High-fat diet enhances stemness and tumorigenicity of intestinal progenitors

Abstract

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

Extended Data Figure 1. HFD alters intestinal morphology and enhances intestinal progenitor proliferation

a–g, In comparison to mice fed a standard chow, mice on a HFD gained on average 50% mass (a, C: n=11, HFD: n=15), had reduced small intestinal mass and length (b,c, C: n=11, HFD: n=15), longer crypts and shorter villi (e,g, n=3 each), and fewer villus enterocytes (f, n=3). HFD did not change the density of crypts (d, n=3) in the proximal jejunum. The proximal jejunum was defined as the length between 6 and 9 cm as measured from the pylorus (the distal portion of the stomach). h–k, HFD enhanced BrdU incorporation in ISCs (or crypt base columnar cells) and progenitor cells (or transit-amplifying cells) in the proximal jejunum (h, n=6) and sorted cell populations (i, n=3) after a 4-hour pulse. HFD increased the total (j, control: n=4, HFD: n=5) and normalized numbers of BrdU-labeled enterocytes compared to controls after a 24-hour pulse. Arrowhead (k) marks the leading edge of migrating BrdU-positive enterocyte. l, m, Representative images of Olfm4 (l, n=3) and Crypt4 (m, n=6) in situ hybridizations from C and HFD mice. n, No significant difference in the number of jejunal caspase3⁺ cells was detected by immunohistochemistry. Images are representative of three separate experiments (n=3); arrows indicate representative caspase3⁺ enterocytes. o, The HFD chow (Research Diets D12492) provides a higher percentage of kilocalories from fat and conversely a lower percentage of kilocalories from protein and carbohydrates compared to standard chow diet (Labdiet RMH3000). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001, scale bars in g=100μm, h,i=50μm, k=100μm and 50μm (inset), l,m= 50μm and 20μm (inset), n=100μm; two separate fields of jejunum (d), and at least 15 crypts (e), 15 villi (f), 10 villi (g), 100 cells (i), 25 villi (j) and 25 crypt-villus units (n) were counted per sample in each independent experiment. ISCs= intestinal stem cells; Olfm4= Olfactomedin 4; Crypt4= Cryptdin 4; C= control; HFD= high fat diet)

Extended Data Figure 2. HFD and PPAR-d signaling have minimal effects on enteroendocrine and goblet cell differentiation but promote intestinal regeneration after 15 Gy irradiation

a, b, Quantification (a, n=3) of immunostains (b, n=3) for chromogranin A revealed no difference in the numbers of jejunal enteroendocrine cells (arrowheads) per crypt-villus unit in HFD-fed mice and GW-treated mice compared to their respective controls. c, d, Quantification (c, n=4) of Alcian blue/PAS staining (d, n=4) showed no difference in mucinous goblet cells (arrowhead) in HFD-fed mice and GW-treated mice compared to their respective controls. e, f, HFD increased the number of regenerating crypts as measured by an increased number of crypts containing at least ten Ki67⁺ (a marker of proliferation) cells (e, n=3) or at least one Olfm4⁺ cell (f, n=3) per 5mm of jejunum by immunohistochemistry (IHC) or in situ hybridization (ISH). Arrows indicate Olfm4⁺ crypts. g, Surviving crypt numbers after ionizing irradiation-induced (XRT) damage. Arrows: regenerating crypts. Asterisks: aborted crypts (n=3). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; scale bars in b, d=100μm, e, f=50μm and 20μm (inset), g=50μm; 50 crypt-villus units per sample were analyzed (a, c) and approximately 50 crypts (e, f, g) were counted per sample in each independent experiment. Olfm4= Olfactomedin 4; Crypt4= Cryptdin 4; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516)

Extended Data Figure 3. A HFD and fatty acids do not activate inflammatory pathways in intestinal crypts and organoids, while HFD and enforced PPAR-d signaling enhance colonic stem-cell function

