Use of arachidonic acid to ameliorate the cytotoxic effects of chemotherapy and radiotherapy.

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 482,280, filed January 30, 2023, entitled "USE OF ARACHIDONIC ACID FOR AMELIORATION OF CYTOTOXIC EFFECTS FROM CHEMOTHERAPY AND RADIATION THERAPY," the entire disclosure of which is incorporated herein by reference.

[0002] Federally sponsored research or development 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 technology]

[0003] Cancer treatment ideally eliminates all cells that could cause cancer recurrence throughout the patient's lifetime. Chemotherapy and radiation therapy are two forms of treatment for some types of cancer. Chemotherapy involves the administration of single- or multi-drug regimens to kill cancer cells and shrink tumors, while radiation therapy uses high doses of radiation to accomplish the same thing. When surgery is used for cancer treatment, combining surgery with chemotherapy or radiation therapy can improve cure rates or allow for more limited surgery. Radiation therapy can be administered before surgery or chemotherapy (neoadjuvant therapy) or after surgery or chemotherapy (adjuvant therapy). Chemotherapy and radiation therapy target cancer cells, but normal cells are often also affected, resulting in harmful side effects and cytotoxic effects on normal cells and tissues, severely impacting the quality of life of cancer patients. Summary of the Invention

[0004] This disclosure illustrates how the oral administration or ingestion of arachidonic acid triglyceride (AATG) described herein enhances intestinal stem cell (ISC) generation or stemness, providing the basis for methods and compositions for reducing, preventing, or ameliorating the cytotoxic / harmful effects of radiation exposure, such as chemotherapy or radiotherapy. In particular, treatment of single cells derived from intestinal organoids with omega-6 family fatty acid (FA) members (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 morphology correlated with improved regenerative stem cell status (stemness) and reduced differentiation, but also leads to a significant increase in size compared to control organoids. Furthermore, omega-6 FAs ​​converge to arachidonic acid (AA) in gastrointestinal cells, and omega-6 fatty acids that converge to arachidonic acid (AA) improve the stemness of mouse and human organoids. Supplementation of arachidonic acid in the form of oral administration of arachidonic acid triglycerides (AA TG) / dietary increase in AA in the gut (e.g., through consumption of a diet high in AA TG or other oral intake) enhances ISC regeneration.

[0005] Chemotherapy or radiotherapy treatments often result in harmful side effects and cytotoxic effects due, at least in part, to damage to normal cells not targeted by treatment. Promoting cellular repair mechanisms is a potential method for preventing damage to normal cells and preventing, reducing, or reversing the harmful side effects or cytotoxic effects of chemotherapy or radiotherapy. For example, intestinal cells can be affected by changes in a subject's diet. One common feature of dietary interventions to promote intestinal resilience is increasing the abundance and metabolism of fatty acids (FAs), either through dietary intake or release from adipose tissue (Novak et al., 2021). However, some clinical and epidemiological studies suggest that increasing the total amount of polyunsaturated fatty acids (PUFAs), including omega-6 fatty acids, increases cancer risk. In short, conclusive evidence regarding the impact of omega-6 on cancer outcomes is lacking. PUFAs, including omega-6 fatty acids such as arachidonic acid, are important structural components of cell membranes, which rapidly proliferating cells require for their growth. Additionally, upon tissue injury, omega-6 fatty acids are released from cell membranes, generating inflammatory bioactive lipid mediators such as prostaglandins, which are implicated in 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). In some embodiments, the present disclosure provides fatty acids (FAs) (e.g., dietary FAs) to a subject in need thereof to prevent, reduce, or reverse adverse side effects or cytotoxic effects of chemotherapy or radiation therapy. In some embodiments, administering to a subject a FA, such as arachidonic acid (AA), at least one precursor of AA (linoleic acid (LA), gamma-linolenic acid (gamma-LA), dihomo-gamma-linolenic acid (dh-gamma-LA), LA and gamma-LA, gamma-LA and dh-gamma-LA, or LA, gamma-LA, and dh-gamma-LA), or a combination of AA and at least one precursor of AA, before the subject begins a course of chemotherapy or radiation therapy, during a course of chemotherapy or radiation therapy, or after the subject has completed a course of chemotherapy or radiation therapy, prevents, reduces, or reverses adverse side effects or cytotoxic effects of chemotherapy or radiation therapy in the subject. In some embodiments, providing AA, at least one precursor of AA, or AA and at least one precursor of AA prevents, reduces, or ameliorates the adverse side effects or cytotoxic effects of chemotherapy or radiation therapy in a subject. In some embodiments, AA or at least one precursor of AA is in the form of triglyceride (TG, AA TG, AA precursor TG).

[0006] In some embodiments, methods of preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject are disclosed. In some embodiments, the methods comprise orally administering at least about 2 g of arachidonic acid triglyceride (AA TG) per day (2 g / day) to a subject in need thereof for a period of time sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject. In some embodiments, the sufficient period is at least about 7 days; (a) administration is initiated no more than 28 days before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated within 28 days after the subject completes a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during a course of chemotherapy or radiation therapy.

[0007] In some embodiments, a sufficient period of time is at least about 14 days. In some embodiments, a sufficient period of time is at least about 21 days. In some embodiments, a sufficient period of time is at least about 28 days. In some embodiments, the course of chemotherapy or radiation therapy lasts for at least about 3 months. In some embodiments, the course of chemotherapy or radiation therapy lasts for at least about six months. In some embodiments, the course of chemotherapy or radiation therapy lasts for at least about 12 months. In some embodiments, the course of chemotherapy or radiation therapy lasts from about 3 months to about 12 months.

[0008] In some embodiments, at least about 3 g of AA TG / day (3 g / day) is administered to the subject. In some embodiments, at least about 20 g of AA TG / day (20 g / day) is administered to the subject. In some embodiments, at least about 30 g of AA TG / day (30 g / day) is administered to the subject. In some embodiments, at least about 60 g of AA TG / day (60 g / day) is administered to the subject. In some embodiments, at least about 90 g of AA TG / day (90 g / day) is administered to the subject. In some embodiments, at least about 100 g of AA TG / day (100 g / day) is administered to the subject.

[0009] In some embodiments, about 2 g of AA TG / day (2 g / day) to about 100 g of AA TG / day (100 g / day) is administered to the subject. In some embodiments, AATG is present in the composition. In some embodiments, the composition comprises at least about 2% by weight of AATG. In some embodiments, the composition comprises about 20% by weight AA TG to about 50% by weight AA TG. In some embodiments, the composition comprises about 40% by weight of AATG. In some embodiments, the composition comprises no more than 5% by weight of arachidonic acid (AA) esters.

[0010] In some embodiments, the composition is an oil. In some embodiments, the oil is extracted from a fungus. In some embodiments, the fungus is Mortierella alpina. In some embodiments, the composition is a liquid or a powder. In some embodiments, the composition is in the form of a food product, capsule, or pill.

[0011] In some embodiments, the composition further comprises at least one precursor of AA. In some embodiments, the precursor of AA is in the form of a triglyceride (TG). In some embodiments, the precursor of AA is linoleic acid (LA), gamma-linolenic acid (gamma-LA), dihomo-gamma-linolenic acid (dh-gamma-LA), LA and gamma-LA, gamma-LA and dh-gamma-LA, or LA, gamma-LA, and dh-gamma-LA. In some embodiments, AA TG increases the intestinal AA levels of a subject, producing a beneficial effect. In some embodiments, administration of AA TG increases plasma AA levels in a subject by at least 2-fold compared to baseline. 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.

[0012] In some embodiments, the adverse side effect is a gastrointestinal side effect. In some embodiments, the adverse side effect is nausea, vomiting, diarrhea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation disease, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation. In some embodiments, the subject is a human. In some embodiments, methods of preventing, reducing, or ameliorating the cytotoxic effects of chemotherapy or radiation therapy in a subject are disclosed.

[0013] In some embodiments, the methods comprise orally administering at least about 2 g of arachidonic acid triglyceride (AA TG) per day (2 g / day) to a subject in need thereof for a period of time sufficient to prevent, reduce, or ameliorate the cytotoxic effects of chemotherapy or radiation therapy in the subject. In some embodiments, the cytotoxic effect is intestinal tissue damage. In some embodiments, the methods include increasing plasma arachidonic acid (AA) levels in a subject, which indicates intestinal arachidonic acid (AA) levels that prevent, reduce, or ameliorate adverse side effects from chemotherapy or radiation therapy.

[0014] In some embodiments, the method includes: (a) measuring the arachidonic acid (AA) level 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 ameliorate adverse side effects from chemotherapy or radiation therapy; and (b) if the AA level is below the predetermined AA level, administering at least about 2 g of AA TG per day (2 g / day) to the subject in (a) for a period sufficient to increase the AA level to or above the predetermined AA level.

[0015] In some embodiments, the method further includes: (c) measuring the AA level resulting from administering AA TG in (b) to determine the AA level; and (d) if the AA level in (b) is not equal to or greater than the predetermined AA level, further administering AA TG to the subject per day in an amount sufficient to result in an intestinal AA level equal to or greater than the predetermined AA level. In some embodiments, the method further comprises repeating (c)-(d) to produce intestinal AA levels in the subject that are equal to or greater than the predetermined AA level.

[0016] In some embodiments, the sample is plasma. In some embodiments, the sample is intestinal tissue. In some embodiments, methods are provided for preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject. In some embodiments, AA in the AA TG is substituted with at least one precursor of AA.

[0017] In some embodiments, the precursor of at least one AA is linoleic acid (LA), gamma-linolenic acid (gamma-LA), dihomo-gamma-linolenic acid (dh-gamma-LA), LA and gamma-LA, gamma-LA and dh-gamma-LA, or LA, gamma-LA, and dh-gamma-LA. In some embodiments, the methods comprise orally administering to a subject in need thereof at least about 2 g per day of at least one precursor of arachidonic acid (AA) (2 g / day) for a period sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject. In some embodiments, the precursor of AA is in the form of triglyceride (TG). In some embodiments, the precursor of at least one AA is linoleic acid (LA), gamma-linolenic acid (gamma-LA), dihomo-gamma-linolenic acid (dh-gamma-LA), LA and gamma-LA, gamma-LA and dh-gamma-LA, or LA, gamma-LA, and dh-gamma-LA.

[0018] In some embodiments, kits are provided for use in preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject. In some embodiments, the kit comprises: (a) one or more supplementation units sufficient to provide a subject in need thereof with at least about 2 g of arachidonic acid triglycerides (AA TG) per day (2 g / day) for at least 7 days; and (b) instructions for preparing and consuming the one or more supplementation units. In some embodiments, one or more supplementation units each contain 500 mg of AA TG, 1 g of AA TG, 2 g of AA TG, or 4 g of AA TG.

[0019] In some embodiments, the number of supplemental units to administer to a subject in need thereof is determined in consultation with a healthcare provider. In some embodiments, the supplement unit is in liquid or powder form. In some embodiments, the supplement unit is in liquid or powder form. In some embodiments, the supplement unit is in the form of a pill or capsule.

[0020] In some embodiments, the supply unit is contained in one or more containers. In some embodiments, the kit includes: (a) one or more supplement units sufficient to provide a subject in need thereof with at least about 2 g per day of at least one precursor of arachidonic acid (AA) (2 g / day) for a sufficient period of time; and (b) instructions for preparing and consuming the one or more supplement units. In some embodiments, the precursor of AA is in the form of triglyceride (TG). In some embodiments, the precursor of at least one AA is linoleic acid (LA), gamma-linolenic acid (gamma-LA), dihomo-gamma-linolenic acid (dh-gamma-LA), LA and gamma-LA, gamma-LA and dh-gamma-LA, or LA, gamma-LA, and dh-gamma-LA. [Brief explanation of the drawings]

[0021] [Figure 1A] Figures 1A-1T show how a fatty acid (FA) screen in mouse and human organoids identifies arachidonic acid (AA) as a regenerative FA. Figures 1A-1D show the types of FA used in the screen (Figure 1A), the time-kinetic analysis of organoid area (Figure 1B), the ratio of spheroid-type organoid structure to budding organoid morphology (spheroid ratio) in mouse intestinal organoids (Figure 1C), and the time-kinetic analysis of organoid area in patient-derived human intestinal organoids (PDO) (n=4, t-test).

[0022] [Figures 1B-1D] Figures 1A-1D show the type of FA used in the screening (Figure 1A), the time-kinetic analysis of the organoid area (Figure 1B), the ratio of spheroid-type organoid structure to budding organoid-type organoid structure (spheroid ratio) in mouse intestinal organoids (Figure 1C), and the time-kinetic analysis of the organoid area in patient-derived human intestinal organoids (PDO) (Figure 1D) (n=4, t-test).

[0023] [Figures 1E-1F] Figure 1E-1F shows the number of Ki67+ cells in vehicle (V) or AA-treated (25 µM) mouse organoids (Figure 1E), and representative images of Ki67 immunostaining in organoids (Figure 1F) (n = 5).

[0024] [Figure 1G-1H] Figures 1G-1J show representative images of V- or AA-treated mouse organoids (Figure 1G), quantification of spheroid ratio (Figure 1H), crypt domains per organoid (Figure 1I), and organoid area (Figure 1J) in V- or AA-treated organoids (n=50).

[0025] [Figures 1I-1J] Figures 1G-1J show representative images of V- or AA-treated mouse organoids (Figure 1G), quantification of spheroid ratio (Figure 1H), crypt domains per organoid (Figure 1I), and organoid area (Figure 1J) in V- or AA-treated organoids (n=50).

[0026] [Figures 1K-1Q] Figures 1K-1N show quantification of spheroid ratio (Figure 1K), organoid area (Figure 1L), number of subcultured (secondary) organoids (Figure 1M), and representative images of secondary organoids (Figure 1N) derived from V- or AA-treated primary mouse organoids (n=6). Figures 1O-1Q show quantification of organoid area (Figure 1O), spheroid ratio (Figure 1P), and representative images of human PDO (Figure 1Q) in V- or AA-treated human PDO (25 μM) (n=7).

[0027] [Figure 1R-1T] Figures 1R-1T show quantification of organoid area (Figure 1R), spheroid ratio (Figure 1S), and representative images of secondary organoids (Figure 1T) derived from primary V- or AA-treated PDO cultures (n = 4). Unless otherwise noted, data in these figures are means ± standard error from n independent experiments. ***P<0.001, ****P<0.0001 (Mann-Whitney test). Scale bars represent 200 µm (Figures 1F, 1G, 1N, 1Q, 1T). See also Figures 8A-8I and Tables 1 and 2.

[0028] [Figure 2A-2B]Figures 2A-2S show how an AA-rich diet (ARD) enhances intestinal regeneration in vivo. Figure 2A shows the nutrient, carbohydrate (Carb.), and protein (Prot.) ratios of an isocaloric (3.8 kcal / g) control diet (control) and an AA-rich diet (ARD). Figures 2B-2C show metabolomic analysis by liquid chromatography coupled to mass spectrometry (LC-MS) of AA abundance in plasma (n = 13) (Figure 2B) and tissues (intestine, n = 7) (Figure 2C) from mice fed either the control or ARD diet.

[0029] [Figures 2C-2G] Figures 2B-2C show metabolomic analysis by liquid chromatography coupled to mass spectrometry (LC-MS) for the abundance of AA in plasma (n = 13) (Figure 2B) and tissues (intestine, n = 7) (Figure 2C) from control or ARD-fed mice. Figures 2D-2E show crypt length (Figure 2D) and representative hematoxylin and eosin-stained (H&E) images (Figure 2E) of the small intestine from control or ARD-fed mice (n = 5). Figures 2F-2G show Ki67+ cells per crypt (Figure 2F) and representative images of Ki67 immunostaining in the small intestine (Figure 2G) from control or ARD-fed mice (n = 5).

[0030] [Figure 2H-2J] Figures 2H-2J show the spheroid ratio (Figure 2H), crypt domains per organoid (Figure 2I), and representative images of organoids (Figure 2J) in organoids derived from control or ARD-fed mice on day 3 (n = 5, Mann-Whitney test). Scale bars represent 100 µm.

[0031] [Figure 2K-2L] Figures 2K-2L show the organoid-initiating ability of sorted Epcam+ cells derived from crypts of control or ARD-fed mice (Figure 2K), and representative images of organoids (Figure 2L) (n = 4). Scale bars represent 100 µm.

[0032] [Figures 2M-2O] Figures 2M-2O show intestinal length (Figure 2M, n = 9) and number of surviving crypts per intestinal area (Figure 2N, n = 5) in control or ARD-treated mice (3 days after 15 Gy gamma irradiation), as well as representative H&E images of the small intestine (Figure 2O). Scale bars represent 50 µm.

[0033] [Figure 2P-2R] Figures 2P-2R show representative confocal microscopy images (Figure 2R) of EdU signal intensity per intestinal area (Figure 2P, n ​​= 4 0 Gy, n = 6 15 Gy), EdU cells per crypt (Figure 2Q, n = 6), and Epcam (green), EdU (red), and DAPI (blue) staining in the intestine in control or ARD-fed mice 3 days after 15 Gy γ-irradiation and a 4-h pulse of EdU. Scale bars represent 50 μm.

[0034] [Figure 2S] Figure 2S shows the intestinal length of control or ARD-fed mice 3 days after 10 μM doxorubicin (Dox) or vehicle (V) injection (n = 8 Veh, n = 6 Dox). Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (ANOVA). See also Figures 9A-9H and Table 3.

[0035] [Figure 3A] Figures 3A-3L show how AA induces stem cell reprogramming gene expression signatures in mouse and human organoids. Figure 3A shows gene set enrichment analysis (GSEA) using bulk RNA sequencing of AA vs. V-treated mouse organoids across time points (n=3 day 1 (D1) and day 3 (D3), n=4 day 6 (D6)). The scale represents the adjusted p-value for enrichment analysis.

[0036] [Figure 3B] Figure 3B is a heatmap showing differentially expressed (DE) genes (rows) involved in stem cell regeneration or differentiation between AA vs. V-treated organoids across time points (columns) (n = 3 D1 and D3, n = 4 D6). Scale represents the log2 fold change in expression between AA vs. V-treated organoids. As abbreviated in the figure, "GC" indicates goblet cells, and "EE" indicates enteroendocrine cells.

[0037] [Figure 3C-3D] Figure 3C shows Western blots for β-catenin from cytoplasmic and nuclear fractions of V- or AA-treated organoids (n=5). Figures 3D-3F show quantification of organoid area (Figure 3D, n=5) and organoid number per well (Figure 3E, n=5) of V- or AA-treated organoids grown with the indicated concentrations of Wnt3a, as well as representative images of V- or AA-treated organoids with (100 ng / ml) or without (0 ng / ml) Wnt3a (Figure 3F). Scale bars represent 100 μm.

[0038] [Figures 3E-3F] Figures 3D-3F show representative images of organoid area (Figure 3D, n=5) and organoid number per well (Figure 3E, n=5) quantification of V- or AA-treated organoids grown with the indicated concentrations of Wnt3a, and V- or AA-treated organoids with (100 ng / ml) or without (0 ng / ml) Wnt3a (Figure 3F). Scale bars represent 100 µm.

[0039] [Figure 3G-3I]Figure 3G is a heatmap showing DE genes involved in the EGFR signaling pathway (receptor and ligand) between AA vs. V-treated organoids across time points (n = 3 on D1 and D3, n = 4 on D6). The scale represents the log2 fold change in expression between AA vs. V-treated organoids. Figures 3H-3I show organoid area quantification (Figure 3H, n = 5) of V- or AA-treated organoids with or without EGF supplementation (40 ng / ml), as well as representative images of organoids (Figure 3I). The scale bar represents 100 μm.

[0040] [Figure 3J] Figure 3J shows GSEA enrichment of various stem cell signatures using bulk RNA sequencing of AA vs. V-treated human PDO (n=4). Scale represents adjusted p-value for enrichment analysis.

[0041] [Figure 3K-3L] Figure 3K is a heatmap showing DE genes involved in stemness, differentiation, or proliferation between AA- and V-treated human PDOs (n = 4). Scale represents the log2 fold change in expression between AA- and V-treated human PDOs. Figure 3L shows the relative expression of CD55, MSLN, NR4A1, NR4A2, L1CAM, and DUSP4 in V- or AA-treated human PDOs (n = 4, t-test). Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (ANOVA). See also Figures 10A-10K.

[0042] [Figure 4A-4B]Figures 4A-4T show single-cell analysis of AA-induced stemness in vivo. Figure 4A shows uniform manifold approximation and projection (UMAP) clustering of single-cell RNA sequencing (scRNA-seq) data 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). Scale represents the difference in single-cell density between ARD and control on UMAP (n = 2 independent experiments). Figures 4B-4D are split violin plots illustrating the single-cell gene expression levels of S100a6 (Figure 4B), Lgr5 (Figure 4C), and Ascl2 (Figure 4D) across various intestinal epithelial cell clusters in control or ARD-fed mice (n = 2, 23,161 cells, Wilcoxon rank-sum test).

[0043] [Figure 4C-4D] Figures 4B-4D are split violin plots illustrating single-cell gene expression levels of S100a6 (Figure 4B), Lgr5 (Figure 4C), and Ascl2 (Figure 4D) across different intestinal epithelial cell clusters in control or ARD-fed mice (n=2, 23,161 cells, Wilcoxon rank-sum test).

[0044] [Figure 4E] Figure 4E shows a UMAP plot from pseudotime trajectory analysis of crypt cells from control or ARD-fed mice. Arrows highlight predicted trajectories within cell clusters (n = 2, 23,161 cells). Scale represents pseudotime.

[0045] [Figure 4F] Figure 4F is a density plot from pseudotime trajectory analysis showing the difference in density along pseudotime in all cells (top), cells in the Stem1 cluster (middle), and cells in the Stem2 cluster (bottom) between control and ARD-fed mice ( n = 2, 23,161 cells, Fisher's test).

[0046] [Figures 4G-4H] Figure 4G consists of line plots showing the expression levels of Lgr5 (top) and Ascl2 (bottom) in control or ARD-fed mice along the indicated pseudotime axis ( n = 2, 23,161 cells, Wilcoxon rank-sum test). Figures 4H-4P show representative confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 4H), Ascl2 (Figure 4K), and S100a6 (Figure 4N) in intestinal crypts from irradiated (15 Gy) (n = 6 mice) or non-irradiated (0 Gy) (n = 4 mice) control or ARD-fed mice. Quantification of sm-FISH signal per crypt unit for Lgr5 (Figure 4I), Ascl2 (Figure 4L), and S100a6 (Figure 4O) and the frequency of Lgr5+ cells (Figure 4J), Ascl2+ cells (Figure 4M), and S100a6+ cells (Figure 4P) at different crypt layer locations are shown.

[0047] [Figures 4I-4K]Figures 4H-4P show representative confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 4H), Ascl2 (Figure 4K), and S100a6 (Figure 4N) in intestinal crypts from irradiated (15 Gy) (n = 6 mice) or non-irradiated (0 Gy) (n = 4 mice) control or ARD-fed mice. Quantification of sm-FISH signals per crypt unit for Lgr5 (Figure 4I), Ascl2 (Figure 4L), and S100a6 (Figure 4O) and the frequency of Lgr5+ cells (Figure 4J), Ascl2+ cells (Figure 4M), and S100a6+ cells (Figure 4P) at different crypt layer locations are shown. Figures 4Q-4T show that S100a6 is an AA induction- and regeneration-related gene regulated by the PGE2-PTGER4-PKA-CREB / YAP pathway. Scale bars in Figures 4H, 4K, and 4N represent 10 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). See also Figures 11A-12T.

