embodiments, the instructions include instructions for treating a patient with an effective amount of AATG, at least one precursor AATG, or AATG and at least one precursor AATG, and optionally one or more additional agents. It should also be understood that the container containing the formulation, whether the container is a bottle, a vial with a septum, an ampoule with a septum, a sealed bottle for edible liquids, etc., may include a mark, such as a conventional mark that changes color when the formulation is autoclaved or otherwise sterilized.

[0171] In some embodiments, the kit is provided or sold as a service bundled with instructions, instructions, or suggestions for consuming one or more supplement units from a health care provider. In some embodiments, the health care provider or consultant is a physician, dietitian, registered dietician, physician assistant, nurse practitioner, or nurse. In some embodiments, the health care provider is an oncologist or surgeon. In some embodiments, the instructions, instructions, or suggestions comprise verbal communication with the health care provider. In some embodiments, the instructions, instructions, or suggestions comprise written instructions.

[0172] In some embodiments, disclosed herein are kits containing one or more supplement units and one or more food compositions. In some embodiments, the kit further comprises instructions for consuming AATG, at least one precursor AATG, or AATG and at least one precursor AATG, and one or more food compositions. In some embodiments, the supplement units of AATG, at least one precursor AATG, or AATG and at least one precursor AATG are packaged separately from the one or more food compositions. In some embodiments, in one or more supplement units, AATG, at least one precursor AATG, or AATG and at least one precursor AATG are premixed with the one or more food compositions.

[0173] Supplement Unit

[0174] In some embodiments, a supplement unit containing AATG, a precursor of at least one AA (e.g., TG form), or both AATG and a precursor of at least one AA (e.g., TG form) is disclosed for administration to a subject in need thereof. In some embodiments, the supplement unit comprises AATG. In some embodiments, the supplement unit comprises a precursor of at least one AA in TG form (precursor AATG). In some embodiments, the supplement unit comprises AATG and a precursor of at least one AA in TG form (precursor AATG). In some embodiments, one supplement unit is administered daily. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 supplement units are administered daily to provide at least about 2 g (2 g / day) of AATG to a subject in need thereof. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 supplement units are administered daily to provide at least about 2 g (2 g / day) of at least one precursor of AA to a subject in need thereof. In some embodiments, the supplement unit comprises an oil comprising an AA TG and a pharmaceutically acceptable excipient. In some embodiments, the supplement unit comprises an oil comprising at least one precursor AA TG and a pharmaceutically acceptable excipient.

[0175] In some embodiments, the supplement unit is in the form of a liquid or powder. In some embodiments, the supplement unit is in the form of a pill or capsule. In some embodiments, the capsule comprises soft gelatin or is a soft gel capsule. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the capsule allows for the regulated release of AATG, at least one precursor AATG, or both AATG and at least one precursor AATG. In some embodiments, the capsule allows for the timed release of AATG, at least one precursor AATG, or both AATG and at least one precursor AATG.

[0176] In some embodiments, one or more supplement units are in one container (e.g., a bottle, a box) or more than one container. In some embodiments, one supplement unit is contained in a plastic pocket of a blister pack. In some embodiments, the blister pack is backed with a cardboard card. In some embodiments, the blister pack contains 10 plastic pockets, each containing one supplement unit. In some embodiments, one blister pack contains enough supplement units to provide at least 2 g (2 g / day) of AATG to a subject in need thereof.

[0177] In some embodiments, precursors of AA and AATG are commercially available to one of ordinary skill in the art. Some non-limiting examples include AA from Cargill (cargill.com / food-bev / na / arachidonic-acid), ARASCO as an oil from DSM, and TM , from DSM powder.

[0178] In some embodiments, one or more supplement units are consumed under medical supervision and are intended for dietary management of a condition in a subject in need thereof. In some embodiments, one or more supplement units can be the sole source of nutrition for a subject in need thereof. In some embodiments, one or more supplement units are intended to supplement or augment the general diet of a subject in need thereof.

[0179] In some embodiments, the supplement unit is a syrup, a liquid, a powder, a concentrated powder, a concentrated powder mixed with a liquid, a swallowable form, a soluble form, an effervescent, a granulated form or an oral liquid solution. In some embodiments, the supplement unit is prepared in any convenient form. In some embodiments, the supplement unit is in the form of a beverage, mayonnaise, salad dressing, margarine, low-fat spreads, dairy products, cheese spreads, processed cheese, dairy desserts, flavored milk, cream, fermented milk products, cheese, butter, condensed milk products, ice cream mixes, soy products, pasteurized liquid eggs, baked products, confectionery products, confectionery bars, chocolate bars, high-fat bars, liquid emulsions, spray-dried powders, freeze-dried powders, ultra-high temperature (ultra-high-temperature, UHT) pudding, pasteurized pudding, gel, jelly, yogurt or the food with fat-based or watery fillings (filling).

[0180] In some embodiments, the supplement unit further comprises water, sucrose, maltodextrin, milk protein concentrate, soybean oil, rapeseed oil, short chain fructooligosaccharides, soy protein isolate, corn syrup, sodium caseinate, and potassium citrate.

[0181] In some embodiments, the supplement unit comprises a flavoring, such as a natural flavoring or an artificial flavoring. In some embodiments, the flavoring is apple, banana, blueberry, caramel, cherry, chocolate, cinnamon, coffee, cranberry, grape, honey, kiwi, lemon, lime, lemon-lime, mango, mint, orange, peach, pineapple, raspberry, strawberry, tangerine, vanilla, or watermelon.

[0182] In some embodiments, the supplement unit comprises fat from one or more additional sources, such as an oil that is not an oil containing a significant amount of AATG. In some embodiments, the oil that does not contain a significant amount of AATG is an oil that does not contain more than 5% AATG per total volume of oil. In some embodiments, the fat from one or more sources promotes energy metabolism. In some embodiments, the supplement unit comprises a fat source that includes one or more of saturated, monounsaturated, and polyunsaturated fatty acids in a ratio that is found in a healthy diet for the subject.

[0183] In some embodiments, the supplement unit comprises about 3% or at least about 3% oil containing about 40%AATG. In some embodiments, the supplement unit comprises about 5% or at least about 5% oil containing about 40%AATG, about 10% or at least about 10% oil containing about 40%AATG, about 15% or at least about 15% oil containing about 40%AATG, or about 20% or at least about 20% oil containing about 40%AATG, or its arbitrary range or combination. In some embodiments, percentage AA oil is calculated as weight / volume percentage. In some embodiments, percentage AA oil is calculated as weight / weight percentage. In some embodiments, percentage AA oil is calculated as volume / volume percentage.

[0184] In some embodiments, one or more supplement units comprise about 5 g or at least about 5 g of an oil containing about 40% AATG. In some embodiments, a supplement unit comprises about 10 g or at least about 10 g of an oil containing about 40% AATG, about 30 g or at least about 30 g of an oil containing about 40% AATG, about 40 g or at least about 40 g of an oil containing about 40% AATG, about 50 g or at least about 50 g of an oil containing about 40% AATG.

[0185] In some embodiments, one supplement unit comprises 50 mg of AATG, 100 mg of AATG, 200 mg of AATG, 300 mg of AATG, 400 mg of AATG, 500 mg of AATG, 1 g of AATG, 2 g of AATG, 4 g of AATG, 5 g of AATG, 10 g of AATG, 15 g of AATG, 20 g of AATG, or any range or combination thereof. In some embodiments, one supplement unit comprises no more than 50 mg of AATG, 100 mg of AATG, 200 mg of AATG, 300 mg of AATG, 400 mg of AATG, 500 mg of AATG, 1 g of AATG, 2 g of AATG, 4 g of AATG, 5 g of AATG, 10 g of AATG, 15 g of AATG, 20 g of AATG. In some embodiments, one supplement unit contains at least 50 mg AATG, 100 mg AATG, 200 mg AATG, 300 mg AATG, 400 mg AATG, 500 mg AATG, 1 g AATG, 2 g AATG, 4 g AATG, 5 g AATG, 10 g AATG, 15 g AATG, 20 g AATG.

[0186] In some embodiments, one supplement unit contains 50 mg of at least one precursor AATG, 100 mg of at least one precursor AATG, 200 mg of at least one precursor AATG, 300 mg of at least one precursor AATG, 400 mg of at least one precursor AATG, 500 mg of at least one precursor AATG, 1 g of at least one precursor AATG, 2 g of at least one precursor AATG, 4 g of at least one precursor AATG, 5 g of at least one precursor AATG, 10 g of at least one precursor AATG, 15 g of at least one precursor AATG, 20 g of at least one precursor AATG, or any range or combination thereof. In some embodiments, one supplement unit contains no more than 50 mg of at least one precursor AATG, 100 mg of at least one precursor AATG, 200 mg of at least one precursor AATG, 300 mg of at least one precursor AATG, 400 mg of at least one precursor AATG, 500 mg of at least one precursor AATG, 1 g of at least one precursor AATG, 2 g of at least one precursor AATG, 4 g of at least one precursor AATG, 5 g of at least one precursor AATG, 10 g of at least one precursor AATG, 15 g of at least one precursor AATG, 20 g of at least one precursor AATG. In some embodiments, one supplement unit contains at least 50 mg of at least one precursor AATG, 100 mg of at least one precursor AATG, 200 mg of at least one precursor AATG, 300 mg of at least one precursor AATG, 400 mg of at least one precursor AATG, 500 mg of at least one precursor AATG, 1 g of at least one precursor AATG, 2 g of at least one precursor AATG, 4 g of at least one precursor AATG, 5 g of at least one precursor AATG, 10 g of at least one precursor AATG, 15 g of at least one precursor AATG, 20 g of at least one precursor AATG.

[0187] Example

[0188] Example 1: Fatty acid (FA) screening in mouse and human organs reveals ω-6 FA as a promoter of stemness

[0189] The present disclosure provides an explanation of how the fatty acids (FA) of different diets affect intestinal stem cell (ISC) function. By screening FA in intestinal organoids of mice and people, the present disclosure characterizes a subgroup of ω-6 family FA, ​​including but not limited to arachidonic acid (AA), which has a robust stemness-enhancing effect. For example, cross-species gene expression analysis reveals the induction of conservative repair-related stem cell reprogramming features in response to AA. In addition, single-cell RNA sequencing (scRNA-seq) is used to identify the new (de novo) stem cell state and dedifferentiation program induced by AA in vivo and in vitro. Without wishing to be bound by theory, it is believed that dietary AA (e.g., AA triglycerides) causes the production of epithelial prostaglandin E2 (PGE2), which activates the Ptger4-cAMP-PKA signal transduction axis to promote the stemness in mice and people. AA (e.g., AA triglycerides) can induce epigenetic reprogramming around stem cell regeneration-related genes in a Ptger4-dependent manner. The data presented herein demonstrate that dietary AA (eg, AA triglycerides) is a conserved promoter of stem cell regeneration that mimics the repair response to tissue injury through PGE2-Ptger4 signaling and downstream epigenetic reprogramming.

[0190] ISCs can undergo frequent symmetrical cell divisions to replenish the intestinal epithelium, one of the most regenerative tissues in mammals, consisting of a single layer of cells with absorptive, secretory, and barrier functions (Barker et al., 2007; Cheng and Leblond, 1974; Leblond and Stevens, 1948; Snippert et al., 2010). The progeny of dividing ISCs give rise to transit amplifying (TA) progenitors, which proliferate and differentiate into multiple lineages of the intestinal epithelium, including absorptive enterocytes and secretory cells, such as mucus-producing goblet cells, hormone-secreting enteroendocrine (EE) cells, chemosensory tuft cells, and Paneth cells (Bankaitis et al., 2018; Clevers, 2013). ISC self-renewal and differentiation can be tightly regulated by microenvironmentally derived signals, such as ligands, growth factors, and cytokines from neighboring Paneth cells (Sato et al., 2011), fibroblasts (Degirmenci et al., 2018; Greicius et al., 2018; Roulis et al., 2020; Shoshkes-Carmel et al., 2018), enteric glia (Van Landeghem et al., 2011), and immune cells (Beyaz et al., 2021a; Biton et al., 2018; Lindemans et al., 2015) surrounding the intestinal crypts (Clevers, 2013). Homeostatic regeneration of the intestinal epithelium can be maintained by ISCs expressing the leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) both in vivo and in clonogenic organoid cultures in vitro (Barker et al., 2007; Sato et al., 2009). When the Lgr5+ stem cell compartment is damaged, plasticity and dedifferentiation of multiple epithelial lineages may enable efficient regeneration and repair of the intestinal epithelium (Clevers, 2013; de Sousa and de Sauvage, 2019; Potten et al., 1978; Tian et al., 2011).Secretory progenitors, EE progenitors, TA progenitors, Paneth cells, and enterocyte progenitors are lineages known to dedifferentiate and acquire stem cell potential in response to intestinal injury caused by radiation, infection, chemotherapy, or depletion of Lgr5+ ISCs using genetic models (Asfaha et al., 2015; Buczacki et al., 2013; Jadhav et al., 2017; Nusse et al., 2018; Schmitt et al., 2018; Tetteh et al., 2016; Tian et al., 2011; van Es et al., 2012; von Moltke et al., 2016; Yan et al., 2017; Yu et al., 2018). Without wishing to be bound by theory, proposed mechanisms for stem cell reprogramming in response to tissue injury include maintenance of accessible chromatin, induction of fetal-like gene expression programs, cytokine signaling, and Notch signaling (Ayyaze et al., 2019; Gregorieff et al., 2015; Jadhav et al., 2017; Murata et al., 2020; Nusse et al., 2018; Yu et al., 2018; Yui et al., 2018).

[0191] Accumulating evidence suggests that nutrients and metabolic pathways not only influence growth and proliferation but can also significantly impact cell function and fate by signaling to transcription factors and altering epigenetic landscapes (Beyaz et al., 2016; Beyaz et al., 2021b; Beyaz and Yilmaz, 2016; Chandel et al., 2016; Chen et al., 2020; Cimmino et al., 2018; Lu and Thompson, 2012). Although recent studies have begun to explore metabolic regulation of ISC activity through fatty acid (FA) oxidation (Chen et al., 2020; Mihaylova et al., 2018; Stine et al., 2019), ketone body signaling (Cheng et al., 2019), mitochondrial pyruvate metabolism (Rodriguez-Colman et al., 2017; Schell et al., 2017), vitamins (Jijon et al., 2018; Lukonin et al., 2020; Peregrina et al., 2015), and microbiome-derived metabolites (Kaiko et al., 2016; Lee et al., 2018), the present disclosure provides insights into how nutrients and their metabolite derivatives influence ISC activity and cellular plasticity in the intestine by inducing epigenetic changes. Different dietary interventions that perturb organismal metabolism (e.g., fasting, calorie restriction, ketogenic, or obesity-promoting high-fat diets (HFDs)) have focused on their ability to enhance ISC activity through both cell-intrinsic mechanisms (Beyaz et al., 2016; Cheng et al., 2019; Fu et al., 2019; Mihaylova et al., 2018; Wang et al., 2018) and microenvironment-mediated extrinsic mechanisms (Igarashi and Guarente, 2016; Yilmaz et al., 2012). One of the common features of these stemness-enhancing dietary interventions is that they can increase the abundance and metabolism of FAs, either through dietary intake or release from adipose tissue (Novak et al., 2021). Fasting and long-term obesogenic lard-based HFD may enhance ISC function in part by activating FA metabolism (Beyaz et al., 2016; Beyaz et al., 2021b; Mihaylova et al., 2018).

[0192] There are several potential mechanisms by which FAs may regulate stem cell fate. First, FAs or their metabolites can bind to and activate FA-sensing transcription factors (TFs) (e.g., PPAR-δ) to directly regulate transcription (Beyaz et al., 2016; Beyaz et al., 2021b; Evans and Mangelsdorf, 2014; Neels and Grimaldi, 2014). Second, FA-derived metabolites (e.g., acetyl-CoA) can be used for histone modification and influence epigenetic status (McDonnell et al., 2016; Schvartzman et al., 2018). Third, alterations in cellular FA abundance can perturb membrane lipid composition and affect signaling pathways (Zhu et al., 2019). Fourth, bioactive lipids derived from FAs can activate G protein-coupled receptors (GPCRs), which stimulate second messengers capable of signaling downstream cascades and multiple TFs that regulate cell fate and function (Brash, 2001). Finally, FA can perturb the microbiome and immune cells, which can influence ISC activity (Beyaz et al., 2021a; Biton et al., 2018). While these results suggest that FA and its metabolism can be linked to stem cell fate and function, it remains unclear how different types of dietary FA influence stemness and epigenetic regulation of gene expression in the intestinal epithelium.

[0193] Intestinal organoids have the potential to recapitulate the compositional and functional characteristics of the mammalian intestine, including stem cell regeneration and differentiation in culture, and therefore provide a reliable system to identify factors that promote stemness (Beyaz et al., 2016; Kaiko et al., 2016; Lukonin et al., 2020; Sato et al., 2009). To explore how various dietary FAs affect intestinal stemness, a live imaging screening platform was developed that monitors organoid formation from single cells and measures features of stem cell activity, including organoid morphology (spheroid vs. branched), size, and number, over a period of five days (Beyaz et al., 2016; Farin et al., 2012; Mustata et al., 2013; Schuijers et al., 2015) ( Figure 8A , see Example 9). A group of 23 FAs was assembled and stratified according to degree of saturation (polyunsaturated, monounsaturated, and saturated), position of double bonds (ω-3, ω-6, ω-7, and ω-9), configuration (cis, trans), chain length (short, medium, long), and number of double bonds (1 to 6) ( Figure 1A , Table 1).

[0194] Table 1: Fatty acids used for screening in mouse and human organs.

[0195]

[0196]

[0197] Since most FAs in vivo are bound to serum albumin to enhance transport and solubility, poorly soluble FAs were conjugated to bovine serum albumin (BSA) (Beyaz et al., 2016; Brash, 2001; McArthur et al., 1999; Spector et al., 1969; Zhu et al., 2019) (see Example 9). Dose-response experiments were performed to define the FA concentration that does not induce lipotoxicity in organoids (Alsabeeh et al., 2018; Brash, 2001) ( Figure 8B FA screening in mouse intestinal organoid-derived single cells revealed that treatment with FAs (belonging to the ω-6 family, such as linoleic acid (LA), γ-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), and arachidonic acid (AA), but not docosatetranoic acid (DA) or the trans FA linoelaidic acid (LEA)) promoted the formation of spheroids lacking budding crypt-like domains ( Figure 1B and 8C This spheroid morphology can be associated with an enhanced regenerative stem cell state and reduced differentiation (Beyaz et al., 2016; Beyaz et al., 2021b; Farin et al., 2012; Mustata et al., 2013; Schuijers et al., 2015). Treatment with these FAs also resulted in a significant increase in size relative to vehicle-treated control organoids starting after approximately 72 hours, which is the approximate duration of symmetry disruption in single-cell-derived organoids (Serra et al., 2019) ( Figure 1C and 8C To assess the human relevance of these results, human patient-derived organoids (PDOs) generated from normal colon sections and obtained from male or female patients of different ancestries (Table 2) were used and FA screening was performed.

[0198] Table 2: Patient information of patient-derived organoids used in the study.

[0199] Patient coding gender age BMI P1 female 51 27 Asian P2 male 50 26 White people P3 female 89 34 Hispanic P4 male 77 23 White people P5 female 66 31 African Americans P6 female 64 31 White people P7 male 31 23 White people

[0200] Similar to the screen in mice, it was possible to treat dissociated single cells from human PDO with a subset of ω-6 family FAs, including dh-γ-LA and AA, to promote growth relative to control organoids ( Figure 1D and 8D FA elongases (Elov15) and desaturases (Fads1 and Fads2) that regulate the biosynthesis of AA from essential FAs (Fan et al., 2012; Moon et al., 2009) are abundantly expressed in organoids of both mice and humans ( Figures 8E to 8F Given that DA does not promote organoid growth, it was hypothesized that the stemness-enhancing effects of ω-6FA could be focused on AA. Indeed, inhibition of FADS1, the rate-limiting desaturase in AA biosynthesis (Fan et al., 2012), attenuated the increase in organoid size in response to ω-6FA ( Figure 8G For these reasons, AA was selected as a target for further functional evaluation of stem cell regeneration. It was determined that AA treatment resulted in more proliferating cells in spheroids that lacked crypt domains and had larger sizes relative to vehicle-treated control organoids ( Figures 1E to 1J Transmission electron microscopy analysis showed that these AA-induced spheroids had smaller microvilli and fewer Paneth cells with granules, indicating reduced differentiation (Crawley et al., 2014; Miyoshi et al., 2017) ( Figures 8H to 8I In addition, subculture experiments were performed to functionally evaluate stem cell activity after AA treatment (Beyazet et al., 2016). When subcultured, primary mouse AA-induced spheroids produced more secondary organoids that were again larger in size relative to controls and had spheroid morphology ( Figures 1K to 1N Similarly, AA treatment increased organoid size and promoted spheroid formation in human intestinal PDO in both primary and secondary cultures ( Figures 1O to 1T These results suggest that ω-6FA concentrated in AA enhances organ stemness in both mice and humans.