a, HFD did not alter the normalized expression levels of inflammatory genes from the gene set enrichment analysis (GSEA) MSigDB (signature M6557) data set in ISCs and progenitors. b, HFD did not induce differential expression of Inflammatory Response genes from Gene Ontology (GO: 0006954) in ISCs (Lgr5-GFP^hi) or progenitors (Lgr5-GFP^low) compared to C. Fold changes of GO Inflammatory Response genes are indicated in red, and fold changes for all other genes are indicated in blue. c, HFD did not activate the NFκB or the STAT-3 pathways in the intestinal crypt. Total and phosphorylated protein levels in crypt lysates were assessed by immunoblots (n=3). For western blot source data, see Supplementary Figure 1. d, HFD did not induce pro-inflammatory gene expression in ISCs (Lgr5-GFP^hi) or progenitors (Lgr5-GFP^low). Relative expression levels compared to Actb were measured by qRT-PCR (n=5). e, Ex vivo PA, L or GW treatment did not induce inflammatory gene expression in crypt-derived organoids compared to V. Relative expression levels compared to Actb were assessed by qRT-PCR (n=4, 12 wells per sample were analyzed). f, HFD boosted the number of BrdU-labeled cells as measured in distal colonic crypts compared to C (C n=6, HFD n=5) after a 4-hour pulse. g, HFD increased the frequency of colonic ISCs (Lgr5-GFP^hi, dark green) and (Lgr5-GFP^low, light green) cells (n=8). h, i, HFD enhanced PPAR-d (h) and β-catenin (i) target gene expression in colonic ISCs and progenitors. Relative expression levels compared to Actb were determined by qRT-PCR (n=5, all fold changes are significant with P<0.05) j–m, Colonic crypts derived from HFD-fed (j, n=4; k, n=4) and GW-treated (l, n=5; m, n=4) mice demonstrated greater primary and secondary organoid-forming capacity compared to their respective controls. Representative day 4 organoids are depicted. (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001, scale bars in f=50μm, j=100μm, l=200μm; 50 crypts per sample were analyzed (f) in each independent experiment. ISCs= intestinal stem cells; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516)

Extended Data Figure 4. Characterization of HFD crypt and ISC-derived organoids

a, HFD organoids contained higher frequencies of ISCs (Lgr5-GFP^hi) compared to C (n=3). b, c, C and HFD organoids demonstrated no differences in morphologic ultrastructure as seen in (b) one micron sections of control (left) and HFD (right) organoids counterstained with Toluidine Blue and (c) electron microscopy images of representative C (left) and HFD (right) organoids (n=3). S=Stem-cell, P=Paneth cell. d, e, Composition of organoids derived from C (d) and HFD (e) crypts as assessed by single-cell gene expression analysis. Organoids on day 5 contained ISCs (Lgr5 and Olfm4, Olfactomedin 4), Paneth cells (Lyz, Lysozyme), enteroendocrine cells (Chga, Chromogranin A), and goblet cells (Muc2, Mucin 2). 48 live cells per group were sorted and single-cell gene expression analysis was performed after pre-amplification using corresponding stem-cell and lineage primers (See Materials and Methods). f, Crypt-derived organoids from control or HFD mice included Chromogranin A-, Mucin 2-, and Lysozyme-positive cells as assessed by immunofluorescence (blue=DAPI, red=cell-specific antibody). Images represent two experiments (n=2). blue: DAPI. red: cell-specific antibody. g, Cultured villi from C and HFD mice lack the ability to form organoids. Images represent two experiments with 6 wells per sample (n=2). h, ISCs from HFD-fed mice harbored greater organoid-forming potential compared to controls. Arrowheads indicate representative organoids at days 4, 7, 10 of culture (n=4). i, Individually dissociated HFD primary organoids that were derived from single ISCs possessed more secondary organoid-forming ability than those from C. (n=4). Representative day 4 secondary organoids are illustrated. j, k, Single-cell gene expression analysis revealed that ISCs from both control (j) and HFD (k) mice can beget Paneth cells (Lyz) within 24 hours in culture (48 cells per group, See Materials and Methods). l, m, Composition of organoids derived from control (l) and HFD (m) ISCs (Lgr5-GFP^hi) as assessed by single-cell gene expression analysis (48 cells per group, See Materials and Methods). Organoids on day 5 contained ISCs (Lgr5 and Olfm4), Paneth cells (Lyz), endocrine cells (Chga) and goblet cells (Muc2). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05; scale bars in b= 20μm, c=2μm, f=50μm, g,i=200μm, h=100μm. C= control; HFD= high fat diet)

