Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche

doi:10.1038/s41586-020-2166-3

. 2020 Apr;580(7804):524-529.

doi: 10.1038/s41586-020-2166-3. Epub 2020 Apr 1.

Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche

Manolis Roulis^#¹, Aimilios Kaklamanos^#², Marina Schernthanner³, Piotr Bielecki³, Jun Zhao^{3

4

5}, Eleanna Kaffe³, Laura-Sophie Frommelt³, Rihao Qu^{3

4

5}, Marlene S Knapp³, Ana Henriques², Niki Chalkidi², Vasiliki Koliaraki², Jing Jiao⁶, J Richard Brewer³, Maren Bacher³, Holly N Blackburn³, Xiaoyun Zhao⁷, Richard M Breyer^{8

9}, Vassilis Aidinis², Dhanpat Jain⁵, Bing Su⁷, Harvey R Herschman⁶, Yuval Kluger^{4

5

10}, George Kollias^{11

12}, Richard A Flavell^{13

14}

Affiliations

¹ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. emmanouil.roulis@yale.edu.
² Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece.
³ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.
⁴ Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
⁵ Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
⁷ Shanghai Institute of Immunology, Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
⁸ Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN, USA.
⁹ Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
¹⁰ Applied Mathematics Program, Yale University, New Haven, CT, USA.
¹¹ Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece. kollias@fleming.gr.
¹² Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece. kollias@fleming.gr.
¹³ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. richard.flavell@yale.edu.
¹⁴ Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA. richard.flavell@yale.edu.

^# Contributed equally.

PMID: 32322056
PMCID: PMC7490650
DOI: 10.1038/s41586-020-2166-3

Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche

Manolis Roulis et al. Nature. 2020 Apr.

. 2020 Apr;580(7804):524-529.

doi: 10.1038/s41586-020-2166-3. Epub 2020 Apr 1.

Authors

Affiliations

¹ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. emmanouil.roulis@yale.edu.
² Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece.
³ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.
⁴ Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
⁵ Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
⁷ Shanghai Institute of Immunology, Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
⁸ Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN, USA.
⁹ Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
¹⁰ Applied Mathematics Program, Yale University, New Haven, CT, USA.
¹¹ Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece. kollias@fleming.gr.
¹² Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece. kollias@fleming.gr.
¹³ Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. richard.flavell@yale.edu.
¹⁴ Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA. richard.flavell@yale.edu.

^# Contributed equally.

PMID: 32322056
PMCID: PMC7490650
DOI: 10.1038/s41586-020-2166-3

Abstract

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts¹. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types^2,3. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E₂ (PGE₂). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE₂ drives the expansion οf a population of Sca-1⁺ reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1⁺ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE₂ promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1⁺ cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE₂-Ptger4. Analyses of patient-derived organoids established that PGE₂-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE₂-Ptger4-Yap signalling axis.

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Figures

**Extended Data Fig. 1 |. Mesenchymal *Ptgs2* expression in the microenvironment of crypt stem cells.**
a, Transmission electron microscopy photograph of the base of a mouse ileum crypt. P, Paneth cell; S, columnar basal stem cell; F, fibroblast. Scale bar, 5 μm. Indicative of independent observations in two experiments. b, Immunostaining for Lgr5–eGFP and Vimentin in the ileum of an *Lgr5*-*eGFP*-*IRES*-*creERT2* mouse. Scale bar, 20 μm. Indicative of independent observations in one experiment. c, *PTGS2* relative gene expression (RE) in intestinal epithelial cells (IECs) and stromal cells isolated from healthy human colonic tissues (n = 6 individuals). Statistical comparison performed with two-tailed Wilcoxon matched-pairs signed-rank test. d, *Ptgs2* gene expression in IECs and stromal cells isolated from the ileum and the colon of wild-type mice (n = 4). Statistical significance was determined by two-tailed paired t-test. e, Biological replicates of the Drop-seq experiment shown in Fig. 1a visualized on the respective t-SNE plot depicting n = 3,179 mesenchymal cells. Mesenchymal cells were independently isolated from two groups of wild-type mice (biological replicates 1 and 2). From each of these isolations up to three independent Drop-seq samples were collected (A to C) for a total of five samples. f, All *Ptgs2*-expressing single cells (n = 1,136) detected in the experiment shown in Fig. 1a, c were analysed separately and re-clustered. Cluster annotations are visualized on a t-SNE plot. Violin plots display the entire distribution of gene expression levels per single cell in each cluster for key mesenchymal marker genes. F, fibroblasts. g, Schematic representation of the arachidonic acid metabolism pathway. For each mesenchymal cluster shown in Fig. 1a, violin plots display the entire distribution of gene expression levels per single cell for six genes involved in the metabolism of arachidonic acid to prostanoids. Data from n = 3,179 single mesenchymal cells are shown. h, Analysis of single-cell RNA-seq data (GSE11434) from the healthy human colonic mesenchyme. Clustering results for n = 4,348 cells and cluster annotations are visualized on a t-SNE plot. The annotations of stromal populations are matched with the ones reported by Kinchen et al. on the basis of the respective markers. Expression levels of *PTGS2* per single cell are visualized on a t-SNE plot. The entire range of gene expression levels per single cell for *PTGS1*, *PTGS2* and key mesenchymal marker genes is displayed in violin plots. Data are mean ± s.e.m.; ns, non-significant; *P < 0.05; **P < 0.01.