[0201] Example 2: AA-rich diet (ARD) promotes intestinal regeneration in vivo

[0202] AA is a bioactive lipid that plays important structural and functional roles in mammalian cells and tissues, including the intestinal epithelium (Brash, 2001; Fan et al., 2016; Fan et al., 2012). The functional significance of dietary AA supplementation in intestinal homeostasis was explored in Calder et al. (2019). To investigate the effects of AA on ISC function in vivo, a new isocaloric (3.8 kcal / g) AA-enriched diet (ARD) model (Teklad, TD190641) was developed with a matched purified control (control) diet (Teklad, TD97184) ( Figure 2A Oil extracted from the fungus Mortierella alpina (M. alpina) was used and contains approximately 40% AA in the form of triglycerides, which is required for a diet containing 3% AA-enriched oil and 4% soybean oil (7% total fat) (Kikukawa et al., 2018). ARD and its matched isocaloric control consisted of equivalent amounts of major nutrients (protein, carbohydrates, and fat) and minor nutrients (minerals and vitamins) (Table 3).

[0203] Table 3: Composition of isocaloric AA-enriched diets and their matched purified control diets.

[0204]

[0205]

[0206]

[0207] Feeding mice with ARD for four weeks did not affect their body weight or plasma glucose levels, but resulted in an increase in the abundance of AA in both plasma and intestine relative to controls ( Figures 2B to 2C Moreover, metabolomic analysis revealed that the amounts of other major metabolites were not significantly altered in the intestines of mice fed with ARD ( Figure 9C Such an increase in AA abundance in the intestine leads to an increase in crypt length and the number of Ki67+ proliferating cells per crypt (Gerdes et al., 1984). Figures 2D to 2GIntestinal crypts were isolated from mice fed an ARD or control diet for functional organoid assays. Intestinal organoid morphology and organoid formation capacity were assessed and determined to be a proxy for stem cell activity (Beumer and Clevers, 2016; Beyazet al., 2016; Mustata et al., 2013; Nusse et al., 2018; Sato et al., 2009; Yui et al., 2018). ARD promoted the formation of regenerative spheroid morphology and resulted in a significant reduction in the number of crypt domains per organoid relative to controls (9 Figures 2H to 2J Sorted Epcam+ intestinal epithelial cells from the crypts of ARD-fed mice generated more organoids than controls and demonstrated that dietary AA promotes stem cell activity in vivo under steady-state conditions ( Figures 2K to 2L ).

[0208] The intestinal epithelium exhibits a rapid regenerative response to a variety of stressors to maintain tissue function and barrier integrity (Bankaitis et al., 2018; Gehart and Clevers, 2019). Ionizing radiation is frequently used to assess stem cell regeneration after intestinal injury (Beyaz et al., 2016; Potten, 1977; Withers and Elkind, 1970). Administration of a clinically relevant 15 Gy of γ-irradiation to control mice induces cytotoxicity in the intestinal epithelium that results in crypt loss and decreased intestinal length (Beyaz et al., 2016; Kirsch et al., 2010) ( Figures 2M to 2O Feeding mice with ARD reversed these effects and enhanced crypt regeneration, as evidenced by an increase in proliferating cells incorporating 5-ethynyl-2'-deoxyuridine (EdU) (Salic and Mitchison, 2008) per unit area of ​​intestine and per crypt relative to controls ( Figures 2P to 2R and 9E). Since cancer chemotherapeutics induce similar cytotoxic effects, it is uncertain whether ARD can promote intestinal regeneration in a separate injury model using doxorubicin, a commonly used antitumor drug with well-characterized adverse effects in the intestine (Dekaney et al., 2009; Ijiri and Potten, 1987). 72 hours after treatment in mice fed a control diet, doxorubicin resulted in a significant decrease in intestinal length. In mice treated with doxorubicin, ARD prevented intestinal shortening and increased the number of EdU+ proliferating crypt cells ( Figure 2S and 9F to 9 hours). This is consistent with the proliferation assessment using Ki67 ( Figure 2F), for both models, crypts in intact ARD-fed mice contained more EdU+ proliferating cells relative to intact controls ( Figure 2Q and 9G Taken together, our data demonstrate that dietary enrichment of intestinal AA enhances ISC regeneration in vivo.

[0209] Example 3: AA can induce conserved stem cell reprogramming gene expression characteristics

[0210] Intestinal organoids can recapitulate regenerative features of the intestinal epithelium (Beyaz et al., 2016; Sato et al., 2009; Serra et al., 2019). To elucidate the mechanism by which AA enhances stemness, temporal dynamic bulk RNA-seq analysis was performed at different mouse organoid developmental stages, including the formation of symmetric vesicles (day 1), symmetry disruption (day 3), and mature organoids with differentiated cells (day 6) (Beyaz et al., 2016; Sato et al., 2009; Serra et al., 2019) ( Figures 10A to 10B AA treatment resulted in a strong upregulation of genes associated with stem cell reprogramming at different time points in response to ablation of Lgr5+ stem cells (Lypd6, Ctgf, Anxa13) (Murata et al., 2020), radiation damage (Ccnd1, Anxa3, Ly6a, Clu) (Ayyaz et al., 2019), and granuloma formation (Il33, S100a6, Areg) (Nusse et al., 2018), or regeneration of a fetal-like state (fetal spheroids) (Cd55, Ereg, Myof, Msln) (Mustata et al., 2013) ( Figures 3A to 3B ). In contrast, organoids downregulated markers of differentiated cells in response to AA, such as Paneth cells (Defa24, Defa21, Lyz1), tuft cells (Dclk1), and enteroendocrine cells (Neurog3) (Haber et al., 2017)( Figure 3B Interestingly, in AA-treated organoids, Lgr5 expression and homeostatic stem cell characteristics (Haber et al., 2017; Munoz et al., 2012) were initially suppressed on days 1 and 3 but were restored on day 6 ( Figures 3A to 3B All previously reported signatures of reprogrammed stem cells significantly overlapped with genes induced by AA, suggesting that AA can induce a gene expression signature that may be central to stem cell regeneration in different experimental models ( Figures 10C to 10DA signature gene list induced by AA was designed, and it included relevant stem cell reprogramming genes (Ly6a, S100a6, Ccnd1, Cd55, Msln) (Ayyaz et al., 2019; Mustata et al., 2013; Nusse et al., 2018) and the CREB target Nr4a1 (Rodon et al., 2019), and their induction in AA-treated organoids was confirmed by qRT-PCR ( Figure 10E ).

[0211] Gene set enrichment analysis (GSEA) of AA-induced genes showed enrichment for wound healing, cell proliferation, the PPAR pathway, as well as lipid metabolism and calcium signaling. In addition, GSEA revealed key regulators of intestinal stemness during homeostasis and regeneration in response to injury, such as the Wnt / β-catenin and Egfr pathways (Beumer and Clevers, 2016) ( Figure 1F ). Therefore, the present disclosure evaluated whether these pathways are functionally involved in the AA-induced stemness phenotype. First, a subset of Wnt / β-catenin targets (including stem cell reprogramming signature genes such as S100a6 (Ayyaz et al., 2019; Mustata et al., 2013; Nusse et al., 2018) and Ccnd1 (Ayyaz et al., 2019; Mustata et al., 2013)) were upregulated in AA-treated organoids ( Figure 10G ), while the nuclear localization of β-catenin increased, which may be an indicator of its activity (Molenaar et al., 1996) ( Figure 3C Titration of exogenous Wnt3a (Beyaz et al., 2016) showed that AA treatment reduced the dependence of organoid formation and growth on Wnt ( Figures 3D to 3F Secondly, AA enhances the expression of Egf family receptors Egfr and its ligands such as Areg and Ereg, which are characteristic genes of stem cell reprogramming (Mustata et al., 2013; Nusse et al., 2018) and are mainly produced by stromal cells to support epithelial repair after injury in a paracrine manner (Gregorieff et al., 2015; Lee et al., 2004; Monticelli et al., 2015; Shao and Sheng, 2010; Van Landeghem et al., 2011; Yang et al., 2017) ( Figure 3B and 3GThis was a prompt to test whether AA-induced autocrine Egfr ligand expression contributes to enhanced stemness. The present disclosure found that replacing Egf (an essential component of organoid culture media (Basak et al., 2017; Oszvald et al., 2020; Sato et al., 2009)) with Areg or Ereg is sufficient to construct organoids from dissociated single cells ( Figures 10H to 10I ). Treatment with AA significantly promoted organoid growth in the absence of Egf, which is required for stem cell proliferation (Basak et al., 2017; Biteau and Jasper, 2011; Jiang and Edgar, 2009) ( Figures 3H to 3I These findings suggest that AA-induced transcriptional reprogramming reduces dependence on exogenous Wnt and Egf signals from the microenvironment and promotes stemness.

[0212] To confirm these observations in humans, human PDO were used and gene expression changes in response to AA were assessed ( Figure 10J GSEA showed that AA-induced genes were enriched for homeostasis and repair-related stemness traits as well as Myc pathway and cell proliferation ( Figure 3J and 10K Similar to mice, genes associated with stem cell reprogramming (CD55, MYOF, MSLN, ANXA3, AREG, CCND1, LYPD6) (Ayyaz et al., 2019; Murata et al., 2020; Musta et al., 2013; Nusse et al., 2018), Wnt / β-catenin targets (L1CAM, TCF4, CCND1) (Beyaz et al., 2016), Egfr ligand (AREG) (Monticelli et al., 2015), and CREB targets (NR4A1, SIK1) (Rodone et al., 2019) were robustly upregulated in AA-treated human PDOs ( Figures 3K to 3L Together, these results highlight that AA can trigger a conserved stem cell regenerative program that is somewhat reminiscent of the repair response to injury.

[0213] Example 4: Single-cell analysis of AA-induced stemness in vivo and in vitro

[0214] To determine the precise cellular states that define AA-induced stemness in vivo, single-cell RNA sequencing (scRNA-seq) was performed. 23,161 single cells from crypts were profiled after filtering and clustering to define intestinal epithelial cell types (Ayyaz et al., 2019; Grun et al., 2015; Haber et al., 2017) ( Figures 11A to 11C Dietary AA generates new stem cell-like clusters (Stem 2) in vivo, which are absent in control crypts and are characterized by high expression of stem cell reprogramming-related markers Ly6a and S100a6 (Ayyaz et al., 2019; Mustata et al., 2013; Nusse et al., 2018)( Figures 4A to 4B and 11A to 11F), rather than high expression of other putative stem cell markers such as Clu and Msi1 (Ayyaz et al., 2019; Wang et al., 2020). S100a6 and Ly6a represent two AA-induced genes that are shared by fetal, radiation-induced, and granuloma-induced repair features ( Figure 10C In addition, ARD leads to upregulation of stem cell marker genes Lgr5 and Aslc2 in transition amplifying (TA) cells, enterocyte progenitors (EP), enteroendocrine cells (EE), and goblet cells, but not in homeostatic stem cells (Stem 1) ( Figures 4C to 4D TA, EP, and EE cells have been shown to dedifferentiate and regenerate intestinal crypts in response to crypt injury (Jadhav et al., 2017; Nusse et al., 2018; Tetteh et al., 2016; Tian et al., 2011; Yan et al., 2017). Ascl2, a stem cell-restricted Wnt / β-catenin target (Schuijers et al., 2015; van der Flier et al., 2009), has recently been shown to orchestrate stem cell regeneration after injury through dedifferentiation of intestinal epithelial cells and secretory progenitor cells (Murata et al., 2020). Consistent with this, a pseudo-time trajectory analysis algorithm was used (Cao et al., 2019), and the present disclosure discovered a dedifferentiation trajectory from TA, EP, and EE to AA-induced stem 2 clusters in vivo ( Figures 4E to 4F Crypt cells from ARD mice showed increased expression of Lgr5, Ascl2, and reprogramming-related markers Ly6a and S100a6 relative to controls during the differentiation pseudo-time trajectory ( Figure 4G and11E Upregulation of Ascl2 in upper crypt cells is a hallmark of intestinal regeneration in response to crypt injury before they dedifferentiate into stem cells (Murata et al., 2020). To assess how dietary AA affects the spatial expression of these key stemness genes, single-molecule fluorescence in situ hybridization (sm-FISH) was performed. Mice fed ARD expressed higher levels of Lgr5 and Ascl2 per crypt, and at steady state, the frequency of cells expressing these markers in the upper crypt layer was increased ( Figures 4H to 4M and 11G to 11H). This is consistent with the observation that ARD mice can exhibit enhanced intestinal regeneration in vivo relative to controls after irradiation ( Figures 3A to 3L ), determined that dietary AA increases Lgr5 and Ascl2 expression in regenerating crypts in response to radiation. Regenerating crypts in irradiated ARD mice contained a higher percentage of cells expressing Lgr5 and Aslcl2 in the upper crypt layer relative to irradiated controls ( Figures 4H to 4M and 11G to 11H). In addition, dietary AA promoted the expression of repair-related stem cell signature genes (e.g., S100a6) in crypt cells both at steady state and in response to radiation ( Figures 4N to 4P Together, these results suggest that dietary AA can promote stemness in the intestine by inducing a regeneration-associated neoblast state and a dedifferentiation program in vivo.

[0215] To assess whether dietary AA induces stem cell reprogramming through epithelial-intrinsic mechanisms, 23,599 single cells from organoids were profiled by scRNA-seq ( Figures 12A to 12C In organoid cultures, AA treatment for three days resulted in the emergence of new stem cell-like states (Stem 2 and Stem 3) marked by signature genes associated with stem cell reprogramming, such as Ly6a, Clu, and S100a6 ( Figures 12A to 12G and 12K). Furthermore, pseudo-temporal analysis of scRNA-seq from organoids revealed a dedifferentiation trajectory toward a nascent stem cell-like state and upregulation of Ly6a and S100a6 in the pseudo-temporal trajectory ( Figures 12L to 12R ). AA-treated organoids showed significant differences in stem / progenitor cells, intestinal epithelial cells, and EE cells ( Figure 12H ) and in the pseudo-time trajectory ( Figure 12S ) showed increased Ascl2 expression relative to controls. Some features differed between crypts and organoids, such as decreased Lgr5 expression in stem and progenitor cell clusters and in the pseudo-temporal trajectory, likely due to in vitro characteristics or temporal aspects of organoid culture (Lukonin et al., 2020; Sato et al., 2009; Serra et al., 2019; Yui et al., 2018). Figure 12I and 12T ), which is consistent with the bulk RNA-seq data of day 3 organoids ( Figure 3B In contrast to a recent report (Wang et al., 2020), no induction of Msi1 in response to AA was observed in vivo or in vitro ( Figure 11D and 12J Together, these results indicate that AA-treated organoids generally exhibit features similar to crypts from ARD mice, suggesting epithelial-intrinsic mechanisms as possible drivers of the AA-induced stemness phenotype.

[0216] Example 5: AA metabolism towards prostaglandin E2 (PGE2) may be necessary and sufficient to promote stem cell reprogramming

[0217] AA exerts its biological activity through several mechanisms, such as by regulating membrane fluidity, ion channels, reactive oxygen species levels, lipid sensing receptors (e.g., PPARs), and by generating a large number of bioactive lipids through non-enzymatic and enzymatic degradation, especially in response to tissue damage (Brash, 2001). Data suggest that dietary AA can promote stemness and induce repair-related stem cell reprogramming characteristics in the absence of any overt intestinal epithelial damage ( Figures 2A to 4P Because AA and AA-derived metabolites can be conserved regulators of wound detection and tissue repair (Fan et al., 2014; Katikaneni et al., 2020; Miyoshi et al., 2017), the present disclosure attempts to evaluate alterations in AA metabolism in our model. Metabolomic analysis showed that AA treatment can lead to changes in in vitro organoids ( Figure 5A ) and crypts in vivo ( Figure 13B To determine whether AA-derived metabolites could be sufficient to promote stem cell activity, we screened in mouse organoids and found that prostaglandin E2 (PGE2) and, to a lesser extent, PGD2, reproduced AA-induced stemness. Figure 5B and 13C ). Use of the nonsteroidal anti-inflammatory drug (NSAID) celecoxib ( Figures 5C to 5D ) or indomethacin ( Figures 13D to 13E ) inhibition of prostaglandin production attenuated AA-induced stemness in organoid assays, suggesting that epithelial PGE2 production may be necessary for the stemness-enhancing effects of AA.

[0218] Paracrine PGE2 production in the intestine contributes to wound repair (Miyoshi et al., 2017; Roulis et al., 2014) and carcinogenesis (Roulis et al., 2020; Wang and DuBois, 2018), but little is known about how diet modulates PGE2 signaling in epithelial cells to regulate stem cell function. In addition to AA-treated organoids, the present disclosure also confirmed PGE2 production by sorted Epcam+ crypt cells in response to AA ( Figure 13F Furthermore, AA treatment resulted in the adaptive upregulation of enzymes regulating prostaglandin production in organoids, such as Ptges and Ptgs2 ( Figure 13A and 13G Single-cell analysis of AA-treated organoids demonstrated adaptive induction of Ptges expression in stem cells, progenitor cells, and intestinal epithelial cells, and highlighted a possible epithelial source of PGE2 in response to AA ( Figure 13H ). PGE2 treatment in organoids triggered a similar effect to that in mice ( Figure 5E ) and people( Figure 5F ) in organoids from both mice and rats. Furthermore, scRNA-seq of mouse organoids in response to PGE2 treatment revealed similar stem cell reprogramming features to those observed in AA-treated organoids, such as the emergence of a nascent stem cell-like state marked by reprogramming-associated genes ( Figures 5G to 5I and 13I to 13L), and upregulation of Ascl2 in stem / progenitor cells ( Figure 5J ). Similar to AA, PGE2 triggered a dedifferentiation trajectory, in which the reprogramming-related genes Ascl2, S100a6, and Ly6a were upregulated in organoids, but Lgr5 was not ( Figures 5K to 5M and 13M to 13N). These data suggest that the stemness-enhancing effects of dietary AA may be mediated by PGE2 signaling in both mice and humans.

[0219] Example 6: Ptger4-cAMP-PKA signaling axis regulates AA-induced stemness in mice and humans

[0220] PGE2 binds to four G protein-coupled receptors (Ptger1 to 4) and activates different downstream pathways to mediate different functions (Breyer et al., 2001; Narumiya et al., 1999). To determine which PGE2 receptor subtype is required for AA-induced stemness, we screened pharmacological inhibitors of each receptor in an organoid assay and found that inhibition of Ptger4 (but not other PGE2 receptors) attenuated AA-induced stemness ( Figures 14A to 14E). Ptger4 is highly expressed in both mouse and human organoids ( Figures 14G to 14H Treatment with AA promoted the expression of Ptger4 (but not other PGE2 receptors) in secretory progenitor cells, TA cells and newborn stem cells (Stem 2 and Stem 3) ( Figures 14I to 14L Furthermore, Ptger4 knockout (Ptger4 KO) organoids were generated and the necessity of PGE2-Ptger4 signaling in mediating the stemness-enhancing effects of AA was demonstrated ( Figures 6A to 6C Inhibition of Ptger4 signaling attenuated the upregulation of signature genes associated with stem cell reprogramming in response to AA or PGE2 ( Figure 6D and 14F ).