Extended Data Figure 5. Ex vivo exposure of mouse and human organoids to fatty acids recapitulates aspects of a HFD

a, b Individually dissociated primary organoids possessed more secondary organoid-forming activity (a, n=4, the mean number of secondary organoids subcloned from each of 5 primary organoids in 4 independent experiments.) and contained a higher frequency of Lgr5-GFP^hi ISCs (b, n=3) after four weeks of treatment with 30μM PA compared to V. c, Exposure of naïve crypts to 30μM OA had no effect on primary organoid formation measured at day 7 (n=6). Representative day 7 organoids depicted. d, Individually dissociated primary organoids possessed more secondary organoid-forming capacity after four weeks of treatment with 30μM OA (n=4, the mean number of secondary organoids subcloned from each of 5 primary organoids in 4 independent experiments.) compared to V (same vehicle cohort used in a and d). e, Lipid mixture composition (Sigma L0288) as described by the manufacturer. f, Ex vivo treatment of human-derived small intestinal crypts (H1–H4) passaged in the presence of L, PA, or GW augmented relative clonogenicity compared to V as shown in representative images from 4 independent experiments. H1: n= 10 (V, PA, GW) and n= 6 (L) wells were analyzed. H2: n= 16 (V), n= 6 (L), n= 12 (PA), and n= 14 (GW) wells were analyzed. H3: n= 10 (V), n= 12 (L, PA), and n= 8 (GW) wells were analyzed. H4: n= 7 (V, GW), n= 6 (L), and n= 9 (PA) wells were analyzed. Age, gender, and BMI are specified to the right of the bar graph panel. g–j, Human crypt-derived organoids after ex vivo treatment with PA, L, or GW induced PPAR-d target gene expression as assessed in passaged cultures with qRT-PCR (n=4, 12 wells per sample were analyzed, all fold changes are significant, P<0.05). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bar in c=100μm, f=500μm. ISCs= intestinal stem cells; V= vehicle; PA= palmitic acid; OA= oleic acid; L= lipid mixture; GW= PPAR-d agonist, GW501516)

Extended Data Figure 6. PPAR-d is the predominant PPAR family member expressed in intestinal progenitors and mediates the effects of HFD

a, PPAR-d is the most abundant PPAR family member in ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) based on RNA-seq data. b, Confirmation of PPAR family member mRNA expression levels in ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) by qRT-PCR (n=5). c, Genes upregulated in HFD ISCs (Lgr5-GFP^hi) versus control ISCs were enriched in PPAR and LXR/RXR motifs. d, GSEA of RNA-seq data identified enrichment of PPAR-d targets in ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) with a HFD. e, Confirmation of induced PPAR-d target gene expression in flow sorted ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) by qRT-PCR (n=5). All fold changes were significant with P<0.05. f, g, Representative images of Olfm4⁺ (ISCs, f) and Crypt4⁺ (Paneth cells, g) in situ hybridization (ISH) from V and GW-treated mice (f, n=3; g, n=4). h, Ex vivo exposure of organoids to PA, L, or GW stimulated PPAR-d and β-catenin target gene expression (n=3, all fold changes were significant with P<0.05). i, j, Injection with tamoxifen (4 injections on alternating days) in Ppard^L/L; Villin-CreERT2 mice led to efficient intestinal deletion (IKO) of Ppard (7 days after the last tamoxifen dose) as assessed by allele-specific deletion PCR (i, n=3) and immunoblot analysis (j, n=3) of crypts. For western blot source data, see Supplementary Figure 1. k, Acute disruption of Ppard (8 days after the last tamoxifen dose) did not perturb ISC and progenitor proliferation as determined 4-hours following BrdU administration (n=3). l, m, Acute Ppard deletion (8 days after the last tamoxifen dose) did not significantly alter Olfm4⁺ ISCs numbers (L/L: n=5, IKO: n=4) (l) or Crypt4⁺ Paneth cell (n=5) (m) numbers as assessed by in situ hybridization. n, Loss of Ppard transcripts in PPAR-d IKO organoids was confirmed by qRT-PCR using deletion-specific primers (n=3). o, PPAR-d is required for the induction of PPAR-d and β-catenin target gene expression in secondary organoids after ex vivo PA, L, or GW treatment (n=5, all fold changes are significant with P<0.05) p, Heat map of differentially expressed genes illustrated induction of a PPAR-d program in HFD-derived ISCs and progenitors relative to controls (C). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, scale bars in f, g, k–m=50μm and 20μm (inset); 50 crypts per sample were analyzed in each independent experiment (f, g, k–m). ISCs= intestinal stem cells; C= control; Olfm4= Olfactomedin 4; Crypt4= Cryptdin4; HFD= high fat diet; V= vehicle; PA= palmitic acid; L= lipid mixture; GW= PPAR-d agonist, GW501516)