**Extended Data Fig. 2 |. Location of major fibroblast populations in the mouse intestine.**
a, Detection of *Pdgfra*-expressing mesenchymal cells in the intestine of adult *Pdgfra*-*H2B*-*eGPF*-knockin mice. Two distinct populations of Pdgfra^high and Pdgfra^low mesenchymal cells were detected in fixed tissue sections by direct eGFP fluorescence (green) and confocal microscopy. Nuclei are stained with DAPI (blue). Pdgfra^high cells are located under the epithelium along the crypt–villus axis and in the muscularis propria. They form clusters at the tips of villi and the apical part of the colonic mucosa. Pdgfra^low cells are located in the inner part of the villi, the pericryptal area and the submucosa. Filled arrows indicate pericryptal Pdgfra^low cells. Open arrows indicate subepithelial Pdgfra^high cells. M, mucosa; V, villus; SM, submucosa; MP, muscularis propria. Scale bars, 20 μm. Data are representative of six independent experiments. b, Detection of Pdgfra^high and Pdgfra^low fibroblasts in the fresh, intact intestine of adult *Pdgfra*-*H2B*-*eGPF*-knockin mice by two-photon microscopy. The cells were detected by direct eGFP fluorescence (green). Pdgfra^high cells are predominant in the muscularis propria, whereas Pdgfra^low cells are predominant in the submucosa. Both populations are present in the mucosa. Data are representative of independent observations from one experiment. Scale bars, 100 μm. c, Detection of Pdgfra^high and Pdgfra^low fibroblasts in the intestine of *Pdgfra*-*H2B*-*eGPF*-knockin embryos on embryonic day 15 (E15.0) and in early postnatal development. E15.0: clusters of Pdgfra^high cells in early villi are indicated by white arrows. Pdgfra^low mesenchymal cells occupy the rest of the mesenchyme (asterisks). P0: Pdgfra^high cells are observed in the villi (V) and Pdgfra^low cells are observed both in the villi and in the rest of the mesenchyme (asterisks). P15: Pdgfra^low cells surround an early crypt (C) and Pdgfra^high cells are located at the edges of the crypt (open white arrows). Pdgfra^low cells occupy the inner mesenchyme (asterisks). Data are representative of independent observations from one experiment per developmental stage. Scale bars, 20 μm. d, The location of *Fgfr2*-expressing mesenchymal cells was determined in the intestine of an *Fgfr2*-*T2A*-*H2B*-*mCherry*-knockin mouse, by detecting direct mCherry fluorescence (red) in the nucleus (blue, DAPI). The arrows indicate pericryptal Fgfr2⁺ fibroblasts. Data are representative of independent observations from one experiment. Scale bars, 20 μm. e, Immunostaining for laminin A1 (encoded by *Lama1*), the epithelial marker E-cadherin and the mesenchymal marker vimentin in the normal mouse intestines shows that laminin A1 is detected specifically at the mesenchymal–epithelial interface. Data are representative of two independent experiments. Scale bars, 5 μm. f, In-situ hybridization analysis showing the location of *Rspo1*-expressing cells in the normal mouse colon. The position of *Rspo1*-expressing cells along the crypt axis was quantified in 40 × 80 μm² sub-epithelial areas at the base, middle and top sections of n = 9 crypts. Unpaired two-tailed Student’s t-test. Mean ± s.e.m. **P < 0.01.