[0048] [Figures 4L-4N]Figures 4H-4P show representative confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 4H), Ascl2 (Figure 4K), and S100a6 (Figure 4N) in intestinal crypts from irradiated (15 Gy) (n = 6 mice) or non-irradiated (0 Gy) (n = 4 mice) control or ARD-fed mice. Quantification of sm-FISH signals per crypt unit for Lgr5 (Figure 4I), Ascl2 (Figure 4L), and S100a6 (Figure 4O) and the frequency of Lgr5+ cells (Figure 4J), Ascl2+ cells (Figure 4M), and S100a6+ cells (Figure 4P) at different crypt layer locations are shown. Figures 4Q-4T show that S100a6 is an AA induction- and regeneration-related gene regulated by the PGE2-PTGER4-PKA-CREB / YAP pathway. Scale bars in Figures 4H, 4K, and 4N represent 10 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). See also Figures 11A-12T.

[0049] [Figures 4O-4P]Figures 4H-4P show representative confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 4H), Ascl2 (Figure 4K), and S100a6 (Figure 4N) in intestinal crypts from irradiated (15 Gy) (n = 6 mice) or non-irradiated (0 Gy) (n = 4 mice) control or ARD-fed mice. Quantification of sm-FISH signals per crypt unit for Lgr5 (Figure 4I), Ascl2 (Figure 4L), and S100a6 (Figure 4O) and the frequency of Lgr5+ cells (Figure 4J), Ascl2+ cells (Figure 4M), and S100a6+ cells (Figure 4P) at different crypt layer locations are shown. Figures 4Q-4T show that S100a6 is an AA induction- and regeneration-related gene regulated by the PGE2-PTGER4-PKA-CREB / YAP pathway. Scale bars in Figures 4H, 4K, and 4N represent 10 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). See also Figures 11A-12T.

[0050] [Figure 4Q-4T] Figures 4Q-4T show that S100a6 is an AA induction- and regeneration-related gene regulated by the PGE2-PTGER4-PKA-CREB / YAP pathway. Scale bars in Figures 4H, 4K, and 4N represent 10 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). See also Figures 11A-12T.

[0051] [Figure 5A-5B]Figures 5A-5M demonstrate how prostaglandin E2 (PGE2) can mediate the stemness-enhancing effects of AA. Figure 5A is a heatmap showing the changes in bioactive lipid mediators between AA vs. V-treated organoids. The scale represents the log2 fold change in metabolite abundance between AA vs. V-treated organoids. Figure 5B shows the spheroid ratio of organoids after treatment with various AA-derived metabolites (n=5, Mann-Whitney test).

[0052] [Figure 5C-5D] Figures 5C-5D show quantification of organoid area for V- or AA-treated organoids grown with the indicated concentrations of celecoxib (Figure 5C, n=5), and representative images of V- or AA-treated organoids with or without celecoxib (15 µM) (Figure 5D). Scale bars represent 200 µm.

[0053] [Figures 5E-5F] Figures 5E-5F show the concordance between AA-induced changes in gene expression and PGE2-induced gene expression assessed by bulk RNA-seq in mouse (Figure 5E, n=3, R2=0.82, P<0.001) and human PDO (Figure 5G, n=4, R2=0.64, P<0.001). DE genes involved in stem cell reprogramming and differentiation are highlighted.

[0054] [Figure 5G-5H] Figure 5G shows UMAP clustering of single cells from V- or PGE2-treated organoids. The scale represents the density difference of single cells between PGE2 vs. V on the UMAP plot (n = 2). Figure 5H is a density UMAP plot showing the changes in gene expression of the fetal spheroid signature between PGE2 vs. V-treated organoids. The scale represents the density difference of gene expression of the fetal spheroid signature in single cells between PGE2 vs. V on the UMAP plot (n = 2).

[0055] [Figures 5I-5K]Figures 5I-5J are split violin plots (n = 2, Wilcoxon rank-sum test) illustrating gene expression levels of S100a6 (Figure 5I) and Ascl2 (Figure 5J) across various intestinal epithelial cell clusters in V- or PGE2-treated organoids. Figure 5K shows a UMAP plot from pseudotime trajectory analysis of cells from V- or PGE2-treated organoids. Arrows highlight predicted trajectories within cell clusters, and the scale represents pseudotime.

[0056] [Figure 5L-5M] Figures 5L-5M are line plots showing the expression levels of Ascl2 (top) and S100a6 (bottom) in V- or PGE2-treated organoids along the indicated pseudotime axis (n=2, Wilcoxon rank-sum test). Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (ANOVA). See also Figures 13A-13N.

[0057] [Figures 6A-6C] Figures 6A-6W show how the Ptger4-cAMP-PKA signaling axis can regulate AA-induced stemness. Figures 6A-6C show the spheroid ratio (Figure 6A, n = 10), organoid area (Figure 6B, n = 10), and representative photographs (Figure 6C) of V, AA, or PGE2-treated wild-type (Ptger4f / f) or Ptger4 knockout (Ptger4 KO) mouse organoids. Scale bars represent 100 µm.

[0058] [Figure 6D] Figure 6D shows the relative expression of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in Ptger4 f / f or Ptger4 KO mouse organoids treated with V, AA, or PGE2 (n = 5).

[0059] [Figures 6E-6G] Figures 6E-6G show the spheroid ratio (Figure 6E, n = 7), organoid area (Figure 6F, n = 5), and representative photographs (Figure 6G) of mouse organoids treated with V or cAMP derivative (8-bromo) (20 µM) (t-test). Scale bars represent 100 µm.

[0060] [Figures 6H-6J] Figure 6H shows the relative expression of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in V- or 8-bromo-treated mouse organoids (n = 4, t-test). Figures 6I-6K show the spheroid ratio (Figure 6I, n = 8), organoid area (Figure 6J, n = 8), and representative photographs (Figure 6K) of V- or AA-treated organoids with or without the PKA inhibitor (H89) (20 μM). Scale bars represent 100 μm.

[0061] [Figures 6K-6L] Figures 6I-6K show the spheroid ratio (Figure 6I, n = 8), organoid area (Figure 6J, n = 8), and representative photographs (Figure 6K) of V- or AA-treated organoids with or without the PKA inhibitor (H89) (20 μM). Scale bars represent 100 μm. Figure 6L shows the relative expression of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in V- or AA-treated organoids with or without H89 (n = 4).

[0062] [Figures 6M-6R]Figures 6M-6N show the organoid area (Figure 6M, n=8) and representative photographs (Figure 6N) of V- or AA-treated human PDO with or without Ptger4 inhibitor (Ptger4i) (10 μM) (n=X). Scale bars represent 100 μm. Figures 6O-6P show the organoid area (Figure 6O, n=5) and representative photographs (Figure 6P) of V- or AA-treated human PDO with or without H89. Scale bars represent 100 μm. Figures 6Q-6R show the organoid area (Figure 6Q, n=5) and representative photographs (Figure 6R) of V- or 8-bromo-treated human PDO. Scale bars represent 100 μm.

[0063] [Figures 6S-6T] Figures 6S-6T show EdU+ cells per GFP+ crypt 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 the intestine (Figure 6T).

[0064] [Figure 6U-6W] Figures 6U-6W show that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit AA-induced stemness. Scale bars represent 50 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). See also Figures 14A-14L.

[0065] [Figure 7A]Figures 7A-7I show how AA can induce epigenetic reprogramming of stemness in a Ptger4-dependent manner. Figure 7A shows pathway enrichment analysis of regions within 5 kb of the transcription start site (TSS) with increased or decreased chromatin accessibility in response to AA treatment in organoids as assessed by ATAC-seq. The scale represents adjusted p-values, and the x-axis represents the normalized enrichment score (NES) from GSEA (n = 2 independent experiments).

[0066] [Figure 7B-7C] Figure 7B is a dot plot showing transcription factor (TF) motifs with the potential to regulate chromatin accessibility changes induced by AA by multivariate analysis. The scale represents the effect size of the motif's presence on the peak accessibility change in AA vs. V-treated organoids. The dot scale represents the adjusted p-value. Figure 7C is a Western blot for cyclic adenosine monophosphate response element-binding protein (CREB)1, phosphorylated CREB1 (pCREB1), YAP1, and CREB-binding protein (CBP) from cytosolic, chromatin, and nuclear fractions of V- or AA-treated organoids (n = 5).

[0067] [Figure 7D] Figure 7D is a heatmap showing genes near regions with significant increases in chromatin accessibility in response to AA (adjusted p-value < 0.01 by negative binomial analysis). The scale represents the calculated z-score of normalized ATAC-seq reads at the peak.

[0068] [Figures 7E-7F]Figure 7E illustrates pathway analysis showing epigenetic reprogramming around genes that are part of the regenerative stem cell signature in AAvs.V-treated organoids, as assessed by Cut&Run assay of the indicated histone marks. The scale represents the adjusted p-value, and the y-scale represents the normalized enrichment score by GSEA. Figure 7F shows a ranked list of log2 fold changes of putative enhancers defined by H3K27ac peaks in AAvs.V-treated organoids around the proximal and distal regulatory regions of genes annotated by the Genomic Region Enrichment Annotation Tool (GREAT). Genes controlling intestinal stemness and differentiation are labeled.

[0069] [Figure 7G] Figure 7G is a scatter plot of genes in AA vs. V-treated organoids that showed a significant increase in H3K4me3 signal approximately 10 kb from the TSS at day 3 and were significantly upregulated in expression at day 6 (negative binomial test adjusted p-value < 0.05 and log2 fold change > 0.58 for both assays).

[0070] [Figure 7H] Figure 7H shows ATAC-seq and Cut&Run (H3K27me3, H3K4me3, H3K4me3) tracks for the AA signature gene S100a6 locus. Below the plot, the gene structure and transcription direction are indicated.

[0071] [Figure 7I] Figure 7I shows the profile plot of the median change in H3K27ac signal in AA vs. vehicle-treated Ptger4 iKO or WT organoids for stem cell regeneration-related gene signatures (see Example 9). ***P<0.001, P value is calculated by the effect of genotype terms in the linear model of H3K27ac difference by distance to TSS. See also Figures 15A-15K.

[0072] [Figure 8A-8B] Figures 8A-8I show time-kinetic FA screening in mouse and human organoids and are related to Figures 1A-1T. Figure 8A shows a schematic of the FA screening approach using live cell imaging. Figure 8B shows the relative number of organoids (clonal potential) in response to increasing doses of various FAs (normalized to the number of organoids at V for each FA) (n=4).

[0073] [Figure 8C] Figure 8C shows representative images of mouse organoids after treatment with various FAs (25 μM) for 120 hours. The scale bar represents 200 μm.

[0074] [Figure 8D] Figure 8D shows representative images of human PDO after treatment with various FAs (25 μM) for 120 hours. The scale bar represents 200 μm.

[0075] [Figures 8E-8F] Figure 8E shows a schematic of AA biosynthesis from LA by desaturase and elongase enzymes. Figure 8F shows the normalized log2-transformed (rlog) counts of gene expression for enzymes controlling AA biosynthesis (Fads1, Fads2, Elovl5) using bulk RNA sequencing of mouse organoids (n = 3) or human PDOs (n = 4). Epcam was used as a reference gene abundant in intestinal epithelial cells.

[0076] [Figure 8G-8I]Figure 8G shows quantification of organoid area after pretreatment with a desaturase inhibitor (sesamin) followed by the indicated FA treatment (n = 4, ANOVA). Figures 8H-8I show the microvilli length (Figure 8H) and representative transmission electron microscopy images (Figure 8I) of V- or AA-treated organoids (n = 3, Mann-Whitney test). Scale bars represent 10 µm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; ***P < 0.001; ****P < 0.0001.

[0077] [Figures 9A-9C] Figures 9A-9H show the in vivo regenerative effects of an AA-rich diet (ARD) and are related to Figures 2A-2S. Figures 9A-9B show the weight (Figure 9A) and blood glucose levels (Figure 9B) of control or ARD-fed mice (n=10, t-test). Figure 9C shows the analysis of polar metabolites by LC-MS using plasma from control or ARD-fed mice. The X-axis represents the log2 fold change in metabolite abundance, and the Y-axis represents the P-value at -log10 (n=5).

[0078] [Figure 9D-9E] Figure 9D shows representative images of the intestines of control (C) or ARD-fed mice that were irradiated (15 Gy) or not (0 Gy) (n = 9). Figure 9E shows representative confocal images of Epcam (green), EdU (red), and DAPI (blue) staining in the intestines of control or ARD-fed mice 3 days after 15 Gy irradiation (n = 5, scale bar represents 10 μm).

[0079] [Figure 9F-9H]Figure 9F shows representative images of intestines from control (C) or ARD-fed mice 3 days after 10 μM doxorubicin (Dox) or vehicle (V) injection (n = 5). Figures 9G-9H show representative confocal images of EdU+ cells per crypt (Figure 9G) and Epcam (green), EdU (red), and DAPI (blue) staining in the intestine in control or ARD-fed mice 3 days after 10 μM doxorubicin (Dox) or vehicle (V) injection (n = 5, ANOVA). Scale bars represent 50 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; ***P < 0.001; ****P < 0.0001.

[0080] [Figures 10A-10B] Figures 10A-10K show AA-mediated induction of stem cell reprogramming gene expression in mouse and human organoids and are related to Figures 3A-3L. Figure 10A shows a schematic of the time-kinetic bulk RNA-seq experiment in organoids. Figure 10B shows principal component analysis (PCA) of time-kinetic bulk RNA-seq data from mouse organoids after treatment with AA for 1 day (D1, n = 3), 3 days (D3, n = 3), or 6 days (D6, n = 4).

[0081] [Figure 10C] Figure 10C is constructed by an upset plot (right) showing overlapping gene sets between AA-induced genes and various stem cell signatures (see Example 9). Box plots (left) show the log2 fold change of the indicated gene set in each row for each time point in AA vs. V-treated organoids.

[0082] [Figure 10D] Figure 10D illustrates the statistical significance of AA-induced upregulation of genes that are part of various stem cell signatures based on a permutation test that calculated the distribution of the difference between two average profiles using 100,000 permutations. The solid line illustrates the empirical null model, and the dotted line indicates the observed value of the measured profile.

[0083] [Figure 10E] Figure 10E shows the relative expression of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in organoids treated with V or AA for 1 day (D1) or 3 days (D3) (n=4, ANOVA).

[0084] [Figures 10F-10G] Figure 10F shows how GSEA can enrich pathways using bulk RNA sequencing of AA vs. V-treated mouse organoids across time points (D1, D3, D6). The color scale represents the adjusted p-value for enrichment analysis. Figure 10G is a heatmap showing DE genes that are targets of Wnt / β-catenin targets between AA vs. V-treated organoids across time points (n=3 D1 and D3, n=4 D6). The scale represents the log2 fold change in expression between AA vs. V-treated organoids.

[0085] [Figure 10H-10I] Figures 10H-10I show the relative number (clonal potential) of organoids in culture sets with Egf or other Egfr ligands Areg and Ereg (normalized to the number of organoids in the Wnt3a-only condition) (Figure 10H) and representative images of organoids (n = 5, ANOVA). Scale bar represents 100 µm.

[0086] [Figures 10J-10K] Figure 10J shows PCA analysis of bulk RNA-seq data from V- or AA-treated human PDO (n=4). Figure 10K shows how GSEA can enrich pathways using bulk RNA sequencing of AA vs. V-treated human PDO. Scales represent adjusted p-values ​​for enrichment analysis. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0087] [Figures 11A-11B] Figures 11A-11I show single-cell analysis of AA-induced stemness in vivo and are related to Figures 4A-4P. Figure 11A is a bubble plot showing cluster-identifying marker gene expression levels from scRNAseq analysis of 23,161 epithelial cells within intestinal crypts (n = 2 independent experiments). Clusters were identified based on the expression of known marker genes. Figure 11B is a stacked bar plot showing the percentage of various epithelial cell clusters identified by scRNAseq from crypts of C (n = 2) or ARD-fed mice (n = 2).

[0088] [Figure 11C] Figure 11C is a bar plot showing the percentage of cells in different epithelial cell clusters identified by scRNAseq from crypts of C (n=2) or ARD-fed mice (n=2).

[0089] [Figure 11D] Figure 11D shows split violin plots illustrating single-cell gene expression levels of Ly6a, Clu, and Msi1 in control or ARD-fed mice across different intestinal epithelial cell clusters (Wilcoxon rank-sum test).

[0090] [Figure 11E] FIG. 11E illustrates line plots showing expression levels of S100a6 (top) and Ly6a (bottom) in control or ARD-fed mice along the indicated pseudotime axis (Wilcoxon rank sum test).

[0091] [Figure 11F] Figure 11F is a density UMAP plot showing changes in gene expression of various stem cell signatures across clusters between ARD-fed versus control mice (see Example 9). The scale represents the density difference in gene expression of the indicated signature in single cells between ARD-fed versus control mice on the UMAP plot (n=2).

[0092] [Figures 11G-11H] Figures 11G-11I show representative low-magnification confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 11G), Ascl2 (Figure 11H), and S100a6 (Figure 11I) in intestinal crypts from irradiated (15 Gy, n = 6 mice) or non-irradiated (0 Gy, n = 4 mice) control or ARD-fed mice. Figures 11G-11I show the frequency of Lgr5+ cells (Figure 11G) and Ascl2+ cells (Figure 11H) at various crypt layer locations. Scale bars represent 50 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0093] [Figure 11I] Figures 11G-11I show representative low-magnification confocal microscopy images of single-molecule fluorescence in situ hybridization (sm-FISH) for Lgr5 (Figure 11G), Ascl2 (Figure 11H), and S100a6 (Figure 11I) in intestinal crypts from irradiated (15 Gy, n = 6 mice) or non-irradiated (0 Gy, n = 4 mice) control or ARD-fed mice. Figures 11G-11I show the frequency of Lgr5+ cells (Figure 11G) and Ascl2+ cells (Figure 11H) at various crypt layer locations. Scale bars represent 50 μm. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0094] [Figure 12A]Figures 12A-12T show single-cell analysis of AA-induced stemness in vitro and are related to Figures 4A-4P. Figure 12A is a bubble plot showing cluster-distinguishing marker gene expression levels obtained from scRNA-seq analysis of 23,599 epithelial cells from V (n=2) or AA-treated (n=2) organoids. Clusters were identified based on the expression of known marker genes (see Example 9).

[0095] [Figures 12B-12C] Figure 12B presents a stacked bar plot showing the percentage of various epithelial cell clusters identified by scRNAseq from V (n = 2) or AA-treated (n = 2) organoids. Figure 12C is a bar plot showing the percentage of cells within various epithelial cell clusters identified by scRNAseq from the crypts of V (n = 2) or AA-treated (n = 2) organoids.

[0096] [Figure 12D] Figure 12D shows UMAP clustering of 23,599 single cells from V- or AA-treated organoids, where the scale represents the difference in density of single-cell clusters between AA vs. V treatments on the UMAP plot (n=2).

[0097] [Figures 12E-12H] Figures 12E-12J are split violin plots illustrating gene expression levels of S100a6 (Figure 12E), Ly6a (Figure 12F), Clu (Figure 12G), Ascl2 (Figure 12H), Lgr5 (Figure 12I), and Msi1 (Figure 12J) across different intestinal epithelial cell clusters in V- or AA-treated organoids (n = 2, Wilcoxon rank-sum test). Figure 12K is a density UMAP plot showing changes in gene expression in different stem cell signatures across clusters between AA- and V-treated organoids. The scale represents the density difference in gene expression of the indicated signatures in single cells between AA and V on the UMAP plot (n = 2).

[0098] [Figures 12I-12J] Figures 12E-12J are split violin plots illustrating gene expression levels of S100a6 (Figure 12E), Ly6a (Figure 12F), Clu (Figure 12G), Ascl2 (Figure 12H), Lgr5 (Figure 12I), and Msi1 (Figure 12J) across different intestinal epithelial cell clusters in V- or AA-treated organoids (n = 2, Wilcoxon rank-sum test).

[0099] [Figure 12K] Figure 12K is the density UMAP plot showing the gene expression changes in different stem cell signatures across clusters between AA vs.V treated organoids.Scale represents the density difference of the gene expression of the indicated signature in single cells between AA vs.V on UMAP plot (n=2).

[0100] [Figure 12L] Figure 12L presents UMAP plots from pseudotime trajectory analysis of cells from V- or AA-treated organoids. Arrows highlight predicted trajectories within cell clusters, and the scale represents pseudotime.

[0101] [Figures 12M-12P] Figures 12M-12P are density plots from pseudotime trajectory analysis showing the difference in density over pseudotime between AA vs. V treated organoids for all cells (Figure 12M), cells in the Stem1 cluster (Figure 12N), cells in the Stem2 cluster (Figure 12O), and cells in the Stem3 cluster (Figure 12P) (Fisher's test).

[0102] [Figure 12Q-12T]Figures 12Q-12T display line plots showing the expression levels of S100a6 (Figure 12Q), Ly6a (Figure 12R), Ascl2 (Figure 12S), and Lgr5 (Figure 12T) in V- or AA-treated organoids along the indicated pseudotime axis (Wilcoxon rank-sum test). Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001).

[0103] [Figures 13A-13C] Figures 13A-13N show that the metabolism of AA to PGE2 leads to stem cell reprogramming and are related to Figures 5A-5M. Figure 13A is a heat map showing DE genes that regulate AA metabolism to bioactive lipid mediators between AA- and V-treated organoids across time points (n=3 D1 and D3, n=4 D6). The color scale represents the log2 fold change in expression between AA- and V-treated organoids. Figure 13B shows the abundance of bioactive lipid mediators from the intestines of control or ARD-fed mice assessed by LC-MS (n=4, ANOVA). Figure 13C shows representative images of organoids treated with the indicated bioactive lipid mediators, and the scale bar represents 200 μm.

[0104] [Figure 13D-13E] Figures 13D-13E show quantification of organoid area of ​​V- or AA-treated organoids grown with the indicated concentrations of indomethacin (Figure 13D), representative images of V- or AA-treated organoids with or without indomethacin (15 μM) (Figure 13E), and the scale bar represents 200 μm.

[0105] [Figures 13F-13H]Figure 13F shows PGE2 levels in Epcam+ intestinal epithelial cells sorted from crypts after treatment with V or AA, assessed by ELISA (n=3, ANOVA). Figure 13G shows the relative expression of enzymes controlling prostaglandin production (Ptges, Ptgs1, Ptgs2) in organoids treated with V, AA, or PGE2 (n=5, ANOVA). Figure 13H shows a split-violin plot illustrating the single-cell gene expression levels of Ptges in V or AA-treated organoids across different intestinal epithelial cell clusters (Wilcoxon rank-sum test).

[0106] [Figures 13I-13J] Figure 13I displays a bubble plot showing cluster-identifying marker gene expression levels from scRNAseq analysis of 23,599 epithelial cells from V (n = 2) or PGE2-treated (n = 2) organoids. Clusters were identified based on the expression of known marker genes (see Example 9). Figure 13J presents a stacked bar plot showing the percentage of various epithelial cell clusters identified by scRNAseq from V (n = 2) or PGE2-treated (n = 2) organoids.

[0107] [Figure 13K] FIG. 13K presents the concordance between AA-induced changes in gene expression and PGE2-induced gene expression as assessed by scRNAseq.

[0108] [Figure 13L] Figure 13L displays a density UMAP plot showing changes in gene expression in various stem cell signatures across clusters between PGE2 vs. V-treated organoids (see Example 9). The color scale represents the density difference in gene expression of the indicated signatures in single cells between PGE2 vs. V on the UMAP plot (n=2).

[0109] [Figure 13M]Figure 13M presents split violin plots illustrating single-cell gene expression levels of Lgr5, Ly6a, and Clu in V- or AA-treated organoids across various intestinal epithelial cell clusters (Wilcoxon rank-sum test).

[0110] [Figure 13N] Figure 13N presents line plots showing the expression levels of Lgr5 and Ly6a in V or PGE2-treated organoids along the indicated pseudotime axis (Wilcoxon rank-sum test). Unless otherwise noted, the data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0111] [Figure 14A] Figures 14A-14L show how AA can promote stem cell reprogramming through the PGE2-Ptger4 signaling pathway and are related to Figures 6A-6T. Figures 14A-14B show quantification of organoid area of ​​V- or AA-treated organoids grown with the indicated inhibitors of the PGE2 receptor (Ptger1i, Ptger2i, Ptger3, Ptger4i) (Figure 14A), and representative images of V- or AA-treated organoids with or without inhibitors (10 μM) (Figure 14B) (n=5). Scale bars represent 200 μm.