[0221] PGE2 signaling through Ptger4 participates in multiple downstream pathways, such as activation of adenylate cyclase to increase cAMP production, activation of phosphatidylinositol 3-kinase (PI3K), β-arrestin, β-catenin, and extracellular signal-regulated kinase (ERK) (Yokoyama et al., 2013). Using a membrane-permeable and stable cAMP derivative (8-bromo) in organoid assays (Tuesta et al., 2017), it was found that elevated cAMP levels were sufficient to reproduce the effects of AA on organoid growth and enhance the expression of AA signature genes ( Figures 6E to 6H Elevated cAMP levels activate downstream effector molecules, including protein kinase A (PKA), exchange protein activated by cAMP (Epac), and cyclic nucleotide-gated ion channels (Sassone-Corsi, 2012). Inhibition of PKA using a potent antagonist (H89) (Chijiwa et al., 1990) attenuated the effects of AA on organoid growth and AA-induced characteristic gene expression ( Figures 6I to 6L Ptger4 is highly conserved between mice and humans (Narumiya et al., 1999). Human PDO was used to assess whether the Ptger4-cAMP-PKA signaling axis regulates AA-induced stemness in the human intestine. Similar to the observations in mouse organoids, Ptger4 and PKA were found to enhance the stemness of AA ( Figures 6M to 6P ) and for increasing cAMP levels to promote stem cell activity in human PDO ( Figures 6Q to 6R ) may be necessary.

[0222] To elucidate whether Ptger4 signaling is required for ARD-induced enhanced intestinal regeneration in vivo, we generated Lgr5-CreERT2-IRES-GFP,Ptger4 f / f mice, which allow inducible ablation of Ptger4 in green fluorescent protein (GFP)-labeled Lgr5+ stem cells and their progeny in intestinal crypts. We found that ARD failed to increase the number of EdU+ cells in Ptger4-deficient crypts under steady-state conditions and after irradiation, highlighting the necessity of Ptger4 signaling in mediating ARD-induced stem cell regeneration in vivo ( Figures 6S to 6T Together, these data identify PGE2 signaling through the Ptger4-cAMP-PKA axis as a conserved mechanism in mice and humans by which dietary AA promotes reprogramming and regeneration of stem cells in the intestine.

[0223] Example 7: AA triggers epigenetic reprogramming around regeneration-related gene loci in a Ptger4-dependent manner

[0224] It has been observed that there is an open chromatin state between adult stem cells and differentiated cells in the intestinal epithelium, with limited differences in epigenetic indicators such as chromatin accessibility, histone and DNA modifications (Jadhav et al., 2016; Kaaij et al., 2013; Kazakevych et al., 2017; Kim et al., 2014; Sheaffer et al., 2014). Although stem cell plasticity and dedifferentiation in response to intestinal injury are partly attributed to this low chromatin barrier between differentiated cells and the stem cell state, little is known about how dietary and metabolic signals that affect stem cell regeneration affect the intestinal epigenome (Verzi and Shivdasani, 2020). To determine whether AA-induced stemness involves epigenetic reprogramming, the present disclosure used sequencing (ATAC-seq) to measure transposase-accessible chromatin, which captures accessible chromatin regions in organoids treated with vehicle or AA (Buenrostro et al., 2013). Differential analysis of accessible chromatin regions between AA-treated vs. vehicle-treated organoids showed that AA promoted, rather than repressed, chromatin accessibility around promoters, enhancers, and intergenic regions (5807 open peaks, 2132 closed peaks, q < 0.01, and absolute log2 fold change > 0.58) ( Figure 15A AA-induced reprogramming of chromatin accessibility around promoters can lead to concomitant upregulation of neighboring genes with open ATAC-seq peaks ( Figure 15BPathway enrichment analysis of regions with increased accessibility in response to AA has highlighted stemness-associated features, including telomerase activity (Hoffmeyer et al., 2012; Montgomery et al., 2011; Schepers et al., 2011) and acetyltransferase complex activity (Sampurno et al., 2013; Yin et al., 2014). Certain pathways are critical for stem cell proliferation and regeneration in response to injury, such as EGF (Basak et al., 2017), calcium signaling and calcium-binding S100 proteins (Bresnick et al., 2015; Deng et al., 2015), stem cell regulators such as β-catenin (Beumer and Clevers, 2016), MYB (Cheasley et al., 2011), CREB and its targets (ID1 and ID2) and chaperones (ATF and JUN) (Nigmatullina et al., 2017; Sampurno et al., 2013; Zhang et al., 2014), all of which are consistent with gene expression and functional data on the stemness-enhancing effects of AA ( Figure 7A On the other hand, regions that lost accessibility through AA treatment were enriched for signatures of enteroendocrine cells, extracellular matrix, negative regulators of wound healing, and PRC2 targets ( Figure 7A To identify TFs with regulatory potential in AA-induced open or closed chromatin regions, a multivariate linear model was constructed based on the peak changes in the presence of TF motifs (Doane et al., 2021). Motifs for NFIC, a suppressor of proliferation and Ccdn1 expression (Eeckhoute et al., 2006), and for the enteroendocrine cell marker NEUROD1 (Li et al., 2019) were enriched in regions closed in AA-treated organoids ( Figure 7BTF motifs associated with stemness and regeneration were identified as enriched in regions that gain chromatin accessibility in response to AA, such as NFE2L1 (NRF1) (Schell et al., 2017), the AP1 family (FOS, JDP2, JUNB) (Haber et al., 2017; Nateri et al., 2005), PPARs (Beyaz et al., 2016; Beyaz et al., 2021b), the YAP complex (TEAD3) (Gregorieff et al., 2015; Yui et al., 2018), the Notch modulator HES1 (Pellegrinet et al., 2011; VanDussen et al., 2012), KLF5 (Nandan et al., 2015), and the CREB complex (CREB1 and ATF4) (Sampurno et al., 2013) ( Figure 7B Among these factors, YAP and CREB are important and conserved regenerative regulators (Deng et al., 2015; Gregorieff et al., 2015; Li and Fan, 2017; Nusse et al., 2018; Sampurno et al., 2013; Yui et al., 2018), which function through their cooperation with multiple stem cell factors including the AP1 family and β-catenin (Goessling et al., 2009; Shaywitz and Greenberg, 1999; Zanconato et al., 2015). In addition, data indicate that the AA-induced stemness phenotype is mediated by Ptger4-PKA signaling ( Figures 6A to 6T ), which can activate and promote the nuclear localization of CREB1 (Sassone-Corsi, 2012; Yokoyama et al., 2013). AA treatment can lead to enhanced CREB1 activity, such as increased nuclear localization and phosphorylation ( Figure 7C ) and upregulation of bona fide CREB1 target genes associated with tissue repair and stemness, such as Nr4a1 and Id2 ( Nigmatullina et al., 2017 ; Wu et al., 2016 ) ( Figure 10E and 10G ). Similarly, AA promoted the nuclear localization of YAP ( Figure 7C) and target gene expression, such as Ly6a (Yui et al., 2018). Finally, regions with the most significant chromatin accessibility gains in response to AA contained nearby genes that are known targets of CREB1 and YAP and are part of AA-induced signatures, such as stem cell reprogramming (S100a6, Ly6a, Msln, Anxa10, Il33, Ccnd1, Ascl2), proliferation (Myc, Max, Mki67, Ccnd1), EGFR pathway (Areg, Egfros), Wnt / β-catenin pathway (Ascl2, Id2, Wnt4, Wnt7b, Jag1, Asap1, Plaur, Cd44), and PGE2 signaling (Ptgs2, Ptger4) (Ayyaz et al., 2019; Murata et al., 2020; Mustata et al., 2013; Nusse et al., 2018) ( Figure 7D Together, these data suggest that AA reprograms chromatin accessibility around regeneration-associated loci, in part by activating CREB1 and YAP.

[0225] Covalent histone modifications are associated with chromatin activity and transcriptional outcomes (Berger et al., 2009). To further define the changes in the epigenetic landscape induced by AA, the present disclosure performed a "Cleavage Under Targets and Release Using Nuclease" (Cut & Run) assay in organoids (Meers et al., 2019) and assessed the genome-wide distribution of histone modifications associated with transcriptional activation (H3K4me3), repression (H3K27me3), or active enhancers (H3K27ac) (Beyaz et al., 2017; Das et al., 2014) Figure 15C AA-induced upregulated genes were found to have significant increases in activation-associated H3K4me3 and H3K27ac marks around their promoters and proximal putative enhancers ( Figures 15D to 15E In contrast, genes downregulated in AA lost these active markers and were associated with increased levels of repressive H3K27me3 ( Figure 14F Genes that are part of the regenerative fetal spheroid (Mustata et al., 2013) and repair-related stem cell regeneration gene signatures (Ayyaz et al., 2019; Nusse et al., 2018) exhibited significant AA-induced epigenetic reprogramming, with increased levels of H3K4me3 and H3K27ac, and decreased levels of H3K27me3 ( Figure 7E Putative enhancers with increased H3K27ac abundance by AA contain stem cell reprogramming signature genes, such as S100a6, whereas enhancers with H3K27ac loss in AA contain differentiation genes, such as Defa17 ( Figure 7F Integration of gene expression data with chromatin status further confirmed these observations and revealed that the upregulated genes with the most significant AA-induced increases in H3K4me3 or H3K27ac abundance could be stem cell reprogramming genes ( Figure 7G and 15G As shown in representative genomic tracks, signature genes such as S100a6, Msln, Anxa10, Ly6a, and Ascl2 accumulated active histone marks around promoters or enhancers in response to AA, and chromatin accessibility was also increased ( Figure 7H and 15I ).

[0226] Since AA-induced upregulation of stem cell reprogramming signature genes is mediated by PGE2-Ptger4 signaling, the necessity of PGE2-Ptger4 signaling in epigenetic regulation of AA-induced stem cell reprogramming was evaluated. Cut&Run analysis of H3K27ac was performed using Ptger4 KO organoids. The data suggest that Ptger4 may be required for AA-induced H3K27ac accumulation around upregulated genes and for repair of the relevant stem cell regeneration gene signature ( Figure 7I and 15J Together, these results highlight the epigenetic basis of AA-induced stem cell reprogramming, which may be dependent on PGE2-Ptger4 signaling.

[0227] Example 8: Discussion

[0228] The intestinal epithelium is one of the most regenerative tissues in mammals, thanks to ISCs, which reside in crypts and replenish the tissue approximately every 3 to 5 days (Clevers, 2013). However, exposure to genotoxic stressors such as radiation and chemotherapy is associated with degeneration of the intestinal epithelium, and there is an unmet need for the development of regenerative therapies. Dietary intake of nutrients, such as FA, can influence the activity of ISCs (Beyaz et al., 2016; Beyaz et al., 2021b), but how specific FAs influence intestinal regeneration remains unclear. Several clinical and epidemiological studies have shown that increased intake of total polyunsaturated fatty acids (PUFAs), including ω-6 fatty acids, can increase cancer risk. However, conclusive evidence for the role of ω-6 fatty acids in cancer outcomes is currently lacking. PUFAs (including ω-6 fatty acids, such as arachidonic acid) are important structural components of cell membranes and are required by rapidly proliferating cells to maintain their growth. In addition, after tissue damage, ω-6 fatty acids are released from cell membranes to produce inflammatory bioactive lipid mediators, such as prostaglandins, which are associated with carcinogenesis (Hanson, et al. Br J Cancer (2020) 122(8): 1260-70; Sakai, et al. BMC Cancer (2012) 12: 606; Liput, et al. Int J Mol Sci (2021) 22(13): 6965; Azrad, et al. Front Oncol (2013) 3: 224).

[0229] Using FA screening methods in ex vivo mouse and human organ cultures, combined with in vivo studies using isocaloric diets with different AA abundances, the present disclosure discovered a conserved nutrient-gene interaction mechanism that regulates the stemness of intestinal epithelial cells. Using a screening platform of mouse or human organs, regenerative dietary nutrients with beneficial (e.g., therapeutic) significance were identified. Using this platform, a causal mechanistic link between dietary AA and intestinal stem cell regeneration was revealed through epigenetic reprogramming, which has broad implications for alleviating intestinal degeneration. Intestinal mucosal damage is one of the most common debilitating side effects of cancer treatments such as radiotherapy and chemotherapy, resulting in decreased quality of life and survival in cancer patients (Kim et al., 2017; Sougiannis et al., 2021). The present disclosure shows that a four-week regenerative dietary AA regimen (3% AA in the form of triglycerides) protects mice from intestinal damage in response to clinically relevant abdominal 15Gy irradiation and doxorubicin treatment, a widely used chemotherapeutic agent that causes intestinal mucositis in patients (Kim et al., 2017; Sougiannis et al., 2021). Based on these findings, prospective clinical studies can be conducted to verify that increasing AA abundance through dietary intervention can improve intestinal regeneration and reduce gastrointestinal side effects in cancer patients receiving radiation therapy or chemotherapy.

[0230] Consistent with data on the regenerative role of dietary AA, mice lacking Fads1, the rate-limiting desaturase for AA biosynthesis, exhibit poor overall survival and impaired intestinal epithelial cell proliferation unless supplemented with exogenous AA (Fan et al., 2016; Fan et al., 2012). AA is abundant in breast milk and is thought to be essential for infant growth and development, but global dietary lipid intake estimates suggest that humans primarily obtain AA through desaturation of dietary LA (Calder et al., 2019; Fan et al., 2012). Multiple polymorphisms in the FA desaturase gene cluster are strongly associated with metabolic traits and diseases, including IBD (Sabatti et al., 2009; Dupuis et al., 2010; Costea et al., 2014). Therefore, further research is needed to determine the importance of dietary FA for human physiology and disease states by considering genetic variation in AA utilization and metabolism genes.

[0231] It is becoming increasingly clear that epithelial plasticity, rather than a reserve stem cell pool, drives regeneration to recover from intestinal injury (Ayyaz et al., 2019; de Sousa and de Sauvage, 2019; Murata et al., 2020; Nusse et al., 2018; Tian et al., 2011; Yan et al., 2017). These findings illuminate a new paradigm linking specific dietary nutrients to stemness reprogramming of adult intestinal epithelial cells. Previous studies using different experimental injury models have captured distinct and differentiated molecular signatures of epithelial plasticity in response to injury or ISC ablation (Ayyaz et al., 2019; Murata et al., 2020; Mustata et al., 2013; Nusse et al., 2018). Notably, in the absence of tissue injury, dietary AA mimicked a conserved repair response program through epithelial PGE2-Ptger4 signaling, a program that shares all previously defined hallmarks of stem cell reprogramming, including targets of Wnt / β-catenin and ligands of Egfr. AA led to upregulation of microenvironment-derived signals in epithelial cells and reduced dependence on Wnt and Egf in organoid culture, all of which suggest that dietary nutrients play an important role in influencing microenvironment-mediated control of intestinal stemness. Similarly, dietary AA-induced activation of PGE2-Ptger4 signaling in the intestinal epithelium has important implications for regeneration and epithelial plasticity. Paracrine PGE2 signaling is known to promote tissue repair after injury, and PGE2 analogs have previously been characterized as having radioprotective effects in the intestine (Hanson and Ainsworth, 1985; Miyoshi et al., 2017). Pharmacological interventions that activate PGE2-Ptger4-cAMP signaling have been explored in clinical studies to promote tissue regeneration (Miyoshi et al., 2017; Taha et al., 2018; Nakase et al., 2010). It will be interesting to elucidate whether dietary AA synergizes with these therapeutic interventions to trigger regenerative stem cell plasticity in the intestine. Furthermore, lipid peroxidation and AA release at the wound site regulate damage detection and tissue repair processes (Katikaneni et al., 2020). Consistent with these observations, these findings suggest that adaptive PGE2-Ptger4 signaling induced by dietary AA can drive epithelial cell plasticity, promote regeneration, and protect the intestine from injury.Thus, the regenerative effects of dietary AA via the PGE2-Ptger4 axis highlight the importance of dietary factors in influencing stem cell reprogramming and represent a new robust and physiologically relevant model to study epithelial cell plasticity in the intestine (de Sousa and de Sauvage, 2019).

[0232] Downstream of Ptger4, cAMP-PKA signaling mediates the stemness-enhancing effects of dietary AA. cAMP-PKA signaling plays a key role in tissue repair and resolution of inflammation. Elevating cAMP signaling in tissues promotes regeneration and blocks or potentially reverses scarring after injury, but the cell-type-specific mechanisms remain unclear (Insel et al., 2012).

[0233] Little is known about the effects of diet and metabolic perturbations on the intestinal epigenome (Verzi and Shivdasani, 2020). The present disclosure suggests that the stemness-enhancing effect of the AA-Ptger4-cAMP-PKA axis in intestinal epithelial cells is likely mediated by the activity of multiple TFs that activate and synergistically orchestrate the establishment of regenerative epigenetic programs downstream of PKA. In fact, the data reveal significant epigenetic reprogramming induced by AA in a Ptger4-dependent manner around regeneration-related loci (including CREB1 and YAP targets). Studies have shown that epithelial stem cells retain functional characteristics from previous exposures (Beyaz et al., 2016; Naik et al., 2017; Ordovas-Montanes et al., 2020). An important significance of this epigenetic reprogramming is that dietary AA can induce regenerative memory in the intestinal epithelium, which protects the tissue from subsequent damage.

[0234] The present disclosure considers the epithelial intrinsic mechanism that controls the stemness enhancing effect of AA. Although acute PGE2 production promotes tissue repair in response to damage, chronic inflammation and dysregulated PGE2 signaling promote tumorigenesis (Wang and DuBois, 2018). The short-term regeneration diet AA regimen used in this study causes elevated levels of PGE2 in the intestine of mice without any obvious safety issues. However, future research is still needed to clarify the dynamics of the stemness of the improvement induced by AA, so as to accurately distinguish the regeneration effect from the possible risk of tumorigenesis. Although the data show the robust AA-induced epigenetic reprogramming around the regeneration-related locus, the epigenetic method used only provides an average population-based analysis and does not retain single cell information.

[0235] Example 9: Reagents and Methods

[0236] Table 4.

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] Animals, diet, and medication

[0243] Mice were housed at Cold Spring Harbor Laboratory. The following strains were obtained from Jackson Laboratory: Ptger4 f / f (strain name: 6.129S6(D2)-Ptger4tm1.1Matb / BreyJ, stock number: 028102) and Lgr5-EGFP-IRES-CreERT2 (strain name: B6.129P2-Lger5tm1(cre / ERT2)Cle / J, stock number: 008875). Animals were housed in a pathogen-free environment and maintained on a 12-hour light / dark cycle. ARD was developed by using an oil (Arasco oil, DSM, 5015002S02) extracted from a fungus (Mortierella alpina) containing approximately 40% AA in the form of triglycerides to prepare a diet (7% total fat) containing 3% AA-rich oil and 4% soybean oil (catalog number TD.190641, Envigo) starting from 8 to 12 weeks of age and continuing for 4 weeks (Table 3). Control mice were matched for age and sex and fed an isocaloric control diet (catalog number TD.97184, Envigo) containing equal amounts of major nutrients and minor nutrients. Food and water were supplied ad libitum. The alleles of Lgr5-EGFP-IRES-CreERT2 (for the generation of stem cell-specific knockout, Lgr5-iKO) mice were removed by administering tamoxifen suspended in corn oil (Cat. No. C8267, Sigma) at a concentration of 20 mg / ml and 100 μl / 25 g body weight, and administered by intraperitoneal injection every other day for a total of 5 injections. All animals used in this study were handled in accordance with ethical procedures approved by the Cold Spring Harbor Laboratory Institutional Care and Use Committee (IACUC).

[0244] The following compounds were used for organoid treatment: dmPGE2 (5 nM, catalog number 14750, Cayman), PGD2 (5 nM, catalog number P5172, Sigma), Celexocib (1 to 45 μM, catalog number 10008672, Cayman), 8-bromo-cAMP (20 μM, catalog number 1140, Tocris), sesamin (20 μM, catalog number SMB00705, Sigma), H89 (20 μM, catalog number 10008672, Cayman), dmPGE2 (5 nM, catalog number P5172, Sigma), dmPGE2 (5 nM, catalog number P5172, Sigma), dmPGE2 (5 nM, catalog number P5172, Sigma), dmPGE2 (5 nM, catalog number P5172, Sigma), dmPGE2 (5 nM, catalog number No. 2910, Tocris), indomethacin (0 to 80 μM, catalog number 17378, Sigma), Ptger1 inhibitor (50 μM, catalog number SC51322, R&D), Ptger2 inhibitor (25 μM, catalog number PF04418948, R&D), Ptger3 inhibitor (50 μM, catalog number L-798,106, R&D), Ptger4 inhibitor (50 μM, catalog number L-161,982, R&D) , 5-HETE (0.5 μM, catalog number 34210, Cayman), 12-HETE (0.5 μM, catalog number 34550, Cayman), 15-HETE (0.5 μM, catalog number 34700, Cayman), 8(9)-EET (0.5 μM, catalog number 50351, Cayman), 11(12)-EET (0.5 μM, catalog number 50511, Cayman), 14(15)-EET (0.5 μM, catalog number 50651, Cayman), LTB4 (0.5 μM, catalog number 20110, Cayman), TXB2 (5 μM, catalog number 19030, Cayman), Wnt3a (10 to 100 ng / ml, catalog number 315-20, Peprotech), amphiregulin (50 ng / ml, catalog number 989-AR, R&D), epiregulin (500 ng / ml, catalog number 1068-EP, R&D).