**Extended Data Fig. 3 |. Mice with fibroblast-specific ablation or fibroblast-restricted expression of Ptgs2.**
a, Immunofluorescence of ileum and colon sections from *Col6-Cre-Rosa26*^tdTomato/+ mice (scale bar, 20 μm) and of a small intestinal tumour section from an *Apc*^Min/+*-Col6-Cre-Rosa26*^tdTomato/+ mouse (scale bar, 150 μm). Data are representative of two experiments. b, Efficiency of *Col6-Cre*-mediated recombination of a lox-stop-lox tdTomato reporter in Pdgfra^high and Pdgfra^low Cd45⁻ cells determined by flow cytometry in intestinal mesenchymal and lamina propria cells isolated from the small intestine and the colon of *Col6-Cre-Rosa26*^tdTomato/+*Pdgfra*^eGFP/+ mice in one experiment. c, *Ptgs2* gene expression in whole tissue, isolated IECs, FACS-sorted *Col6Cre*⁺ fibroblasts (CD45⁻tdTomato⁺) and *Col6Cre*⁻ mesenchymal cells (CD45⁻tdTomato⁻) from the small intestine of *Col6-Cre-Rosa26*^tdTomato/+ mice (n = 3, pooled). Representative of two experiments. d, Efficiency of *Col6-Cre*-mediated *Ptgs2* gene ablation in Col6-Cre⁺ mesenchymal cells determined by RT–qPCR analysis of *Ptgs2* expression in FACS-sorted *Col6-Cre*⁺ fibroblasts (eGFP⁺) from the small intestine of *Col6-Cre-Rosa26*^mT/mGPtgs2^f/+ (n = 3) and *Col6-Cre-Rosa26*^mT/mGPtgs2^f/f (n = 3) mice. Unpaired two-tailed Welch’s t-test. e, Expression of the *Ptgs2* gene in whole tissue ileum of littermate *Ptgs2*^f/f and *Ptgs2*^ΔFibr mice (n = 7 each). Two-tailed t-test. f, Spleen weight of 5.5-month-old *Apc*^Min/+*Ptgs2*^f/f (n = 8) and *Apc*^Min/+*Ptgs2*^ΔFibr (n = 6) mice. Average spleen weight of (n = 6) normal littermates (*Ptgs2*^f/f) is displayed for comparison. Two-tailed t-test. g, Survival analysis of *Apc*^Min/+*Ptgs2*^f/f (n = 12) and *Apc*^Min/+*Ptgs2*^ΔFibr (n = 12) mice. A two-tailed P = 0.00009687 was calculated by log-rank test. h, Size of 274 adenomas from 5.5-month-old *Apc*^Min/+*Ptgs2*^f/f (n = 16) and *Apc*^Min/+*Ptgs2*^ΔFibr (n = 18) mice. The whiskers extend from minimum to maximum and the box extends from the 25th to 75th percentiles with the median indicated. Two-tailed Mann–Whitney test. i, Generation of knockin mice bearing a lox-stop-lox cassette insertion in intron-3 of the *Ptgs2* gene which prevents its expression (*Ptgs2*^OFF). *Col6-Cre*-mediated excision of the lox-stop-lox cassette reactivates *Ptgs2* expression specifically in fibroblasts (*Ptgs2*^FibrON). The orange box depicts an frt site remaining from the flp-mediated removal of an frt-flanked PGK-neomycin selection cassette (see Methods). j, *Ptgs2*^f/f (n = 30) and *Ptgs2*^ΔFibr (n = 24) mice were subjected to 10 weekly intraperitoneal injections with 10 mg kg⁻¹ azoxymethane as displayed. Quantification of the number of dysplastic foci and microadenomas per mouse and quantification of tumour size is shown. Statistical significance was tested by two-tailed Mann–Whitney test. k, Quantification of intestinal epithelial populations in the ileum of littermate *Ptgs2*^f/f and *Ptgs2*^ΔFibr mice (n = 3–5 per genotype). Immunostaining was performed for markers of Paneth cells (lysozyme), tuft cells (Dclk1), enteroendocrine cells (chromogranin A) and stem cells (Olfm4). Goblet cells were identified by periodic acid Schiff (PAS) staining and enterocytes were identified by detecting alkaline phosphatase enzymatic activity. Incorporation and immunohistochemical detection of BrdU was used to determine the numbers of cycling cells. Data for each mouse represent mean number of positive cells per crypt or crypt–villus unit as indicated. N = 400–822 crypts and villi were evaluated per staining. Statistical comparisons were performed with two-tailed unpaired t-test except for Olfm4⁺ cells for which unpaired t-test with Welch’s correction was applied. Scale bars, 50 μm. All data represent mean ± s.e.m. unless otherwise indicated. ns, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001.