[0112] [Figures 14B-14C] Figures 14A-14B present quantification of organoid area of ​​V- or AA-treated organoids grown with the indicated inhibitors of the PGE2 receptor (Ptger1i, Ptger2i, Ptger3, Ptger4i) (Figure 14A), representative images of V- or AA-treated organoids with or without inhibitors (10 μM) (Figure 14B) (n=5), and the scale bar represents 200 μm.

[0113] [Figure 14D-14E]Figures 14C-14E ​​show the organoid area (Figure 14C, n = 10), spheroid ratio (Figure 14D, n = 10), and representative photographs (Figure 14E) of V, AA, or PGE2-treated mouse organoids with or without Ptger4 inhibitor (Ptger4i) (10 μM). The scale bar represents 200 μm.

[0114] [Figure 14F] Figure 14F shows the relative expression of AA-induced signature genes (Cd55, Ly6a, Msln, Nr4a1, S100a6) in V, AA, or PGE2-treated organoids with or without Ptger4i (n=4).

[0115] [Figures 14G-14H] Figures 14G-14H show rlog counts of gene expression for PGE2 receptors (Ptger1, Ptger2, Ptger3, Ptger4) using bulk RNA sequencing of mouse organoids (Figure 14G, n=3) or human PDO (Figure 14H, n=4).

[0116] [Figures 14I-14J] Figures 14I-14L present split violin plots (Wilcoxon rank-sum test) illustrating single-cell gene expression levels of Ptger1 (Figure 14I), Ptger2 (Figure 14J), Ptger3 (Figure 14K), and Ptger4 (Figure 14L) in V- or AA-treated organoids across various intestinal epithelial cell clusters. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001.

[0117] [Figures 14K-14L]Figures 14I-14L present split violin plots (Wilcoxon rank-sum test) illustrating single-cell gene expression levels of Ptger1 (Figure 14I), Ptger2 (Figure 14J), Ptger3 (Figure 14K), and Ptger4 (Figure 14L) in V- or AA-treated organoids across various intestinal epithelial cell clusters. Unless otherwise noted, data in these figures are means ± standard error from n independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001.

[0118] [Figure 15A] Figures 15A-15K show Ptger4-dependent epigenetic reprogramming of stemness by AA and are related to Figures 7A-7I. Figure 15A presents the number of open and closed chromatin peaks in AA vs. vehicle-treated organoid ATACseq annotated by gene regions, FANTOM enhancers, and CpG islands (negative binomial test adjusted p<0.01, log2 fold change>0.58).

[0119] [Figures 15B-15C] Figure 15B shows the gene expression difference of differentially accessible peaks by ATACseq and RNAseq integration for AA vs. V-treated cells (negative binomial test adjusted p-value < 0.01, log2 fold change > 0.58). Genes are associated with peaks within 2.5 kb of the TSS (closing vs. stable: p = 4.9e-6, opening vs. stable: p < 2.2e-16, closing vs. opening: p = 6.4e-9, Wilcoxon rank sum test). Figure 15C presents the number of differential peaks for the indicated histone marks in AA vs. V-treated organoids (negative binomial test adjusted p-value < 0.01, log2 fold change > 0.58).

[0120] [Figure 15D-15E]Figures 15D-15F present profile plots of the median reads per genome coverage (RPGC) of histone marks H3K4me3 (Figure 15D), H3K27ac (Figure 15E), and H3K27me3 (Figure 15F) near the TSS of differentially up- or down-regulated genes in V- or AA-treated organoids. P-values ​​were calculated by using GSEA to find enrichment of significantly up- and down-regulated gene signatures within all genes ranked by Cut&Run signal changes within 2.5 kb of the TSS.

[0121] [Figure 15F] Figures 15D-15F present profile plots of the median reads per genome coverage (RPGC) of histone marks H3K4me3 (Figure 15D), H3K27ac (Figure 15E), and H3K27me3 (Figure 15F) near the TSS of differentially up- or down-regulated genes in V- or AA-treated organoids. P-values ​​were calculated by using GSEA to find enrichment of significantly up- and down-regulated gene signatures within all genes ranked by Cut&Run signal changes within 2.5 kb of the TSS.

[0122] [Figure 15G] Figure 15G presents a scatter plot of genes that showed a significant increase in H3K4me3 signal approximately 10 kb from the TSS at day 3 and were significantly upregulated in expression at day 3 in AA vs. V-treated organoids (negative binomial test adjusted p-value < 0.05 and log2 fold change > 0.58 for both assays).

[0123] [Figure 15H]Figure 15H presents a scatter plot of genes that showed a significant increase in H327ac signal at approximately 25 kb of the TSS at day 3 (left) and were significantly upregulated in expression at day 3 (left) or day 6 (right) in AA vs. V-treated organoids (negative binomial test adjusted p-value < 0.05 and log2 fold change > 0.58 for both assays).

[0124] [Figure 15I] Figure 15I shows ATAC-seq and Cut&Run (H3K27me3, H3K4me3, H3K4me3) tracks for the regeneration-related AA signature genes Msln, Anxa10, Ly6a, and Ascl2. Below the plots, the gene structure and transcriptional direction are indicated.

[0125] [Figure 15J] Figure 15J presents a profile plot of median reads per genome coverage (RPGC) of H3K27ac enrichment for AA and vehicle-treated cells around the TSS (±5.0 kb) of the differentially up- or down-regulated gene list from AA vs. V-treated RNAseq. Two conditions are shown: WT (top) and Ptger4 iKO (bottom). P-values ​​were calculated by using GSEA to find enrichment of significantly up- and down-regulated gene signatures within all genes ranked by Cut&Run signal change within 2.5 kb of the TSS.

[0126] [Figure 15K] Figure 15K displays a profile plot of the median change in H3K27ac signal in AA vs. vehicle-treated Ptger4 iKO or WT organoids for up- or down-regulated genes. ***P<0.001, P-value calculated by the effect of genotype terms in a linear model of H3K27ac differences by distance to TSS.

[0127] [Figure 16A] 16A-16E show schematic diagrams detailing potential mechanisms of stem cell reprogramming in response to AA in vitro and in vivo. [Figure 16B] 16A-16E show schematic diagrams detailing potential mechanisms of stem cell reprogramming in response to AA in vitro and in vivo. [Figure 16C] 16A-16E show schematic diagrams detailing potential mechanisms of stem cell reprogramming in response to AA in vitro and in vivo. [Figure 16D] 16A-16E show schematic diagrams detailing potential mechanisms of stem cell reprogramming in response to AA in vitro and in vivo. [Figure 16E] 16A-16E show schematic diagrams detailing potential mechanisms of stem cell reprogramming in response to AA in vitro and in vivo.

[0128] [Figure 17A] Figures 17A-17F show that YAP, CREB1, and CBP are required for the regenerative effects of dietary AA. [Figure 17B] Figures 17A-17F show that YAP, CREB1, and CBP are required for the regenerative effects of dietary AA. [Figures 17C-17D] Figures 17A-17F show that YAP, CREB1, and CBP are required for the regenerative effects of dietary AA. [Figures 17E-17F] Figures 17A-17F show that YAP, CREB1, and CBP are required for the regenerative effects of dietary AA.

[0129] [Figure 18A] Figures 18A-18C show that loss of S100a6 slows stem cell renewal. [Figures 18B-18C]Figures 18A-18C show that loss of S100a6 slows stem cell renewal.

[0130] [Figures 19A-19C] Figures 19A-19C show that an AA-rich diet (ARD, also called FA1) does not increase tumor burden in the small intestine (Figure 19B), colon (Figure 19B), or whole body (Figure 19C) in a tumor-prone mouse model. Figure 19A outlines the experimental procedure.

[0131] [Figures 20A-20C] Figures 20A-20C show that an AA-rich diet (ARD) does not reduce survival (Figure 20B) or increase metastasis (Figure 20C) in a mouse model of metastatic colon cancer compared with a control diet (C). Figure 20A outlines the experimental procedure.

[0132] [Figure 21A] Figures 21A-21B show the kinetics of AA plasma levels in mice fed a 4-week control diet, a 4-week AA-enriched diet (ARD) (Arasco), or a 2-week ARD followed by a 2-week control diet (ArascoRev). Figure 21A shows plasma lipid levels on days 3, 7, or 14 in mice fed the control or AA-enriched diet. [Figure 21B] FIG. 21B shows plasma lipid levels after 4 weeks in all three groups.

[0133] [Figures 22A-22B] Figures 22A-22C show the effect of diet on AA levels (Figure 22B) and intestinal cell proliferation after irradiation (Figures 22C-22D) observed in a mouse model. Figure 22A shows an overview of the experimental protocol. [Figure 22C] Figures 22A-22C show the effect of diet on AA levels (Figure 22B) and intestinal cell proliferation after irradiation (Figures 22C-22D) observed in a mouse model.

[0134] [Figure 23A] Figures 23A-23B show changes in gene expression in mice fed a 4-week control diet, a 4-week AA-enriched diet (ARD), or a 2-week ARD followed by a 2-week control diet. Figure 23A shows an overview of the experimental protocol. [Figure 23B] Figure 23B shows the results of some comparisons.

[0135] [Figures 24A-24B] Figures 24A-24B show the in vivo regenerative effects of an AA-rich diet (ARD) in mice treated with various doses of 5-fluorouracil (5-FU). Figure 24A shows a schematic diagram of the experimental design. Figure 24B shows the % weight loss in control or ARD-fed mice treated with 250 mg / kg of 5-FU on days 1 to 3 (D1, D2, D3) after 5-FU treatment.

[0136] [Figures 25A-25B] Figures 25A-25B show the in vivo regenerative effects of an AA-rich diet (ARD) in mice treated with a multidrug regimen of 5-fluorouracil (5-FU) and oxaliplatin. Figure 25A shows a schematic diagram of the experimental procedure. Figure 25B shows the percent weight change in control or ARD-fed mice treated with a regimen of 100 mg / kg 5-FU and 6 mg / kg oxaliplatin once a week for two weeks, followed by a two-week recovery period, and then repeating the regimen once a week for two weeks. DETAILED DESCRIPTION OF THE INVENTION

[0137] The harmful side effects of chemotherapy or radiotherapy often include digestive upset caused by intestinal tissue damage.In some embodiments, the present disclosure relates to providing an amount (beneficial dose) of arachidonic acid (AA) or its precursor in the form of triglyceride to a subject undergoing or exposed to a course of chemotherapy or radiotherapy before starting a course of chemotherapy or radiotherapy, thereby preventing or reducing intestinal tissue damage.In some embodiments, AA or its precursor in the form of triglyceride (TG) is administered during a course of chemotherapy or radiotherapy.In some embodiments, AA or its precursor is administered in the form of TG before and during a course of chemotherapy or radiotherapy.In some embodiments, the present disclosure relates to administering or supplementing AA or its precursor in the form of triglyceride to promote tissue regeneration in a subject exposed to a course of chemotherapy or radiotherapy.

[0138] Accumulating evidence suggests that nutrients and metabolic pathways can not only affect cell growth and proliferation, but also influence cell function and fate by altering signaling pathways for transcription factors and the epigenetic landscape (Beyaz et al., 2016; Beyaz et al., 2021b; Beyaz and Yilmaz, 2016; Chandel et al., 2016; Chen et al., 2020; Climmino et al., 2018; Lu and Thompson, 2012). Recent studies have explored the metabolic regulation of intestinal stem cell (ISC) activity via 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 that dietary AA and AA precursors affect stemness and epigenetic regulation of gene expression, for example, in the intestinal epithelium. This research provides a basis for using AA and AA precursors, which may be in the form of triglycerides (TG), to prevent, reduce, or reverse the harmful side effects or cytotoxic effects of chemotherapy or radiation therapy in subjects in need thereof.

[0139] Arachidonic acid (AA) and precursors of AA (precursor AA) In some embodiments, methods for preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject are disclosed. In some embodiments, the methods include administering arachidonic acid (AA) in the form of triglycerides (AA TG) to a subject in need thereof for a period sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject. In some embodiments, the methods include orally administering at least one precursor of arachidonic acid (AA) (precursor AA) to a subject in need thereof for a period sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject.

[0140] Arachidonic acid (AA) is a 20-carbon chain fatty acid with four methylene-interrupted 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 AA TG). 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 AA PL). In some embodiments, AA PL is used in the compositions, methods, and kits disclosed herein. In some embodiments, AA is not associated with a triglyceride (TG) or a phospholipid (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), alpha-linoleic acid (ALA), gamma-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA); LA and ALA; LA and gamma-LA; LA and dh-γ-LA; ALA and gamma-LA; ALA and dh-γ-LA; gamma-LA and dh-γ-LA; LA, ALA, gamma-LA; LA, ALA, gamma-LA, and dh-γ-LA; ALA, gamma-LA, and dh-γ-LA; LA, gamma-LA, and dh-γ-LA; γ-LA, ALA, and gamma-LA; LA, gamma-LA, and dh-γ-LA; or LA, ALA, gamma-LA, and dh-γ-LA. In some embodiments, the AA precursor is in the form of triglyceride (TG). In some embodiments, the AA or AA precursor is in the form of phospholipid (PL, AA PL, precursor AA PL).

[0141] In some embodiments, the composition comprises AA TG. 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. Pat. 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.

[0142] In some embodiments, the oil contains about or at least about 10% AATG, about or at least about 15% AATG, about or at least about 20% AATG, about or at least about 25% AATG, about or at least about 25% AATG, about or at least about 30% AATG, about or at least about 35% AATG, about or at least about 35% AATG, about or at least about 40% AATG, about or at least about 45% AATG, about or at least about 50% AATG, about or at least about 55% AATG, about or at least about 60% AATG. In some embodiments, the oil contains about 20% AATG to about 60% AATG. In some embodiments, the oil contains about 20% AATG to about 50% AATG. In some embodiments, the oil contains about 30% AATG to about 50% AATG. In some embodiments, the oil comprises at least or about 40% AA TG. In some embodiments, the AA TG percentage is calculated as a volume / volume percentage. In some embodiments, the AA TG percentage is calculated as a weight / volume percentage. In some embodiments, the AA TG percentage is calculated as a weight / weight percentage.

[0143] In some embodiments, a method for preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy is disclosed. In some embodiments, the method comprises orally administering to a subject in need thereof a composition comprising: (a) an oil comprising arachidonic acid triglyceride (AATG); and (b) an oil other than the oil of (a), wherein the ratio of the oil of (a) to the oil of (b) is about 3:4, and the composition is administered at least 7 days before the subject begins a course of chemotherapy or radiation therapy, thereby preventing, reducing, or ameliorating adverse side effects in the subject caused by chemotherapy or radiation therapy.

[0144] Administration In some embodiments, AA TG is administered to a subject in need thereof to prevent, reduce, or ameliorate adverse side effects or cytotoxic effects of chemotherapy or radiation therapy in the subject. In some embodiments, a beneficial dose of AA TG 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, the administration is or includes supplementation. In some embodiments, the administration is or includes supplementation.

[0145] In some embodiments, an amount of AA TG is administered to a subject in need thereof per day. In some embodiments, about or at least about 2 g of AA TG is administered to a subject per day. In some embodiments, about 2.5 g or at least about 2.5 g of AA TG is administered to a subject per day. In some embodiments, about 3 g or at least about 3 g of AA TG is administered to a subject per day. In some embodiments, about 4 g or at least about 4 g of AA TG is administered to a subject per day. In some embodiments, about 5 g or at least about 5 g of AA TG is administered to a subject per day. In some embodiments, about 6 g or at least about 6 g of AA TG is administered to a subject per day. In some embodiments, about 7 g or at least about 7 g of AA TG is administered to a subject per day. In some embodiments, about 8 g or at least about 8 g of AA TG is administered to a subject per day. In some embodiments, about 9 g or at least about 9 g of AA TG is administered to a subject per day. In some embodiments, about 10 g or at least about 10 g of AA TG is administered to a subject per day. In some embodiments, about 15 g or at least about 15 g of AA TG is administered to a subject per day. In some embodiments, about 20 g or at least about 25 g of AA TG is administered to a subject per day. In some embodiments, about 30 g or at least about 30 g of AA TG is administered to a subject per day. In some embodiments, about 40 g or at least about 40 g of AA TG is administered to a subject per day. In some embodiments, about 50 g or at least about 50 g of AA TG is administered to a subject per day. In some embodiments, about 60 g or at least about 60 g of AA TG is administered to a subject per day. In some embodiments, about 70 g or at least about 70 g of AA TG is administered to the subject per day. In some embodiments, about 80 g or at least about 80 g of AA TG is administered to the subject per day.In some embodiments, about 90 g or at least about 90 g of AA TG is administered to the subject per day. In some embodiments, about 100 g or at least about 100 g of AA TG is administered to the subject per day.

[0146] In some embodiments, about 2 g to about 100 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 90 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 80 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 70 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 60 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 50 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 40 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 30 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 20 g of AA TG is administered to a subject per day. In some embodiments, about 2 g to about 10 g of AATG is administered to the subject per day.

[0147] In some embodiments, about 5 g to about 100 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 90 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 80 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 70 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 60 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 50 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 40 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 30 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 20 g of AA TG is administered to a subject per day. In some embodiments, about 5 g to about 10 g of AATG per day is administered to the subject.

[0148] 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 of AA TG / kg body weight, about 100 mg or at least about 100 mg of AA TG / kg body weight, about 150 mg or at least about 150 mg of AA TG / kg body weight, about 200 mg or at least about 200 mg of AA TG / kg body weight, about 300 mg or at least about 300 mg of AA TG / kg body weight, about 400 mg or at least about 400 mg of AA TG / kg body weight, about 500 mg or at least about 500 mg of AA TG / kg body weight, about 600 mg or at least about 600 mg of AA TG / kg body weight, about 700 mg or at least about 700 mg of AA TG / kg body weight, about 800 mg or at least about 800 mg of AA TG / kg body weight, about 900 mg or at least about 900 mg of AA TG / kg body weight, about 1 g or at least about 1 g of AA TG / kg body weight. TG / kg body weight, about 1.5g or at least about 1.5g AA TG / kg body weight, about 2g or at least about 2g AA TG / kg body weight, or any range or combination thereof.

[0149] In some embodiments, the amount of AATG administered to a subject in need thereof takes into account one or more of the subject's age, sex, height, concomitant medication(s), and existing diseases. In some embodiments, when the subject is a pediatric patient, the amount of AATG administered is adjusted based on the age of the pediatric patient and the amount of AATG administered to an adult, as disclosed herein, as a daily amount or amount per kg of body weight. A non-limiting example of determining a pediatric dose is Young's law, according to the following formula: [age / (age+12)] × recommended adult dose = pediatric dose. (See, for example, ncbi.nlm.nih.gov / books / NBK554603 / , which is readily available to those skilled in the art.) If the age of the pediatric patient is unknown, Young's law can be used. If the age of the pediatric patient is known, Clark's law or body surface area law can be applied. (See, for example, ncbi.nlm.nih.gov / books / NBK541104 / , which is readily available to those skilled in the art.)

[0150] In some embodiments, administration of AA TG increases the AA level in the subject's blood or blood components (for example, plasma) compared to the baseline.In some embodiments, the AA level increases in the subject's intestine compared to the baseline.In some embodiments, the AA level increases in the subject's plasma and intestine compared to the baseline.

[0151] In some embodiments, the AA level is measured in the subject's blood or blood components (for example, plasma).In some embodiments, the AA level is measured in the subject's intestine.In some embodiments, the AA level is measured in the subject's intestine and in the subject's blood or blood components (for example, plasma).

[0152] In some embodiments, administration of AATG to a subject in need thereof increases the expression of markers of stemness, such as increased expression of genes associated with stemness, compared to a baseline. 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), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (Kromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln). In some embodiments, administration of AATG increases the expression of genes associated with stemness by about 75%, 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, compared to the reference standard. In some embodiments, the expression of genes associated with stemness is increased in cells (e.g., epithelial cells, intestinal cells, etc.) obtained from a subject. In some embodiments, expression of genes associated with stemness is increased in a sample such as blood, a component of blood (e.g., plasma, serum, etc.), or tissue (e.g., intestinal tissue) obtained from a subject.

[0153] In some embodiments, administering AA TG to a subject in need thereof increases the AA level in the subject (e.g., a sample from the subject) compared to a reference level. In some embodiments, administering AA TG to a subject in need thereof increases the AA level by about 2-fold or at least about 2-fold compared to a reference level. In some embodiments, administering AA TG to a subject in need thereof increases the AA level 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, compared to a reference level. In some embodiments, administration of AA TG increases AA levels in a subject in need thereof by about 3-fold to about 20-fold compared to a baseline. In some embodiments, administration of AA TG increases AA levels in a subject in need thereof by about 3-fold to about 15-fold compared to a baseline. In some embodiments, administration of AA TG increases AA levels in a subject in need thereof by about 3-fold to about 10-fold compared to a baseline. In some embodiments, administration of AA TG increases AA levels in a subject in need thereof by about 1.5-fold to about 3-fold compared to a baseline.

[0154] In some embodiments, the AA TG, at least one precursor AA TG or AA TG, and at least one precursor AA TG are administered prior to a course of chemotherapy, radiation therapy, or a combination of chemotherapy and radiation therapy. In some embodiments, the AA TG, at least one precursor AA TG or AA TG, and at least one precursor AA TG are administered a total 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. In some embodiments, the AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG is administered 2 to 4 weeks prior to the course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG is administered 1 to 3 weeks, 3 to 5 weeks, 4 to 6 weeks, or 5 to 7 weeks prior to the course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0155] A subject may complete one course of chemotherapy or one course of radiation therapy, or may complete 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 depends on the needs of the subject.

[0156] In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof for a sufficient period of time to prevent, reduce, or ameliorate the adverse side effects or cytotoxic effects of chemotherapy or radiotherapy in the subject.In some embodiments, the sufficient period of time is at least about 3 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiotherapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiotherapy; or (c) administration is initiated at any time during the course of chemotherapy or radiotherapy.In some embodiments, the sufficient period of time is at least about 5 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiotherapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiotherapy; or (c) administration is initiated at any time during the course of chemotherapy or radiotherapy. In some embodiments, the sufficient period of time is at least about 7 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during the course of chemotherapy or radiation therapy. In some embodiments, the sufficient period of time is at least about 14 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during the course of chemotherapy or radiation therapy. In some embodiments, the sufficient period of time is at least about 21 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during the course of chemotherapy or radiation therapy.In some embodiments, the sufficient period of time is at least about 28 days; and (a) administration is initiated before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during a course of chemotherapy or radiation therapy.

[0157] 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) administration is initiated before the subject begins a course of chemotherapy or radiation therapy; (b) administration is initiated after the subject has completed a course of chemotherapy or radiation therapy; or (c) administration is initiated at any time during a course of chemotherapy or radiation therapy.

[0158] In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered over the following periods: one cycle comprising or about one week of administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, followed by one week or at least one week of not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG; one cycle comprising or about two weeks or at least two weeks of administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, followed by one week or at least two weeks of not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG; one cycle comprising or about three weeks or at least three weeks of administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, followed by one week or at least two weeks of not administering AA TG, at least one precursor AA TG. or one cycle comprising about or at least three weeks of not administering AA TG, or AA TG and at least one precursor AA TG; or about or at least four weeks of administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, followed by about or at least four weeks of not administering AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG.In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG is administered for about or at least one week, about or at least about two weeks, about or at least about three weeks, or about or at least four weeks, followed by no administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG for about or at least one week, about or at least about two weeks, about or at least about three weeks, or about or at least four weeks.