[0245] Intestinal crypt isolation and flow cytometry

[0246] Intestinal crypt isolation was performed as previously reported (Beyaz et al., 2016). Briefly, the whole intestine was extracted and cleaned of fat, connective tissue, and blood vessels and rinsed with ice-cold 1× PBS. After positioning, the small intestine was cut into 3 to 5 cm segments and incubated in 1× PBS / EDTA (7.5 mM) at 4°C for 30 minutes with gentle agitation. The crypts were mechanically separated from the tissue and filtered through a 70 μm filter to remove villi and tissue debris. The crypts were then washed with ice-cold PBS and centrifuged at 300 g for 5 minutes.

[0247] IEC separation was performed by dissociating the crypt suspension into single cells using TrypLE Express (Cat. No. 12604-013, Invitrogen). The dissociated single cells were labeled with a mixture of antibodies containing EPCAM-APC (1:400, Cat. No. 17-5791-82, eBioscience, G8.8), CD24-PE-Cy7 (1:400, Cat. No. 25-0242-82, eBioscience) and CD45-Alexafluor 488 (1:400, Cat. No. 12-0451-83, eBioscience). Dead cells were excluded from analysis using viability dye SYTOX (Cat. No. S34857, Life Technologies). IEC were separated into Epcam using BD FACS AriaII SORP cell sorter. + CD45 - SYTOX - , and placed in supplemented crypt culture medium for culture or in TRIzol reagent (Cat. No. 15596018, Thermo Fisher) for gene expression analysis.

[0248] Crypt and isolated cell culture media

[0249] Isolated crypts were counted and embedded in Matrigel (Cat. No. 356231, Corning growth factor reduced) at a ratio of 1:4, 5 to 10 crypts per μl. The matrigel was allowed to solidify at 37°C for 8 to 12 minutes, and the solidified domes were then incubated in crypt culture medium containing Advanced DMEM (catalog number 12634010, Gibco) medium supplemented with recombinant mouse EGF 40 ng / ml (catalog number 315-09, PeproTech), recombinant mouse Noggin 50 ng / ml (catalog number 250-38, PeproTech), R-spondin 62.5 ng / ml (catalog number 3474-RS, R&D Systems), N-acetyl-L-cysteine ​​1 μM (Sigma-Aldrich), CHIR-9902 15 μM (catalog number 4423, Tocris), Y-27632 20 ng / ml (catalog number 1254, Tocris), B27 1X (catalog number 17504044, Gibco), N2 1X (catalog number 17502048, Gibco), 1% GlutaMAX (catalog number 35050061, Gibco), 1% penicillin-streptomycin (pen / strep) (catalog number P4333, Sigma-Aldrich). The crypt culture medium was changed every other day and maintained at 37°C in a fully humidified chamber containing 5% CO2. The clonogenic capacity (colony formation efficiency) was determined by plating 50 to 300 crypts per well and assessing the formation of organoids after 3 to 7 days.

[0250] The IEC cells separated were centrifuged at 300 g for 5 minutes and resuspended in the crypt culture medium of appropriate volume (500 to 1,000 cells / μl). Then, cells were seeded on the matrigel in flat-bottomed plates (catalog number (Cat. No.) 3548, Corning). Crypt culture medium was added after matrigel and cells solidified. Crypt culture medium was supplemented every other day. Unless otherwise stated, organoids were quantitatively analyzed at the 1st, 3rd and 6th day of cultivation. In the second experiment, single primary organoids were mechanically dissociated in TrypLE Express at 37°C for 6 minutes, centrifuged and resuspended in cold crypt culture medium, mixed with matrigel, and incubated until solidified. Fresh crypt culture medium was supplemented every other day, and maintained at 37°C containing 5% CO2 in a completely humidified box.

[0251] Fatty acid (FA) BSA conjugation

[0252] Fatty acids (FA) supplemented in powder form were reconstructed in ethanol. The fatty acid solution was then added to 0.01 M NaOH to prepare a 12 mM solution and stirred at 70 ° C for 30 minutes. 10% fatty acid-free BSA (Cat. No. 68700, Proliant Biologicals) was then added to the solution to a concentration of 3 mM and stirred at 37 ° C for 1 hour. The BSA-conjugated fatty acids were filtered through 0.22 μm and stored in glass containers (Cat. No. B7999-2A, Thermo Fisher) at -20 ° C.

[0253] FA screening

[0254] 10,000 cells separated from mouse or human intestinal organoids were seeded in 48-well plates and incubated for 6 hours at 37°C in crypt culture medium containing 5% CO for recovery, followed by fatty acid treatment. The fatty acid screening library was composed of 23 different fatty acids as shown in Table 1. After incubation for 6 hours, culture medium was replaced with crypt culture medium containing a specified concentration of fatty acid (both mouse and human organ were 25 μM). After processing for 24 hours, Cytation7 and BioSpa platforms (Agilent BioTek, Winooski VT) were used, at 37°C, 5% CO, and imaging was performed every 6 hours to each hole (16 z sections, 54.8 μm step length, and focal length fixed to 1719 μm above the plate carrier). Imaging terminated at the 120th hour. Then, focus stacking was used to obtain Z-direction projection. Digital phase contrast technology was applied, and 100 μm structural element size was used to filter the image. Spheroids were detected to create a new population by defining low internal signal targets gated by the new metrics greater than <= 0.95 and circularity > 0.2. This subpopulation was normalized to the total biomass area.

[0255] Human study participants and crypt isolation from patient biopsies

[0256] Human colon tissue samples were obtained from patients who provided informed consent for surgical resection at Huntington Hospital. The study protocol was reviewed and approved by Northwell Health Biospecimen Repository (Protocol No. 1810). Tissue samples were stored in RPMI medium (Cat. No. 10-040-CV, Corning) until processing. Patient metadata are provided in Table 2.

[0257] First, cut the tissue sample into approximately 0.5 cm 2The small pieces were separated and incubated for 15 minutes at 4°C in an antibiotic mixture consisting of 100 μg / mL Normocin (Catalog No. ant-nr-1, Invivogen), 50 μg / mL Gentamycin (Catalog No. E737, Amresco) and 1X Pen / Strep (Catalog No. 15070063, ThermoFisher) in 1×PBS. Next, the small pieces were washed with 1×PBS and then incubated on a shaker in 5mM EDTA solution at 4°C for 75 minutes. After incubation, the tissue samples were washed again with 1×PBS. The crypts were then released from the tissue by shaking the small pieces in a tube filled with ice-cold 1×PBS. The isolated crypts were transferred to a new tube and spun at 100g for 5 minutes at 4°C.

[0258] Human organ passaging and maintenance

[0259] The isolated crypts were then embedded in Matrigel at a 1:4 ratio. Matrigel was polymerized at 37°C for 8 to 12 minutes, and then human crypt culture medium was added to each well, wherein the culture medium consisted of the following: Advanced DMEM (Cat. No. 12634028, Life Technologies), 1X Glutamax (Cat. No. 35050061, Life Technologies), 10mM HEPES (Cat# 15630080, Thermo Fisher Scientific), 50% WRN conditioned medium from the L-WRN cell line (ATCC, CRL-3276), 1X B27 (Cat. No. 12587010, Life Technologies), 1X N2 (Cat. No. 17502048, Life Technologies), 10mM nicotinamide (Cat. No. N0636, Sigma Aldrich), 1mM N-acetylcysteine ​​(Cat. No. A9165, Sigma Aldrich), 100μg / mL Primocin (Cat. No. ant-pm-1, Invivogen), 10μM Organoids were harvested by culturing in 5% CO2 using SB202190 (Cat. No. S7067, Sigma Aldrich), 10 μM Y-27632 (Cat. No. 1254, Tocris), 10 nM Gastrin I (Cat. No. G9020, Sigma Aldrich), 50 ng / mL EGF (Cat. No. AF-100-15, Peprotech) and 500 nM A83-01 (Cat. No. SML0788, Sigma Aldrich). Culture medium was changed every 2 to 3 days, and organoids were passaged approximately every 8 days. Organoids were harvested by removing matrigel using cell recovery solution (Cell Recovery Solution, CRS) (Cat. No. 354253, Corning). Once matrigel is dissolved, organoids were rotated 5 minutes at 4°C with 500 g, and incubated in TryplE Express (Cat. No. 12604039, ThermoFisher) until single cells were seen under a microscope. The cells were then centrifuged at 500 g for 5 minutes at 4°C and replated in Matrigel as described above. Organoids were typically passaged at a 1:6 ratio.

[0260] ELISA assay

[0261] Sorted Epcam from control mice + CD45 - SYTOX -Cell is plated with 25,000 cells per well, and is incubated at 37 DEG C for 6 hours for recovery.Then, the crypt culture medium supplemented with vehicle or AA (25 μM) is replaced.After 24 hours, the supernatant of the organoid processed from vehicle or AA is collected, and centrifuged at 300g to remove any cell or matrigel residue.PGE2 Elisa test kit (catalog number ADI-900-001, Enzo Life Sciences) is used to measure the presence of PGE2 metabolites according to the instructions of manufacturers.

[0262] Nuclear isolation and Western blotting

[0263] Organoids grown in crypt culture for 5 days were treated with vehicle (BSA-ethanol) or 50 μM AA for 4 hours. Organoids were then recovered from the matrigel and washed with PBS. To prepare cytoplasmic extracts, organoids were lysed in buffer A (10 mM HEPES pH-7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1 mM PMSF, 1 mM DTT, 0.1% TritonX-100, PhosSTOP (cat. no. 04906845001, Roche) and protease inhibitors (cat. no. 11873580001, Sigma) and incubated on ice for 15 min with occasional gentle pipetting. Cytoplasmic extracts were collected after centrifugation at 1300 rcf for 5:30 min. To prepare nuclear extracts, nuclei were washed several times with buffer A to remove any residual cytoplasmic proteins and washed twice with buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, PhosSTOP protease inhibitors), and then incubated on ice for 30 to 40 minutes, gently tapping every 5 minutes. The nuclear extract was collected after centrifugation at 1700rcf for 5 minutes. The remaining insoluble chromatin was washed several times with buffer B to remove nuclear protein contamination; the chromatin pellet was then resuspended in Laemmli buffer and sonicated with 30 seconds on / 15 seconds off. The samples were then run on a 10% Tris-HCl gel and transferred to a nitrocellulose membrane. The membrane was blocked with a 5% skim milk solution for 1 hour at room temperature and then incubated with the primary antibody in 5% skim milk at 4°C overnight. After incubation, the membrane was washed with 1× PBST (PBS, 0.1% Tween-20) and then incubated with HRP-linked secondary antibody diluted in 5% skim milk for 1 hour at room temperature. Pierce ECLWestern blotting substrate (catalog number 32106, ThermoFisher Scientific) was used with Mini-Med The signal was detected by the 90 (AFP Manufacturing) imaging system.

[0264] Immunohistochemistry, smISH, and immunofluorescence combination

[0265] The organoids treated with vehicle or AA were harvested by removing the matrigel using CRS solution and washed twice with 1×PBS. Subsequently, the organoids were fixed with 4% paraformaldehyde (PFA) (Cat. No. 15714, Electron Microscopy Sciences) at room temperature for 30 minutes. After removing PFA, the organoids were washed with 1×PBS and centrifuged at 400g for 3 minutes. The precipitated organoids were embedded in 2% agarose gel and made into 10 μm slices.

[0266] Intestinal tissue from control and ARD-fed mice was Swiss-rolled and fixed in 10% formalin solution (catalog number HT501128, Sigma Aldrich). Formalin-fixed tissue samples were processed in a Thermo Excelsior ES processor and embedded with a Thermo HistoStar embedding system according to the manufacturer's protocol. Paraffin-embedded samples were cut into 5 μm thick sections and mounted on positively charged slides (catalog number 48311-703, VWR superfrost plus microslides). HE staining was performed using a Leica Multistainer (ST5020, Leica) at the CSHL Tissue Imaging Facility. Briefly, after dewaxing and rehydration, the slides were stained in hematoxylin (Hematoxylin 560MX, Leica) for 1 minute, then destained in Define MX-aq (Leica) for 30 seconds and bluing in Blue Buffer 8 (Leica) for 1 minute; the slides were then stained in eosin (EOSIN 515LT, Leica) for 30 seconds. After dehydration, the slides were mounted using an automatic mounting slide machine (Leica CV5030).

[0267] For IHC, formalin-fixed, paraffin-embedded tissue sections were dewaxed. Antigen retrieval was performed with 0.1 mM citrate buffer (pH 6) and boiled at 96°C for 6 minutes. After peroxidase blocking, the tissues were blocked with appropriate serum and then incubated overnight with anti-Ki-67 antibody (1:100, Thermo Fisher, clone SP6). A biotin-conjugated secondary antibody from Vector Labs was used. Visualization was performed with diaminobenzidine (DAB) and counterstained with hematoxylin and eosin (VectorLabs). Washes were performed with PBST between each step.

[0268] Single-molecule in situ hybridization (smISH) was performed for Ascl2 (Cat. No. 412211, ACD), Lgr5 (Cat. No. 312171, ACD) and S100a6 (Cat. No. 412981, ACD) using the Advanced Cell Diagnostics RNAscope 2.5HD Detection Kit-Red (Cat. No. 322350, ACD) according to the manufacturer's instructions and combined with subsequent immunostaining for Epcam. For immunostaining, after the smISH step except for DAPI staining, the slides were incubated with anti-Epcam antibody (1:100, Cell Signaling, clone E6V8Y) at 4°C overnight. The slides were then washed with PBST and incubated with secondary antibody (1:500, Alexa Fluor Plus 488, Cat. No. A32766, Invitrogen) for 1 hour at room temperature in the dark. The slides were washed again with PBST and stained with DAPI. Then, the slides were washed with ProLong TM Gold Antifade mounting medium (Cat. No. P36930, Invitrogen) was used for mounting. Images were acquired using a confocal microscope (Zeiss LSM 710, Germany) and processed using ImageJ. smISH signals were quantified using Imaris (Oxford Instruments).

[0269] EdU merged

[0270] 4 hours before mouse euthanasia, EdU (Sigma) was administered intraperitoneally at a dosage of 5 μg / g. After tissue dewaxing and antigen retrieval as described above, according to the manufacturer's instructions, Click-iT EdU cell proliferation imaging kit Alexa Fluor 647 (catalog number C10640, Invitrogen) was used to incorporate the intestinal epithelial cells that detect proliferation by EdU. Epcam and DAPI staining were then performed. Images were collected using a confocal microscope (Zeiss LSM 710, Germany) and processed by ImageJ. The EdU+ cells in each crypt were quantified in a blinded manner. Biotek (Agilent, USA) was used to detect the EdU signal in each Swiss roll.

[0271] Doxorubicin-induced intestinal injury

[0272] Doxorubicin hydrochloride (Cat. No. D1515, Millipore, Sigma) was administered to 8- to 12-week-old mice via a single intraperitoneal injection at a concentration of 20 mg / kg body weight (Carr et al., 2017; Cray et al., 2020). Mice were euthanized in a CO2 chamber and analyzed 72 hours later.

[0273] Irradiation-induced intestinal injury

[0274] Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg / kg) and dexmedetomidine (Dexdomitor) (10 mg / kg). The mice were transferred to a lead shielded unit and exposed only to 15 Gy of ionizing radiation from a 137-cesium source (GammaCell). Mice were sacrificed 72 hours later. The number of viable crypts was counted from hematoxylin-eosin-stained sections (Tustison et al., 2001).

[0275] RNA extraction, cDNA preparation and RT-qPCR

[0276] Total RNA was extracted using the Direct-zol RNA Isolation Kit (Cat. No. R2051, Zymogen) and reverse transcribed using SuperScript IV Vilo (Cat. No. 11756050, Thermo Fisher) as described by the manufacturer. RT-qPCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) with the probes listed in Table 5. Hsp90ab1 was used as an internal control, and the qRT-PCR results were analyzed for relative quantification using the ΔΔCt method.

[0277] Table 5. List of probes used for qRT-PCR

[0278] name Code Target Ccnd1 Mm00432359_m1 mice Ptgs1 Mm00477214_m1 mice Ptgs2 Mm00478374_m1 mice Ptges Mm00452105_m1 mice Cd55 Mm00438377_m1 mice S100a6 Mm00771682_g1 mice Ly6a Mm04337234_mH mice Msln Mm00450773_g1 mice Hsp90ab1 Mm00833431_g1 mice Nr4a1 Mm01300401_m1 mice NR4A1 Hs00374226_m1 people NR4A2 Hs01117527_g1 people MSLN Hs00245879_m1 people L1CAM Hs01109748_m1 people CD55 Hs00892618_m1 people DUSP4 Hs01027785_m1 people HSP90AB Hs03043878_g1 people

[0279] Lentivirus production and generation of Ptger4 knockout organoids

[0280] Lentiviral particles were produced in 293FT cells by co-transfection of the Puro.Cre empty vector (Addgene plasmid #17408 (Kumar et al., 2008)) with a second-generation lentiviral system (pCMV-VSVG, Addgene plasmid #8454), psPAX2 (Addgene plasmid #12260) using the transfection reagent polyethyleneimine (PEI) (Cat. No. 23966, Polysciences). Briefly, 293FT cells were plated in 10 cm dishes at 75% to 80% confluence one day before transfection. 10 μg of Puro.Cre plasmid, 7.5 μg of spPAX2, and 5 μg of pCMV-VSVG were mixed with DMEM-F12 medium at room temperature and incubated for 15 minutes. The transfection mixture was added dropwise to the confluent 293FT cells. After 6 hours, the culture medium was removed and fresh DMEM-F12 culture medium supplemented with 10% FBS and 1% pen / strep was added. The supernatant containing the viral particles was collected after 24, 48 and 72 hours and centrifuged at 300g for 5 minutes to remove any residual cell particles. The supernatant was filtered through a 0.45 μm filter and concentrated by adding (1 / 3 of the total volume of the supernatant) Lenti-X concentrator (Cat. No. 631232, Takara). The mixture was centrifuged at 1500g for 60 minutes at 4°C, and the viral pellet was then resuspended in mouse crypt culture medium. For viral infection, Ptger4 cells cultured for 4 days were added. f / f Organoids were rescued from matrigel and dissociated into single cells as described above. Concentrated lentiviral particles supplemented with polybrene were mixed with 1,000,000 single cells and transferred to 48-well plates, then centrifuged at 600 g for 1 hour at room temperature and incubated at 37 ° C for 4 hours. The infected cells were resuspended in mouse crypt culture medium and plated into 4 wells of 12-well plates until they formed organoids. The infected organoids were selected with puromycin (1 μg / ml, catalog number A1113803, Thermo Fisher) after 3 days.

[0281] Electron microscopy

[0282] The intestinal organoids cultured in 12-well plates were fixed overnight at 4 ° C in 2.5% glutaraldehyde in 0.1M sodium cacodylate solution (pH 7.4). The sample was washed three times with 0.1M sodium cacodylate and post-fixed at room temperature for 1 hour with 1% osmium tetroxide (OsO4) solution. The sample was rinsed three times with distilled water and dehydrated for 10 minutes with a gradient ethanol wash series (50%, 60%, 70%, 80%, 90%, 95%, 100% respectively). After dehydration, the sample was incubated overnight with 812EMed resin and 100% ethanol. For polymerization, the sample was embedded in 812EMed resin and incubated at 60 ° C until the resin was fully polymerized. 45Diamond DiATOME Histo knife was used to cut slices of 60 to 90 nm. The slices were stained with UranyLess for two minutes and then thoroughly washed with ddH2O. Samples were visualized using a H7000 Hitachi transmission electron microscope.