**Extended Data Fig. 4 |. PGE₂-driven spheroids contain more functional stem cells.**
a, Crypts isolated from the small intestine of wild-type mice were grown into organoids by 3D culture with OGM or OGM that was supplemented daily with 0.1 μM 16,16-dimethyl PGE₂ (dmPGE₂). Indicative images and quantification of the absolute numbers of organoids and spheroids grown per 3D structure are shown. n = 6 3D cultures were evaluated per condition. Scale bar, 100 μm. b, Assessment of stem cell activity in organoids or PGE₂-driven spheroids grown as in a by dissociation into single cells and 3D culture in OGM. Growth of crypts and organoids from the same initial number of cells was quantified on day 14. The results are indicative of five independent experiments starting from independent crypt isolations. c, Normal crypts were grown into organoids with OGM in a 3D co-culture with primary mouse intestinal fibroblasts with or without 10 μM ONO-AE3–208 (Ptger4/EP4 inhibitor). Indicative images and quantification of the absolute numbers of organoids and spheroids grown per 3D structure are shown. n = 6 3D co-cultures were evaluated per condition. Scale bar, 200 μm. d, Separation of n = 2,192 fibroblasts and epithelial cells in single-cell RNA-seq data from fibroblast–crypt organotypic cultures on the basis of the expression of key marker genes. Expression of intestinal epithelial marker genes (*Epcam*, *Atp1b1* and *Krt8*) and fibroblast marker genes (*Sparc*, *Col1a1* and *Col3a1*) in single cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures is shown projected onto t-SNE plots. **e–j**, Single-cell data from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures as shown in Fig. 3d, visualized on the respective t-SNE plot depicting n = 1,585 epithelial cells. e, Expression of epithelial population-specific signatures (metagenes) per single epithelial cell. Population signatures were calculated on the basis of single-cell profiling of the mouse intestinal epithelium. f, Cell cycle analysis of single epithelial cells projected onto the t-SNE plot. **g–j**, Expression levels of metagenes for the signatures or transcriptional programs of RSC (g), β-catenin (h), Yap (i) and early (non-tumour) *Apc*^min/+ (j) tumorigenesis per single epithelial cell projected onto t-SNE plots. k, Data from n = 1,585 single epithelial cells, visualized in violin plots for each co-culture condition (Ptger4-ON or Ptger4-OFF). The entire range of metagene expression levels per single epithelial cell for the signatures or transcriptional programs of RSCs, β-catenin, Yap and early *Apc*^min/+ tumorigenesis is displayed. In a, c, two-way ANOVA. Data are mean ± s.e.m. ****P < 0.0001.