Extended Data Figure 7. HFD and PPAR-d signaling boost nuclear β-catenin localization and activity in intestinal progenitors

a, b, HFD-derived ISCs (a, Lgr5-GFP^hi) and progenitors (b, Lgr5-GFP^low) required less Wnt3a and R-spondin to initiate organoids compared to C ISCs as measured by comparing organoid-formation in complete ENRW media, which includes EGF, Noggin, R-spondin, and Wnt3a, versus EN media, which includes EGF and Noggin but lacks Wnt3a and R-spondin (n=3). C-derived progenitors, in contrast to HFD-derived progenitors, rarely formed organoids in either ENRW or EN media. c–f, HFD increased nuclear β-catenin localization in flow sorted ISCs and progenitors from HFD (c, n=5) and GW-treated (d, n=4) mice as determined by immunofluorescence (red=DAPI, cyan=non-phosphorylated β-catenin, CST 8814S). At least 100 cells per sample were quantified. Representative images are shown in (e, f). g–i, HFD (g) and GW (h) treatment increased the numbers of ISCs and progenitors with β-catenin⁺ nuclei as assessed by immunostains (n=4 each). Representative images are shown in (i); arrowheads indicate representative nuclear β-catenin in ISCs (red) and progenitors (black). j–l, Association of PPAR-d and β-catenin in C and HFD derived intestinal crypts as shown by immunoprecipitation (IP) (n=3). For western blot source data, see Supplementary Figure 1. (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in e,f=50μm i=20μm; organoid assays: 2–4 wells per sample analyzed (a, b), 50 crypts per sample were analyzed in each independent experiment (g, h). ISCs= intestinal stem cells; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516)

Extended Data Figure 8. HFD-mediated alterations in β-catenin target gene expression in single ISCs and progenitors

a, Heat map representation of β-catenin target gene expression in single ISCs (Lgr5-GFP^hi, 24 cells) and progenitors (Lgr5-GFP^low, 72 cells; See Materials and Methods). b, Stem-cell signature genes were identified by comparing target gene expression in C ISCs (Lgr5-GFP^hi) to C progenitors (Lgr5-GFP^low). c, HFD signature genes were identified by comparing target gene expression in HFD to C ISCs (Lgr5-GFP^hi). d, t-Distributed stochastic neighbor embedding (t-SNE) analysis of single cells using all β-catenin target genes. e, tSNE analysis of single cells using stem-cell signature genes. f, tSNE analysis of single cells using HFD signature genes. g, h, Lgr5 expression was similar in HFD ISCs (Lgr5-GFP^hi) (g) and progenitors (Lgr5-GFP^low) (h) as compared to their respective controls. i, j, HFD increased the percentage of ISCs (Lgr5-GFP^hi) (i) and progenitors (Lgr5-GFP^low) (j) with elevated Jag1 and Jag2 expression compared to their respective controls. k, l, HFD (k, n=3) and GW treatment (l, n=4) augmented Jag1 expression compared to C and V treatments, respectively, as assayed by single-molecule in situ hybridization: Jag1 is broadly expressed throughout the crypt. (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05; scale bars in k, l=50μm and 20μm (inset); more than 50 crypts per sample were analyzed in each independent experiment (k, l). ISCs= intestinal stem cells; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516)

Extended Data Figure 9. Characterization of obese db/db mouse intestines

a–f, At 4–5 months of age, homozygous db/db gained on average 50% more mass (a, n=9), had increased small intestinal mass and length (b, c n=9), shallower crypts (d, n=4) and longer villi (e, f n=5) when compared to control db/+ mice. g–h, Immunostains for Olfm4 (n=6) and lysozyme (n=6) revealed a slight reduction in the number of Olfm4⁺ ISCs and Paneth cells, respectively, in db/db mice compared to db/+ controls. i, Organoid-forming capacity of db/db crypts trended higher (P=0.095) compared to db/+ controls (n=7). j, Single-cell gene expression analysis revealed no induction of PPAR-d or β-catenin target gene expression in live, enriched stem and progenitor cells that are depleted of secretory cells (7-AAD⁻Epcam⁺CD24⁻c⁻kit⁻ cells, 48 cells per group; See Materials and Methods) from db/db intestines compared to control intestines. (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in f=100μm, g–h=50μm and 20μm (inset), i=200μm; and at least 30 crypts (d); 20 villi (e); 100 crypts (g, h) were assessed per sample in each independent experiment. All db/db and db/+ mice were fed a standard chow diet. ISCs= intestinal stem cells; OLFM4= Olfactomedin 4)