[0283] Metabolomics analysis by liquid chromatography coupled to mass spectrometry (LC-MS)

[0284] Quick-frozen tissue samples are cut and weighed into the Precellys tube pre-installed with ceramic beads (Bertin Instruments). Add the extraction solution (30% acetonitrile, 50% methanol and 20% water) of accurate volume, to obtain 40mg tissue sample of every ml extraction solution. Use Precellys 24 homogenizers (Bertin Instruments) to crack tissue samples, and the suspension is incubated at -20 DEG C for 60 minutes. Samples are mixed, and in Thermomixer (Eppendorf, Germany), at 4 DEG C, incubate 15 minutes, then centrifuge (16,000g, 4 at DEG C 15 minutes). Collect supernatant and transfer to automatic sampler glass bottle, be stored at -80 DEG C until further analysis. For organoid experiment, culture medium is collected three days after vehicle or AA treatment, and extract sample and carry out metabolomics analysis.

[0285] For the analysis of polar metabolites and arachidonic acid derivatives, samples were randomized to avoid bias due to instrument drift and were blinded. LC-MS analysis was performed using a Vanquish Horizon UHPLC system coupled to a Q Exactive HF mass spectrometer (both from Thermo Fisher Scientific). Sample extracts (5 μL) were injected onto a Sequant ZIC-pHILIC column (150 mm × 2.1 mm, 5 μm) from Merck Millipore and a guard column (20 mm × 2.1 mm, 5 μm) maintained at 45°C. The mobile phase consisted of 20 mM ammonium carbonate and 0.1% ammonium hydroxide in water (solvent A) and acetonitrile (solvent B). Analytes were eluted at 200 μl / min using a previously described gradient (Mackay et al., 2015). The mass spectrometer was operated in full MS and polarity switching modes. Acquired spectra were analyzed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Fisher Scientific) with reference to an in-house compound library. Quality control and normalization (with QuantileNorm, LogNorm, and MeanCenter options) were performed using MetaboAnalystR (v3.0.3, (Pang et al., 2020)). Differential abundance analysis was performed using FC.Anal.unpaired.

[0286] Bulk RNA sequencing

[0287] Total RNA was isolated from vehicle-treated and AA-treated mouse intestinal organoids on days 1, 3, and 6, and from PDO on day 6, using the Zymo RNA isolation kit according to the manufacturer's instructions. Starting with 250 ng of total RNA, the rRNA depletion protocol was performed according to the manufacturer's recommended guidelines. Strand-specific RNAseq libraries were prepared using the NebNext Ultra II kit and sequenced on an Illumina NextSeq.

[0288] Reads were trimmed using cutadapt (v2.10) and aligned to the GRCm38.p6 / Gencode annotation (version M24) using STAR (v2.7.2b, (Dobin et al., 2013)) and quantified using `--quantModeGeneCounts'. Read and alignment quality was analyzed using rseqc (v3.0, (Wang et al., 2012)) and summarized using multiqc (v1.9, (Ewels et al., 2016). Sequences were analyzed using DEseq2 (v1.28, (Love et al., 2013)). Differential gene expression between vehicle- and arachidonic acid-treated samples was assessed using the methods (Ritchie et al., 2014). A model with a fixed effect of sequencing batch and treatment was fitted. Treatment contrasts were extracted, and transcripts with an absolute fold change greater than log2(1.5) and an adjusted p-value less than 0.05 were considered differentially expressed. Differential gene expression was assessed independently at each time point. Unless otherwise stated, the union of differentially expressed genes across time points was used in all further analyses. For visualization purposes, sequencing batch effects were adjusted using limma (v3.46, (Ritchie et al., 2015)) before rlog normalization of size-normalized read counts. Heatmaps and UpSet plots were generated using ComplexHeatmap (v2.6.2, (Gu et al., 2016), and volcano plots were generated using EnhancedVolcano (v1.8.0).

[0289] gene sets

[0290] The data were analyzed in the context of previously identified gene signatures and target gene lists. The gene lists are briefly described below:

[0291] Genetic characteristics of fetal spheroids

[0292] 317 differentially upregulated genes were evaluated (microarray) in regenerating spheroids and organoids derived from embryos / mice at different embryonic and postnatal stages (E16, E18, or P0) (Mustata et al., 2013).

[0293] Genetic signature of granuloma induction

[0294] We evaluated 131 differentially expressed genes (bulk RNA-seq) in the granulomatous (Gr) and non-granulomatous (NonGr) crypt epithelium of mice infected with the spiral nematode H. polygyrus for six days (Nusse et al., 2018).

[0295] Radiation-induced genetic signatures

[0296] Fifty differentially expressed genes were assessed in regenerating enterocyte clusters from scRNA sequencing data of irradiated intestinal epithelium versus intact crypts (single-cell RNA-seq) (Ayyaz et al., 2019).

[0297] Gene signatures of regeneration induction

[0298] Relative to intact (resting) ISCs at the bottom of intestinal crypts, regenerating Ascl2 + 316 differentially expressed genes were assessed in upper crypt cells (bulk RNA-seq) (Murata et al., 2020).

[0299] Stem cell homeostasis gene signature

[0300] Differentially expressed genes were assessed in Lgr5+ ISCs (Haber et al., 2017).

[0301] Single-cell RNA sequencing

[0302] In order to carry out single cell sequencing to the organoid of mouse intestine, organoid is collected using cell recovery solution.Then organoid is dissociated into single cell suspension with TrypLE.After organoid dissociation, single cell is precipitated, washed and resuspended in FACS buffer (1 × PBS, 10 μM Y-27632, 1% FBS, 0.5mM EDTA), and passed through 100 μm FlowMi cell strainer (Sigma). Cell viability assessment is performed using DAPI. DAPI negative cells are sorted by Sony SH800S sorter, and single cell droplets are prepared according to manufacturer's instructions on the 10X Chromium of Cold Spring Harbor Laboratory single cell facility. Single cell library is prepared according to manufacturer's instructions using 10X Genomics Chromium controller (catalog number 120223, 10X Genomics) and 10X Genomics Chromium Next GEM single cell 3 ' gene expression kit (catalog number 1000268, 10X Genomics). Adjust the cell suspension to yield 8,000 cells per sample.

[0303] Single-cell RNA-Seq data analysis

[0304] The single-cell datasets for each experiment were independently assessed for data quality following the guidelines described in (Amezquita et al., 2020; Luecken and Theis, 2019). Cells with more than 15% mitochondrial transcripts and cells with fewer than 2,000 feature counts or expressing fewer than 1,000 genes were removed. After quality control, normalization, graph-based clustering, and differential expression analysis were performed using Seurat (v3.2.1, (Butler et al., 2018)). Each dataset was normalized using SCTransform, and the 3000 most variable genes were identified using SelectIntegrationFeatures. Organoid (vehicle-, AA-, and PGE2-treated) and tissue datasets (intestinal tissue from control and Arasco diet-fed mice) were integrated into one organoid and one tissue dataset (Cao et al., 2019; Levine et al., 2015; Qiu et al., 2017; Stuart et al., 2019; Trapnell et al., 2014). RunPCA was used on the integrated dataset to identify the top 10 principal components (PCs) for UMAP analysis and clustering. Louvain clustering with a resolution of 0.3 and 0.6 was used for the organoid and tissue datasets, respectively. Clusters were labeled using expression levels of enterocyte subtype signatures identified by (Roulis et al., 2017); stem 2 and stem 3 clusters were labeled using signatures identified by (Roulis et al., 2020) (see gene list description above).

[0305] To determine whether AA and PGE2 treatments acted synergistically, differential expression analysis was performed between each treatment group and the control using the FindMarkers function and the MAST method (Finak et al., 2015). Wilcoxon rank sum tests were performed using the wilcox.test function in stats (v4.1.0, (R Core Team, 2021)) to determine whether gene expression was significant. Monocle3 (v0.2.3, (Cao et al., 2019; Levine et al., 2015; Qiu et al., 2017; Trappnel et al., 2014) was used for trajectory analysis in both the organoid and in vivo datasets.

[0306] Cut&Run

[0307] Sample preparation

[0308] CutnRun was performed according to Henikoff et al. (Skene et al., 2018) with some minor modifications. Briefly, intestinal organoids treated with vehicle or 25 μM AA for 3 or 6 days were evaluated. 500,000 cells per replicate were counted and washed with 1 ml of 1× PBS, followed by another wash with 1 ml of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and a protease inhibitor cocktail). 10 μl of BioMag Concanvilin A beads were resuspended and washed twice with 1 ml of binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2). The pelleted cells were resuspended in 1 ml of wash buffer, and the bead suspension was added to the cells and incubated on a nutator for 10 minutes at room temperature. The sample was placed on a magnetic stand until the solution was clear, and then the liquid was replaced with antibody buffer with the following antibodies at a ratio of 1:100: H3K4me3 (Cat. No. ab8580, Abcam), H3K27me3 (Cat. No. 07-449, Millipore), H3K27Ac (Cat. No. ab4729, Abcam) and incubated overnight at 4°C on a nutator. The next day, the cells were washed twice with 1 ml of digitonin buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% digitonin, and protease inhibitor cocktail) and resuspended in 150 μl of digitonin buffer. Homemade pA-MNase was added at a concentration of 700 ng / ml, mixed gently, and incubated at 4°C on a nutator for 1 hour. After this, the sample was washed twice with 1 ml of digitonin buffer. Cells are resuspended in 150 μ l digitonin buffer, then placed on the heating block on wet ice for 5 minutes, so that it is cooled to 0 ℃.Add 3 μ l 100mM CaCl 2 and mix gently, and put back immediately on 0 ℃ of blocks and hatch 30 minutes.Terminate digestion with 100 μ l 2X stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% digitonin, 50mg / ml RNase A, 50mg / ml glycogen and 4pg / ml yeast heterologous spike-in DNA).Sample is hatched 10 minutes under 37 μ C, and centrifuged 5 minutes with 16000rcf at 4 ℃.Sample is placed on magnetic rack, and when not stirring sediment, supernatant is transferred to new Eppendorf tube.2 μ l 10% SDS and 1.5 μ l Proteinase K are added to each sample, and hatched 10 minutes under 70 ℃ subsequently.40min.Add 200 μ l PCI after this, and with full speed vortex mixing 3 seconds, be transferred in the phase-locked tube then, and with 16,000rcf rotation 5 minutes.The liquid above the gel is transferred in the new 1.5ml eppendorf tube that contains 2.5 μ l 3mg / ml GlycoBlue (catalog number (Cat. No.) AM9515, Invitrogen), and with 100% ethanol mixing, hatched on ice 10 minutes then, and with 16,000rcf centrifugal 30 minutes.With 70% ethanol flushing sediment, and with maximum speed rotation 10 minutes.After last washing step, abandon ethanol, and remove all remaining ethanol droplets with vacuum.Remaining sediment is dissolved in 15 μ l TE buffer (1mM Tris-HCl pH=8,0.1mM EDTA).

[0309] Library preparation

[0310] Libraries were prepared using the ThruPLEX DNA-seq kit (catalog number R400427, Takara) according to the manufacturer's instructions using the following PCR conditions: 72°C for 3 minutes, 85°C for 2 minutes, 98°C for 2 minutes, (98°C for 20 seconds, 67°C for 20 seconds, 72°C for 30 seconds) × 4 cycles, (98°C for 20 seconds, 72°C for 20 seconds) 7 PCR cycles. Short fragments (0.5x to 1.7x) were selected using AMPure XP beads (catalog number A63880, Beckman Coulter) to remove residual adapters and large DNA fragments. The library was sequenced on a NextSeq500 using the 150-cycle v2 high-output SBS kit using the CSHL Sequencing Technology Shared Resource. 10% PhiX was added to the control sample, and sequencing was performed using double-end sequencing 76bp in length with index reads. The reads were demultiplexed using the barcodes using the bcl2fastq2 tool.

[0311] analyze

[0312] Cut&Run libraries for H3K27me3, H3K27ac, H3K4me3, and IgG controls were paired-end sequenced with 76-bp read length, yielding an average of 13 × 2 million reads per sample. Sequencing data were aligned and processed using CUT&RUNtools using all read fragments (without read length filtering (Zhu et al., 2019)). Peaks were called individually for each replicate using SEACR in stringent mode, and consensus peaks across replicates were called based on their overlap under different conditions (Meers et al., 2019). Reads within peaks were quantified using featureCounts (Liao et al., 2014). Differential binding between AA and control was analyzed for each chromatin mark using a negative binomial model using DESeq2, and peaks were filtered to an adjusted p-value of 0.01 (Love et al., 2014). Pathway analysis was performed by assigning peaks to genes based on their distance from their canonical transcription start sites, using distance thresholds of 25 kb, 25 kb, and 10 kb for H3K27me3, H3K27ac, and H3K4me3, respectively. Multiple-to-one annotations of peaks to genes were collapsed into one-to-one annotations by taking the peak with the largest change within the distance cutoffs. For longer distances, the Genomic Region Annotation Enrichment Tool (GREAT) was used to annotate genes to putative distal enhancers and regulatory regions using a base-plus-expansion approach (McLean et al., 2010). Genes were ranked based on the log2 fold-change value or Wald statistic of differential calls. Pathway and gene set enrichment was calculated using GSEA and the MSigDB database, as well as gene sets from RNAseq and other published literature as described in the text (Liberzon et al., 2011; Subramanian et al., 2005). Significantly altered pathways were selected by applying an adjusted p-value cutoff of 0.05.

[0313] The histone mark profile was calculated by binning the genome into 100-bp bins centered at ±5 kb of the TSS of the target gene set and extracting the normalized reads (RPGC) within these bins. For plotting purposes, bins at a specific distance from the TSS were aggregated into median values. For the AA vs. vehicle profile, the same process was repeated for replicates of both conditions, and the differences between the AA vs. vehicle treated samples were calculated and aggregated using the median.

[0314] ATAC-seq

[0315] Nuclear separation

[0316] ATAC-seq was performed as previously described by Buenrostro et al. (Buenrostro et al., 2013) and the Kaestner lab. Briefly, 50,000 sorted live cells from organoids were washed with 1 ml of cold 1× PBS. The cells were incubated on ice for 3 minutes with 50 μl of lysis buffer (Tris-HCl, pH 7.5 (final 10 mM), NaCl (10 mM), MgCl2 (3 mM), NP-40 (0.1% v / v), Tween-20 (0.1% v / v), digitonin (0.1% v / v)). 1 ml of wash buffer (Tris-HCl, pH 7.5 (final concentration 10 mM), NaCl (10 mM), MgCl2 (3 mM), Tween-20 (0.1% v / v)) was added to the cells. The cells were then centrifuged at 500 g for 10 minutes at 4°C, and the supernatant was discarded. The pellet containing the nuclei was retained for further experiments.

[0317] Library preparation

[0318] ATAC-seq libraries were generated as previously described by Buenrostro et al. and the Kaestner laboratory. Briefly, DNA was purified using DNAClean & Concentrator 5 (D4013; Zymo Research). Libraries were prepared using NEBNext High-Fidelity 2×PCR Master Mix (NEB, M0541S) according to the manufacturer's standard protocol. DNA fragments were pre-amplified for 5 cycles of PCR, and 5 μl of partially amplified libraries were used for qPCR amplification (20 cycles). A graph showing R vs. cycle number was generated to determine the number of cycles required for each sample to reach 1 / 3 of the maximum R. qPCR amplification was then repeated using the calculated additional cycles. After the amplification step, size selection was performed using Agencourt AMPure XP beads (A63881; Beckman Coulter) to create the final library (Ackermann AM et al). Library quality was determined using an Agilent high-sensitivity DNA bioanalyzer (5067-4626; Agilent Technologies).

[0319] ATACseq analysis

[0320] DNA libraries were sequenced using paired-end sequencing with a read length of 76 bp, yielding an average of 46 × 2 million reads per sample. Sequence adapters were trimmed using Trim Galore! (github.com / FelixKrueger / TrimGalore) and the resulting reads were aligned to the mm10 genome reference using BWA (Li and Durbin, 2009). Duplicate reads were marked using Picard (“Picard Toolkit.” 2019. Broad Institute, GitHub Repository. (broadinstitute.github.io / picard / ). After alignment, mitochondrial reads, duplicate reads, reads in blacklisted regions, secondary alignments and multimapping reads, reads with insert sizes greater than 2 kb or misorientation of sequence pairs, and orphan reads were filtered. Peak calling was performed using MACS2 narrow peak calling with a −75 base pair offset and a 150 base pair extension (Liu, 2014). Consensus peaks were called by obtaining overlap in the region covered by at least two samples. Reads within peaks were quantified using featureCounts (Liao et al., 2014). Data were normalized using a variance stabilizing transformation (vst) for visualization and unsupervised clustering (Anders and Huber, 2010). Counts in AA-treated samples were compared to counts in control-treated samples using negative binomial comparisons using DESeq2, filtering for results with an absolute log2 fold change of 0.58 (minimum change 50%) and an FDR-adjusted p-value of 0.01 (Love et al., 2014). Peaks were annotated and collapsed to genes by taking the peak with the largest change within 5 kb of each transcription start site and assigning it to the respective gene via its promoter or putative proximal enhancer. Genes were ranked by log2 fold change or Wald statistic and entered into GSEA using the MSigDB database and gene signatures compiled from RNAseq experiments and the literature. The resulting gene set was filtered using an adjusted p-value of 0.05 (Liberzon et al., 2011; Subramanian et al., 2012). al., 2005). Correlation between RNAseq and ATACseq was performed as follows: all ATACseq peaks within 2.5 kb of the transcription start site of each gene were taken and genes were divided into differentially open or closed or non-differential categories, and the log2 fold change of expression differences was plotted.

[0321] The regulatory potential of transcription factors was calculated by comparing all mammalian motifs in JASPAR (Castro-Mondragon et al., 2022) within accessible peak sequences. In a multivariate manner, the log2 fold change in peak accessibility in AAvs vehicle-treated cells was modeled for the presence of such motifs, while correcting for GC content, i.e., log2FC~β0+β1*x1+β2*x2+…+β N *GC, where x i represents the presence of TF motifs in the peak (binary variable), and GC represents the GC content of the peak. Effect sizes and p-values ​​for each term were obtained after multivariate linear modeling using ordinary least squares regression with robust standard errors.

[0322] statistics

[0323] Statistical analysis was performed using Graphpad Prism 9.0. Results were analyzed using one of the following statistical tests: ANOVA, Wilcoxon rank-sum test, Mann-Whitney test, or two-tailed t-test, as indicated in the figure legends. Bar graphs are presented as mean ± SEM. P values ​​< 0.05 were considered statistically significant and are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

[0324] Data and code availability

[0325] RNAseq, scRNAseq, CutnRun, and Atac-sec data are available from Gene Expression Omnibus (GEO) with the following accession numbers: GSE188213

[0326] Example 10: Regulation of cell fate by FA and its function in physiological and disease states

[0327] The present disclosure explores how members of the omega-6 family of fatty acids, such as arachidonic acid (AA), can promote intestinal regeneration and ameliorate the degenerative effects of the intestine caused by genotoxic insults, such as radiation therapy or chemotherapy. Dietary AA supplementation may have therapeutic implications for cancer patients receiving chemotherapy or radiation therapy, who often suffer from gastrointestinal side effects.

[0328] Example 11: Kinetics of AA-induced cellular changes in the intestine

[0329] Mice fed an AA-enriched diet for 1, 2, or 4 weeks were analyzed to determine the kinetics of AA-induced intestinal regeneration. Approximately 60 mice (10C and 10ARD) were evaluated at three time points. Histological parameters were assessed, and metabolomics, specifically AA levels, were also evaluated.

[0330] Example 12: Ideal Scenario for AA-Mediated Protection from Genotoxic Stress

[0331] The effects of various dietary regimens of AA supplementation (e.g., continuous (AA ON), reversal (AA OFF), or cyclical (AA ON / OFF / ON / OFF) feeding) were evaluated in mice. Forty mice were divided into four groups of 10: C, ARD ON, ARD OFF, and ARD ON / OFF / ON / OFF. Histology was assessed. Metabolic testing assessed AA levels, and these four groups were further analyzed using eight scRNAseq libraries (n = 2).

[0332] Example 13: Effect of Dietary AA Supplementation in Response to Chemotherapy Against Cancer

[0333] A mouse model of cancer was established to evaluate the beneficial (e.g., therapeutic) effects of dietary AA supplementation in: (1) ameliorating the gastrointestinal side effects of chemotherapy; and (2) improving overall survival in tumor-bearing mice. In a subset of animals, blood was assessed for the effect of diet on chemotherapy-induced cytopenias. Approximately 20 mice were characterized in two groups: 10C and 10ARD. Histological parameters and metabolomics, particularly AA levels, were assessed.