[0159] In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof daily. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are not administered to a subject in need thereof daily. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof every other day. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof at least once a day. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof every other day. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are administered to a subject in need thereof two, three, or four times per day.

[0160] In some embodiments, in the absence of administration of an amount of AA TG that increases the AA level in a subject in need thereof above the predetermined AA level, the AA level in a sample from the subject in need thereof is less than the predetermined AA level. In some embodiments, the predetermined AA level is a 2-fold increase in the AA level measured in a sample from the subject compared to the AA level measured in a sample from the subject before administration of AA TG. 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 a sample from the subject compared to the AA level measured in a sample from the subject before administration of AA TG. 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 ameliorate the adverse side effects of chemotherapy or radiotherapy. In some embodiments, the predetermined AA level is the lowest AA level at which beneficial effects are observed in a subject.

[0161] In some embodiments, the beneficial effect is an increase in expression of a marker of stemness, such as an increase in expression of a gene associated with stemness, compared to a baseline. 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), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (Kromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln).

[0162] In some embodiments, a beneficial effect is observed when the expression of genes associated with stemness is increased by about 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, compared to a reference in a subject in need thereof. In some embodiments, expression of genes associated with stemness is increased in cells (eg, epithelial cells, intestinal cells) obtained from the subject. In some embodiments, a beneficial effect is the prevention, reduction, or amelioration of adverse side effects from chemotherapy or radiation therapy in a subject compared to a baseline, as determined by a healthcare provider (e.g., a physician). Illustratively, the healthcare provider may determine that one or more symptom measures, including but not limited to, diarrhea frequency, volume, amount, or presence, blood in the stool, calprotectin in the stool, vomiting, nausea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation disease, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation, are reduced compared to the baseline. In some embodiments, the criteria is the frequency, volume, amount, or presence of chemotherapy- or radiation-induced diarrhea, blood in the stool, calprotectin in the stool, vomiting, nausea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation disease, radiation enteritis, abdominal pain, rectal bleeding, bloating, or constipation experienced by a subject in need thereof before the subject is administered AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG.

[0163] In some embodiments, the beneficial effect is the prevention, reduction, or reversal of cytotoxic effects of chemotherapy or radiation therapy compared to a baseline, as determined by a healthcare provider (e.g., a physician). Illustratively, the healthcare provider may determine this by comparison to a baseline. In some embodiments, intestinal tissue damage is prevented, reduced, or reversed in a subject in need thereof after administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG compared to a baseline. In some embodiments, the baseline is the degree of intestinal tissue damage before administration of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG to a subject in need thereof. Chemotherapy-induced intestinal damage is discussed in Sougiannis, et al. Am J Physiol Gastrointest Liver Physiol (2021) 320(5):G712-G719, which is available to those skilled in the art and is incorporated herein by reference in its entirety. In some embodiments, the cytotoxic effect is intestinal tissue damage.

[0164] In some embodiments, methods of prevention or prevention refers to the clinical failure to observe one or more adverse side effects that would be expected in a subject undergoing a similar course of chemotherapy or radiation therapy.

[0165] 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 stool obtained from a subject in need thereof. In some embodiments, the beneficial effect is an increase in AA levels in a subject in need thereof compared to a reference standard. In some embodiments, the beneficial effect is an increase of about 2-fold or at least 2-fold in AA levels in a subject in need thereof compared to a reference standard. In some embodiments, the beneficial effect is an increase of 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, in AA levels in a subject in need thereof compared to a reference standard. In some embodiments, the beneficial effect is an increase of about 3-fold to about 15-fold in AA levels in a subject in need thereof compared to a reference standard. In some embodiments, the beneficial effect is an increase of about 3-fold to about 10-fold in AA levels in a subject in need thereof compared to a reference standard. In some embodiments, the beneficial effect is about a 1.5-fold to about a 3-fold increase in AA levels in a subject in need thereof compared to a baseline.

[0166] In some embodiments, the reference is the AA level in a subject before being exposed to a course of chemotherapy, radiotherapy, or both chemotherapy and radiotherapy. In some embodiments, the reference is the level of the AA population in a sample from a subject before being exposed to a course of chemotherapy, radiotherapy, or both chemotherapy and radiotherapy. In some embodiments, the reference is the AA level in a subject before being administered AA TG to the subject. In some embodiments, the reference is the AA level in a subject who has not been exposed to a course of chemotherapy, radiotherapy, or both chemotherapy and radiotherapy. In some embodiments, the reference is the AA level in a subject who has the same condition as a subject who is exposed to a course of chemotherapy or radiotherapy, but who has the same condition but has not been exposed to a course of chemotherapy or radiotherapy. In some embodiments, the condition is a condition that is treated with a course of chemotherapy, radiotherapy, or chemotherapy and radiotherapy. In some embodiments, the condition is cancer. In some embodiments, the reference is the intracellular AA level before being exposed to a course of chemotherapy or radiotherapy.

[0167] In some embodiments, the AA TG is orally administered to a subject in need thereof. In some embodiments, the AA TG is administered orally. In some embodiments, the AA TG is administered via a nasogastric tube. In some embodiments, the AA TG is administered to the stomach, such as via a gastric tube (G-tube) or injection. In some embodiments, the AA TG is administered via a nasoduodenal or nasojejunal tube. In some embodiments, the AA TG is administered to the small intestine via a jejunostomy (J-tube). Administration can be via various parenteral routes. In some embodiments, the AA TG is not administered via intragastric injection. In some embodiments, the AA TG is present 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, the AA is administered rectally, for example, using a suppository, as a free fatty acid bound to a carrier protein (e.g., albumin).

[0168] The effect of administering AA TG can be evaluated by comparing the degree of one or more adverse side effects, the degree of cytotoxicity, or both after administration of AA TG with the degree of the same one or more adverse side effects, the degree of cytotoxicity, or both in a subject in need thereof before administration of AA TG. Similarly, the effect of administering at least one precursor AA TG (a precursor of AA in TG form), or AA TG and at least one precursor AA TG (a precursor of AA in TG form), can be evaluated by comparing the degree of one or more adverse side effects, the degree of cytotoxicity, or both after administration of at least one precursor AA TG (a precursor of AA in TG form), or AA TG and at least one precursor AA TG (a precursor of AA in TG form), with the degree of the same one or more adverse side effects, the degree of cytotoxicity, or both in a subject in need thereof before administration of at least one precursor AA TG (a precursor of AA in TG form), or before administration of AA TG and at least one precursor AA TG (a precursor of AA in TG form).

[0169] subject 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 domestic animal (e.g., dog, 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. 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, radiation therapy. In some embodiments, the subject in need thereof is a subject with cancer that is being exposed to or treated with a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AA TG before a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AA TG during chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the subject in need thereof is administered AA TG before and during chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0170] Numerous 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 populations for the treatments described herein. In some embodiments, the methods include increasing plasma arachidonic acid (AA) levels in a subject, which is an indicator of intestinal arachidonic acid (AA) levels, which prevents, reduces, or ameliorates adverse side effects of chemotherapy or radiation therapy.

[0171] In some embodiments, the method includes: (a) measuring the arachidonic acid (AA) level 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 ameliorate adverse side effects from chemotherapy or radiation therapy; and (b) if the AA level is below the predetermined AA level, administering at least about 2 g of AA TG per day (2 g / day) to the subject in (a) for a period sufficient to increase the AA level to or above the predetermined AA level.

[0172] In some embodiments, the method further includes: (c) measuring the AA level resulting from administering the AA TG in (b) to determine the AA level; and (d) if the AA level in (b) is not equal to or greater than the predetermined AA level, further administering to the subject an amount of AA TG per day sufficient to result in an intestinal AA level equal to or greater than the predetermined AA level.

[0173] In some embodiments, the method further comprises repeating (c)-(d) to produce intestinal AA levels in the subject that are equal to or greater than the predetermined AA level.

[0174] In some embodiments, disclosed herein is a method for increasing the AA level of a subject to prevent or reduce tissue damage caused by chemotherapy or radiotherapy.In some embodiments, the method comprises: (a) measuring the AA level in a sample obtained from the subject; (b) determining whether the AA level of the subject in (a) is below a predetermined AA level; (c) administering an amount of AA TG sufficient to increase the AA level to or above the predetermined AA level, wherein the increase in the AA level in (c) prevents or reduces the tissue damage of the subject caused by chemotherapy, radiotherapy, or chemotherapy and radiotherapy.In some embodiments, the method disclosed herein further comprises: (d) measuring the AA level in a sample obtained from the subject after administering AA TG in (c), and determining whether the AA level in the sample in (d) is above the predetermined AA level.In some embodiments, if the AA level is below the predetermined AA level, the amount of AA TG administered to the subject is adjusted to be sufficient to increase the AA level of the subject to above the predetermined AA level.

[0175] 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 stool. In some embodiments, the tissue is intestinal tissue. In some embodiments, the AA in the AA level is free AA fatty acid. In some embodiments, the AA fatty acid is associated with a carrier protein (e.g., albumin). In some embodiments, the AA in the AA level is AA PL.

[0176] In some embodiments, the AA in the sample obtained from the subject is measured by detecting the AA in the sample.In some embodiments, the method for measuring the AA level in the sample obtained from the subject includes but is 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), nuclear magnetic resonance (NMR).

[0177] Preventing or reducing tissue damage In some embodiments, methods for preventing or reducing tissue damage are disclosed. In some embodiments, the methods include administering AA in the form disclosed herein to a subject before exposure to chemotherapy, radiation therapy, or chemotherapy and radiation therapy, to prevent or reduce tissue damage caused by chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0178] In some embodiments, preventing or reducing tissue damage comprises predicting tissue damage in a subject and administering AA TG prophylactically to the subject before the subject is exposed to a course of chemotherapy, radiotherapy, or chemotherapy and radiotherapy.In some embodiments, preventing or reducing tissue damage further comprises observing less tissue damage in the subject compared to the level of tissue damage that occurred or existed before the administration of AA TG, or compared to a baseline.

[0179] In some embodiments, preventing or reducing tissue damage means about 10% or at least 10% less tissue damage, about 15% or at least 15% less tissue damage, about 20% or at least 20% less tissue damage, about 25% or at least 25% less tissue damage, about 30% or at least 30% less tissue damage, about 35% or at least 35% less tissue damage, about 40% or at least 40% less tissue damage, about 45% or at least 45% less tissue damage compared to the degree of tissue damage that occurred or existed before administration of the AATG or compared to a baseline. , about 50% or at least 50% less tissue damage, about 55% or at least 55% less tissue damage, about 60% or at least 60% less tissue damage, about 65% or at least 65% less tissue damage, about 70% or at least 70% less tissue damage, about 75% or at least 75% less tissue damage, about 80% or at least 80% less tissue damage, about 85% or at least 85% less tissue damage, or about 90% or at least 90% less tissue damage. In some embodiments, preventing or reducing tissue damage includes preventing all tissue damage relative to a baseline. In some embodiments, the baseline is tissue damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy without administration of an AATG (e.g., prophylactic administration). In some embodiments, preventing or reducing tissue damage is determined by a healthcare provider (e.g., a physician). By way of illustration, a healthcare provider may determine that a symptom measure, including but not limited to diarrhea, blood in the stool, or calprotectin in the stool, is reduced in the subject compared to a baseline.

[0180] In some embodiments, the tissue damage is damage expected from chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the tissue damage is measured histologically. In some embodiments, the tissue damage is measured or evaluated as understood by those skilled in the art. In some embodiments, the tissue damage is inferred from clinical symptoms in a subject. In some embodiments, the tissue damage is inferred from gastrointestinal symptoms such as nausea, vomiting, diarrhea, weight loss, etc. In some embodiments, the prevention or reduction of tissue damage is indicated by a reduction in clinical symptoms in a subject receiving chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0181] Tissue regeneration In some embodiments, a method for promoting tissue regeneration is disclosed, comprising administering to a subject with tissue damage caused by chemotherapy, radiotherapy, or chemotherapy and radiotherapy, an AA TG that increases the AA level in the subject by at least 2 times compared to the baseline, so as to promote tissue regeneration in the subject.Tissue regeneration includes the regrowth of tissue that has been damaged.Tissue damage includes, but is not limited to, damage to the subject's tissue caused by a course of chemotherapy or radiotherapy.In some embodiments, the damage is caused by a course of chemotherapy and radiotherapy, or a course that includes both chemotherapy and radiotherapy.

[0182] In some embodiments, promoting the regeneration of tissue damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy includes observing tissue damage, administering AA TG, and observing less tissue damage. In some embodiments, tissue regeneration includes, in each case, 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 tissue damage, compared to the baseline. In some embodiments, the baseline is tissue damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy without administration of AA TG (e.g., prophylactic administration). In some embodiments, the baseline is tissue damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy before administration of AA TG. In some embodiments, the regeneration of damaged tissue is determined by a healthcare provider (e.g., a physician). By way of illustration, a healthcare provider may determine that a symptom measure, including but not limited to diarrhea, blood in the stool, or calprotectin in the stool, is reduced in the subject compared to a baseline.

[0183] In some embodiments, the tissue damage is the damage expected from chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, the tissue damage is measured histologically. In some embodiments, the tissue damage is measured or evaluated as understood by those skilled in the art. In some embodiments, the tissue damage is inferred from clinical symptoms in a subject. In some embodiments, the tissue damage is inferred from gastrointestinal symptoms such as nausea, vomiting, diarrhea, weight loss, etc. In some embodiments, the regeneration of damaged tissue is indicated by a reduction in clinical symptoms in a subject receiving chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

[0184] In some embodiments, regeneration of damaged tissue comprises increased expression of markers of stemness, such as increased expression of genes associated with stemness, compared to a baseline. 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), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (Chromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln). In some embodiments, the expression of genes associated with stemness increases 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, compared to the reference. In some embodiments, the reference is the expression level of genes associated with stemness in tissue damaged by chemotherapy, radiation therapy, or combined chemotherapy and radiation therapy without the administration of AATG (e.g., prophylactic administration). In some embodiments, the reference is the expression level of genes associated with stemness in tissue damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy and before the administration of AATG. In some embodiments, the regeneration of damaged tissue is determined by a medical provider (e.g., a physician). By way of illustration, a healthcare provider may determine that a symptom measure, including but not limited to diarrhea, blood in the stool, or calprotectin in the stool, is reduced in the subject compared to a baseline.

[0185] 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 that have been damaged by a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy with an AA TG that increases AA levels by at least 2-fold compared to the baseline inside the damaged or injured cells or the environment surrounding the damaged or injured cells, thereby promoting the regeneration of the damaged or injured cells. In some embodiments, the damaged or injured cells are isolated from tissues that have been damaged by chemotherapy, radiation, or chemotherapy and radiation.

[0186] In some embodiments, promoting regeneration in damaged or injured cells comprises observing damage in the cells, administering AATG, and observing a reduction in damage in the cells. 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 compared to a baseline. In some embodiments, the baseline is cells damaged or injured by chemotherapy, radiation therapy, or combined chemotherapy and radiation therapy without administration of AATG (e.g., prophylactic administration). In some embodiments, prevention or reduction of cell damage or cell injury is determined by a healthcare provider.

[0187] In some embodiments, regeneration in damaged cells comprises increased expression of markers of stemness, such as increased expression of genes associated with stemness, compared to a baseline. 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), annexin A13 (Anxa13), cyclin D1 (Ccnd1), annexin A3 (Anxa3), interleukin 33 (Il33), clusterin (Clu), amphiregulin (Areg), CD55 molecule (Chromer blood group) (Cd55), epiregulin (Ereg), myoferlin (Myof), mesothelin (Msln). In some embodiments, the expression of genes associated with stemness 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, compared to the reference standard. In some embodiments, the reference standard is the expression level of genes associated with stemness in tissue damaged by chemotherapy, radiotherapy, or combined chemotherapy and radiotherapy without the administration of AATG (e.g., prophylactic administration). In some embodiments, the reference standard is the expression level of genes associated with stemness in tissue damaged by a course of chemotherapy, radiotherapy, or chemotherapy and radiotherapy, and before the administration of AATG.

[0188] In some embodiments, the damaged or injured cells are or comprise damaged or injured epithelial cells. In some embodiments, the damaged or injured cells are or comprise damaged or injured intestinal cells. In some embodiments, the damaged or injured cells are or comprise damaged or injured oral cells. In some embodiments, the damaged or injured cells are or comprise damaged or injured skin cells.

[0189] cell In some embodiments, the present disclosure relates to methods of preventing or reducing cell damage or injury comprising contacting cells with AA TG prior to exposure to chemotherapy, radiation therapy, or a combination of chemotherapy and radiation therapy. 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 cultured cell. In some embodiments, the cell is or comprises a constituent 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 part of a tissue. In some embodiments, the tissue is or comprises an 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.

[0190] In some embodiments, AA level is 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 the reference level.In some embodiments, AA level is measured intracellularly.In some embodiments, AA level is evaluated in the environment surrounding cell.

[0191] In some embodiments, the AA level is increased in cells by 1.5 times, about 1.5 times, or at least 1.5 times; 2 times, about 2 times, or at least 2 times; 3 times, about 3 times, or at least 3 times; 4 times, about 4 times, or at least 4 times; 5 times, about 5 times, or at least 5 times; 6 times, about 6 times, or at least 6 times; 7 times, about 7 times, or at least 7 times; 8 times, about 8 times, or at least 8 times; 9 times, about 9 times, or at least 9 times; 10 times, about 10 times, or at least 10 times; 11 times, about 11 times, or at least 11 times; 12 times, about 12 times, or at least 12 times; 13 times, about 13 times, or at least 13 times; 14 times, about 14 times, or at least 14 times; 15 times, about 15 times, or at least 15 times. In some embodiments, the reference is the AA level in cells before administration of AA TG or in the same cells. In some embodiments, the reference is the AA level in a control sample of cells.

[0192] cancer Cancer (malignant tumor) is a type of disease in which a group of cells exhibits the characteristics of uncontrolled proliferation (growing and dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other parts of the body via lymph or blood). Most cancers form tumors, but some, such as leukemia, do not. In some embodiments, the cancer is selected from the group consisting of colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic carcinoma, medullary carcinoma, Bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioblastoma, neurocytoma, craniopharyngioma, schwannoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, and lymphoma lymphoma, acute lymphocytic leukemia and acute myeloid polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, rectal cancer, urinary tract cancer, uterine cancer, oral cancer, skin cancer, stomach cancer, brain tumor, liver cancer, laryngeal cancer, esophageal cancer, breast tumor, childhood-onset acute lymphocytic leukemia Hematologic malignancies include: hematopoietic leukemia (ALL), thymic ALL, B-cell ALL, acute myeloid leukemia, myelomonocytic leukemia, acute megakaryocytic leukemia, Burkitt's lymphoma, acute myeloid leukemia, chronic myeloid leukemia, and T-cell leukemia, small cell lung cancer and large cell non-small cell lung cancer, acute granulocytic leukemia, germ cell tumors, endometrial cancer, gastric cancer, head and neck cancer, chronic lymphocytic leukemia, hairy cell leukemia, or thyroid cancer.

[0193] Cancer treatment In some embodiments, AA TG is administered to a subject undergoing or intending to undergo chemotherapy or radiation therapy. In some embodiments, chemotherapy comprises the administration of one or more pharmaceutical compositions, including but not limited to alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, antimitotic agents, and corticosteroids. In some embodiments, the chemotherapeutic agent is altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, nitrosoureas, azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, a combination of trifluridine and tipiracil, daunorubicin, doxorubicin, thiazolinone ... The agent is one or more selected from sorbic acid, liposomal doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin C, mitoxantrone, irinotecan, liposomal irinotecan, topotecan, etoposide (VP-16), teniposide, taxanes, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinca alkaloids, vinblastine, vincristine, liposomal vincristine, vinorelbine, prednisone, methylprednisone, dexamethasone, all-trans retinoid acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane omacetaxine, pegaspargase, procarbazine, romidepsin, or vorinostat.

[0194] In some embodiments, the subject is undergoing or intends to undergo radiation therapy. In some embodiments, the radiation therapy comprises external beam radiation therapy. In some embodiments, the radiation therapy comprises three-dimensional conformal radiation therapy (3D-CRT). In some embodiments, the radiation therapy comprises intensity-modulated radiation therapy (IMRT). In some embodiments, the radiation therapy comprises proton beam therapy. In some embodiments, the radiation therapy comprises image-guided radiation therapy (IGRT). In some embodiments, the radiation therapy comprises stereotactic radiation therapy (SRT). In some embodiments, the radiation therapy comprises internal radiation therapy. In some embodiments, the internal radiation therapy comprises permanent implants. In some embodiments, the internal radiation therapy comprises temporary internal radiation therapy. In some embodiments, the radiation therapy comprises intraoperative radiation therapy (IORT). In some embodiments, the radiation therapy comprises system radiation therapy. In some embodiments, the radiation therapy comprises radioimmunotherapy. In some embodiments, the radiation therapy comprises a radiosensitizer and a radioprotector. In some embodiments, the radiation therapy comprises neoadjuvant radiation therapy. In some embodiments, the radiation therapy comprises adjuvant radiation therapy. In some embodiments, the radiation therapy comprises palliative radiation therapy.

[0195] kit In some embodiments, a kit is disclosed for use in preventing, reducing, or ameliorating the adverse side effects of chemotherapy or radiation therapy in a subject. In some embodiments, the kit comprises: (a) one or more supplement units sufficient to provide a subject in need thereof with at least about 2 g of arachidonic acid triglyceride (AA TG) per day (2 g / day) for at least 7 days; and (b) instructions for preparing and consuming one or more supplement units. In some embodiments, the kit comprises: (a) one or more supplement units sufficient to provide a subject in need thereof with at least about 2 g of at least one precursor of arachidonic acid (AA) per day (2 g / day) for a sufficient period of time; and (b) instructions for preparing and consuming one or more supplement units. In some embodiments, the number of supplemental units to administer to a subject in need thereof is determined in consultation with a healthcare provider.

[0196] In some embodiments of the present invention, the kit may include a preparation vial, a preparation diluent vial, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG and additional agent(s). The diluent vial contains a diluent, such as an edible composition, for diluting AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, which may be a solution or powder (such as a concentrated solution or a lyophilized powder). In some embodiments, the edible composition is a fruit or vegetable puree. In some embodiments, the edible composition is a nutritional shake, etc.

[0197] In some embodiments, the instructions include instructions for mixing a specific amount of diluent with a specific amount of concentrated solution or lyophilized powder, thereby preparing the final formulation for administration. In some embodiments, the instructions include instructions for use with a syringe or other administration device. In some embodiments, the instructions include instructions for treating a patient with an effective amount of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, and any additional agent(s). It will 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 of edible liquid, or the like, can contain indicia, such as a conventional marking that changes color when the formulation is autoclaved or otherwise sterilized.

[0198] In some embodiments, the kit is provided or sold as a service packaged with guidance, instructions, or recommendations from a healthcare provider for consuming one or more supplementation units. In some embodiments, the healthcare provider or advisor is a physician, nutritionist, registered dietitian, physician assistant, nurse practitioner, or nurse. In some embodiments, the healthcare provider is an oncologist or surgeon. In some embodiments, the guidance, instructions, or recommendations comprise oral communication with the healthcare provider. In some embodiments, the guidance, instructions, or recommendations comprise written instructions.

[0199] In some embodiments, the present disclosure provides a kit comprising one or more supplement units and one or more food compositions. In some embodiments, the kit further comprises instructions for ingesting AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG, and one or more food compositions. In some embodiments, the supplement units of AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are packaged separately from one or more food compositions. In some embodiments, AA TG, at least one precursor AA TG, or AA TG and at least one precursor AA TG are premixed with one or more food compositions in one or more supplement units.