**Extended Data Fig. 5 |. Expression of PGE₂ receptors in mouse and human tissues.**
a, RT–qPCR analysis for *Ptger1*, *Ptger2*, *Ptger3* and *Ptger4* genes across 12 mouse tissues. Expression relative to *B2m* is displayed as 2^−ΔCt. Data represent one experiment. b, RT–qPCR analysis for *Ptger1*, *Ptger2*, *Ptger3* and *Ptger4* genes in isolated IECs and matched stromal fractions from the small intestine (ileum) and the colon of wild-type mice (n = 4). Statistical comparisons were performed by two-tailed paired t-test. c, Expression levels of *Ptger1*, *Ptger2*, *Ptger3* and *Ptger4* genes determined by RNA-seq in FACS-sorted intestinal epithelial cell populations in 2 or 3 biological replicates and displayed as FPKM (fragments per kilobase of transcript per million mapped reads). Data retrieved from the GSE83394 GEO dataset. d, Expression levels of the human *PTGER1*, *PTGER2*, *PTGER3* and *PTGER4* genes in matched normal colon and tumour tissues from colorectal cancer patients (n = 41), determined by RNA-seq and displayed as FPKM. Data retrieved from The Cancer Genome Atlas for colon adenocarcinoma (TCGA–COAD dataset). Statistical comparisons were performed by two-tailed Wilcoxon matched-pairs signed-rank test. e, Analysis of single-cell RNA-seq data (GSE117783) from crypts isolated from the small intestine of normal mice (blue) and mice treated with 12 Gy irradiation (red). n = 6,644 single cells are visualized on t-SNE plots based on the experimental condition (normal, n = 2,882; irradiated, n = 3,762) and the clustering results. Violin plots represent the entire distribution of *Ptger4* expression levels per single cell in each cluster and in each condition. The annotations of epithelial populations are matched with the ones reported by Ayyaz et al. on the basis of the respective markers. **b–d**, Mean ± s.e.m.; ns, non-significant; *P < 0.05; **P < 0.01.

**Extended Data Fig. 6 |. PGE₂–Ptger4 drive the induction of Yap target genes in intestinal organoids.**
a, Volcano plot displaying the results of differential gene-expression analysis performed in single epithelial cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures (n = 1,585). *Yap1* and Yap target genes are indicated. Moderated t-test with false-discovery rate (Benjamini–Hochberg) correction. b, Expression levels of the genes indicated in single epithelial cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures (n = 1,585), projected onto t-SNE plots. c, Experimental setup for data shown in d, e. Crypts were grown into organoids or spheroids by 3D culture in OGM or OGM that was supplemented daily with 0.1 μM dmPGE₂ for 7 days. Gene expression levels were measured by RT–qPCR on day 7. d, Relative expression of Yap target genes (*Ly6a*, *Clu*, *Il1rn*, *Msln* and *Cxcl16*) in day 7 organoids and PGE₂-driven spheroids developed from wild-type crypts. N = 3 3D cultures per condition. Two-tailed Welch’s t-test. e, Relative expression of Yap target genes in day 7 organoids and PGE₂-driven spheroids developed from crypts isolated from *Ptger4*^f/f and *Ptger4*^ΔIEC mice. n = 3 3D cultures per genotype and condition. One-way ANOVA. f, Correlation between the expression levels of metagenes of a Yap transcriptional program and an early (non-tumour) *Apc*^min/+ tumorigenesis transcriptional program in single epithelial cells (n = 1,585) from the Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures of Fig. 3. g, Small intestinal crypts were grown into organoids or spheroids with OGM or OGM that was supplemented daily with 0.1 μM dmPGE₂. Western blot analysis for Yap1 and β-actin was performed in total lysates from untreated organoids, organoids treated with 0.1 μM dmPGE₂ for 16 h and untreated spheroids. Data from one organoid and three independent spheroid cultures. h, Relative expression of the *Yap1* gene and Yap target genes in wild-type organoid cultures treated with 0.1 μM dmPGE₂ for 13 h, as determined by RT–qPCR. n = 3–5 cultures per condition. Statistical comparisons were performed with unpaired two-tailed t-test. For *Ly6a*, Welch’s correction was applied. i, Western blot analysis for Ser127 pYap and total Yap performed in total lysates from wild-type organoids stimulated with 0.1 μM dmPGE₂ for the indicated time-points. Indicative of five independent experiments. j, Relative expression of Yap target genes in wild-type organoids treated with 1 μM verteporfin and 0.1 μM dmPGE₂ for 13 h. n = 3–4 cultures per condition. Statistical comparisons were performed with unpaired two-tailed t-test, two-tailed Welch’s t-test or Mann–Whitney test on the basis of the criteria described in Methods. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