[0334] Example 14: Effects of AA on human intestinal tissue in response to chemotherapy

[0335] Human intestinal organoid models were established. These models were used to evaluate the effects of AA treatment on damage from doxorubicin. Human PDO (n=2, V / AA treatment + / - doxorubicin).

[0336] Example 15: A diet rich in AA does not increase the risk of colorectal cancer in a tumor-prone mouse model

[0337] Tumor models

[0338] Tumor-prone mice (villinCreERt2APC L / +) were treated with tamoxifen and fed an AA-enriched diet (ARD, also known as FA1) or a control diet as described in the previous example. After 3 to 4 months of feeding the ARD or control diet, mice were sacrificed and tumor burden was assessed. Figure 19A The experimental process is shown in Figure 2. Figure 19B As shown in Figure 2, ARD did not increase the number of tumors found in the small intestine or colon. Figure 19C As shown, the total tumor burden in mice fed ARD was no higher than that in mice fed the control diet.

[0339] Metastatic cancer models

[0340] like Figure 20A As shown, C57BL6 / J mice were injected with AKPS (APC KO , KRAS G12D 、P53 KO SMAD4 KO ) cells. After one week, the presence of tumors was confirmed. Subsequently, the mice were fed a diet enriched with AA or a control diet. The survival of the mice was Figure 20B After sacrifice, mice were dissected and evaluated for primary tumors and metastases. Figure 20C As shown, mice fed an AA-enriched diet had the same metastasis rate as mice fed a control diet. As shown in this example, an AA-enriched diet did not increase metastasis rate or decrease survival compared to a control diet.

[0341] Example 16: Kinetics of the Effects of an AA-Enriched Diet in Mice

[0342] Plasma AA levels

[0343] To assess plasma levels of fatty acids over time, mice were fed i) a control diet for 4 weeks, ii) an AA-enriched diet (Arasco) for 4 weeks, or iii) an AA-enriched diet for 2 weeks followed by a switch to a control diet for 2 weeks (ArascoRev). Plasma lipid levels were assessed on days 3, 7, and 14 in mice fed the control diet or the Arasco diet; the results are presented in Table 1. Figure 21A Plasma lipid levels were assessed in all three conditions at 4 weeks; the results are shown in Figure 21B Shown in.

[0344] ARD-induced stem cell regeneration

[0345] Mice were fed a control diet (C) or an AA-enriched diet (ARD) for 3 days, 1 week, or 2 weeks. Mice were then exposed to 15 Gy abdominal irradiation. Figure 22A A schematic diagram of the experimental protocol is shown. Each mouse was evaluated for indicators, histology, and metabolomics. Figure 22B The levels of arachidonic acid observed under each condition are shown. Figure 22C Representative histological images of mice fed a control diet or ARD are shown (left), along with a graph showing the number of EdU+ cells per crypt. EdU (5-ethynyl-2'-deoxyuridine) is a marker of cell proliferation, and in irradiated mice fed ARD for 7 or 14 days, a higher number of EdU+ cells per crypt indicates increased cell proliferation.

[0346] Gene expression

[0347] Mice were fed one of the three diets described in the first part of this example. Figure 23A A schematic diagram of the experimental protocol is shown. Subsequently, gene expression was assessed by scRNAseq and scATACseq, as described in the previous examples; the results are shown in FIG. Figure 23B shown.

[0348] Example 17: Protective Effect of AA in Response to 5-Fluorouracil Chemotherapy

[0349] Mice were fed an AA-enriched diet (ARD) or a control diet for 2 weeks as described in the previous examples. Mice were then treated with vehicle control or a single dose of 50 mg / kg, 250 mg / kg, or 500 mg / kg of 5-fluorouracil (5-FU).

[0350] Figure 24A A schematic diagram showing the experimental process. Figure 24B It was shown that mice fed the ARD diet and dosed with 250 mg / kg 5-FU lost less weight than mice fed the control diet and dosed with 250 mg / kg 5-FU.

[0351] Example 18: Protective Effect of AA in Response to Multiple-Dose Chemotherapy

[0352] Mice (8 weeks old) were fed a control diet or ARD for two weeks and then injected with 5-FU (100 mg / kg) and oxaliplatin (6 mg / kg) once a week for two weeks, then allowed to recover for two weeks and then repeated the once a week regimen for two weeks.

[0353] Figure 25A A schematic diagram showing the experimental procedure. Figure 25B showed that mice fed the ARD diet lost less weight than mice fed a control diet.

[0354] References

[0355] Alsabeeh, N., Chausse, B., Kakimoto, PA, Kowaltowski, AJ, and Shirihai, O. (2018). Cell culture models of fatty acid overload: Problems and solutions. Biochim Biophys Acta Mol Cell Biol Lipids 1863, 143-151.

[0356] Asfaha,S.,Hayakawa,Y.,Muley,A.,Stokes,S.,Graham,T.A.,Ericksen,R.E.,Westphalen,C.B.,von Burstin,J.,Mastracci,T.L.,Worthley,D.L.,et al.(2015).Krt19(+) / Lgr5(-)Cells Are Radioresistant Cancer-Initiating Stem Cells in theColon and Intestine.Cell Stem Cell16,627-638.

[0357] Ayyaz,A.,Kumar,S.,Sangiorgi,B.,Ghoshal,B.,Gosio,J.,Ouladan,S.,Fink,M.,Barutcu,S.,Trcka,D.,Shen,J.,et al.(2019).Single-cell transcriptomes of theregenerating intestine reveal a revival stem cell.Nature 569,121-125.

[0358] Bankaitis,E.D.,Ha,A.,Kuo,C.J.,and Magness,S.T.(2018).Reserve StemCells in Intestinal Homeostasis and Injury.Gastroenterology 155,1348-1361.

[0359] Barker,N.,van Es,J.H.,Kuipers,J.,Kujala,P.,van den Born,M.,Cozijnsen,M.,Haegebarth,A.,Korving,J.,Begthel,H.,Peters,P.J.,et al.(2007).Identification of stem cells in small intestine and colon by marker geneLgr5.Nature 449,1003-1007.

[0360] Basak,O.,Beumer,J.,Wiebrands,K.,Seno,H.,van Oudenaarden,A.,andClevers,H.(2017).Induced Quiescence of Lgr5+Stem Cells in IntestinalOrganoids Enables Differentiation of Hormone-Producing EnteroendocrineCells.Cell Stem Cell 20,177-190e174.

[0361] Berger,S.L.,Kouzarides,T.,Shiekhattar,R.,and Shilatifard,A.(2009).Anoperational definition of epigenetics.Genes Dev 23,781-783.

[0362] Beumer,J.,and Clevers,H.(2016).Regulation and plasticity ofintestinal stem cells during homeostasis and regeneration.Development 143,3639-3649.

[0363] Beyaz,S.,Chung,C.,Mou,H.,Bauer-Rowe,K.E.,Xifaras,M.E.,Ergin,I.,Dohnalova,L.,Biton,M.,Shekhar,K.,Eskiocak,O.,et a1.(2021a).Dietarysuppression of MHC class II expression in intestinal epithelial cellsenhances intestinal tumorigenesis.Cell Stem Cell 28,1922-1935e1925.

[0364] Beyaz,S.,Kim,J.H.,Pinello,L.,Xifaras,M.E.,Hu,Y.,Huang,J.,Kerenyi,M.A.,Das,P.P.,Barnitz,R.A.,Herault,A.,et al.(2017).The histone demethylaseUTX regulates the lineage-specific epigenetic program of invariant naturalkiller T cells.Nat Immunol18,184-195.Beyaz,S.,Mana,M.D.,Roper,J.,Kedrin,D.,Saadatpour,A.,Hong,S.J.,Bauer-Rowe,K.E.,Xifaras,M.E.,Akkad,A.,Arias,E.,et al.(2016).High-fat diet enhances stemness and tumorigenicity of intestinalprogenitors.Nature 531,53-58.

[0365] Beyaz,S.,Mana,M.D·,and Yilmaz,O.H.(2021b).High-fat diet activates aPPAR-delta program to enhance intestinal stem cell function.Cell Stem Cell28,598-599.

[0366] Beyaz,S.,and Yilmaz,O.H.(2016).Molecular Pathways:Dietary Regulationof Stemness and Tumor Initiation by the PPAR-delta Pathway.Clin Cancer Res22,5636-5641.

[0367] Biteau,B.,and Jasper,H.(2011).EGF signaling regulates theproliferation of intestinal stem cells in Drosophila.Development 138,1045-1055.

[0368] Biton,M.,Habet,A.L.,Rogel,N.,Burgin,G.,Beyaz,S.,Schnell,A.,Ashenberg,O.,Su,C.W.,Smillie,C.,Shekhar,K.,et al.(2018).T Helper Cell CytokinesModulate Intestinal Stem Cell Renewal and Differentiation.Cell 175.1307-1320e1322.

[0369] Brash,A.R.(2001).Arachidonic acid as a bioactive molecule.J ClinInvest 107,1339-1345.Bresnick,A.R.,Weber,D.J.,and Zimmer,D.B.(2015).S100proteins in cancer.Nat Rev Cancer 15,96-109.

[0370] Breyer,R.M.,Bagdassarian,C.K.,Myers,S.A.,and Breyer,M.D.(2001).Prostanoid receptors:subtypes and signaling.Annu Rev Pharmacol Toxicol 41,661-690.

[0371] Buczacki,S.J.,Zecchini,H.I.,Nicholson,A.M.,RuSsell,R.,Vermeulen,L,Kemp,R.,and Winton,D.J.(2013).Intestinal label-retaining cells are secretoryprecursors expressing Lgr5.Nature 495,65-69.

[0372] Buenrostro,J.D.,Giresi,P.G.,Zaba,L.C.,Chang,H.Y.,and Greenleaf,W.J.(2013).Transposition of native chromatin for fast and sensitive epigenomicprofiling of open chromatin,DNA-binding proteins and nucleosome position.NatMethods 10,1213-1218.

[0373] Calder,P.C.,Campoy,C.,Eilander,A.,Fleith,M.,Forsyth,S.,Larsson,P.O.,Schelkle,B.,Lohner,S.,Szommer,A.,van de Heijning,B.J.M.,et al.(2019).Asystematic review of the effects of increasing arachidonic acid intake onPUFA status,metabolism and health-related outcomes in humans.Br J Nutr 121,1201-1214.

[0374] Cao,J.,Spielmann,M.,Qiu,X.,Huang,X.,Ibrahim,D.M.,Hill,A.J.,Zhang,F.,Mundlos,S.,Christiansen,L.,Steemers,F.J.,et al.(2019).The single-celltranscriptional landscape of mammalian organogenesis.Nature 566,496-502.

[0375] Chandel,N.S.,Jasper,H.,Ho,T.T.,and Passegue,E.(2016).Metabolicregulation of stem cell function in tissue homeostasis and organismalageing.Nat Cell Biol 18,823-832.

[0376] Cheasley,D.,Pereira,L.,Lightowler,S.,Vincan,E.,Malaterre,J.,andRamsay,R.G.(2011).Myb controls intestinal stem cell genes and self-renewal.Stem Cells 29,2042-2050.

[0377] Chen,L.,Vasoya,R.P.,Toke,N.H.,Parthasarathy,A.,Luo,S.,Chiles,E.,Flores,J.,Gao,N.,Bonder,E.M.,Su,X.,et al.(2020).HNF4 Regulates Fatty AcidOxidation and Is Required for Renewal of Iutestinal Stem Cells inMice.Gastroenterology 158,985-999 e989.

[0378] Cheng,C.W.,Biton,M.,Haber,A.L.,Gunduz,N.,Eng,G.,Gaynor,L.T.,Tripathi,S.,Calibasi-Kocal,G.,Rickelt,S.,Butty,V.L,et al.(2019).Ketone Body SignalingMediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.Cell 178,1115-1131e1115.

[0379] Cheng,H.,Huang,H.,Guo,Z.,Chang,Y.,and Li,Z.(2021).Role ofprostaglandin E2 in tissue repair and regeneration.Theranostics 11,8836-8854.

[0380] Cheng,H.,and Leblond,C.P.(1974).Origin,differentiation and renewal ofthe four main epithelial cell types in the mouse small intestine.I.Columnarcell.Am J Anat 141,461-479.

[0381] Chijiwa,T.,Mishima,A.,Hagiwara,M.,Sano,M.,Hayashi,K.,Inoue,T.,Naito,K.,Toshioka,T.,and Hidaka,H.(1990).Inhibition of forskolin-induced neuriteoutgrowth and protein phosphorylation by a newly synthesized selectiveinhibitor of cyclic AMP-dependent protein kinase,N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide(H-89),of PC12D pheochromocytoma cells.J Bio1Chem 265,5267-5272.

[0382] Cimmino,L.,Neel,B.G.,and Aifantis,I.(2018).Vitamin C in Stem CellReprogramming and Cancer.Trends Cell Biol28,698-708.

[0383] Clevers,H.(2013).The intestinal crypt,a prototype stem cellcompartment.Cell 154,274-284.

[0384] Costea,I.,Mack,D.R.,Lemaitre,R.N.,Israel,D.,Marcil,V.,Ahmad,A.,andAmre,D.K.(2014).Interactions between the dietary polyunsaturated fatty acidratio and genetic factors determine susceptibility to pediatric Crohn′sdisease.Gastroenterology 146,929-931.

[0385] Crawley,S.W.,Mooseker,M.S.,and Tyska,M.J.(2014).Shaping theintestinal brush border.J Cell Biol207,441-451.

[0386] Das,P.P.,Shao,Z.,Beyaz,S.,Apostolou,E.,Pinello,L.,De Los Angeles,A.,O′Brien,K.,Atsma,J.M.,Fujiwara,Y.,Nguyen,M.,et al.(2014).Distinct andcombinatorial functions of Jmjd2b / Kdm4b and Jmjd2c / Kdm4c in mouse embryonicstem cell identity.Mol Cell 53,32-48.

[0387] de Sousa,E.M.F.,and de Sauvage,F.J.(2019).Cellular Plasticity inIntestinal Homeostasis and Disease.Cell Stem Cell 24,54-64.

[0388] Degirmenci,B.,Valenta,T.,Dimitrieva,S.,Hausmann,G.,and Basler,K.(2018).GLI1-expressing mesenchymal cells form the essential Wnt-secretingniche for colon stem cells.Nature 558,449-453.

[0389] Dekaney,C.M.,Gulati,A.S.,Garrison,A.P.,Helmrath,M.A.,and Henning,S.J.(2009).Regeneration of intestinal stem / progenitor cells following doxorubicintreatment of mice.Am J Physiol Gastrointest Liver Physiol297,G461-470.

[0390] Deng,H.,Gerencser,A.A.,and Jasper,H.(2015).Signalintegration by Ca(2+)regulates intestinal stem-cell activity.Nature 528,212-217.

[0391] Doane,A.S.,Chu,C.S.,Di Giammartino,D.C.,Rivas,M.A.,Hellmuth,J.C.,Jiang,Y.,Yusufova,N.,Alonso,A.,Roeder,R.G.,Apostolou,E.,et al.(2021).OCT2pre-positioning facilitates cell fate transition and chromatin architecturechanges in humoralimmunity.Nat Immunol22,1327-1340.

[0392] Dupuis,J.,Langenberg,C.,Prokopenko,I.,Saxena,R.,Soranzo,N.,Jackson,A.U.,Wheeler,E.,Glazer,N.L.,Bouatia-Naji,N.,Gloyn,A.L.,et al.(2010).Newgenetic loci implicated in fasting glucose homeostasis and their impact ontype 2diabetes risk.Nat Genet 42,105-116.

[0393] Eeckhoute,J.,Carroll,J.S.,Geistlinger,T.R.,Torres-Arzayus,M.I.,andBrown,M.(2006).A cell-type-specific transcriPtional network required forestrogen regulation of cyclin D1 and cell cycle progression in breastcancer.Genes Dev 20,2513-2526.

[0394] Evans,R.M.,and Mangelsdorf,D.J.(2014).Nuclear Receptors,RXR,and theBig Bang.Cell 157,255-266.

[0395] Fan,Y.Y.,Callaway,E.,J,M.M.,J,S.G.,Yang,P.,Vincent,L.,and R,S.C.(2016).A New Model to Study the Role of Arachidonic Acid in Colon CancerPathophysiology.Cancer Prev Res(Phila)9,750-757.

[0396] Fan,Y.Y.,Davidson,L.A.,Callaway,E.S.,Goldsby,J.S.,and Chapkin,R.S.(2014).Differential effects of 2-and 3-series E-prostaglandins on in vitroexpansion of Lgr5+colonic stem cells.Carcinogenesis 35,606-612.

[0397] Fan,Y.Y.,Monk,J.M.,Hou,T.Y.,Callway,E.,Vincent,L.,Weeks,B.,Yang,P.,and Chapkin,R.S.(2012).Characterization of an arachidonic acid-deficient(Fads1 knockout)mouse model.J Lipid Res 53,1287-1295.

[0398] Farin,H.F.,Van Es,J.H.,and Clevers,H.(2012).Redundant sources of Wntregulate intestinal stem cells and promote formation of Panethcells.Gastroenterology 143,1518-1529e1517.

[0399] Fu,T.,Coulter,S.,Yoshihara,E.,Oh,T.G.,Fang,S.,Cayabyab,F.,Zhu,Q.,Zhang,T.,Leblanc,M.,Liu,S.,et al.(2019).FXR Regulates Intestinal Cancer StemCell Proliferation·Cell 176,1098-1112e1018.

[0400] Gehart,H.,and Clevers,H.(2019).Tales from the crypt:new insights intointestinal stem cells.Nat Rev Gastroenterol Hepatol 16,19-34.

[0401] Gerdes,J.,Lemke,H.,Baisch,H.,Wacker,H.H.,Schwab,U.,and Stein,H.(1984).Cell cycle analysis of a cell proliferation-associated human nuclearantigen defined by the monoclonal antibody Ki-67.J Immunol133,1710-1715.

[0402] Goessling,W.,North,T.E.,Loewer,S.,Lord,A.M.,Lee,S.,Stoick-Cooper,C.L.,Weidinger,G.,Puder,M.,Daley,G.Q.,Moon,R.T.,et a1.(2009).Geneticinteraction of PGE2 and Wnt signaling regulates developmental specificationof stem cells and regeneration.Cell 136,1136-1147.

[0403] Gregorieff,A.,Liu,Y.,Inanlou,M.R.,Khomchuk,Y.,and Wrana,J.L.(2015).Yap-dependent reprogramming of Lgr5(+)stem cells drives intestinalregeneration and cancer.Nature 526,715-718.

[0404] Greicius,G.,Kabiri,Z.,Sigmundsson,K.,Liang,C.,Bunte,R.,Singh,M.K.,andVirshup,D.M.(2018).PDGFRalpha(+)pericryptal stromal cells are the criticalsource of Wnts and RSPO3 for murine intestinal stem cells in vivo.Proc NatlAcad Sci U S A 115,E3173-E3181.

[0405] Grun,D.,Lyubimova,A.,Kester,L.,Wiebrands,K.,Basak,O.,Sasaki,N.,Clevers,H.,and van Oudenaarden,A.(2015).Single-cell messenger RNA sequencingreveals rare intestinal cell types.Nature 525,251-255.

[0406] Haber,A.L.,Biton,M.,Rogel,N.,Herbst,R.H.,Shekhar,K.,Smillie,C.,Burgin,G.,Delorey,T.M.,Howitt,M.R.,Katz,Y.,et al.(2017).A single-cell surveyof the small intestinal epithelium.Nature 551,333-339.

[0407] Hanson,W.R.,and Ainsworth,E.J.(1985).16,16-Dimethyl prostaglandin E2induces radioprotection in murine intestinal and hematopoietic stemcells.Radiat Res 103,196-203.

[0408] Hoffmeyer,K.,Raggioli,A.,Rudloff,S.,Anton,R.,Hierholzer,A.,Del Valle,I.,Hein,K.,Vogt,R.,and Kemler,R.(2012).Wnt / beta-catenin signaling regulatestelomerase in stem cells and cancer cells.Science 336,1549-1554.

[0409] Igarashi,M.,and Guarente.L.(2016).mTORC1 and SIRT1 Cooperate toFoster Expansion of Gut Adult Stem Cells during Calorie Restriction.Cell 166,436-450.