Extended Data Figure 10. PPAR-d activation bestows adenoma-initiating capacity to Apc-null progenitors

a, b, Representative optical endoscopy images (top) from Figure 5 with H&E (middle), and β-catenin (immunohistochemistry, bottom) sections of adenomas derived from orthotopic transplantation of Apc-null ISCs (a, Lgr5-GFP^hi) and progenitors (b, Lgr5-GFP^low) from V and GW-treated mice 4 days post Apc deletion. Tumors exhibited hyperchromasia, lack of maturation, nuclear crowding, and nuclear β-catenin positivity. Two independent pathologists blinded to treatment groups interpreted the results. c, d. Apc deletion was confirmed in sorted small intestinal ISCs and progenitors from V and GW-treated Apc^L/L; Lgr5-EGFP-IRES-CreERT2 mice 4 days after tamoxifen administration (c, n=3) and in isolated tumors (d, n=3) by PCR amplification using allele-specific deletion primers targeting exon14. (Unless otherwise indicated, n represents independent experiments; scale bars in a (20X)=50μm and (60X)=20μm. ISCs= intestinal stem cells; V= vehicle; GW= PPAR-d agonist, GW501516)

Figure 1. High Fat Diet augments ISC numbers and function

a, Quantification of Olfm4⁺ ISCs (n=3) and Crypt4⁺ Paneth cells (n=6) in the proximal jejunum of C and HFD mice by in situ hybridization. b, BrdU incorporation in ISCs (crypt base columnar cells) and progenitors (transit-amplifying cells) after a 4-hour pulse (n=6). c, d, e, Organoid per crypt (c, n=4) and crypt domain (d, n=7) quantification from C and HFD mice (d, n=4). Representative images: Day 7 organoids (e). Arrows: organoids. Asterisks: aborted crypts. f, Number of secondary organoids per dissociated crypt-derived primary organoid (n=9 primary organoids, 3 primary organoids per sample were individually subcloned in 3 independent experiments). g, h Frequencies of ISCs (Lgr5-GFP^hi, dark green) and progenitors (Lgr5-GFP^low, light green) in the entire small intestine (g, n=10) and colon (h, n=8) as measured by flow cytometry. i, j, Organoid-initiating capacity of C and HFD ISCs cultured +/− Paneth cells (i, n=4). Representative images: Day 5 primary organoids (arrows, j). k, Number of secondary organoids per dissociated ISC-derived primary organoid (n=4). l, m, Crypts (l) and ISCs (m) isolated from HFD mice that were reverted to a standard chow diet (HFNC) retained augmented organoid-forming capacity for one week (red; n=4) but not for one month (blue; n=4) when compared to their HFD counterparts (n=6 crypts, n=4 ISCs). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in a,b=20μm, e,j=100μm; histological analysis: a, Olfm4: 10 crypts/group, Crypt4: 50 crypts/group; b, 50 crypts/group in each experiment. ISCs= intestinal stem cells; Olfm4= Olfactomedin 4; Crypt4= Cryptdin 4; C= control; HFD= high fat diet)

Figure 2. Ex vivo exposure of intestinal organoids to palmitic acid recapitulates aspects of a HFD

a, Clonogenicity of naïve crypts cultured with 30μM PA in primary organoid cultures (n=5). Representative images: Day 4 organoids. b, c, Secondary organoid formation of one thousand sorted live primary organoid cells after four weeks of 30μM PA treatment (b, n=3). Representative images: Day 4 secondary organoids (arrows, c). d, e, Frequency (d) and organoid initiation (e) of ISCs (Lgr5-GFP^hi) after four weeks of 30μM PA exposure (d, n=3; e, n=4). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01; scale bars in a=100μm, c=100μm, 50μm (inset). ISCs= intestinal stem cells; V= vehicle; PA= palmitic acid)