**Extended Data Fig. 7 |. Genetic ablation of Yap prevents spheroid formation and Sca-1⁺ stem cell expansion in fibroblast–crypt organotypic co-cultures.**
a, Crypts isolated from the small intestines of *Yap1*^f/f and *Yap1*^ΔIEC mice were grown into organoids by 3D culture with OGM supplemented with 0.5 mg ml⁻¹ recombinant mouse epiregulin (Ereg) as previously described, or in a co-culture with wild-type primary mouse intestinal fibroblasts with OGM without Ereg supplementation. Indicative images and quantification of the percentages of crypts, organoids and spheroids grown per 3D structure are shown. n = 2 cultures per condition. Data are representative of two independent experiments. Scale bars, 100 μm. Two-way ANOVA. Data represent mean ± s.e.m. ****P < 0.0001. b, Intestinal crypts isolated from the small intestines of *Yap1*^f/f and *Yap1*^ΔIEC mice were co-cultured with wild-type primary mouse intestinal fibroblasts. On day 4, these co-cultures and control *Yap1*^f/f organoid cultures were processed into single-cell suspensions and analysed by flow cytometry for Sca-1 expression in Cd24⁺ epithelial cells. n = 2 cultures per condition. Scale bars, 100 μm. FSC, forward scatter. Unpaired two-tailed t-test. Mean ± s.e.m. ***P < 0.001.

**Extended Data Fig. 8 |. Ptger4 ablation does not affect epithelial lineage differentiation and stem cell function.**
a, *Ptger4* gene expression in crypts isolated from the ileum of littermate *Ptger4*^f/f and *Ptger4*^ΔIEC mice (n = 3 mice per genotype) and in organoids grown from these crypts (n = 3 cultures per genotype) determined by RT–qPCR analysis. Two-tailed unpaired t-test. b, Single-cell RNA-seq (Drop-seq) was performed in crypt epithelial cells isolated from littermate *Ptger4*^f/f and *Ptger4*^ΔIEC mice. Data for 2,439 single epithelial cells are shown in a t-SNE plot. c, Biological replicates visualized on a t-SNE plot. Crypt epithelial cells were independently isolated from two groups of mice per genotype (biological replicates 1 and 2). From the first biological replicate, two independent Drop-seq samples were collected (A and B) for a total number of three samples per genotype. d, Clustering and cluster assignments of 2,439 single epithelial cells displayed on a t-SNE plot. e, Proportion of each epithelial cluster among total crypt epithelial cells in *Ptger4*^f/f and *Ptger4*^ΔIEC mice. f, Violin plots showing the entire range of expression levels for a metagene of the β-catenin transcriptional program in n = 2,439 single epithelial cells from *Ptger4*^f/f and *Ptger4*^ΔIEC mice. g, Analysis of all *Ptger4*-expressing single cells detected (n = 478). Re-clustering results of *Ptger4*-expressing single cells with cluster annotations are visualized on a t-SNE plot. The expression levels of key marker genes for these clusters are visualized on t-SNE plots. h, Lineage tracing of *Ptger4* heterozygous (*Ptger4-*HET) and *Ptger4*-knockout (*Ptger4-*KO) Lgr5⁺ stem cells. The small intestines of *Lgr5-CreERT2-Rosa26*^tdTomato/+*Ptger4*^f/+ (*Ptger4-*HET) and *Lgr5-CreERT2-Rosa26*^tdTomato/+*Ptger4*^f/f (*Ptger4-*KO) mice were examined for direct tdTomato fluorescence 5 days after a single injection of 2 mg tamoxifen per mouse. The results shown are representative of independent observations from one experiment. Scale bars, 70 μm. i, Quantification of intestinal epithelial populations in the ileum of littermate *Ptger4*^f/f (n = 5) and *Ptger4*^ΔIEC (n = 5) mice. Immunostaining was performed for markers of Paneth cells (lysozyme), tuft cells (Dclk1), enteroendocrine cells (chromogranin A) and stem cells (Olfm4). Scale bars, 20 μm. Goblet cells were identified by PAS staining and enterocytes were detected by alkaline phosphatase enzymatic activity. Scale bars, 50 μm. Incorporation and immunohistochemical detection of BrdU was used to determine the numbers of cycling cells. Scale bars, 50 μm. Data for each mouse represent mean number of positive cells per crypt or crypt–villus unit as indicated. n = 217–565 crypts and villi were evaluated per staining. Statistical comparisons were performed with two-tailed unpaired t-test except for PAS⁺ cells, for which unpaired Welch’s t-test was applied. Mean ± s.e.m.; ns, non-significant; **P < 0.01.