[0410] Ijiri,K.,and Potten,C.S.(1987).Further studies on the response ofintestinal crypt cells of different hierarchical status to eighteen differentcytotoxic agents.Br J Cancer 55,113-123.

[0411] Insel,P.A.,Murray, F.,Yokoyama,U.,Romano,S..Yun,H.,Brown,L.,Snead,A.,Lu,D.,and Aroonsakool,N.(2012).cAMP and Epac in the regulation of tissuefibrosis.Br J Pharmacol166,447-456.

[0412] Jacobi,S.K.,Moeser,A.J.,Corl,B.A.,Harrell,R.J.,Blikslager,A.T.,andOdle,J.(2012).Dietary long-chain PUFA enhance acute repair of ischemia-injured intestine of suckling pigs.J Nutr 142,1266-1271.

[0413] Jadhav,U.,Nalapareddy,K.,Saxena,M.,O′Neill,N.K.,Pinello,L.,Yuan,G.C.,Orkin,S.H.,and Shivdasani,R.A.(2016).Acquired Tissue-Specific PromoterBivalency Is a Basis for PRC2 Necessity in Adult Cells.Cell 165,1389-1400.

[0414] Jadhav,U.,Saxena,M.,O′Neill,N.K.,Saadatpour,A.,Yuan,G.C.,Herbert,Z.,Murata,K.,and Shivdasani,R.A.(2017).Dynamic Reorganization of ChromatinAccessibility Signatures during Dedifferentiation of Secretory Precursorsinto Lgr5+Intestinal Stem Cells.Cell Stem Cell 21,65-77e65.

[0415] Jiang,G.L.,Im,W.B.,Donde,Y.,and Wheeler,L.A.(2009).EP4 agonistalleviates indomethacin-induced gastric 1esions and promotes chronic gastriculcer healing.World J Gastroenterol 15,5149-5156.

[0416] Jiang,H.,and Edgar,B.A.(2009).EGFR signaling regulates theproliferation of Drosophila adult midgut progenitors.Development 136,483-493.

[0417] Jijon, H. B. Suarez-Lopez, L., Diaz, OE, Das, S., De Calisto, J., Parada-Kusz, M., Yaffe, MB, Pittet, M. J., Mora, J.R., B.Y. al.(2018).Intestinalepithelial cell-specific RARalpha depletion results in aberrant epithelialcell homeostasis and underdeveloped immune system.Mucosal Immunol 11,703-715.

[0418] Kaaij, LT, van de Wetering, M., Fang, F., Decato, B., Molaro, A., van deWerken, H. J., van Es, J. H., Schuijers, J., de Wit, E. de Laat, W. al.(2013).DNAmethylation dynamics during intestinal stem cell differentiation revealsenhancers driving gene expression in the villus.Genome Biol 14,R50.

[0419] Kaiko, GE, Ryu, SH, Koues, OI, Collins, PL, Solnica-Krezel, L., Pearce, EJ, Pearce, EL, Oltz, EMThe, and Stappenbeck, TS (2016). Microbiota-Derived Metabolites.Cell 165,1708-1720.

[0420] Katikaneni,A.,Jelcic,M.,Gerlach,G.F.,Ma,Y.,Overholtzer,M.,andNiethammer,P.(2020).Lipid peroxidation regulates long-range Wound detectionthrough 5-lipoxygenase in zebrafish.Nat Cell Biol22,1049-1055.

[0421] Kazakevych,J.,Sayols,S.,Messner,B.,Krienke,C.,and Soshnikova,N.(2017).Dynamic changes in chromatin states during specification anddifferentiation of adult intestinal stem cells.Nucleic Acids Res 45.5770-5784.

[0422] Kikukawa,H.,Sakuradani,E.,Ando,A.,Shimizu,S.,and Ogawa,J.(2018).Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4:A review.J Adv Res 11,15-22.

[0423] Kim,C.K.,Yang,V.W.,and Bialkowska,A.B.(2017).The Role of IntestinalStem Cells in Epithelial Regeneration Following Radiation-Induced GutInjury.Curr Stem Cell Rep 3,320-332.

[0424] Kim,T.H.,Li,F.,Ferreiro-Neira,I.,Ho,L.L.,Luyten,A.,Nalapareddy,K.,Long,H.,Verzi,M.,and Shivdasani,R.A.(2014).Broadly permissive intestinalchromatin underlies lateral inhibition and cell plasticity.Nature 506,511-515.

[0425] Kirsch,D.G.,Santiago,P.M.,di Tomaso,E.,Sullivan,J.M.,Hou,W.S.,Dayton,T.,Jeffords,L.B.,Sodha,P.,Mercer,K.L.,Cohen,R.,et al.(2010).p53 controlsradiation-induced gastrointestinal syndrome in mice independent ofapoptosis.Science 327,593-596.

[0426] Leblond,C.P.,and Stevens,C.E.(1948).The constant renewal of theintestinal epithelium in the albino rat.Anat Rec 100,357-377.

[0427] Lee,D.,Pearsall,R.S.,Das,S.,Dey,S.K.,Godfrey,V.L.,and Threadgill,D.W.(2004).Epiregulin is not essential for development of intestinal tumors butis required for protection fromintestinal damage.Mol Cell Biol24,8907-8916.

[0428] Lee,Y.S.,Kim,T.Y.,Kim,Y.,Lee,S.H.,Kim,S.,Kang,S.W.,Yang,J.Y.,Baek,I.J.,Sung,Y.H.,Park,Y.Y.,et al.(2018).Microbiota-Derived Lactate AcceleratesIntestinal Stem-Cell-Mediated Epithelial Development.Cell Host Microbe 24,833-846e836.

[0429] Li,H.J.,Ray,S.K.,Pan,N.,Haigh,J.,Fritzsch,B.,and Leiter,A.B.(2019).Intestinal Neurodl expression impairs paneth cell differentiation andpromotes enteroendocrine lineage specification.Sci Rep 9,19489.

[0430] Li,L.,and Fan,C.M.(2017).A CREB-MPP7-AMOT Regulatory Axis ControlsMuscle Stem Cell Expansion and Self-Renewal Competence.Cell Rep 21,1253-1266.

[0431] Libioulle,C.,Louis,E.,Hansoul,S..Sandor,C.,Farnir,F.,Franchimont,D.,Vermeire,S.,Dewit,O.,de Vos,M.,Dixon,A.,et al.(2007).Novel Crohn diseaselocus identified by genome-wide association maps to a gene desert on5p13.1and modulates expression of PTGER4.PLoS Genet 3,e58.

[0432] Lin,X.,Gaudino,S.J.,Jang,K.K.,Bahadur,T.,Singh,A.,Banerjee,A.,Beaupre,M.,Chu,T.,Wong,H.T.,Kim,C.K.,et al.(2022).IL-17RA-signaling in Lgr5(+)intestinal stem cells induces expression of transcription factor ATOH1 topromote secretory cell lineage commitment.Immunity 55,237-253e238.

[0433] Lindemans,C.A.,Calafiore,M.,Mertelsmann,A.M.,O′Connor,M.H.,Dudakov,J.A.,Jenq,R.R.,Velardi,E.,Young,L.F.,Smith,O.M.,Lawrence,G.,et al.(2015).Interleukin-22promotes intestinal-stem-cell-mediated epithelialregeneration.Nature 528,560-564.

[0434] Lu,C.,and Thompson,C.B.(2012).Metabolic regulation ofepigenetics.Cell Metab 16,9-17.

[0435] Lukonin,I.,Serra,D.,Challet Meylan,L.,Volkmann,K.,Baaten,J.,Zhao,R.,Meeusen,S.,Colman,K.,Maurer,F.,Stadler,M.B.,et al.(2020).Phenotypic landscapeof intestinal organoid regeneration.Nature 586,275-280.

[0436] Martinez,F.J.,Calverley,P.M.,Goehring,U.M.,Brose,M.,Fabbri,L.M.,andRabe,K.F.(2015).Effect of roflumilast on exacerbations in patients withsevere chronic obstructive pulmonary disease uncontrolled by combinationtherapy(REACT):a multicentre randomised controlled trial.Lancet 385,857-866.

[0437] McArthur,M.J.,Atshaves,B.P.,Frolov,A.,Foxworth,W.D.,Kier,A.B.,andSchroeder,F.(1999).Cellular uptake and intracellular trafficking of longchain fatty acids.J Lipid Res 40,1371-1383.

[0438] McDonnell,E.,Crown,S.B.,Fox,D.B.,Kitir,B.,Ilkayeva,O.R.,Olsen,C.A.,Grimsrud,P.A.,and Hirschey,M.D.(2016).Lipids Reprogram Metabolism to Become aMajor Carbon Source for Histone Acetylation.Cell Rep 17,1463-1472.

[0439] Meers,M.P.,Bryson,T.D.,Henikoff,J.G.,and Henikoff,S.(2019).ImprovedCUT&RUN chromatin profiling tools.Elife8.

[0440] Mihaylova,M.M.,Cheng,C.W.,Cao,A.Q.,Tripathi,S.,Mana,M.D.,Bauer-Rowe,K.E.,Abu-Remaileh,M.,Clavain,L.,Erdemir,A.,Lewis,C.A.,et al.(2018).FastingActivates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Functionduring Homeostasis and Aging.Cell Stem Cell 22,769-778e764.

[0441] Miyoshi,H.,VanDussen,K.L.,Malvin,N.P.,Ryu,S.H.,Wang,Y.,Sonnek,N.M.,Lai,C.W.,and Stappenbeck,T.S.(2017).Prostaglandin E2 promotes intestinalrepair through an adaptive cellular response of the epithelium.EMBO J 36,5-24.

[0442] Molenaar,M.,van de Wetering,M.,Oosterwegel,M.,Peterson-Maduro,J.,Godsave,S.,Korinek,V.,Roose,J.,Destree,O.,and Clevers,H.(1996).XTcf-3transcription factor mediates beta-catenin-induced axis formation in Xenopusembryos.Cell 86,391-399.

[0443] Montgomery,R.K.,Carlone,D.L.,Richmond,C.A.,Farilla,L.,Kranendonk,M.E.,Henderson,D.E.,Baffour-Awuah,N.Y.,Ambruzs,D.M.,Fogli,L.K.,Algra,S.,eta1.(2011).Mouse telomerase reverse transcriptase(mTert)expression marksslowly cycling intestinal stem cells.Proc Natl Acad Sci U S A 108,179-184.

[0444] Monticelli,L.A.,Osborne,L.C.,Noti,M.,Tran,S.V.,Zaiss,D.M.,and Artis,D.(2015).IL-33promotes an innate immune pathway of intestinal tissueprotection dependent on amphiregulin-EGFR interactions.Proc Natl Acad Sci U SA 112,10762-10767.

[0445] Moon,Y.A.,Hammer,R.E.,and Horton,J.D.(2009).Deletion of ELOVL5 leadsto fatty liver through activation of SREBP-1c in mice.J Lipid Res 50,412-423.

[0446] Munoz,J.,Stange,D.E.,Schepers,A.G.,van de Wetering,M.,Koo,B.K.,Itzkovitz,S.,Volckmann,R.,Kung,K.S.,Koster,J.,Radulescu,S.,et al.(2012).TheLgr5 intestinal stem cell signature:robust expression of proposed quiescent′+4′cell markers.EMBO J 31,3079-3091.

[0447] Murata,K.,Jadhav,U.,Madha,S.,van Es,J.,Dean,J.,Cavazza,A.,Wucherpfennig,K.,Michor,F.,C1evers,H.,and Shivdasani,R.A.(2020).Ascl2-Dependent Cell Dedifferentiation Drives Regeneration of Ablated IntestinalStem Cells.Cell Stem Cell 26,377-390e376.

[0448] Mustata,R.C.,Vasile,G.,Fernandez-Vallone,V.,Strollo,S.,Lefort,A.,Libert,F.,Monteyne,D.,Perez-Morga,D.,Vassart,G.,and Garcia,M.I.(2013).Identification of Lgr5-independent spheroid-generating progenitors of themouse fetal intestinal epithelium.Cell Rep 5,421-432.

[0449] Naik,S.,Larsen,S.B.,Gomez,N.C.,Alaverdyan,K.,Sendoel,A.,Yuan,S.,Polak,L.,Kulukian,A.,Chai,S.,and Fuchs,E.(2017).Inflammatory memorysensitizes skin epithelial stem cells to tissue damage.Nature 550,475-480.

[0450] Nakase,H.,Fujiyama,Y.,Oshitani,N.,Oga,T.,Nonomura,K.,Matsuoka,T.,Esaki,Y.,Murayama,T.,Teramukai,S.,Chiba,T.,et al.(2010).Effect of EP4 agonist(ONO-4819CD)for patients with mild to moderate ulcerative colitis refractoryto 5-aiminosalicylates:a randomized phase II,placebo-controlled trial.InflammBowel Dis 16,73l-733.

[0451] Nandan,M.O.,Ghaleb,A.M.,Bialkowska,A.B.,and Yang,V.W.(2015).Kruppel-like factor5is essential for proliferation and survival of mouse intestinalepithelial stem cells.Stem Cell Res 14,10-19.

[0452] Narumiya,S.,Sugimoto,Y.,and Ushikubi,F.(1999).Prostanoid receptors:structures,properties,and functions.Physiol Rev 79,1193-1226.

[0453] Nateri,A.S.,Spencer-Dene,B.,and Behrens,A.(2005).Interaction ofphosphorylated c-Jun with TCF4 regulates intestinal cancer development.Nature437,281-285.

[0454] Neels,J.G.,and Grimaldi,P.A.(2014).Physiological functions ofperoxisome proliferator-activated receptor beta.Physiol Rev 94,795-858.

[0455] Nigmatullina,L.,Norkin,M.,Dzama,M.M.,Messner,B.,Sayols,S.,andSoshnikova,N.(2017).Id2 controls specification of Lgr5(+)intestinal stem cellprogenitors during gut development.EMBO J 36,869-885.

[0456] Novak,J.S.S.,Baksh,S.C.,and Fuchs,E.(2021).Dietary interventions asregulators of stem cell behavior in homeostasis and disease.Genes Dev 35,199-211.

[0457] Nusse,Y.M.,Savage,A.K.,Marangoni,P.,Rosendah1-Huber,A.K.M.,Landman,T.A.,de Sauvage,F.J.,Locksley,R.M.,and Klein,O.D.(2018).Parasitic helminthsinduce fetal-like reversion in the intestinal stem cell niche.Nature 559,109-113.

[0458] Ordovas-Montanes,J.,Beyaz,S.,Rakoff-Nahoum,S.,and Shalek,A.K.(2020).Distribution and storage of inflammatory memory in barrier tissues.Nat RevImmunol 20,308-320.

[0459] Oszvald,A.,Szvicsek,Z.,Sandor,G.O.,Kelemen,A.,Soos,A.A.,Paloczi,K.,Bursics,A.,Dede,K.,Tolgyes,T.,Buzas,E.I.,et al.(2020).Extracellular vesiclestransmit epithelial growth factor activity in the intestinal stem cellniche.Stem Cells 38,291-300.

[0460] Palla,A.R.,Ravichandran,M.,Wang,Y.X.,AlexandroVa,L,Yang,A.V.,Kraft,P.,Holbrook,C.A.,Schurch,C.M.,Ho,A.T.V.,and Blau,H.M.(2021).Inhibition ofprostaglandin-degrading enzyme 1 5-PGDH rejuvenates aged muscle mass andstrength.Science 371.

[0461] Patankar,J.V.,Muller,T.M.,Kantham,S.,Acera,M.G.,Mascia,F.,Scheibe,K.,Mahapatro,M.,Heichler,C.,Yu,Y.,Li,W.,et al.(2021).E-type prostanoid receptor4drives resolution of intestinal inflammation by blocking epithelialnecroptosis.Nat Cell Biol 23,796-807.

[0462] Pellegrinet,L.,Rodilla,V.,Liu,Z.,Chen,S.,Koch,U.,Espinosa,L.,Kaestner,K.H.,Kopan,R.,Lewis,J.,and Radtke,F.(2011).Dll1-and dll4-mediatednotch signaling are required for homeostasis of intestinal stemcells.Gastroenterology 140,1230-1240e1231-1237.

[0463] Peregrina,K.,Houston,M.,Daroqui,C.,Dhima,E.,Sellers,R.S.,andAugenlicht,L.H.(2015).Vitamin D is a determinant of mouse intestinal Lgr5stem cell functions.Carcinogenesis 36,25-31.

[0464] Potten,C.S.(1977).Extreme sensitivity of some intestinal crypt cellsto X and gamma irradiation.Nature 269,518-521.

[0465] Potten,C.S.,Hume,W.J.,Reid,P.,and Caims,J.(1978).The segregation ofDNA in epithelial stem cells.Cell 15,899-906.

[0466] Rodon,L.,Svensson,R.U.,Wiater,E.,Chun,M.G.H.,Tsai,W.W.,Eichner,L.J.,Shaw,R.J.,and Montminy,M.(2019).The CREB coactivator CRTC2 promotesoncogenesis in LKB1-mutant non-small cell lung cancer.Sci Adv 5,eaaw6455.

[0467] Rodriguez-Colman,MJ,Schewe,M.,Meerlo,M.,Stigter,E.,Gerrits,J., Pras-Raves,M.,Sacchetti,A.,Hornsveld,M.,Oost,KC,Snippert,HJ,et al.(2017).Interplay between metabolic identities in the intestinal crypt supports stemcell function.Nature 543,424-427.

[0468] Roulis, M., Kaklamanos, A., Schernthanner, M., Bielecki, P., Zhao, J., Kaffe, E., Frommelt, L., Qu, R., Knapp, MS, Henrique, A. al.(2020).Paracrineorchestration of intestinal tumorigenesis by a mesenchymal niche.Nature 580,524-529.

[0469] Roulis, M., Nikolaou, C., Kotsaki, E., Kaffe, E., Karagianni, N., Koliaraki, V., Salpea, K. Ragoussis, J., Aidinis, V. Martini, E. Martin. al.(2014).Intestinalmyofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing toepithelial homeostasis.Proc Natl Acad Sci USA 111,E4658-4667.

[0470] Sabatti,C.,Service,SK,Hartikainen,AL,Pouta,A.,Ripatti,S.,Brodsky,J.,Jones,CG,Zaitlen,NA,Varilo,T.,Kaakinen,M.,et al.(2009).Genome-wide association analysis of metabolic traits in a birth cohort from a founderpopulation.Nat Genet 41,35-46.

[0471] Salic,A.,and Mitchison,TJ(2008).A chemical method for fast andsensitive detection of DNA synthesis in vivo.Proc Natl Acad Sci USA 105,2415-2420.

[0472] Sampurno,S.,Bijenhoff,A.,Cheasley,D.,Xu,H.,Robine,S.,Hilton,D.,Alexander,WS,Pereira,L.,Mantamadiotis,T.,Malaterre,J.,et al.(2013).The Myb-p300-CREB axis modulates intestinal homeostasis,radiosensitivity andtumorigenesis.Cell Death Dis 4,e605.

[0473] Sassone-Corsi,P.(2012).The cyclic AMP pathway.Cold Spring HarbPerspect Biol 4.

[0474] Sato,T.,van Es,JH,Snippert,HJ,Stange,DE,Vries,RG,van denBorn,M.,Barker,N.,Shroyer,NF,van de Wetering,M. Clevers,H.(2011).Panethcells constitute the niche for Lgr5 stem cells in intestinal crypts.Nature469,415-418.

[0475] Sato, T., Vries, RG, Snippert, H. J., van de Wetering, M., Barker, N., Stange, DE, van Es, JH, Abo, A. Kujala, P. Peters, P. J. et. al.(2009).Single Lgr5 stemcells build crypt-villus structures in vitro without a mesenchymalniche.Nature 459,262-265.

[0476] Schell,JC,Wisidagama,DR,Bensard,C.,Zhao,H.,Wei,P.,Tanner,J.,Flores,A.,Mohlman,J.,Sorensen,ElK,Carl,et. al.(2017).Control ofintestinal stem cell function and proliferation by mitochondrial pyruvatemetabolism.Nat Cell Biol19,1027-1036.

[0477] Schepers,AG,Vries,R.,van den Born,M.,van de Wetering,M.,andClevers,H.(2011).Lgr5 intestinal stem cells have high telomerase activity andrandomly segregate their chromosomes.EMBO J 1004-1094.