[0200] Supply unit In some embodiments, a supplementation unit containing AA TG, at least one precursor of AA (e.g., TG form), or both AA TG and at least one precursor of AA (e.g., TG form) for administration to a subject in need thereof is disclosed. In some embodiments, the supplementation unit contains AA TG. In some embodiments, the supplementation unit contains at least one precursor of AA in TG form (precursor AA TG). In some embodiments, the supplementation unit contains AA TG and at least one precursor of AA in TG form (precursor AA TG). In some embodiments, one supplementation unit is administered per day. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 supplementation units are administered per day, providing a subject in need thereof with at least about 2 g of AA TG per day (2 g / day). In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 supplementation units are administered per day to provide a subject in need thereof with at least about 2 g of at least one precursor of AA per day (2 g / day). In some embodiments, the supplementation units comprise an oil comprising AA TG and a pharmaceutically acceptable excipient. In some embodiments, the supplementation units comprise an oil comprising at least one precursor AA TG and a pharmaceutically acceptable excipient.

[0201] 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 modified release of AA TG, at least one precursor AA TG, or both AA TG and at least one precursor AA TG. In some embodiments, the capsule allows for timed release of AA TG, at least one precursor AA TG, or both AA TG and at least one precursor AA TG. In some embodiments, one or more supplementation units are in a single container (e.g., a bottle, package, etc.) or multiple containers. In some embodiments, one supplementation unit is contained in a plastic pocket of a blister pack. In some embodiments, the blister pack is lined with a paperboard card. In some embodiments, the blister pack contains 10 plastic pockets, each containing one supplementation unit. In some embodiments, one blister pack contains enough supplementation units to provide a subject in need thereof with at least 2 g of AATG per day (2 g / day).

[0202] In some embodiments, precursors of AA and AA TG are commercially available to those skilled in the art. Non-limiting examples include AA from Cargill (cargill.com / food-bev / na / arachidonic-acid), ARASCO™ as an oil from DSM, and ARASCO® powder from DSM.

[0203] In some embodiments, the one or more supplement units are taken under the supervision of a physician and are intended for the dietary management of a condition of a subject in need thereof. In some embodiments, the one or more supplement units can be the sole source of nutrients for a subject in need thereof. In some embodiments, the one or more supplement units supplement or are intended to supplement the general diet of a subject in need thereof. In some embodiments, the supplement unit is in the form of syrup, liquid, powder, concentrated powder, concentrated powder mixed with liquid, swallowable form, dissolvable form, effervescent, granular form, or oral liquid solution.In some embodiments, the supplement unit is formulated in any convenient form.In some embodiments, the supplement unit is in the form of beverage, mayonnaise, salad dressing, margarine, low-fat spread, dairy product, cheese spread, processed cheese, dairy dessert, flavored milk, cream, cultured milk product, cheese, butter, condensed milk product, ice cream mix, soy product, pasteurized liquid egg, bakery product, confectionery product, confectionery bar, chocolate bar, high-fat bar, liquid emulsion, spray-dried powder, freeze-dried powder, ultra-high temperature (UHT) pudding, pasteurized pudding, gel, jelly, yogurt, or fat-based or water-containing filled food. In some embodiments, the supplement unit further comprises water, sucrose, maltodextrin, milk protein concentrate, soybean oil, canola oil, short chain fructooligosaccharides, soy protein isolate, corn syrup, sodium caseinate, and potassium citrate.

[0204] In some embodiments, the refill unit contains a flavoring, such as a natural or artificial flavor, hi 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.

[0205] In some embodiments, the supplementation unit comprises additional fat source(s), such as oil that does not contain significant amount of AATG. In some embodiments, the oil that does not contain significant amount of AATG is oil that does not contain more than 5% AATG per total volume of oil. In some embodiments, the fat source promotes energy metabolism. In some embodiments, the supplementation unit comprises a fat source that comprises one or more of saturated fatty acids, mono-unsaturated fatty acids, and poly-unsaturated fatty acids in a proportion that is found in a healthy diet of a subject.

[0206] In some embodiments, the supplementation unit contains about 3% or at least about 3% of oil containing about 40% AA TG. In some embodiments, the supplementation unit contains about 5% or at least about 5% of oil containing about 40% AA TG, about 10% or at least about 10% of oil containing about 40% AA TG, about 15% or at least about 15% of oil containing about 40% AA TG, or about 20% or at least about 20% of oil containing about 40% AA TG, or any range or combination thereof. In some embodiments, the percentage of AA oil is calculated as a weight / volume percentage. In some embodiments, the percentage of AA oil is calculated as a weight / weight percentage. In some embodiments, the percentage of AA oil is calculated as a volume / volume percentage.

[0207] In some embodiments, one or more supplementation units contain about 5 g or at least about 5 g of oil containing about 40% AA TG, hi some embodiments, the supplementation units contain about 10 g or at least about 10 g of oil containing about 40% AA TG, about 30 g or at least about 30 g of oil containing about 40% AA TG, about 40 g or at least about 40 g of oil containing about 40% AA TG, or about 50 g or at least about 50 g of oil containing about 40% AA TG.

[0208] In some embodiments, one supplement unit contains 50 mg of AA TG, 100 mg of AA TG, 200 mg of AA TG, 300 mg of AA TG, 400 mg of AA TG, 500 mg of AA TG, 1 g of AA TG, 2 g of AA TG, 4 g of AA TG, 5 g of AA TG, 10 g of AA TG, 15 g of AA TG, 20 g of AA TG, or any range or combination thereof. In some embodiments, one supplement unit contains 50 mg or less of AA TG, 100 mg or less of AA TG, 200 mg or less of AA TG, 300 mg or less of AA TG, 400 mg or less of AA TG, 500 mg or less of AA TG, 1 g or less of AA TG, 2 g or less of AA TG, 4 g or less of AA TG, 5 g or less of AA TG, 10 g or less of AA TG, 15 g or less of AA TG, 20 g or less of AA TG. In some embodiments, one supplement unit contains at least 50 mg of AA TG, at least 100 mg of AA TG, at least 200 mg of AA TG, at least 300 mg of AA TG, at least 400 mg of AA TG, at least 500 mg of AA TG, at least 1 g of AA TG, at least 2 g of AA TG, at least 4 g of AA TG, at least 5 g of AA TG, at least 10 g of AA TG, at least 15 g of AA TG, or at least 20 g of AA TG.

[0209] In some embodiments, one supplement unit contains 50 mg of at least one precursor AA TG, 100 mg of at least one precursor AA TG, 200 mg of at least one precursor AA TG, 300 mg of at least one precursor AA TG, 400 mg of at least one precursor AA TG, 500 mg of at least one precursor AA TG, 1 g of at least one precursor AA TG, 2 g of at least one precursor AA TG, 4 g of at least one precursor AA TG, 5 g of at least one precursor AA TG, 10 g of at least one precursor AA TG, 15 g of at least one precursor AA TG, 20 g of at least one precursor AA TG, or any range or combination thereof. In some embodiments, one supplement unit contains 50 mg or less of at least one precursor AA TG, 100 mg or less of at least one precursor AA TG, 200 mg or less of at least one precursor AA TG, 300 mg or less of at least one precursor AA TG, 400 mg or less of at least one precursor AA TG, 500 mg or less of at least one precursor AA TG, 1 g or less of at least one precursor AA TG, 2 g or less of at least one precursor AA TG, 4 g or less of at least one precursor AA TG, 5 g or less of at least one precursor AA TG, 10 g or less of at least one precursor AA TG, 15 g or less of at least one precursor AA TG, or 20 g or less of at least one precursor AA TG. In some embodiments, one supplement unit contains at least 50 mg of at least one precursor AA TG, 100 mg of at least one precursor AA TG, 200 mg of at least one precursor AA TG, 300 mg of at least one precursor AA TG, 400 mg of at least one precursor AA TG, 500 mg of at least one precursor AA TG, 1 g of at least one precursor AA TG, 2 g of at least one precursor AA TG, 4 g of at least one precursor AA TG, 5 g of at least one precursor AA TG, 10 g of at least one precursor AA TG, 15 g of at least one precursor AA TG, 20 g of at least one precursor AA TG.

[0210] example Example 1: Fatty acid (FA) screening in mouse and human organoids can reveal omega-6 fatty acids as promoters of stemness. This disclosure provides an explanation for how diverse dietary fatty acids (FAs) affect intestinal stem cell (ISC) function. Through FA screening in mouse and human intestinal organoids, this disclosure characterized a subset of omega-6 family FAs (including, but not limited to, arachidonic acid (AA)) with robust stemness-enhancing effects. Illustratively, cross-species gene expression analysis revealed that AA induces a conserved repair-related stem cell reprogramming signature. Furthermore, single-cell RNA sequencing (scRNA-seq) was used to identify the 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) induces the production of epithelial prostaglandin E2 (PGE2), which activates the Ptger4-cAMP-PKA signaling axis to promote stemness in mice and humans. AA (e.g., AA triglyceride) can induce epigenetic reprogramming around stem cell regeneration-related genes in a Ptger4-dependent manner. The data provided herein demonstrate that dietary AA (e.g., AA triglyceride) is a conserved promoter of stem cell regeneration that mimics the repair response to tissue injury through the PGE2-Ptger4 signaling pathway and downstream epigenetic reprogramming.

[0211] ISCs can undergo frequent, symmetric cell divisions to replenish the intestinal epithelium, one of the most regenerative tissues in mammals, consisting of a monolayer of cells with absorptive, secretory, and barrier functions (Barker et al., 2007; Cheng and Leblond, 1974; Leblond and Stevens, 1948; Snippert et al., 2010). Dividing ISC progeny give rise to transient outgrowth (TA) precursors that proliferate and differentiate into various lineages of the intestinal epithelium, including absorptive and secretory cells such as mucus-producing goblet cells, hormone-secreting enteroendocrine (EE) cells, chemosensory brush cells, and Paneth cells (Bankaitis et al., 2018; Clevers, 2013). ISC self-renewal and differentiation can be tightly controlled by niche-derived signals such as ligands, growth factors, and cytokines emanating 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 crypt (Clevers, 2013). Homeostatic regeneration of the intestinal epithelium can be sustained by ISCs expressing leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) both in vivo and in vitro in clonogenic organoid cultures (Barker et al., 2007; Sato et al., 2009). When the Lgr5+ stem cell compartment is damaged, plasticity and dedifferentiation of multiple epithelial cell lineages can enable effective 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 intestinal epithelial cell progenitors are some of the lineages known to dedifferentiate and acquire stem cell potential in response to intestinal injury due to irradiation, 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). While not wishing to be bound by theory, proposed mechanisms of stem cell reprogramming in response to tissue injury encompass the maintenance of accessible chromatin, induction of a fetal-like gene expression program, cytokine signaling systems, and Notch signaling systems ( Ayyaz 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 ).

[0212] Accumulating evidence suggests that nutrients and metabolic pathways not only affect growth and proliferation, but can also significantly influence cell function and fate by altering signaling pathways for transcription factors and 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). While recent studies have begun to explore the metabolic control 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), this disclosure provides insight into how nutrients and their metabolite derivatives affect ISC activity and cellular plasticity in the gut by inducing epigenetic changes. Various dietary interventions that perturb an organism's metabolism (e.g., fasting, calorie restriction, ketogenic diets, or obesogenic high-fat diets (HFDs)) converge in their ability to enhance ISC activity through cell-intrinsic ( Beyaz et al., 2016 ; Cheng et al., 2019 ; Fu et al., 2019 ; Mihaylova et al., 2018 ; Wang et al., 2018 ) and niche-mediated extrinsic ( Igarashi and Guarente, 2016 ; Yilmaz et al., 2012 ) mechanisms.One common feature of these stemness-enhancing dietary interventions is their ability to increase FA abundance and metabolism, either through dietary intake or release from adipose tissue (Novak et al., 2021). Fasting and a long-term fat-based obesogenic HFD can enhance ISC function, in part, through activation of FA metabolism (Beyaz et al., 2016; Beyaz et al., 2021b; Mihaylova et al., 2018).

[0213] There are several possible mechanisms by which FAs may control stem cell fate. First, FAs or their metabolites can bind to and activate FA-sensing transcription factors (TFs), such as 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, such as acetyl-CoA, can be utilized for histone modifications and affect epigenetic states (McDonnell et al., 2016; Schvartzman et al., 2018). Third, changes in cellular FA abundance can disrupt membrane lipid composition and affect signaling pathways (Zhu et al., 2019). Fourth, bioactive lipids generated from FAs can activate G protein-coupled receptors (GPCRs), which stimulate second messengers capable of transmitting signals to downstream cascades, as well as various TFs that control cell fate and function (Brash, 2001). Finally, FAs can disrupt the microbiome and immune cells, which affect ISC activity (Beyaz et al., 2021a; Biton et al., 2018). While these findings suggest that FAs and their metabolism may be linked to stem cell fate and function, it remains unclear how diverse dietary FAs affect stemness and epigenetic regulation of gene expression in the intestinal epithelium.

[0214] Intestinal organoids are capable of recapitulating the compositional and functional characteristics of the mammalian intestine, including stem cell renewal and differentiation in culture, and therefore represent a reliable system for identifying factors that promote stemness (Beyaz et al., 2016; Kaiko et al., 2016; Lukonin et al., 2020; Sato et al., 2009). To explore how diverse dietary FAs affect intestinal stemness, a live-imaging screening platform was developed to monitor organoid formation starting from single cells and measure informative traits regarding stem cell activity, including organoid morphology (spheroid vs. branched), size, and number, over a 5-day period (Beyaz et al., 2016; Farin et al., 2012; Mustata et al., 2013; Schuijers et al., 2015) (Figure 8A, see Example 9). A panel of 23 FAs was assembled and stratified by degree of saturation (polyunsaturated, monounsaturated, and saturated), double bond position (omega-3, omega-6, omega-7, omega-9), configuration (cis, trans), chain length (short, medium, long), and number of double bonds (1–6) ( Figure 1 A, Table 1 ).

[0215] Table 1: Fatty acids used for screening mouse and human organoids. [Table 1]

[0216] Because most FAs in the body 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 FA concentrations that did not induce lipotoxicity in organoids (Alsabeeh et al., 2018; Brash, 2001) (Figure 8B). FA screening in single cells derived from mouse intestinal organoids revealed that treatment with omega-6 FAs, such as linoleic acid (LA), γ-linolenic acid (γ-LA), dihomo-γ-linolenic acid (dh-γ-LA), and arachidonic acid (AA), but not docosatetraonic acid (DA) or the trans-FA linoelaidic acid (LEA), promoted the formation of spheroids lacking adult crypt-like domains (Figures 1B and 8C). This spheroid morphology may correlate with enhanced regenerative stem cell status 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 led to a significant increase in size compared to vehicle-treated control organoids, beginning at approximately 72 hours, the approximate time period for symmetry breaking in single-cell-derived organoids (Serra et al., 2019) (Figures 1C and 8C). To assess the human relevance of these results, FA screening was performed on human patient-derived organoids (PDOs) generated from normal portions of the colon and obtained from male or female patients across diverse origins (Table 2).

[0217] Table 2: Patient information for patient-derived organoids used in the study. [Table 2]

[0218] Similar to the mouse screen, treatment of single cells isolated from human PDO with a subset of omega-6 family FAs, including dh-γ-LA and AA, promoted growth compared to control organoids (Figures 1D and 8D). FA elongase (Elovl5) and desaturases (Fads1 and Fads2), which control AA biosynthesis from essential FAs (Fan et al., 2012; Moon et al., 2009), were highly expressed in both mouse and human organoids (Figures 8E-8F). Given that DA did not promote organoid growth, we hypothesized that the stemness-enhancing effects of omega-6 FAs ​​might converge on AA. Indeed, inhibiting FADS1, the rate-limiting desaturase for AA biosynthesis (Fan et al., 2012), blunted the increase in organoid size in response to omega-6 FAs ​​(Figure 8G). For these reasons, AA was selected as a target for further functional assessment of stem cell regeneration. We found that AA treatment led to more proliferative cells in spheroids lacking cryptdomains and larger in size compared to vehicle-treated control organoids (Figures 1E-1J). Transmission electron microscopy analysis showed that these AA-induced spheroids had smaller microvilli and fewer granular Paneth cells, suggesting reduced differentiation (Crawley et al., 2014; Miyoshi et al., 2017) (Figures 8H-8I). Furthermore, subculture experiments were performed to functionally assess stem cell activity after AA treatment (Beyaz et al., 2016). When subcultured, primary mouse AA-induced spheroids generated more secondary organoids, which again retained spheroid morphology but were larger in size compared to controls (Figures 1K-1N). Similarly, AA treatment increased the size of human intestinal PDO organoids and promoted spheroid formation in both primary and secondary cultures (Figures 1O-1T). These results demonstrate that omega-6 FAs, converging with AA, enhance the stemness of mouse and human organoids.

[0219] Example 2: A diet rich in AA (ARD) can promote intestinal regeneration in vivo. AA are bioactive lipids that can play essential 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 has been explored in Calder et al. (2019). To study the effects of AA on ISC function in vivo, a novel isocaloric (3.8 kcal / g) AA-enriched diet (ARD) model (TekLAd, TD190641) was developed along with a corresponding purified control (control) diet (TekLAd, TD97184) (Figure 2A). Oil extracted from M. alpina fungus was used, containing approximately 40% AA in the form of triglycerides, which was necessary to formulate a diet containing 3% AA-rich oil and 4% soybean oil (7% total fat) (Kikukawa et al., 2018). The ARD and its corresponding isocaloric control were composed of equal amounts of macronutrients (protein, carbohydrates, and fat) and minor nutrients (minerals and vitamins) (Table 3).

[0220] Table 3: Composition of isocaloric AA-rich diets and their corresponding purified control diets. [Table 3]

[0221] Feeding mice a 4-week ARD diet did not affect body weight or plasma glucose levels, but did increase AA levels in both plasma and intestine compared with controls (Figures 2B-2C and 9A-9B). Furthermore, metabolomic analysis revealed no significant changes in the levels of other major metabolites in the intestine of ARD-fed mice (Figure 9C). Increased AA levels in the intestine resulted in increased crypt length and the number of Ki67+ proliferative cells per crypt (Figures 2D-2G). Intestinal crypts were isolated from mice fed the ARD or control diet, and functional organoid assays were performed. We assessed the morphology and organoid formation capacity of intestinal organoids to establish proxies for stem cell activity (Beumer and Clevers, 2016; Beyaz et 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 decrease in crypt domains per organoid compared to controls (Figures 2H-2J). Epcam+ intestinal epithelial cells sorted from crypts of ARD-fed mice gave rise to more organoids than controls, suggesting that dietary AA promotes stem cell activity under homeostatic conditions in vivo (Figures 2K-2L).

[0222] The intestinal epithelium exhibits rapid regenerative responses to numerous stressors, maintaining 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 clinically relevant 15 Gy gamma irradiation to control mice caused cytotoxicity in the intestinal epithelium, leading to crypt loss and reduced intestinal length (Beyaz et al., 2016; Kirsch et al., 2010) (Figures 2M-2O and 9D). Feeding mice an ARD reversed these effects and enhanced crypt regeneration, as evidenced by an increase in proliferative cells incorporating 5-ethynyl-2'-deoxyuridine (EdU) (Salic and Mitchison, 2008) per unit area of ​​intestine and per crypt compared with controls (Figures 2P-2R and 9E). Because cancer chemotherapy induces similar cytotoxic effects, it was unclear whether ARDs could promote intestinal regeneration in a separate injury model using doxorubicin, a commonly used antitumor drug with well-characterized intestinal toxicity (Dekaney et al., 2009; Ijiri and Potten, 1987). Doxorubicin resulted in a significant decrease in intestinal length 72 hours after treatment in mice receiving a control diet. ARDs prevented intestinal shortening and increased the number of EdU+ proliferative crypt cells in doxorubicin-treated mice (Figures 2S and 9F-9H). Consistent with the assessment of proliferation using Ki67 ( Figure 2F ), crypts contained more EdU+ proliferative cells in uninjured ARD-fed mice compared with uninjured controls in both models ( Figures 2Q and 9G ). Collectively, the data indicate that dietary increases in AA in the intestine can enhance ISC regeneration in vivo.

[0223] Example 3: AA can induce a preserved stem cell reprogramming gene expression signatureIntestinal organoids can recapitulate the 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, we performed time-kinetic bulk RNA-seq analysis across various developmental stages of mouse organoids, including symmetric cyst formation (day 1), symmetry breaking (day 3), and mature organoids with differentiated cells (day 6) (Beyaz et al., 2016; Sato et al., 2009; Serra et al., 2019) (Figures 10A-10B). Across time points, AA treatment led to a strong upregulation of genes associated with stem cell reprogramming in response to Lgr5+ stem cell loss (Lypd6, Ctgf, Anxa13) (Murata et al., 2020), radiation injury (Ccnd1, Anxa3, Ly6a, Clu) (Ayyaz et al., 2019), and granuloma (Il33, S100a6, Areg) (Nusse et al., 2018), or regenerative fetal-like states (fetal spheroids) (Cd55, Ereg, Myof, Msln) (Mustata et al., 2013) (Figures 3A-3B). In contrast, organoids downregulated markers of differentiated cells, such as Paneth cells (Defa24, Defa21, Lyz1), Tuft cells (Dclk1), and enteroendocrine cells (Neurog3) in response to AA (Haber et al., 2017) (Figure 3B). Interestingly, Lgr5 expression and homeostatic stem cell signatures (Haber et al., 2017; Munoz et al., 2012) were suppressed in AA-treated organoids on days 1 and 3 but recovered by day 6 (Figures 3A-3B). All previously reported reprogrammed stem cell signatures significantly overlapped with AA-induced genes, suggesting that AA may induce gene expression signatures that may be central to stem cell regeneration across various experimental models (Figures 10C-10D).An AA-induced signature gene list was devised, encompassing 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 ).

[0224] Gene set enrichment analysis (GSEA) of AA-induced genes revealed enrichment in wound healing, cell proliferation, the PPAR pathway, and lipid metabolism, as well as 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, this study assessed 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)) was upregulated in AA-treated organoids (Figure S10G), accompanied by increased nuclear localization of β-catenin (Molenaar et al., 1996) (Figure 3C), which may serve as a proxy for its activity. Titration of exogenous Wnt3a (Beyaz et al., 2016) demonstrated that AA treatment reduced Wnt dependency on organoid formation and growth (Figures S3D-S3F). Second, AA enhances the expression of the Egf family receptor Egfr, one of the stem cell reprogramming signature genes (Mustata et al., 2013; Nusse et al., 2018), and its ligands, such as Areg and Ereg, which are primarily produced by stromal cells and paracrinely support epithelial repair after injury (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) (Figures 3B and 3G). This prompted us to examine whether AA-induced autocrine Egfr ligand expression contributes to the enhanced stemness.In this disclosure, we found that replacing Egf, an essential component of organoid culture medium (Basak et al., 2017; Oszvald et al., 2020; Sato et al., 2009), with Areg or Ereg can be sufficient to establish organoids from dissociated single cells (Figure 10H-10I). Treatment with AA significantly boosted organoid growth even in the absence of Egf, which is necessary for stem cell proliferation (Basak et al., 2017; Biteau and Jasper, 2011; Jiang and Edgar, 2009) (Figure 3H-3I). These findings suggest that AA-induced transcriptional reprogramming can reduce dependence on niche-derived exogenous Wnt and Egf signals and promote stemness.

[0225] To confirm these human observations, we used human PDO to assess gene expression changes in response to AA (Figure S10J). GSEA demonstrated that AA-induced genes were enriched in both homeostasis- and repair-related stemness signatures, as well as the Myc pathway and cell proliferation (Figures 3J and S10K). Similar to mice, genes associated with stem cell reprogramming (CD55, MYOF, MSLN, ANXA3, AREG, CCND1, LYPD6) (Ayyaz et al., 2019; Murata et al., 2020; Mustata 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) (Rodon et al., 2019) were robustly upregulated in AA-treated human PDO (Figures 3K-3L). Collectively, these results highlight that AA can induce a conserved stem cell regeneration program that is partially reminiscent of the repair response to injury.