Figure 3. Activated PPAR-d in ISCs mediates the effects of a HFD

a, Immunoblots of PPAR-d target proteins in flow sorted ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) from C, HFD, V, and GW mice(n=2). b, Quantification of Olfm4⁺ ISCs (n=4) and Crypt4⁺ Paneth cells (n=3) by in situ hybridization in proximal jejunal crypts. c, BrdU incorporation in ISCs (crypt base columnar cells adjacent to Paneth cells) and progenitors (transit-amplifying cells not adjacent to Paneth cells) after a 4-hour pulse (n=4). d, Organoid per crypt from V and GW-treated mice (n=3). e, Frequencies of flow sorted ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) (n=5) from the entire small intestine of V and GW-treated mice. f, Organoid-initiating capacity of ISCs derived from V and GW-treated mice. Representative images: Day 12 organoids (n=5). g, Frequency of ISCs (Lgr5-GFP^hi) in organoids after 14 days of ex vivo GW exposure (n=3). h, i, j, Primary (h, n=5) and secondary (i, j, n=5; normalized to V) organoid-forming capacity of control and PPAR-d intestinal knockout (IKO) mice upon ex vivo treatment with V, PA, L, and GW. Representative images: Day 4 secondary organoids (j). k, Normalized clonogenicity of human-derived intestinal organoids after ex vivo treatment with PA, L, and GW in secondary culture (n=4, See Materials and Methods). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in b,d=20μm, f=200μm, j=100μm; histological analysis: b, Olfm4: 15 crypts/group, Crypt4: 50 crypts/group; c, 50 crypts/group in each experiment. For western blot source data, see Supplementary Figure 1. ISCs= intestinal stem cells; Olfm4= Olfactomedin 4; Crypt4= Cryptdin 4; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516; PA= palmitic acid; L= lipid mixture)

Figure 4. HFD-induced PPAR-d signaling induces expression of a subset of β-catenin target genes

a, b, Organoid per crypt quantification from HFD-fed (a) and GW-treated (b) mice with indicated concentrations of Wnt3a. Representative images: Day 4 organoids (n=5). c, d, e, f, Violin plots for most induced β-catenin target genes in ISCs (Lgr5-GFP^hi) from HFD-fed (c) and GW-treated mice (d) (24 single cells/group), and in progenitors (Lgr5-GFP^low) from HFD-fed (e) and GW-treated mice (f) (72 single cells/group; See Materials and Methods). (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in a,b=100μm. ISCs= intestinal stem cells; C= control; HFD= high fat diet; V= vehicle; GW= PPAR-d agonist, GW501516)

Figure 5. PPAR-d activation confers organoid and tumor-initiating capacity to non-stem-cells

a, b, Representative spontaneous intestinal tumor from a HFD mouse: gross image (a) and microscopic H&E image (b). c, Incidence of spontaneous intestinal low-grade dysplastic lesions (adenoma) and carcinomas in C (n=19) and HFD (n=25) mice. d, e, Organoid-initiating capacity of progenitors (Lgr5-GFP^low) from HFD (n=7) and GW-treated (n=5) mice. Representative images: Day 7 organoids (e). f, Schematic assessing in vitro and in vivo adenoma-initiating capacity of Apc-null ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) from V and GW-treated mice (Tam=tamoxifen). g, Numbers and representative day 5 images of adenomatous organoids from Apc-null ISCs (Lgr5-GFP^hi) and progenitors (Lgr5-GFP^low) treated +/− GW in EN media (EGF and Noggin only) (n=6). h, Optical colonoscopy of tumors formed after orthotopic transplantation of Apc-null ISCs (Lgr5-GFP^hi) and Apc-null progenitors (Lgr5-GFP^low) from V and GW-treated mice (n=5). i, A model of intestinal adaptation to HFD: mechanistically, HFD activates a PPAR-d-mediated program that augments the organoid and tumor-initiating capacity of intestinal progenitors. A feature of the PPAR-d program includes induction of a subset of β-catenin target genes. S= stem-cell, P= Paneth cell, Pr= progenitor cell, T= tumor cell, red dotted line= Apc-null cells with tumor-forming capability. (Unless otherwise indicated, data reflect mean ± s.d. from n independent experiments; *P<0.05, **P<0.01, ***P<0.001; scale bars in b=50μm, e=200μm, g=200μm (upper) and 50μm (lower). ISCs= intestinal stem cells; C= control; HFD= high fat diet; V=vehicle; GW= PPAR-d agonist, GW501516)