[0478] Schmitt,M.,Schewe,M.,Sacchetti,A.,Feijtel,D.,van de Geer,W.S.,Teeuwssen,M.,Sleddens,H.F.,Joosten,R.,van Royen,M.E.,van de Werken,H.J.G.,etal.(2018).Paneth Cells Respond to Inflammation and Contribute to TiSSueRegeneration by Acquiring Stem-like Features through SCF / c-Kit Signaling.CellRep 24,2312-2328e2317.

[0479] Schuijers,J.,Junker,J.P.,Mokry,M.,Hatzis,P.,Koo,B.K.,Sasselli,V.,vander Flier,L.G.,Cuppen,E.,van Oudenaarden,A.,and Clevers,H.(2015).Ascl2 actsas an R-spondin / Wnt-responsive switch to control stemness in intestinalcrypts.Cell Stem Cell 16,158-170.

[0480] Schvartzman,J.M.,Thompson,C.B.,and Finley,L.W.S.(2018).Metabolicregulation of chromatin modifications and gene expression.J Cell Biol 217,2247-2259.

[0481] Serra,D.,Mayr,U.,Boni,A.,Lukonin,I.,Rempfler,M.,Challet Meylan,L.,Stadler,M.B.,Strnad,P.,Papasaikas,P.,Vischi,D.,et al.(2019).Self-organizationand symmetry breaking in intestinal organoid development.Nature 569,66-72.

[0482] Shao,J.,and Sheng,H.(2010).Amphiregulin promotes intestinalepithelial regeneration:roles ofintestinal subepithelialmyofibroblasts.Endocrinology 151,3728-3737.

[0483] Shaywitz,A.J.,and Greenberg,M.E.(1999).CREB:a stimulus-inducedtranscription factor activated by a diverse array of extracellularsignals.Annu Rev Biochem 68,821-861.

[0484] Sheaffer,K.L.,Kim,R.,Aoki,R.,Elliott,E.N.,Schug,J.,Burger,L,Schubeler,D.,and Kaestner,K.H.(2014).DNA methylation is required for thecontrol of stem cell differentiation in the small intestine.Genes Dev 28.652-664.

[0485] Shoshkes-Carmel,M.,Wang,Y.J.,Wangensteen,K.J.,Toth,B.,Kondo,A.,Massasa,E.E.,Itzkovitz,S.,and Kaestner,K.H.(2018).Subepithelial telocytes arean important source of Wnts that supports intestinal crypts.Nature 557,242-246.

[0486] Snippert,H.J.,van der Flier,L.G.,Sato,T.,van Es,J.H.,van den Born,M.,Kroon-Veenboer,C.,Barker,N.,Klein,A.M.,van Rheenen,J.,Simons,B.D.,et al.(2010).Intestinal crypt homeostasis results from neutral competition betweensymmetrically dividing Lgr5 stem cells.Cell 143,134-144.

[0487] Sougiannis,A.T.,VanderVeen,B.N.,Davis,J.M.,Fan,D.,and Murphy,E.A.(2021).Understanding chemotherapy-induced intestinal mucositis and strategiesto improve gut resilience.Am J Physiol Gastrointest Liver Physiol320,G712-G719.

[0488] Spector,A.A.,John,K.,and Fletcher,J.E.(1969).Binding of long-chainfatty acids to bovine serum albumin.J Lipid Res 10,56-67.

[0489] Stine,R.R.,Sakers,A.P.,TeSlaa,T.,Kissig,M.,Stine,Z.E.,Kwon,C.W.,Cheng,L.,Lim,H.W.,Kaestner,K.H.,Rabinowitz,J.D.,et al.(2019).PRDM16 MaintainsHomeostasis of the Intestinal Epithelium by Controlling Region-SpecificMetabolism.Cell Stem Cell 25,830-845e838.

[0490] Taha,A.S.,McCloskey,C.,McSkimming,P.,and McConnachie,A.(2018).Misoprostol for small bowel ulcers in patients with obscure bleeding takingaspirin and non-steroidal anti-inflammatory drugs(MASTERS):a randomised,double-blind,placebo-controlled,phase 3trial.Lancet Gastroenterol Hepatol 3,469-476.

[0491] Tetteh,P.W.,Basak,O.,Farin,H.F.,Wiebrands,K.,Kretzschmar,K.,Begthel,H.,van den Born,M.,Korving,J.,de Sauvage,F.,van Es,J.H.,et al.(2016).Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of TheirEnterocyte-Lineage Daughters.Cell Stem Cell18,203-213.

[0492] Tian,H.,Biehs,B.,Warming,S.,Leong,K.G.,Rangell,L.,Klein,O.D.,and deSauvage,F.J.(2011).A reserve stem cell population in small intestine rendersLgr5-positive cells dispensable.Nature 478,255-259.

[0493] Tuesta,LM.,Chen,Z.,Duncan,A.,Fowler,CD,Ishikawa,M.,Lee,BR,Liu,XA,Lu,Q.,Cameron,M.,Hayes,et al., MRGLP on acts 2017-17. habenularavoidance circuits to control nicotine intake.Nat Neurosci 20,708-716.

[0494] van der Flier, LG, van Gijn, ME, Hatzis, P., Kujala, P., Haegebarth, A., Stange, DE, Begthel, H., van den Born, M., Guryev, V. I. Oving, et. al.(2009).Transcription factor achaete scute-like 2controls intestinal stem cellfate.Cell 136,903-912.

[0495] van Es, JH, Sato, T., van de Wetering, M., Lyubimova, A., Yee Nee, AN, Gregorieff, A., Sasaki, N., Zeinstra, L, van den Born, M. J. K. al.(2012).Dll1+secretory progenitor cells revert to stem cells upon crypt damage.NatCell Biol14,1099-1104.

[0496] Van Landeghem,L.,Chevalier,J.,Mahe,M.M.,Wedel,T.,Urvil,P.,Derkinderen,P.,Savidge,T.,and Neunlist,M.(2011).Enteric glia promoteintestinal mucosal healing via activation of focal adhesion kinase andrelease of proEGF.Am J Physiol Gastrointest Liver Physiol 300,G976-987.

[0497] VanDussen,K.L.,Carulli,A.J.,Keeley,T.M.,Patel,S.R.,Puthoff,B.J.,Magness,S.T.,Tran,I.T.,Maillard,I.,Siebel,C.,Kolterud,A.,et al.(2012).Notchsignaling modulates proliferation and differentiation of intestinal cryptbase columnar stem cells.Development139,488-497.

[0498] Verzi,M.P.,and Shivdasani,R.A.(2020).Epigenetic regulation ofintestinal stem cell differentiation.Am J Physiol Gastrointest Liver Physiol319,G189-G196.

[0499] von Moltke,J.,Ji,M.,Liang,H.E.,and Locksley,R.M.(2016).Tuft-cell-derived IL-25regulates an intestinal ILC2-epithelial response circuit.Nature529,221-225.

[0500] Wang,B.,Rong,X.,Palladino,E.N.D.,Wang,J.,Fogelman,A.M.,Martin,M.G.,Alrefai,W.A.,Ford,D.A.,and Tontonoz,P.(2018).Phospholipid Remodeling andCholesterol Availability Regulate Intestinal Stemness and Tumorigenesis.CellStem Cell 22,206-220e204.

[0501] Wang,D.,and DuBois,R.N.(2018).Role of prostanoids in gastrointestinalcancer.J Clin Invest 128.2732-2742.

[0502] Wang,Q.,Lin,Y.,Sheng,X.,Xu,J.,Hou,X.,Li,Y.,Zhang,H.,Guo,H.,Yu,Z.,andRen,F.(2020).Arachidonic Acid Promotes Intestinal Regeneration by ActivatingWNT Signaling.Stem Cell Reports 15,374-388.

[0503] Withers,H.R.,and Elkind,M.M.(1970).Microcolony survival assay forcells of mouse intestinal mucosa exposed to radiation.Int J Radiat Biol RelatStud Phys Chem Med 17,261-267.

[0504] Wu,H.,Li,X.M.,Wang,J.R.,Gan,W.J.,Jiang,F.Q.,Liu,Y.,Zhang,X.D.,He,X.S.,Zhao,Y.Y.,Lu,X.X.,et al.(2016).NUR77 exerts a protective effect againstinflammatory bowel disease by negatively regulating the TRAF6 / TLR-IL-1Rsignalling axis.J Pathol238,457-469.

[0505] Yan,K.S.,Gevaert,O.,Zheng,G.X.Y.,Anchang,B.,Probert,C.S.,Larkin,K.A.,Davies,

[0506] P.S.,Cheng,Z.F.,Kaddis,J.S.,Han,A.,et al.(2017).IntestinalEnteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible StemCell Activity.Cell Stem Cell 21,78-90e76.

[0507] Yang,Y.P.,Ma,H.,Starchenko,A.,Huh,W.J.,Li,W.,Hickman,F.E.,Zhang,Q.,Franklin,J.L,Mortlock,D.P.,Fuhrmann,S.,et al.(2017).A Chimeric Egfr ProteinReporter Mouse Reveals Egfr Localization and Trafficking In Vivo.Cell Rep19,1257-1267.

[0508] Yilmaz,O.H.,Katajisto,P.,Lamming,D.W.,Gultekin,Y.,Bauer-Rowe,K.E.,Sengupta,S.,Birsoy,K.,Dursun,A.,Yilmaz,V.O.,Selig,M.,et al.(2012).mTORC1 inthe Paneth cell niche couples intestinal stem-cell function to calorieintake.Nature 486,490-495.

[0509] Yin,X.,Farin,H.F.,van Es,J.H.,Clevers,H.,Langer,R.,and Karp,J.M.(2014).Niche-independent high-purity cultures of Lgr5+intestinal stem cellsand their progeny.Nat Methods 11,106-112.

[0510] Yokoyama,U.,Iwatsubo,K.,Umemura,M.,Fujita,T.,and Ishikawa,Y.(2013).The prostanoid EP4 receptor and its signaling pathway.Pharmacol Rev 65,1010-1052.

[0511] Yu,S.,Tong,K.,Zhao,Y.,Balasubramanian,I.,Yap,G.S.,Ferraris,R.P.,Bonder,E.M.,Verzi,M.P.,and Gao,N.(2018).Paneth Cell Multipotency Induced byNotch Activation following Injury.Cell Stem Cell 23,46-59e45.

[0512] Yui,S.,Azzolin,L.,Maimets,M·,Pedersen,M.T.,Fordham,R.P.,Hansen,S.L.,Larsen,H.L.,Guiu,J.,Alves,M.R.P.,Rundsten,C.F.,et al.(2018).YAP / TAZ-DependentReprogramming of Colonic Epithelium Links ECM Remodeling to TissueRegeneration.Cell Stem Cell 22,35-49e37.

[0513] Zanconato,F.,Forcato,M.,Battilana,G.,Azzolin,L.,Quaranta,E.,Bodega,B.,Rosato,A.,Bicciato,S.,Cordenonsi,M.,and Piccolo,S.(2015).Genome-wideassociation between YAP / TAZ / TEAD and AP-1 at enhancers drives oncogenicgrowth.Nat Cell Biol17,1218-1227.

[0514] Zhang,N.,Yantiss,R.K.,Nam,H.S.,Chin,Y.,Zhou,X.K.,Scherl,E.J.,Bosworth,B.P.,Subbaramaiah,K.,Dannenberg,A.J.,and Benezra,R.(2014).ID1is afunctional marker for intestinal stem and progenitor cells required fornormal response to injury.Stem Cell Reports3,716-724.

[0515] Zhu, 74, 45-58e47.

[0516] Equivalent schemes and scope

[0517] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein.The scope of the present invention is not limited to the above description, but is instead defined by the claims appended hereto.

[0518] Claims or descriptions that include "or" between one or more members of a group are considered to satisfy the following: for a given product or process, one, more than one, or all of the group members are present, used, or otherwise relevant, unless otherwise indicated or otherwise obvious from the context. The invention includes embodiments in which exactly one group member is present, used, or otherwise relevant for a given product or process. The invention includes embodiments in which more than one or all of the group members are present, used, or otherwise relevant for a given product or process. Furthermore, it should be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims are introduced into another claim. For example, any claim dependent on another claim can be amended to include one or more limitations found in any other claim dependent on the same base claim. Furthermore, if a claim recites a composition, it should be understood that methods of using the composition to achieve any of the objects disclosed herein are also included, unless otherwise indicated or a contradiction or inconsistency is obvious to one of ordinary skill in the art. Methods of preparing the composition according to any method of preparation disclosed herein or otherwise known in the art are also included.

[0519] In the case of giving a range, the endpoints are included. In addition, it should be understood that unless otherwise indicated or obvious from the context and the understanding of those skilled in the art, the numerical values ​​expressed in ranges can assume any specific value or sub-range within the range in different embodiments of the present invention, accurate to one-tenth of the unit of the lower limit of the range, unless the context dictates otherwise.

[0520] In addition, it should be understood that any specific embodiment of the present invention that is in the prior art may be explicitly excluded from any one or more claims. Because such embodiments are deemed to be known to those skilled in the art, they may be excluded even if not expressly excluded herein. Any specific embodiment of the composition of the present invention may be excluded from any one or more claims for any reason, regardless of whether it is related to the existence of prior art.

[0521] All references cited herein, including patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

Claims

1. A method for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject, comprising: At least about 2 grams per day (2 g / day) of arachidonic acid triglyceride (AATG) is orally administered to a subject in need thereof for a period of time sufficient to prevent, reduce, or reverse adverse side effects caused by chemotherapy or radiation therapy in the subject.

2. The method of claim 1, wherein the sufficient time is at least about 7 days; and (a) administration is initiated no earlier than 28 days before the subject begins a course of chemotherapy or radiotherapy; (b) administration begins no later than 28 days after the subject completes a course of chemotherapy or radiation therapy; or (c) Administration is initiated at any time during the course of chemotherapy or radiotherapy.

3. The method of claim 1 or claim 2, wherein the sufficient time is at least about 14 days.

4. The method of claim 1 or claim 2, wherein the sufficient time is at least about 21 days.

5. The method of claim 1 or claim 2, wherein the sufficient time is at least about 28 days.

6. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiation therapy lasts for at least about 3 months.

7. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiation therapy lasts for at least about 6 months.

8. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiation therapy continues for at least about 12 months.

9. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiation therapy lasts from about 3 months to about 12 months.

10. The method of any one of claims 1 to 9, wherein at least about 3 g AA TG / day (3 g / day) is administered to the subject.

11. The method of any one of claims 1 to 9, wherein at least about 20 g AA TG / day (20 g / day) is administered to the subject.

12. The method of any one of claims 1 to 9, wherein at least about 30 g AA TG / day (30 g / day) is administered to the subject.

13. The method of any one of claims 1 to 9, wherein at least about 60 g AA TG / day (60 g / day) is administered to the subject.

14. The method of any one of claims 1 to 9, wherein at least about 90 g AA TG / day (90 g / day) is administered to the subject.

15. The method of any one of claims 1 to 9, wherein at least about 100 g AA TG / day (100 g / day) is administered to the subject.

16. The method of any one of claims 1 to 15, wherein about 2 g AATG / day (2 g / day) to about 100 g AATG / day (100 g / day) is administered to the subject.

17. The method of any one of claims 1 to 15, wherein the AATG is in a composition.

18. The method of claim 17, wherein the composition comprises at least about 2% by weight AATG.

19. The method of claim 17 or claim 18, wherein the composition comprises from about 20% by weight AATG to about 50% by weight AATG.

20. The method of claim 17 or claim 18, wherein the composition comprises about 40% by weight AATG.

21. The method of any one of claims 17 to 20, wherein the composition comprises no more than 5% by weight arachidonic acid (AA) ester.

22. The method of any one of claims 17 to 21, wherein the composition is an oil.

23. The method of claim 22, wherein the oil is extracted from a fungus.

24. The method of claim 23, wherein the fungus is Mortierella alpina.

25. The method of any one of claims 17 to 24, wherein the composition is a liquid or a powder.

26. The method of any one of claims 22 to 25, wherein the composition is in food, in a capsule, or in a pill.

27. The method of any one of claims 1 to 26, wherein the AA TG increases intestinal AA levels in the subject, which produces a beneficial effect.

28. The method of any one of claims 1 to 27, wherein administration of AATG increases plasma AA levels in the subject by at least 2-fold compared to a reference.

29. The method of claim 28, wherein the reference is the AA level in plasma or intestinal tissue from the subject before administration of AA TG, or a predetermined AA level in plasma or intestinal tissue.

30. The method of any one of claims 1 to 29, wherein the adverse side effect is a gastrointestinal side effect.

31. The method of any one of claims 1 to 29, wherein the adverse side effect is nausea, vomiting, diarrhea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation sickness, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation.

32. The method of any one of claims 1 to 31, wherein the subject is a human.

33. A method for preventing, reducing or reversing the cytotoxic effects of chemotherapy or radiation therapy in a subject, comprising: At least about 2 grams per day (2 g / day) of arachidonic acid triglyceride (AATG) is orally administered to a subject in need thereof for a period of time sufficient to prevent, reduce, or reverse the cytotoxic effects of chemotherapy or radiation therapy in the subject.

34. The method of claim 33, wherein the cytotoxic effect is intestinal tissue damage.

35. A method of increasing arachidonic acid (AA) levels in a subject, said arachidonic acid (AA) levels being indicative of intestinal AA levels to prevent, reduce or reverse adverse side effects due to chemotherapy or radiation therapy, comprising: (a) measuring the level of arachidonic acid (AA) in a sample from a subject in need thereof and determining whether the AA level is below a predetermined AA level sufficient to prevent, reduce or reverse adverse side effects caused by chemotherapy or radiation therapy; and (b) if the AA level is below the predetermined AA level, administering to the subject of (a) at least about 2 g per day (2 g / day) of AATG for a period of time sufficient to increase the AA level to or above the predetermined AA level.

36. The method of claim 35, further comprising: (c) measuring the AA level produced by the administration of the AA TG in (b) and determining the AA level; and (d) if the AA level in (b) is not at or above the predetermined AA level, further administering to the subject a sufficient amount of AATG per day to bring the intestinal AA level to or above the predetermined AA level.

37. The method of claim 36, further comprising repeating (c) through (d) to produce an intestinal AA level in the subject at or above the predetermined AA level.

38. The method of any one of claims 35 to 37, wherein the sample is plasma.

39. The method of any one of claims 35 to 37, wherein the sample is intestinal tissue.

40. The method of any one of claims 1 to 39, wherein the AA in the AATG is replaced with at least one AA precursor.

41. The method of claim 40, wherein the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

42. A method for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject, comprising: At least about 2 grams per day (2 g / day) of at least one arachidonic acid (AA) precursor is orally administered to a subject in need thereof for a period of time sufficient to prevent, reduce, or reverse adverse side effects caused by chemotherapy or radiation therapy in the subject.

43. The method of claim 42, wherein the AA precursor is in the form of triglycerides (TG).

44. The method of claim 43, wherein the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

45. A kit for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject, comprising: (a) one or more supplement units sufficient to provide at least about 2 grams per day (2 g / day) of arachidonic acid triglyceride (AATG) to a subject in need thereof for at least 7 days; and (b) instructions for preparation and consumption of the one or more supplement units.

46. ​​The kit of claim 45, wherein the one or more supplement units each comprise 500 mg AATG, 1 g AA TG, 2 g AA TG, or 4 g AA TG.

47. The kit of claim 45 or claim 46, wherein the number of supplement units administered to a subject in need thereof is determined in consultation with a healthcare provider.

48. The kit of claim 45 or claim 46, wherein the supplement unit is in the form of a liquid or a powder.

49. The kit of claim 45 or claim 46, wherein the supplement unit is in the form of a liquid or powder.

50. The kit of claim 45 or claim 46, wherein the supplement unit is in the form of a pill or capsule.

51. The kit of claim 50, wherein the supplement units are in one or more containers.

52. A kit for preventing, reducing or reversing adverse side effects caused by chemotherapy or radiation therapy in a subject, comprising: (a) one or more supplement units sufficient to provide at least about 2 grams per day (2 g / day) of at least one arachidonic acid (AA) precursor to a subject in need thereof for a sufficient period of time; and (b) instructions for preparation and consumption of the one or more supplement units.

53. The kit of claim 52, wherein the AA precursor is in the form of triglycerides (TG).

54. The kit of claim 53, wherein the at least one AA precursor is linoleic acid (LA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), LA and γ-LA, γ-LA and dh-γ-LA, or LA, γ-LA and dh-γ-LA.

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