[0226] Example 4: In vivo and in vitro single-cell analysis of AA-induced stemness To determine the precise cellular states that define AA-induced stemness in vivo, we performed single-cell RNA sequencing (scRNA-seq). 23,161 single cells from the crypts were filtered, clustered, and then profiled to define intestinal epithelial cell types (Ayyaz et al., 2019; Grun et al., 2015; Haber et al., 2017) (Figures 11A-11C). Dietary AA resulted in a de novo stem-like cluster (Stem2) in vivo that was absent in control crypts and was characterized by high expression of Ly6a and S100a6, markers associated with stem cell reprogramming (Ayyaz et al., 2019; Mustata et al., 2013; Nusse et al., 2018) (Figures 4A-4B and 11A-11F), but not 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 shared among fetal, radiation-induced, and granuloma-induced repair signatures (Figure 10C). Additionally, ARD led to upregulation of the stem cell marker genes Lgr5 and Aslc2 in transit-amplifying (TA) cells, intestinal epithelial progenitor cells (EP), enteroendocrine cells (EE), and goblet cells, but not in homeostatic stem cells (Stem1) in vivo (Figures 4C-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 upon injury through dedifferentiation of intestinal epithelial cells and secretory precursors ( Murata et al., 2020 ).Consistent with this, using a pseudotime trajectory analysis algorithm (Cao et al., 2019), we found a dedifferentiation trajectory through the TA, EP, and EE toward the AA-induced stem2 cluster in vivo (Figures 4E-4F). Crypt cells from ARD mice exhibited elevated expression of Lgr5, Ascl2, and the reprogramming-related markers Ly6a and S100a6 during the differentiation pseudotime trajectory compared with controls (Figures 4G and 11E). Upregulation of Ascl2 in upper crypt cells was a hallmark of intestinal regeneration in response to crypt injury prior to dedifferentiation into stem cells (Murata et al., 2020). To assess how dietary AA affects the spatial expression of these key stemness genes, we performed single-molecule fluorescence in situ hybridization (sm-FISH). ARD-fed mice expressed higher levels of Lgr5 and Ascl2 per crypt and increased the frequency of cells expressing these markers in the upper crypt layer at steady state (Figures 4H-4M and 11G-11H). Consistent with the observation that ARD mice could exhibit enhanced in vivo intestinal regeneration after irradiation compared with controls (Figures 3A-3L), we found that dietary AA could increase the expression of Lgr5 and Ascl2 in regenerating crypts in response to irradiation. Regenerating crypts from irradiated ARD mice contained a higher percentage of cells expressing Lgr5 and Ascl2 in the upper crypt layer compared with irradiated controls (Figures 4H-4M and 11G-11H). In addition, dietary AA boosted the expression of repair-related stem cell signature genes, such as S100a6, in crypt cells both at steady state and in response to irradiation (Figures 4N-4P and 11I). Collectively, these results suggested that dietary AA could promote intestinal stemness by inducing a regeneration-associated de novo stem cell state and dedifferentiation program in vivo.

[0227] To assess whether dietary AA induces stem cell reprogramming through an epithelial-specific mechanism, we profiled 23,599 single cells from organoids by scRNA-seq (Figures 12A-12C). In organoid cultures, 3-day AA treatment led to the emergence of a de novo stem-like state (Stem2 and Stem3) marked by stem cell reprogramming-associated signature genes, such as Ly6a, Clu, and S100a6 (Figures 12A-12G and 12K). Furthermore, pseudo-temporal analysis of scRNA-seq from organoids revealed a trajectory of dedifferentiation toward a de novo stem-like state and upregulation of Ly6a and S100a6 over the pseudo-temporal trajectory (Figures 12L-12R). AA-treated organoids showed increased expression of Ascl2 compared to controls in stem / progenitor cells, intestinal epithelial cells, and EE cells (Figure 12H), and throughout the pseudo-time trajectory (Figure 12S). AA-treated organoids showed increased expression of Ascl2 compared to controls in stem / progenitor cells, intestinal epithelial cells, and EE cells (Figure 12H), and throughout the pseudo-time trajectory (Figure 12S). Consistent with bulk RNA-seq data for day 3 organoids (Figure 3B), likely due to the 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), there were several distinct features between crypts and organoids, including reduced expression of Lgr5 in stem and progenitor cell clusters and across the pseudo-time trajectory (Figures 12I and 2T). In contrast to a recent report (Wang et al., 2020), induction of Msi1 in response to AA was not observed either in vivo or in vitro (Figures S11D and S12J). Overall, these results suggest that AA-treated organoids generally exhibit a profile similar to that of crypts from ARD mice, suggesting an epithelial-intrinsic mechanism as a likely driver of the AA-induced stemness phenotype.

[0228] Example 5: Metabolism of AA to prostaglandin E2 (PGE2) may be necessary and sufficient to promote stem cell reprogramming AA exerts its biological activity through several mechanisms, including modulation of membrane fluidity, ion channels, reactive oxygen species levels, and lipid-sensing receptors (e.g., PPARs), as well as the generation of numerous bioactive lipids through non-enzymatic and enzymatic degradation, particularly in response to tissue injury (Brash, 2001). Our data demonstrated that dietary AA can promote stemness and induce repair-associated stem cell reprogramming signatures, even in the absence of obvious damage to the intestinal epithelium (Figures 2A-4P). Because AA and AA-derived metabolites may be conserved regulators of wound detection and tissue repair (Fan et al., 2014; Katikaneni et al., 2020; Miyoshi et al., 2017), this study aimed to assess changes in AA metabolism in our model. Metabolomic analysis revealed that AA treatment can lead to increased production of bioactive lipid mediators in organoids in vitro (Figure 5A) and crypts in vivo (Figure 13B). To determine whether AA-derived metabolites might be sufficient to promote stem cell activity, we performed a screen of mouse organoids and found that prostaglandin E2 (PGE2), and to a lesser extent, PGD2, recapitulated AA-induced stemness (Figures 5B and 13C). Inhibiting prostaglandin production using the nonsteroidal anti-inflammatory drugs (NSAIDs) celecoxib (Figures 5C-5D) or indomethacin (Figures 13D-13E) blunted AA-induced stemness in organoid assays, suggesting that epithelial PGE2 production may be required for the stem cell-enhancing effect of AA.

[0229] 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 dietary modulation of the PGE2 signaling pathway in epithelial cells modulates stem cell function. In addition to AA-treated organoids, this study confirmed PGE2 production by Epcam+ crypt cells sorted in response to AA (Figure 13F). Furthermore, AA treatment resulted in adaptive upregulation of enzymes controlling prostaglandin production in organoids, such as Ptges and Ptgs2 (Figures 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, highlighting a potential epithelial source of PGE2 in response to AA (Figure 13H). PGE2 treatment of organoids induced a gene expression program consistent with the AA-induced stem cell reprogramming signature in both mouse (Figure 5E) and human (Figure 5F) organoids. Furthermore, scRNA-seq of mouse organoids in response to PGE2 treatment revealed similar stem cell reprogramming features to those observed in AA-treated organoids, including the emergence of a de novo stem-like state marked by reprogramming-related genes (Figures 5G-5I and 13I-13L) and upregulation of Ascl2 in stem / progenitor cells (Figure 5J). Similar to AA, PGE2 induced a dedifferentiation trajectory in organoids accompanied by upregulation of the reprogramming-related genes Ascl2, S100a6, and Ly6a, but not Lgr5 (Figures 5K-5M and 13M-13N). These data indicated that the stemness-enhancing effect of dietary AA can be mediated by PGE2 signaling in both mice and humans.

[0230] Example 6: The Ptger4-cAMP-PKA signaling axis can regulate AA-induced stemness in mice and humans PGE2 binds to four G protein-coupled receptors (Ptger1-4) and activates diverse downstream pathways to mediate various functions (Breyer et al., 2001; Narumiya et al., 1999). To determine which PGE2 receptor subtype is required for AA-induced stemness, pharmacological inhibitors for each receptor were screened in organoid assays. We found that inhibition of Ptger4, but not other PGE2 receptors, attenuated AA-induced stemness (Figures 14A-14E). Ptger4 was highly expressed in both mouse and human organoids (Figures 14G-14H). Treatment with AA boosted Ptger4 (but not the expression of other PGE2 receptors in secretory progenitor cells), TA cells, and de novo stem cells (Stem2 and Stem3) (Figures 14I-14L). Furthermore, Ptger4 knockout (Ptger4 KO) organoids were generated, demonstrating the requirement for PGE2-Ptger4 signaling in regulating the stemness-enhancing effect of AA (Figures 6A-6C). Inhibition of Ptger4 signaling blunted the upregulation of signature genes associated with stem cell reprogramming in response to AA or PGE2 (Figures 6D and 14F).

[0231] Ptger4-mediated PGE2 signaling pathways contribute to diverse downstream pathways, including activation of adenylyl cyclase, phosphatidylinositol 3-kinase (PI3K), β-arrestin, β-catenin, and extracellular signal-regulated kinase (ERK), leading to increased cAMP production (Yokoyama et al., 2013). Using a membrane-permeable and stable cAMP derivative (8-bromo) (Tuesta et al., 2017) in organoid assays, we found that elevated cAMP levels were sufficient to recapitulate the effects of AA on organoid growth and enhance the expression of AA signature genes (Figures 6E-6H). Increased cAMP levels activated downstream effector molecules, such as protein kinase A (PKA), cAMP-activated exchange protein (Epac), and cyclic nucleotide-gated ion channels (Sassone-Corsi, 2012). Inhibiting PKA using a potent antagonist (H89) (Chijiwa et al., 1990) blunted the effects of AA on organoid growth and AA-induced signature gene expression (Figures 6I-6L). Ptger4 is highly conserved between mice and humans (Narumiya et al., 1999). Using human PDO, we assessed whether the Ptger4-cAMP-PKA signaling axis regulated AA-induced stemness in the human intestine. Similar to the observations in mouse organoids, we found that Ptger4 and PKA may be required for the stemness-enhancing effect of AA (Figures 6M-6P) and for increasing cAMP levels to promote stem cell activity in human PDO (Figures 6Q-6R).

[0232] 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 loss of Ptger4 in green fluorescent protein (GFP)-tagged Lgr5 stem cells and their progeny within intestinal crypts. We found that ARD failed to increase the number of EdU+ cells in Ptger4-deficient crypts both at steady state and after irradiation, highlighting the requirement for Ptger4 signaling in mediating ARD-induced stem cell regeneration in vivo (Figures 6S-6T). Collectively, these data demonstrate that PGE2 signaling via the Ptger4-cAMP-PKA axis is a conserved mechanism in mice and humans through which dietary AA promotes intestinal stem cell reprogramming and regeneration.

[0233] Example 7: AA induced epigenetic reprogramming around regeneration-related loci in a Ptger4-dependent manner A permissive chromatin state with limited differences in epigenetic proxies, such as chromatin accessibility, histones, and DNA modifications, between adult stem cells and differentiated cells in the intestinal epithelium has been observed (Jadhav et al., 2016; Kaaij et al., 2013; Kazakevych et al., 2017; Kim et al., 2014; Sheaffer et al., 2014). Stem cell plasticity and dedifferentiation in response to intestinal injury are partially attributed to this low chromatin barrier between differentiated and stem cell states, but little is known about how dietary and metabolic signals that influence stem cell renewal affect the intestinal epithelial epigenome (Verzi and Shivdasani, 2020). To determine whether AA-induced stemness involves epigenetic reprogramming, the present disclosure performed an assay for transposase-accessible chromatin using accessible chromatin region capture sequencing (ATAC-seq) in vehicle- or AA-treated organoids (Buenrostro et al., 2013). Differential analysis of accessible chromatin regions between AA-treated vs. vehicle-treated organoids revealed that AA promoted, rather than inhibited, chromatin accessibility around promoters, enhancers, and intergenic regions (5807 start peaks, 2132 end peaks, q<0.01, absolute log2 fold change>0.58) (Figure 15A). AA-induced reprogramming of chromatin accessibility around promoters may lead to concomitant upregulation of nearby genes along with the start ATAC-seq peak (Figure 15B). Pathway enrichment analysis of regions with increased accessibility in response to AA highlighted stemness-associated signatures encompassing telomerase activity ( Hoffmeyer et al., 2012 ; Montgomery et al., 2011 ; Schepers et al., 2011 ) and activity of acetyltransferase complexes ( Sampurno et al., 2013 ; Yin et al., 2014 ).Certain pathways are crucial 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), and stem cell regulators such as β-catenin (Beumer and Clevers, 2016), MYB (Cheasley et al., 2011), CREB and its targets (ID1 and ID2) and partners (ATF and JUN) (Nigmatullina et al., 2017; Sampurno et al., 2013; Zhang et al., 2014), all of which were consistent with the gene expression and functional data on the stemness-enhancing effects of AA (Figure 7A). On the other hand, regions that lost accessibility due to AA treatment were enriched for signatures of enteroendocrine cells, extracellular matrix, negative regulation of wound healing, and PRC2 targets (Figure 7A). To identify TFs with regulatory potential in AA-induced open or closed chromatin regions, we constructed a multivariate linear model of the peaks shifting 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 NEUROD1, an enteroendocrine cell marker (Li et al., 2019), were enriched in closed regions in AA-treated organoids (Figure 7B).NFE2L1 (NRF1) (Schell et al., 2017), AP1 family (FOS, JDP2, JUNB) (Haber et al., 2017; Nateri et al., 2005), PPAR (Beyaz et al., 2016; Beyaz et al., 2021b), 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., Enriched TF motifs associated with stemness and regeneration, such as ATPase inhibitors (TAIs), were identified in regions with increased chromatin accessibility in response to AA (Figure 7B). Among these factors, YAP and CREB are important and conserved regulators of regeneration through partnerships with numerous stem cell factors, including the AP1 family and β-catenin (Goessling et al., 2009; Shaywitz and Greenberg, 1999; Zanconato et al., 2015) (Deng et al., 2015; Gregorieff et al., 2015; Li and Fan, 2017; Nusse et al., 2018; Sampurno et al., 2013; Yui et al., 2018). Furthermore, the data demonstrated that the AA-induced stemness phenotype is mediated by the Ptger4-PKA signaling system (Figures 6A-6T), which can activate and promote the nuclear localization of CREB1 (Sassone-Corsi, 2012; Yokoyama et al., 2013).AA treatment could lead to enhanced CREB1 activity, as assessed by increased nuclear localization and phosphorylation (Figure 7C), and upregulation of bona fide CREB1 target genes, such as Nr4a1 and Id2, which are associated with tissue repair and stemness (Nigmatullina et al., 2017; Wu et al., 2016) (Figures S10E and S10G). Similarly, AA boosted the nuclear localization of YAP and target gene expression, such as Ly6a (Figure 7C). Finally, the regions with the most significant increase in chromatin accessibility in response to AA harbored nearby genes that are known targets of CREB1 and YAP, as well as some of the AA-induced signatures, such as those involved in stem cell reprogramming (S100a6, Ly6a, Msln, Anxa10, Il33, Ccnd1, Ascl2), proliferation (Myc, Max, Mki67, Ccnd1), the EGFR pathway (Areg, Egfros), the Wnt / β-catenin pathway (Ascl2, Id2, Wnt4, Wnt7b, Jag1, Asap1, PLAur, Cd44), and the PGE2 signaling system (Ptgs2, Ptger4) (Ayyaz et al., 2019; Murata et al., 2020; Mustata et al., 2013; Nusse et al., 2018) (Figure 7D). Altogether, these data indicate that AA can reprogram chromatin accessibility around regeneration-associated loci, in part through activation of CREB1 and YAP.

[0234] Covalent histone modifications are associated with chromatin activity and transcriptional outcomes (Berger et al., 2009). To further define AA-induced changes in the epigenetic landscape, we performed a "Cleavage Under Targets and Release Using Nuclease" (Cut&Run) assay (Meers et al., 2019) in organoids to assess the genome-wide distribution of histone modifications correlated 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 significantly increased activation-associated H3K4me3 and H3K27ac marks around promoters and proximal putative enhancers (Figures 15D-15E). In contrast, genes downregulated by AA lost these activation marks and were associated with elevated levels of repressive H3K27me3 (Figure 14F). Genes that are part of regenerative fetal spheroids (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 that gained abundant H3K27ac upon AA encompass stem cell reprogramming signature genes, such as S100a6, while enhancers that lost H3K27ac upon AA encompass differentiation genes, such as Defa17 (Figure 7F). Integrating gene expression data into chromatin states further corroborated these observations, revealing that AA-induced upregulated genes with the most significant increases in H3K4me3 or H3K27ac abundance may be stem cell reprogramming genes ( Figures 7G and 15G-15H ).As shown in the representative genome tracks, signature genes such as S100a6, Msln, Anxa10, Ly6a, and Ascl2 accumulated active histone marks concomitant with increased chromatin accessibility around their promoters or enhancers in response to AA ( Figures 7H and 15I ).

[0235] Because AA-induced upregulation of stem cell reprogramming signature genes is mediated through the PGE2-Ptger4 signaling pathway, the necessity of the PGE2-Ptger4 signaling pathway in the epigenetic regulation of AA-induced stem cell reprogramming was evaluated. Cut&Run analysis for H3K27ac was performed using Ptger4 KO organoids. The data suggested that Ptger4 may be required for AA-induced accumulation of H3K27ac around upregulated genes and repair-related stem cell regeneration gene signatures (Figures 7I and 15J-15K). Collectively, these results highlighted an epigenetic basis for AA-induced stem cell reprogramming that may depend on the PGE2-Ptger4 signaling pathway.

[0236] Example 8: Discussion 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 3–5 days (Clevers, 2013). However, exposure to genotoxic stressors, such as radiation and chemotherapy, is associated with degeneration of the intestinal epithelium, creating an unmet need for the development of regenerative therapies. Dietary intake of nutrients, such as FAs, influences ISC activity (Beyaz et al., 2016; Beyaz et al., 2021b), but how specific FAs affect intestinal regeneration is not fully understood. Several clinical and epidemiological studies suggest that increasing total polyunsaturated fatty acids (PUFAs), including omega-6 fatty acids, increases cancer risk. However, conclusive evidence regarding the impact of omega-6 on cancer outcomes is currently lacking. PUFAs, including omega-6 fatty acids such as arachidonic acid, are important structural components of cell membranes, which rapidly proliferating cells require for their growth. Additionally, upon tissue injury, omega-6 fatty acids are released from cell membranes, generating inflammatory bioactive lipid mediators such as prostaglandins, which are implicated in 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).

[0237] This study uses an FA screening approach in ex vivo mouse and human organoid cultures, combined with in vivo studies utilizing isocaloric diets differing in AA abundance, to discover conserved mechanisms of nutrient-gene interactions regulating stemness in the intestinal epithelium. The screening platform with mouse or human organoids identified regenerative dietary nutrients with beneficial (e.g., therapeutic) implications. Using this platform, a mechanistic causal relationship between dietary AA and intestinal stem cell regeneration was revealed through epigenetic reprogramming, which has broad implications for mitigating intestinal degeneration. Damage to the intestinal mucosa is one of the most common debilitating side effects of cancer treatments, such as radiation therapy and chemotherapy, leading to reduced quality of life and decreased survival in cancer patients (Kim et al., 2017; Sougiannis et al., 2021). This disclosure has shown that a 4-week regenerative dietary AA regimen (3% AA in triglyceride form) prevented intestinal damage in mice in response to clinically relevant 15 Gy abdominal irradiation and treatment with doxorubicin, a widely used chemotherapy agent that causes intestinal mucositis in patients (Kim et al., 2017; Sougiannis et al., 2021). Based on the findings, prospective clinical studies may be conducted to demonstrate that increasing AA abundance through dietary intervention can improve intestinal regeneration and ameliorate gastrointestinal side effects in cancer patients undergoing radiation therapy or chemotherapy.

[0238] Consistent with data on the regenerative effects of dietary AA, mice lacking Fads1, the rate-limiting desaturase in AA biosynthesis, exhibit overall poor survival and impaired proliferation in the intestinal epithelium unless supplemented with exogenous AA (Fan et al., 2016; Fan et al., 2012). Although AA are abundant in breast milk and considered essential for infant growth and development, global estimates of dietary lipid intake indicate that humans ingest AA primarily through desaturated dietary LA (Calder et al., 2019; Fan et al., 2012). Numerous 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 studies are needed to elucidate the significance of dietary FA in human physiological and pathological conditions, taking into account human genetic variation in AA utilization and metabolic genes.

[0239] It is becoming increasingly clear that epithelial cell plasticity, rather than a reserve stem cell pool, drives regeneration for intestinal injury recovery (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). This finding represents a new paradigm linking specific dietary nutrients to stemness reprogramming in the adult intestinal epithelium. Previous studies using various experimental injury models have captured distinct and diverse molecular signatures of epithelial cell plasticity in response to ISC injury or loss (Ayyaz et al., 2019; Murata et al., 2020; Mustata et al., 2013; Nusse et al., 2018). Strikingly, in the absence of tissue injury, dietary AA mimicked a conserved repair response program through the epithelial PGE2-Ptger4 signaling system, which shares features of all previously defined stem cell reprogramming signatures, including targeting of Wnt / β-catenin and Egfr ligands. AA upregulated niche-derived signals in epithelial cells and reduced reliance on Wnt and Egfr in organoid cultures, all of which suggested a critical role for dietary nutrients in influencing niche-mediated stemness regulation in the intestine. Similarly, dietary AA-induced activation of the PGE2-Ptger4 signaling system in the intestinal epithelium was critically involved in regeneration and epithelial plasticity. The paracrine PGE2 signaling system is known to promote tissue repair after injury, and PGE2 analogs have previously been characterized to have radioprotective effects in the intestine (Hanson and Ainsworth, 1985; Miyoshi et al., 2017).Pharmacological interventions that activate the PGE2-Ptger4-cAMP signaling pathway 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 determine whether dietary AA synergizes with these therapeutic interventions to elicit the plasticity of regenerative stem cells in the intestine. Furthermore, lipid peroxidation and AA release in wounds regulated injury detection and tissue repair processes (Katikaneni et al., 2020). Consistent with these observations, the aforementioned findings demonstrate that dietary AA-induced adaptive PGE2-Ptger4 signaling pathway generates epithelial cell plasticity, boosts regeneration, and protects the intestine from injury. Thus, the regenerative effect of dietary AA through the PGE2-Ptger4 axis highlights the importance of dietary factors in influencing stem cell reprogramming and represents a new, robust physiological model to study epithelial cell plasticity in the intestine ( de Sousa and de Sauvage, 2019 ).

[0240] Downstream of Ptger4, the cAMP-PKA signaling pathway mediated the stemness-enhancing effects of dietary AA. The cAMP-PKA signaling pathway played a central role in tissue repair and inflammation resolution. Elevated cAMP signaling in tissues can promote regeneration and prevent or reverse scarring after injury, although the cell type-specific mechanisms are not fully understood (Insel et al., 2012).

[0241] Little is known about the effects of dietary and metabolic perturbations on the intestinal epigenome (Verzi and Shivdasani, 2020). This study demonstrates that the stemness-enhancing effect of the AA-Ptger4-cAMP-PKA axis in intestinal epithelial cells is likely mediated by the activity of several TFs activated downstream of PKA, which coordinately regulate the establishment of a regenerative epigenetic program. Indeed, data revealed significant AA-induced epigenetic reprogramming around regeneration-related loci, including targets of CREB1 and YAP, in a Ptger4-dependent manner. Epithelial stem cells have been shown to retain functional traits from past exposure (Beyaz et al., 2016; Naik et al., 2017; Ordovas-Montanes et al., 2020). An important implication of this epigenetic reprogramming is that dietary AA can trigger a regenerative memory in the intestinal epithelium, protecting the tissue from subsequent damage.

[0242] This disclosure explores epithelial-specific mechanisms that regulate the stemness-enhancing effects of AA. Rapid PGE2 production promotes tissue repair in response to injury, while chronic inflammation and dysregulation of the PGE2 signaling pathway promote tumor development (Wang and DuBois, 2018). The short-term regenerative dietary AA regimen used in this study resulted in increased PGE2 levels in the intestine without any apparent safety concerns in mice. However, further research is needed to elucidate the kinetics of AA-induced increases in stemness and precisely separate the regenerative effects from potential risks of carcinogenesis initiation. While the data demonstrated robust AA-induced epigenetic reprogramming around regeneration-related loci, the utilized epigenetic approach only provided average population-based analysis and did not preserve single-cell information.

[0243] Example 9: Reagents and Methods Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0244] Animals, diets, and drug treatments Mice were bred at Cold Spring Harbor Laboratory. The following strains were obtained from The Jackson Laboratory: Ptger4 f / f (strain: 6.129S6(D2)-Ptger4tm1.1Matb / BreyJ, stock number: 028102) and Lgr5-EGFP-IRES-CreERT2 (strain: B6.129P2-Lgr5tm1(cre / ERT2)Cle / J, stock number: 008875). Animals were housed in a pathogen-free environment and maintained on a 12-h light / dark cycle. The ARD was developed by formulating a diet containing 3% AA-rich oil and 4% soybean oil (total fat 7%) (Cat# TD.190641, Envigo) for 4 weeks, starting from 8–12 weeks of age, using oil extracted from the fungus Mortierella alpina (Arasco Oil, DSM, 5015002S02), which contains approximately 40% AA in the form of triglycerides (Table 3). Control mice were age- and sex-matched and received an isocaloric control diet containing equivalent amounts of macronutrients and minor nutrients (Cat# TD.97184, Envigo). Food and water were provided ad libitum. The allele was crossed with Lgr5-EGFP-IRES-CreERT2 (to generate stem cell-specific knockout, Lgr5-iKO) mice and then treated with tamoxifen suspended in corn oil (Cat# C8267, Sigma) at a concentration of 20 mg / ml and 100 μl per 25 g of body weight, administered intraperitoneally five times every other day. 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).

[0245] Organoids were treated with the following compounds: dmPGE2 (5 nM, Cat# 14750, Cayman), PGD2 (5 nM, Cat# P5172, Sigma), celexosib (1–45 μM, Cat# 10008672, Cayman), 8-bromo-cAMP (20 μM, Cat# 1140, Tocris), sesamin (20 μM, Cat# SMB00705, Sigma), H89 (20 μM, Cat# 2910, Tocris), indomethacin (0–80 μM, Cat# 17378, Sigma), Ptger1 inhibitor (50 μM, Cat# SC51322, R&DD), Ptger2 inhibitor (25 μM, Cat# PF04418948, R&D), Ptger3 inhibitor (50μM, Cat# L-798,106, R&D), Ptger4 inhibitor (50μM, Cat# L-161,982, R&D), 5-HETE (0.5μM, Cat# 34210, Cayman), 12-HETE (0.5μM, Cat# 34550, Cayman), 15-HETE(0.5μM, Cat# 34700, Cayman), 8(9)-EET(0.5μM, Cat# 50351, Cayman), 11(12)-EET(0.5μM, Cat# 50511, Cayman), 14(15)-EET(0.5μM, Cat# 50651, Cayman), LTB4 (0.5μM, Cat# 20110, Cayman), TXB2 (5μM, Cat# 19030, Cayman), Wnt3a (10~100ng / ml, Cat# 315-20, Peprotech), amphiregulin (50ng / ml, Cat# 989-AR, R&D), Epiregulin (500ng / ml, Cat# 1068-EP, R&D).

[0246] Intestinal crypt isolation and flow cytometry Intestinal crypt isolation was performed as previously reported (Beyaz et al., 2016). Briefly, the entire intestine was removed, cleaned of fat, connective tissue, and blood vessels, and washed with ice-cold 1X PBS. After lateralization, the small intestine was cut into 3-5 cm pieces and incubated in 1X PBS / EDTA (7.5 mM) with gentle agitation at 4°C for 30 min. Crypts were mechanically separated from the tissue and filtered through a 70-micron strainer to remove villi and tissue debris. The crypts were then washed with ice-cold PBS and centrifuged at 300 g for 5 min. IEC isolation was performed by dissociating the crypt suspension into single cells using TrypLE Express (Cat# 12604-013, Invitrogen). Dissociated single cells were labeled with an antibody cocktail containing EPCAM-APC (1:400, Cat# 17-5791-82, eBioscience, G8.8), CD24-PE-Cy7 (1:400, Cat# 25-0242-82, eBioscience), and CD45-Alexa fluor 488 (1:400, Cat# 12-0451-83, eBioscience). Dead cells were excluded from analysis using the dead cell stain SYTOX (Cat# S34857, Life Technologies). IECs were sorted using a BD FACS Aria II SORP cell sorter with Epcam APC. + CD45-SYTOX- were isolated in supplemented crypt culture medium for culture or TRIzol reagent (Cat# 15596018, Thermo Fisher) and subjected to gene expression analysis.

[0247] Culture media for crypt and isolated cells Isolated crypts were counted and embedded in Matrigel (Cat# 356231, Corning Growth Factor Reduced) at a ratio of 1:4, 5-10 per μl. The Matrigel was solidified at 37°C for 8–12 minutes, and the solidified domes were then coated with a mixture of recombinant mouse EGF 40ng / ml (Cat# 315-09, PeproTech), recombinant mouse Noggin 50ng / ml (Cat# 250-38, PeproTech), R-spondin 62.5ng / ml (Cat# 3474-RS, R&D Systems), N-acetyl-L-cysteine ​​1μM (Sigma-Aldrich), CHIR-99021 5μM (Cat# 4423, Tocris), Y-27632 20ng / ml (Cat# 1254, Tocris), B27 1X (Cat# 17504044, Gibco), N2 1X (Cat# 17502048, Gibco), and 1% GlutaMAX (Cat# Cells were cultured in crypt medium containing Advanced DMEM (Cat# 12634010, Gibco) supplemented with 1% penicillin-streptomycin (Cat# 35050061, Gibco) and 1% penicillin-streptomycin (pen / strep) (Cat# P4333, Sigma-Aldrich). Crypt medium was changed every other day and maintained at 37°C in a humidified chamber containing 5% CO2. Clonogenicity (colony formation efficiency) was determined by seeding 50-300 crypts per well and assessing organoid formation after 3-7 days. Isolated IEC cells were centrifuged at 300 g for 5 minutes and suspended in an appropriate volume of crypt culture medium (500–1,000 cells / µl). The cells were then seeded on Matrigel in flat-bottom plates (Corning, Cat# 3548). Crypt medium was added after the Matrigel and cells had solidified. Crypt medium was replenished every other day. Unless otherwise specified, organoid bodies were quantified on days 1, 3, and 6 of culture. In secondary experiments, individual primary organoids were mechanically dissociated in TrypLE Express at 37°C for 6 minutes, centrifuged, resuspended in chilled crypt medium, mixed with Matrigel, and incubated until solidified. Fresh crypt medium was replenished every other day and maintained at 37°C in a humidified chamber containing 5% CO2.

[0248] Fatty acid (FA) BSA conjugation Fatty acids (FA) supplemented as powder were reconstituted with ethanol. The fatty acid solution was then added to 0.01 M NaOH to make a 12 mM solution and stirred at 70°C for 30 minutes. 10% fatty acid-free BSA (Cat# 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 FA was filtered through a 0.22 μm filter and stored in a glass container (Cat# B7999-2A, Thermo Fisher Scientific) at -20°C.

[0249] FA screening Ten thousand dissociated cells from mouse or human intestinal organoids were seeded onto 48-well plates and incubated in crypt medium at 37°C and 5% CO2 for 6 hours for recovery before proceeding to fatty acid treatment. The fatty acid screening library consisted of 23 different fatty acids, as shown in Table 1. After 6 hours of incubation, the medium was replaced with crypt medium containing the indicated concentrations of fatty acids (25 μM for both mouse and human organoids). After 24 hours of treatment, images (16 z-slices at 54.8 μm steps, fixed focal height of 1719 μm on the plate carrier) were taken from each well at 6-hour intervals at 37°C and 5% CO2 using a Cytation 7 and BioSpa platform (Agilent BioTek, Winooski, VT). Imaging was terminated at 120 hours. Z-projections were then acquired using the focus stacking function. Digital phase contrast was applied, and images were filtered for a structural element size of 100 μm. Spheroids were detected by defining low-internal-signal objects gated by a new metric <=0.95 and circularity >0.2, creating a new subpopulation. This subpopulation was normalized to the entire biological region.

[0250] Crypt isolation from biopsies of human study participants and patients Human colon tissue samples were obtained from patients undergoing surgical resection at Huntington Hospital who provided informed consent. The study protocol was reviewed and approved by the Northwell Health Biospecimen Repository (Protocol Number: 1810). Tissue samples were stored in RPMI medium (Cat# 10-040-CV, Corning) until processing. Patient metadata is presented in Table 2.

[0251] First, tissue samples were cut into approximately 0.5 cm pieces and incubated in an antibiotic mixture consisting of 100 μg / mL normocin (Cat# ant-nr-1, Invivogen), 50 μg / mL gentamicin (Cat# E737, Amresco), and 1X Pen / Strep (Cat# 15070063, ThermoFisher) in 1X PBS for 15 minutes at 4°C. The pieces were then washed with 1X PBS before being incubated in 5 mM EDTA solution on a rocker for 75 minutes at 4°C. After incubation, the tissue samples were washed once more with 1X PBS. The crypts were then separated from the tissue by shaking the pieces in a tube containing ice-cold 1X PBS. The isolated crypts were transferred to a new tube and spun down at 100 g for 5 minutes at 4°C.

[0252] Passaging and Maintenance of Human Organoids Isolated crypts were embedded in Matrigel at a ratio of 1:4. Before adding human crypt medium to each well, Matrigel was pre-cultured in Advanced DMEM (Cat# 12634028, Life Technologies), 1X Glutamax (Cat# 35050061, Life Technologies), 10 mM HEPES (Cat# 15630080, Thermo Fisher Scientific), 50% WRN conditioned medium from L-WRN cell line (ATCC, CRL-3276), 1X B27 (Cat# 12587010, Life Technologies), 1X N2 (Cat# 17502048, Life Technologies), 10 mM nicotinamide (Cat# N0636, Sigma-Aldrich), 1 mM N-acetylcysteine ​​(Cat# A9165, Sigma-Aldrich), 100 μg / mL primocin (Cat# Organoids were polymerized for 8–12 min at 37°C in culture medium consisting of ant-pm-1 (Invivogen), 10 μM SB202190 (Cat# S7067, Sigma-Aldrich), 10 μM Y-27632 (Cat# 1254, Tocris), 10 nM gastrin I (Cat# G9020, Sigma-Aldrich), 50 ng / mL EGF (Cat# AF-100-15, Peprotech), and 500 nM A83-01 (Cat# SML0788, Sigma-Aldrich). Culture medium was replaced every 2–3 days, and organoids were passaged approximately every 8 days. Organoids were harvested by removing the Matrigel using cell recovery solution (CRS) (Cat# 354253, Corning). Once the Matrigel had dissolved, the organoids were spun at 500g for 5 minutes at 4°C and incubated in TryplE Express (Cat# 12604039, ThermoFisher) until single cells were visible under a microscope. The cells were then centrifuged at 500g for 5 minutes at 4°C before being replated in Matrigel as described above. Organoids were typically passaged at a ratio of 1:6.

[0253] ELISA assay Epcam selected from control mice + CD45-SYTOX- cells were seeded at 25,000 cells per well and incubated at 37°C for 6 hours for recovery. The medium was then replaced with crypt medium supplemented with vehicle or AA (25 μM). Supernatants from vehicle- or AA-treated organoids were collected after 24 hours and centrifuged at 300 g to remove any cellular or Matrigel residues. The presence of PGE2 metabolites was measured using a PGE2 Elisa kit (Cat# ADI-900-001, Enzo Life Sciences) according to the manufacturer's instructions.

[0254] Nuclear fractionation and Western blot Organoids grown in Crypt medium for 5 days were treated with vehicle (BSA-ethanol) or 50 μM AA for 4 hours. Organoids were then removed from the Matrigel and washed with PBS. To prepare cytosolic extracts, organoids were lysed in buffer A (10 mM HEPES pH-7.9, 10 mM KCl, 1.5 mM MgCl, 0.34 M sucrose, 10% glycerol, 0.1 mM PMSF, 1 mM DTT, 0.1% Triton X-100, PhosSTOP (Cat# 04906845001, Roche) and protease inhibitor (Cat# 11873580001, Sigma)) and incubated on ice for 15 minutes with occasional gentle pipetting. Cytosolic extracts were collected after centrifugation at 1300 rcf for 5 minutes and 30 seconds. To prepare nuclear extracts, nuclei were washed several times with buffer A to remove all residual cytosolic proteins, lysed in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, PhosSTOP protease inhibitor), and then incubated on ice for 30–40 min with gentle tapping every 5 min. Nuclear extracts were collected after centrifugation at 1700 rcf for 5 min. The remaining insoluble chromatin was washed several times with buffer B to remove contaminating nuclear proteins. The chromatin pellet was then resuspended in Laemmli buffer and sonicated for 30 s on / 15 s off. Samples were then run on a 10% Tris-HCl gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk solution for 1 h at room temperature and then incubated with primary antibody in 5% skim milk overnight at 4°C. Following incubation, the membrane was washed with 1X 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. Signals were detected using Pierce ECL Western blotting Substrate (Cat#32106, ThermoFisher Scientific) with a Mini-Med 90 (AFP Manufacturing) documentation system.

[0255] smISH combined with immunohistochemistry and immunofluorescence Vehicle- or AA-treated organoids were harvested by removing the Matrigel using CRS solution and washed twice with 1X PBS. Subsequently, organoids were fixed with 4% paraformaldehyde (PFA) (Cat# 15714, Electron Microscopy Sciences) at room temperature for 30 minutes. Following removal of PFA, organoids were washed with 1X PBS and centrifuged at 400g for 3 minutes. Pelleted organoids were embedded in 2% agarose gel and sectioned at 10μm. Intestinal tissue from control or ARD-fed mice was Swiss-roll sectioned and fixed in 10% formalin solution (Cat# HT501128, Sigma-Aldrich). Formalin-fixed tissue samples were processed in a Thermo Excelsior ES processor and embedded using a Thermo HistoStar system according to the manufacturer's protocol. Paraffin-embedded samples were cut into 5 μm-thick sections and mounted on positively charged slides (Cat# 48311-703, VWR superfrost plus micro slides). HE staining was performed at the CSHL Tissue Imaging Facility using a Leica Multistainer (ST5020, Leica). Briefly, after deparaffinization and rehydration, slides were stained with hematoxylin (Hematoxylin 560 MX, Leica) for 1 minute, subsequently destained with Define MX-aq (Leica) for 30 seconds, blued with Blue Buffer 8 (Leica) for 1 minute, and then stained with eosin (EOSIN 515 LT, Leica) for 30 seconds. After dehydration, slides were coverslipped with a robotic coverslipper (Leica CV5030).

[0256] For IHC, formalin-fixed, paraffin-embedded tissue sections were deparaffinized. Antigen retrieval was performed by boiling at 96°C for 6 minutes using 0.1 mM citrate buffer (pH: 6). Following peroxidase blocking, tissues were blocked with appropriate serum and incubated overnight with anti-Ki-67 antibody (1:100, Thermo Fisher, clone SP6). Biotin-conjugated secondary antibodies were from Vector Labs. Diaminobenzidine (DAB) was used for visualization, and counterstaining was performed with hematoxylin and eosin (Vector Labs). PBST was used for washing between each step.

[0257] Single-molecule in situ hybridization (smISH) was performed for Ascl2 (Cat# 412211, ACD), Lgr5 (Cat# 312171, ACD), and S100a6 (Cat# 412981, ACD) using the Advanced Cell Diagnostics RNAscope 2.5 HD Detection Kit-Red (Cat# 322350, ACD) according to the manufacturer's instructions, combined with subsequent immunostaining for Epcam. For immunostaining, after performing the smISH step excluding DAPI staining, slides were incubated overnight at 4°C with anti-Epcam antibody (1:100, Cell Signaling, clone E6V8Y). Slides were then washed with PBST and incubated with secondary antibody (1:500, Alexa Fluor Plus 488, Cat# A32766, Invitrogen) in the dark for 1 hour at room temperature. The slides were washed again with PBST and stained with DAPI. Then, the slides were mounted using ProLong™ Gold Antifade Mountant (Cat# P36930, Invitrogen). Images were acquired using a confocal microscope (Zeiss LSM 710, Germany) and processed through ImageJ. smISH signals were quantified using Imaris (Oxford Instruments).

[0258] EdU Integration EdU (Sigma) was administered intraperitoneally at a dose of 5 μg / g 4 hours before euthanasia of mice. After tissue deparaffinization and antigen retrieval as described above, proliferating intestinal epithelial cells were detected by EdU incorporation using the Click-iT EdU Cell Proliferation Imaging Kit, Alexa Fluor 647 (Cat# C10640, Invitrogen) according to the manufacturer's instructions. Epcam and DAPI staining were then performed. Images were acquired using a confocal microscope (Zeiss LSM 710, Germany) and processed via ImageJ. EdU+ cells per crypt were quantified in a blinded manner. Edu signal per Swiss roll section was detected by Biotek (Agilent, USA).

[0259] Doxorubicin-induced intestinal injury Mice aged 8–12 weeks were administered a single intraperitoneal injection of doxorubicin hydrochloride (Cat# D1515, Millipore, Sigma) 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.

[0260] radiation-induced intestinal damage Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg / kg) and dexodomitol (10 mg / kg). Mice were placed in a lead shielding device, and only the lower abdominal / pelvic region was exposed to 15 Gy of ionizing radiation from a cesium-137 source (GammaCell). Mice were sacrificed 72 hours later. The number of remaining crypts was counted from hematoxylin and eosin-stained sections.

[0261] RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted using the Direct-zol RNA Isolation Kit (Cat# R2051, Zymogen), and reverse transcription was performed using SuperScript IV Vilo (Cat# 11756050, Thermo Fisher Scientific) as described by the manufacturer. RT-qPCR was performed with TaqMan Fast Advanced Master Mix (Applied Biosystems) using the probes listed in Table 5. qRT-PCR results were analyzed by the ΔΔCt method for relative quantification, using Hsp90ab1 as an internal control.

[0262] Table 5. List of probes used for qRT-PCR [Table 5]

[0263] Lentivirus production and generation of Ptger4 knockout organoids Lentiviral particles were produced in 293FT cells by cotransfection of the Puro.Cre empty vector (Addgene plasmid #17408 (Kumar et al., 2008)) with a second-generation lentiviral system (pCMV-VSVG, Addgene plasmid #8454) and psPAX2 (Addgene plasmid #12260) using the transfection reagent polyethyleneimine (PEI) (Cat# 23966, Polysciences). Briefly, 293FT cells were seeded in 10 cm dishes at 75%–80% confluency one day before transfection. 10 μg of Puro.Cre plasmid, 7.5 μg of spPAX2, and 5 μg of pCMV-VSVG were mixed in DMEM-F12 medium at room temperature and incubated for 15 minutes. The transfection mixture was added dropwise to confluent 293FT cells. The medium was removed after 6 hours, and fresh DMEM-F12 medium was supplemented with 10% FBS and 1% pen / strep. The supernatant containing viral particles was collected after 24, 48, and 72 hours and centrifuged at 300 g for 5 minutes to remove any remaining cell particles. The supernatant was filtered through a 0.45 μm filter and concentrated by adding a Lenti-X concentrator (Cat# 631232, Takara) (1 / 3 of the total supernatant volume). The mixture was centrifuged at 1500 g for 60 minutes at 4°C, and the viral pellet was then resuspended in mouse cytopenic medium. For viral infection, day 4-grown Ptger4 cells were cultured in PBS. f / f Organoids were removed from Matrigel and dissociated into single cells as described above. Concentrated lentivirus particles supplemented with polybrene were mixed with 1,000,000 single cells, transferred to a 48-well plate, centrifuged at 600 g for 1 hour at room temperature, and incubated at 37 ° C for 4 hours. Infected cells were resuspended in Mouse Script medium and seeded into 4 wells of a 12-well plate until organoids formed. Infected organoids were selected after 3 days using puromycin (1 μg / ml, Cat# A1113803, Thermo Fisher Scientific).

[0264] Electron microscopy Intestinal organoids grown in 12-well plates were fixed overnight at 4°C with 2.5% glutaraldehyde in 0.1M sodium cacodylate solution (pH 7.4). Samples were washed three times with 0.1M cacodylic acid and post-fixed for 1 hour at room temperature with 1% osmium tetroxide (OsO4). Samples were rinsed three times with distilled water and dehydrated for 10 minutes in a graded ethanol wash series (50%, 60%, 70%, 80%, 90%, 95%, and 100%). Following dehydration, samples were incubated overnight with 812EMed resin and 100% ethanol. For polymerization, samples were embedded in 812EMed resin and incubated at 60°C until the resin was fully polymerized. 60-90 nm sections were cut using a 45 Diamond DiATOME Histo Knife. Sections were stained with UranyLess for 2 minutes followed by extensive washing with ddH 2 O. A H7000 Hitachi transmission electron microscope was used to visualize the samples.

[0265] Metabolomic analysis by liquid chromatography coupled with mass spectrometry (LC-MS) Snap-frozen tissue specimens were cut and weighed into Precellys tubes pre-filled with ceramic beads (Bertin Instruments). The correct volume of extraction solution (30% acetonitrile, 50% methanol, and 20% water) was added to obtain 40 mg of tissue specimen per mL of extraction solution. Tissue samples were lysed using a Precellys 24 homogenizer (Bertin Instruments), and the suspension was incubated at -20°C for 60 minutes. The samples were mixed and incubated at 4°C for 15 minutes in a thermomixer (Eppendorf, Germany), followed by centrifugation (16,000g, 4°C for 15 minutes). The supernatant was collected, transferred to glass vials in an autosampler, and stored at -80°C until further analysis. For organoid experiments, media was collected 3 days after vehicle or AA treatment, and samples were extracted for metabolomic analysis. For analysis of polar metabolites and arachidonic acid derivatives, samples were randomized and processed blindly to avoid bias due to instrument drift. LC-MS analysis was performed using a Vanquish Horizon UHPLC system coupled with 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) and guard column (20 mm × 2.1 mm, 5 μm) from Merck Millipore, maintained at 45°C. The mobile phase consisted of 20 mM ammonium carbonate with 0.1% ammonium hydroxide in water (solvent A) and acetonitrile (solvent B). Analytes were eluted at 200 μl / min with a previously described gradient (Mackay et al., 2015). The mass spectrometer was operated in full MS and polarity switching mode. Acquired spectra were analyzed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Fisher Scientific) against an internal library of compounds. MetaboAnalyst® (v3.0.3, (Pang et al., 2020)) was used for quality control and normalization (using the options QuantileNorm, LogNorm, and MeanCenter). Differential abundance analysis was performed using FC.Anal.unpaired.

[0266] Bulk RNA sequencing Total RNA was isolated from vehicle- and AA-treated mouse intestinal organoids on days 1, 3, and 6, as well as from day 6 PDO, using the Zymo RNA isolation kit according to the manufacturer's instructions. Starting with 250 ng of total RNA, the rRNA removal protocol was performed according to the manufacturer's recommended guidelines. Strand-specific RNA-seq libraries were prepared using the NebNext Ultra II kit and sequenced on an Illumina NextSeq. Reads were trimmed using cutadapt (v2.10), aligned to the GRCm38.p6 / Gencode annotation (M24 release) using STAR (v2.7.2b, (Dobin et al., 2013)), and quantified using '--quantMode GeneCounts'. Read and alignment quality was analyzed using rseqc (v3.0, (Wang et al., 2012)) and summarized using multiqc (v1.9, (Ewels et al., 2016)). Differential gene expression between vehicle and arachidonic acid-treated samples was assessed using DEseq2 (v1.28, (Love et al., 2014)) and fitted to a model with sequencing batch and treatment as fixed effects. Treatment contrasts were extracted, and transcripts with absolute fold changes greater than log2(1.5) and adjusted p-values ​​less than 0.05 were considered differentially expressed. Differential gene expression was assessed independently at each time point. Unless otherwise specified, the union of differentially expressed genes between time points was used in all subsequent analyses. For visualization purposes, limma (v3.46, (Ritchie et al., 2015)) was used to adjust for sequencing batch effects before log-normalization of size-factor-normalized read counts. Heatmaps and upset plots were generated using ComplexHeatmap (v2.6.2, (Gu et al., 2016)) and Volcano plots using EnhancedVolcano (v1.8.0).

[0267] Gene Set The data were analyzed in the context of previously identified gene signatures and target gene lists, which are briefly described below.

[0268] Fetal spheroid gene signature 317 differentially upregulated genes (microarray) were assessed in regenerated spheroids versus organoids derived from embryos / mice at different embryonic and postnatal stages (E16, E18, or P0) ( Mustata et al., 2013 ).

[0269] Granuloma-inducing gene signature 131 differentially expressed genes (bulk RNA-seq) were assessed in granulomatous (Gr) versus non-granulomatous (NonGr) crypt epithelium of mice infected with H. polygyrus for 6 days ( Nusse et al., 2018 ).

[0270] Radiation-induced gene signature Fifty differentially expressed genes (single-cell RNA-seq) were assessed in clusters of regenerating enterocytes from scRNAseq data of irradiated intestinal epithelium versus uninjured crypts ( Ayyaz et al., 2019 ).

[0271] Regeneration-inducing gene signature 316 differentially expressed genes (bulk RNA-seq) were analyzed for regenerating Ascl2 during DT exposure. + was assessed in upper crypt cells compared to uninjured (quiescent) ISCs at the base of the crypt ( Murata et al., 2020 ).

[0272] Homeostatic stem cell gene signature Differentially expressed genes were assessed in Lgr5+ ISCs ( Haber et al., 2017 ).

[0273] Single-cell RNA sequencing For single-cell sequencing of mouse intestinal organoids, organoids were collected using cell recovery solution. Organoids were then dissociated into single-cell suspensions using TrypLE. After organoid dissociation, single cells were pelleted, washed, resuspended in FACS buffer (1X PBS, 10 μM Y-27632, 1% FBS, 0.5 mM EDTA) and passed through a 100 μm FlowMi cell strainer (Sigma). DAPI was used to assess viability. DAPI-negative cells were sorted using a Sony SH800S sorter, and single-cell droplets were immediately prepared using 10X Chromium according to the manufacturer's instructions at the Cold Spring Harbor Laboratory Single Cell Facility. Single-cell libraries were prepared using the 10X Genomics Chromium Controller (Cat #120223, 10X Genomics) and the 10X Genomics Chromium Next GEM Single Cell 3' Gene Expression Kit (Cat #1000268, 10X Genomics) according to the manufacturer's instructions. Cell suspensions were adjusted to produce 8,000 cells per sample.

[0274] Single-cell RNA-Seq data analysis Single-cell datasets from each experiment were independently assessed for data quality according to the guidelines described by (Amezquita et al., 2020; Luecken and Theis, 2019). Cells with more than 15% mitochondrial transcripts were removed, as were cells with fewer than 2,000 feature counts or expressing fewer than 1,000 genes. After QC, Seurat (v3.2.1, (Butler et al., 2018)) was used for normalization, graph-based clustering, and differential expression analysis. Each dataset was normalized using SCTransform, and the 3,000 most variable genes were identified using SelectIntegrationFeatures. Organoid (vehicle, AA, and PGE2-treated) and tissue datasets (intestinal tissue from mice on control and Arasco diets) were merged into a single organoid and tissue dataset (Cao et al., 2019; Levine et al., 2015; Qiu et al., 2017; Stuart et al., 2019; Trapnell et al., 2014). RunPCA on the merged dataset was used to identify the top 10 principal components (PCs) used for UMAP analysis and clustering. Louvain clustering at resolutions of 0.3 and 0.6 was used for the organoid and tissue datasets, respectively. Clusters were labeled according to the expression levels of enterocyte subtype signatures identified by (Haber et al., 2017); stem2 and stem3 clusters were labeled using signatures identified by (Roulis et al., 2020) (see gene list description above).

[0275] To determine whether AA and PGE2 treatments acted concordantly, differential expression analysis was performed between each treatment and the control using the FindMarkers function with the MAST method (Finak et al., 2015). Wilcoxon rank-sum tests to determine whether gene expression was significant were performed using the wilcox.test function in Statistics (v4.1.0, (R Core Team, 2021)). Monocle 3 (v0.2.3, (Cao et al., 2019; Levine et al., 2015; Qiu et al., 2017; Trapnell et al., 2014)) was utilized for trajectory analysis of both organoid and in vivo datasets.

[0276] Cut&Run Sample preparation CutnRun was performed according to Henikoff et al.'s (Skene et al., 2018) paper 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 1X PBS, followed by an additional wash with 1 ml of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and protease inhibitor cocktail). 10 μl of BioMag Concanvilin A beads were resuspended in 1 ml of binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2) and washed twice. 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 at room temperature for 10 minutes. The sample was placed on a magnetic rack until the solution became clear. The liquid was then replaced with antibody buffer containing the following antibodies at a 1:100 ratio: H3K4me3 (Cat# ab8580, Abcam), H3K27me3 (Cat# 07-449, Millipore), and H3K27Ac (Cat# 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, protease inhibitor cocktail) and resuspended in 150 μl of digitonin buffer. In-house produced pA-MNase was added at a concentration of 700 ng / ml, mixed gently, and incubated on a shaker at 4°C for 1 hour. The sample was then washed twice with 1 ml digitonin buffer. The cells were resuspended in 150 μl digitonin buffer and then cooled to 0°C by placing on a heat block over ice for 5 minutes. 3 μl of 100 mM CaCl2 was added with gentle mixing and immediately placed back on the 0°C block for 30 minutes of incubation.Digestion was stopped with 100 μl of 2X stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 mg / ml RNase A, 50 mg / ml glycogen, and 4 pg / ml yeast heterologous spike-in DNA). Samples were incubated at 37 μC for 10 minutes and centrifuged at 16,000 rcf for 5 minutes at 4°C. Samples were placed on a magnetic rack, and the supernatant was transferred to a new Eppendorf tube without disturbing the pellet. 2 μl of 10% SDS and 1.5 μl of proteinase K were added to each sample, followed by incubation at 70°C for 10 minutes. 200 μl of PCI was then added, mixed on a vortex at full speed for 3 seconds, then transferred to a phase lock tube and centrifuged at 16,000 rcf for 5 minutes. The liquid above the gel was transferred to a new 1.5 ml Eppendorf tube containing 2.5 μl of 3 mg / ml GlycoBlue (Cat# AM9515, Invitrogen) and mixed with 100% ethanol, followed by incubation on ice for 10 minutes and centrifugation at 16,000 rcf for 30 minutes. The pellet was washed with 70% ethanol and spun at maximum speed for 10 minutes. After the final washing step, the ethanol was discarded and any remaining ethanol droplets were removed by vacuum. The remaining pellet was dissolved in 15 μl of TE buffer (1 mM Tris-HCl pH=8, 0.1 mM EDTA).

[0277] Library preparation Libraries were prepared using the ThruPLEX DNA-seq kit (Cat# R400427, Takara) according to the manufacturer's instructions under the following PCR conditions: 3 min at 72°C, 2 min at 85°C, 2 min at 98°C, 4 cycles of 20 s at 98°C, 20 s at 67°C, and 30 s at 72°C, followed by 7 cycles of 20 s at 98°C and 20 s at 72°C. AMPure XP beads (Cat# A63880, Beckman Coulter) were used to select short fragments (0.5x–1.7x) and remove residual adapters and large DNA fragments. Libraries were sequenced on a NextSeq500 using a 150-cycle v2 High-Throughput SBS kit from CSHL The Sequencing Technologies Shared Resource. In the control sample, 10% PhiX was added, and sequencing was performed as paired-end 76-length sequences with index reads. Reads were demultiplexed by barcoding via the bcl2fastq2 tool.

[0278] analysis Cut&Run libraries for H3K27me3, H3K27ac, H3K4me3, and IgG control were sequenced paired-end with a 76-bp read length, resulting in an average of 13x2 million reads per sample. Sequencing data were aligned and processed by the CUT&RUN tool using all read fragments without filtering for read length (Zhu et al., 2019). SEACR was used in stringent mode, calling peaks from each replicate individually, and consensus peak sets were called by overlap across replicates under different conditions (Meers et al., 2019). Reads within peaks were quantified using featureCounts (Liao et al., 2014). Differential binding analysis between AA and control was performed separately for each chromatin mark using a negative binomial model with DESeq2, and the resulting peaks were filtered with an adjusted p-value of 0.01 (Love et al., 2014). Pathway analysis was performed by assigning peaks to genes by their distance to canonical transcription start sites, using distance thresholds of 25 kb, 25 kb, and 10 kb for H3K27me3, H3K27ac, and H3K4me3, respectively. Many-to-one peak-to-gene annotations were merged into one-to-one annotations by taking the peak with the greatest variation within the distance cutoff. For longer distances, genes were annotated to putative distal enhancers and regulatory regions using the Genomic Region Enrichment Annotation Tool (GREAT) with the base-plus-extension method (McLean et al., 2010). Genes were ranked by log2 fold-change values ​​or Wald statistics from differential calling. Pathway and gene set enrichment was calculated using gene sets from the GSEA and MSigDB databases, as well as RNAseq and other published literature mentioned 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. Profile plots for histone marks were calculated by binning the genome into 100-bp bins centered ±5 kb around the TSS of the gene set of interest and obtaining the normalized reads (RPGC) within those bins. Bins at specific distances from the TSS were aggregated to their median for plotting purposes. For AA vs. vehicle profile plots, the same process was repeated for replicates of both conditions, and then the difference between AA and vehicle-treated samples was taken before aggregation by median.

[0279] ATAC-seq Nuclei isolation ATAC-seq was performed as previously described by Buenrostro et al. (Buenrostro et al., 2013) and the Kaestner laboratory. Briefly, 50,000 viable cells sorted from organoids were washed with 1 ml of cold 1X PBS. Cells were incubated on ice for 3 minutes in 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 10 mM), NaCl (10 mM), MgCl2 (3 mM), Tween-20 (0.1% v / v)) was added onto the cells. It was then centrifuged at 500 g for 10 minutes at 4° C. and the supernatant was discarded. The pellet containing the nuclei was kept for further experiments.

[0280] Library preparation ATAC-seq libraries were generated as previously described by Buenrostro et al. and the Kaestner laboratory. Briefly, DNA was purified using DNA Clean & Concentrator 5 (D4013; Zymo Research). Libraries were prepared using NEBNext High-Fidelity 2x PCR Master Mix (NEB, M0541S) according to the manufacturer's standard protocol. DNA fragments were PCR pre-amplified for five cycles, and 5 μl of the partially amplified library was used for qPCR amplification (20 cycles). Plots showing R vs. cycle number were generated to determine the number of cycles required to reach one-third of the maximum R for each sample. qPCR amplification was then repeated with the calculated additional cycles. Size selection was performed using Agencourt AMPure XP beads (A63881; Beckman Coulter) following the amplification step to establish the final library (Ackermann AM et al.). Library quality was determined using an Agilent High-Sensitivity DNA Bioanalyzer (5067-4626; Agilent Technologies).

[0281] ATACseq analysis DNA libraries were sequenced using an average of 46x2 million paired-end reads per sample with a read length of 76 bp. Sequencing 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, duplicated reads, reads in blacklisted regions, secondary alignment and multi-mapping reads, reads with insert sizes larger than 2 kb, reads with incorrect pair orientation, or orphan reads were filtered. Peaks were called using MACS2 narrow peak calling with a -75 base pair shift and a 150 base pair extension (Liu, 2014). Consensus peaks were called by obtaining overlapping regions covered in at least two samples. Reads within peaks were quantified using featureCounts (Liao et al., 2014). A variance-stabilizing transformation (VST) was used to normalize data for visualization and unsupervised clustering (Anders and Huber, 2010). Counts from AA-treated samples were compared to control-treated samples by negative binomial comparison using DESeq2, filtered with an absolute log2 fold-change value of 0.58 (minimum 50% change) and an FDR-adjusted p-value of 0.01 (Love et al., 2014). Peaks were annotated and aggregated into genes by obtaining the maximally altered peaks within 5 kb of each transcription start site and assigning them to each gene by promoter or putative proximal enhancer.Genes were ranked by log2 fold change or Wald statistic and entered into GSEA using gene signatures curated from the MSigDB database and RNAseq experiments and literature. An adjusted p-value of 0.05 was used to filter the resulting gene sets (Liberzon et al., 2011; Subramanian et al., 2005). Correlation between RNAseq and ATACseq was performed by taking all ATACseq peaks within 2.5 kb of the transcription start site of all genes, binning genes into open or closed differential and no differential categories, and plotting the log2 fold change of expression differences.

[0282] The regulatory potential of transcription factors was calculated by comparing all mammalian motifs in JASPAR (Castro-Mondragon et al., 2022) within the sequences of accessible peaks. The log2 fold change in peak accessibility in AA vs. vehicle-treated cells was multivariately 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 denotes the presence of the TF motif in the peak as a binary variable, and GC is the GC content of the peak. Effect sizes and p-values ​​for each term are obtained after multivariate linear modeling using ordinary least squares regression with robust standard errors.

[0283] statistics Statistical analysis was performed using Graphpad Prism 9.0. Results were analyzed using one of the following statistical tests, as indicated in the figure legends: ANOVA, Wilcoxon rank-sum test, Mann-Whitney test, or two-tailed t-test. Bars are presented as mean ± SEM. P values ​​of <0.05 were considered statistically significant and are represented as follows: *P<0.05, **P<0.01, ***P<0.005, and ****P<0.001.

[0284] Data and code availability RNAseq, scRNAseq, CutnRun, and Atac-sec data are accessible from Gene Expression Omnibus (GEO) under the following accession number: GSE188213

[0285] Example 10: FA cell fate control and function in physiological and disease states This 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 genotoxic insults such as radiation and chemotherapy. Dietary AA supplementation may be therapeutically important in cancer patients undergoing chemotherapy and radiation therapy, who often suffer from gastrointestinal side effects.

[0286] Example 11: Kinetics of AA-induced cellular changes in the intestine Mice receiving an AA-enriched diet for 1, 2, or 4 weeks will be analyzed to define the kinetics of AA-induced intestinal regeneration. Approximately 60 mice (10C and 10ARD) will be assessed at three time points. Histological proxies will be assessed, as well as metabolomics, particularly AA levels.

[0287] Example 12: Optimal regimen for AA-mediated protection from genotoxic stress To assess the effects of various dietary regimens with AA supplementation, including continuous (AA on), reversed (AA off), or cyclic (AA on / off / on / off) feeding, in mice, 40 mice were divided into four groups of 10 each (C, ARD on, ARD off, ARD on / off / on / off). Histology was assessed. AA levels were assessed through metabolic testing, and the four groups were further analyzed through eight scRNA-seq libraries (n=2).

[0288] Example 13: Effect of dietary AA supplementation in response to chemotherapy for cancer A cancer mouse model will be established to assess the beneficial (e.g., therapeutic) significance of dietary AA supplementation in (1) ameliorating the gastrointestinal side effects of chemotherapy and (2) improving overall survival in tumor-bearing mice. Blood will be assessed in a subset of animals for the impact of diet on chemotherapy-induced cytopenias. Approximately 20 mice will be characterized, divided into two groups: 10C and 10ARD. Histological proxies and metabolomics, particularly AA levels, will be assessed.

[0289] Example 14: Effect of AA on human intestinal tissue in response to chemotherapy Human intestinal organoid models are established. These models are used to assess the effect of AA treatment on doxorubicin-induced injury. Human PDO (n=2, V / AA treatment + / - doxorubicin).

[0290] Example 15: A diet high in AA does not increase colon cancer risk in tumor-prone mouse models Tumor model Tumor-prone mice (VillinCreERt2 APC L / +) were treated with tamoxifen and fed an AA-enriched diet (ARD, also referred to as FA1) or the control diet described in the previous example. After 3-4 months of ARD or control diet, mice were sacrificed and tumor burden was assessed. Figure 19A shows a schematic diagram of the experimental procedure. As shown in Figure 19B, ARD did not increase the number of tumors identified in the small intestine or colon. As shown in Figure 19C, the total tumor burden in mice fed the ARD was not higher than that in mice fed the control diet.

[0291] Metastatic cancer model As shown in Figure 20A, C57BL6 / J mice were treated with AKPS (APC KO , KRAS G12D ,P53 KO , SMAD4 KO) cells were injected. One week later, the presence of tumors was confirmed. Subsequently, the mice were fed an AA-rich diet or a control diet. The survival rates of the mice are shown in Figure 20B. After sacrifice, the mice were dissected and assessed for primary tumors and metastasis. As shown in Figure 20C, the metastasis rate was the same between mice fed an AA-rich diet and mice fed a control diet. As demonstrated in this example, an AA-rich diet does not increase the rate of metastasis or decrease survival rates compared to a control diet.

[0292] Example 16: Kinetics of the effects of an AA-rich diet in mice Plasma AA levels To assess plasma levels of fatty acids over time, mice were fed i) a control diet for 4 weeks, ii) an AA-rich diet (Arasco) for 4 weeks, or iii) an AA-rich diet for 2 weeks followed by a switch to a control diet (ArascoRev) for 2 weeks. Plasma lipid levels were assessed on days 3, 7, and 14 for mice fed the control diet or the Arasco diet; the results are shown in Figure 21A. Plasma lipid levels were assessed at week 4 for all three conditions; the results are shown in Figure 21B.

[0293] ARD-induced stem cell regeneration Mice were fed a control diet (C) or an AA-enriched diet (ARD) for 3 days, 1 week, or 2 weeks. They were then exposed to 15 Gy of abdominal irradiation. Figure 22A shows a schematic diagram of the experimental protocol. Metrics, histology, and metabolomics were assessed for each mouse. The arachidonic acid levels observed under each condition are shown in Figure 22B. Figure 22C shows representative histological images (left) of mice fed the control diet or the ARD, as well as a graph showing the number of EdU+ cells / crypt. EdU (5-ethynyl-2'-deoxyuridine) is a marker of cell proliferation, and a greater number of EdU+ cells per crypt indicates increased cell proliferation in irradiated mice fed the ARD for 7 or 14 days.

[0294] Gene expression Mice were fed one of three diets as described in the first section of this example. Figure 23A shows a schematic diagram of the experimental protocol. Gene expression was then assessed by scRNAseq and scATACseq as described in the previous example; the results are shown in Figure 23B.

[0295] Example 17: Protective effect of AA in response to 5-fluorouracil chemotherapy Mice were fed an AA-enriched diet (ARD) or a control diet for 2 weeks as described in the previous example, and 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).

[0296] Figure 24A shows a schematic of the experimental procedure, and Figure 24B shows that mice fed the ARD diet administered 250 mg / kg of 5-FU lost less weight than mice fed the control diet administered 250 mg / kg of 5-FU.

[0297] Example 18: Protective effect of AA in response to high-dose chemotherapy Mice (8 weeks old) were fed a control diet or ARD for 2 weeks, then injected with 5-FU (100 mg / kg) and oxaliplatin (6 mg / kg) at a dose administered once per week for 2 weeks, followed by a 2-week recovery period, and then the dose administered once per week for 2 weeks was repeated. Figure 25A shows a schematic of the experimental procedure, and Figure 25B shows that mice fed the ARD diet lost less weight compared to mice fed the control diet.

[0298] References: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0299] Equivalence and Scope 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 intended to be limited to the above description, but rather is set forth in the appended claims.

[0300] A claim or description including "or" between one or more members of a group is considered satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless stated to the contrary or apparent from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes aspects in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it should be understood that the invention covers all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more enumerated claims introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends from the same base claim. Furthermore, when a claim recites a composition, it is understood to encompass methods of using the composition for any purpose disclosed herein, and methods of making the composition according to any manufacturing method disclosed herein or other methods known in the art, unless otherwise indicated or unless a contradiction or inconsistency would arise to one of ordinary skill in the art.

[0301] Where ranges are specified, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can take any particular value or subrange within the ranges defined in various aspects of the invention, down to one-tenth of the unit of the lower limit of the range, unless the context dictates otherwise.

[0302] In addition, it should be understood that any particular aspect of the present invention that falls within the prior art may be expressly excluded from any one or more claims. Such aspects may be excluded even if the exclusion is not explicitly stated herein, because they are deemed to be known to those skilled in the art. Any particular aspect of the compositions of the present invention may be excluded from any one or more claims for any reason, whether or not related to the existence of prior art.

[0303] All references cited herein, including patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

Claims

1. 1. A method of preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject, comprising: Orally administering at least about 2 g of arachidonic acid triglyceride (AATG) per day (2 g / day) to a subject in need thereof for a period of time sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject; The method.

2. the sufficient period of 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 within 28 days of the subject completing a course of chemotherapy or radiation therapy; or (c) The method of claim 1, wherein administration is initiated at any time during the course of chemotherapy or radiation therapy.

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

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

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

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

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

8. 6. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiotherapy lasts for at least about 12 months.

9. 6. The method of any one of claims 1 to 5, wherein the course of chemotherapy or radiotherapy lasts for at least about 3 months to about 12 months.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

29. 29. The method of claim 28, wherein the reference is an 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. 30. The method of any one of claims 1 to 29, wherein the adverse side effect is a gastrointestinal side effect.

31. 30. The method of any one of claims 1-29, wherein the adverse side effect is nausea, vomiting, diarrhea, weight loss, intestinal tissue damage, radiation colitis, radiation mucositis, pelvic radiation disease, 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. 1. A method of preventing, reducing, or ameliorating the cytotoxic effects of chemotherapy or radiation therapy in a subject, comprising: Oral administration of at least about 2 g of arachidonic acid triglyceride (AATG) per day (2 g / day) 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; The method.

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

35. 1. A method of increasing arachidonic acid (AA) levels in a subject, the method comprising: (a) measuring arachidonic acid (AA) levels in a sample from a subject in need thereof to determine whether the AA level is below a predetermined AA level sufficient to prevent, reduce, or ameliorate adverse side effects from 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 of AA TG per day (2 g / day) for a period sufficient to increase the AA level to or above the predetermined AA level.

36. (c) measuring the AA level resulting from administering the AA TG in (b) to determine the AA level; and 36. The method of claim 35, further comprising: (d) administering to the subject, if the AA level in (b) is not equal to or greater than the predetermined AA level, an amount of AA TG per day sufficient to result in an intestinal AA level equal to or greater than the predetermined AA level.

37. 37. The method of claim 36, further comprising repeating steps (c) to (d) to produce an intestinal AA level in the subject that is equal to or greater than 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. 40. The method of any one of claims 1 to 39, wherein AA in the AA TG is substituted by at least one precursor of AA.

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

42. 1. A method of preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject, comprising: orally administering to a subject in need thereof at least about 2 g per day of at least one precursor of arachidonic acid (AA) (2 g / day) for a period of time sufficient to prevent, reduce, or ameliorate adverse side effects of chemotherapy or radiation therapy in the subject; The method.

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

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

45. 1. A kit for use in preventing, reducing, or ameliorating adverse side effects of chemotherapy or radiation therapy in a subject, comprising: (a) one or more supplementation units sufficient to provide a subject in need thereof with at least about 2 g of arachidonic acid triglyceride (AA TG) per day (2 g / day) for at least 7 days; and (b) containing instructions for the preparation and consumption of one or more supplement units; The kit.

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

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

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

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

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

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

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

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

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

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