**Extended Data Fig. 9 |. Nuclear localization of Yap and activation of Yap target genes in *Apc*^Min/+ and azoxymethane-induced tumorigenesis.**
a, Immunostaining for Yap in the small intestine of five-month-old *Apc*^Min/+ and wild-type littermate control mice. Nuclear localization of Yap is displayed on the basis of colocalization with DAPI. Normal (N) and Tumour (T) areas of the *Apc*^Min/+ intestine are indicated. Scale bars, 70 μm. Data are indicative of at least ten different tumour areas. b, Immunostaining for Sca-1 and the epithelial marker E-cadherin in normal and tumour areas of the small intestine of five-month-old *Apc*^Min/+ mice. Indicative of two independent experiments. c, Immunostaining for Yap in the colon of wild-type mice subjected to 10 weekly intraperitoneal injections with 10 mg kg⁻¹ azoxymethane as indicated and in untreated controls. Nuclear localization of Yap is displayed on the basis of colocalization with DAPI. Scale bars, 20 μm. Data indicative of three mice analysed. d, Relative expression of the Yap target gene *Clu* in normal and tumour areas of the colon of wild-type mice (n = 8) subjected to 10 weekly intraperitoneal injections with 10 mg kg⁻¹ azoxymethane as shown in c. Two-tailed Mann–Whitney test. e, Relative expression of *Yap1* and Yap target genes in the small intestine of 5-week-old *Ptger4*^f/f (n = 3), *Apc*^Min/+*Ptger4*^f/f (n = 6) and *Apc*^Min/+*Ptger4*^ΔIEC (n = 8) mice. Statistical comparisons were performed with two-tailed t-test for *Yap1* and *Il1rn* and with two-tailed Mann–Whitney test for *Ly6a*. f, Spleen weight of 5.5-month-old *Apc*^Min/+*Ptger4*^f/f (n = 8) and *Apc*^Min/+*Ptger4*^ΔIEC (n = 7) mice. Average spleen weight of (n = 2) normal littermates (*Ptger4*^f/f) is displayed for comparison. Two-tailed t-test. g, Size of 72 adenomas from 5.5-month-old *Apc*^Min/+*Ptger4*^f/f (n = 6) and *Apc*^Min/+*Ptger4*^ΔIEC (n = 4) mice. The whiskers extend from minimum to maximum and the box extends from the 25th to 75th percentiles with the median indicated. Two-tailed Mann–Whitney test. Mean ± s.e.m.; ns, non-significant; *P < 0.05; **P < 0.01.

**Extended Data Fig. 10 |. PGE ₂–PTGER4 controls stem cell function in human colonic crypts and YAP displays a nuclear localization in human colorectal tumours.**
a, Human colonic crypts were grown into organoids by 3D culture with OGM or OGM supplemented daily with 0.1 μM dmPGE₂, with or without 10 μM ONO-AE3–208 (PTGER4–EP4 inhibitor). Images indicative of three independent experiments with crypts isolated from three patients are shown. Scale bar, 100 μm. b, Immunostaining for YAP in sections of human colorectal adenomas or adenocarcinomas and neighbouring normal tissue areas. Nuclear localization of Yap is displayed on the basis of colocalization with DAPI. Clearly defined normal (N) and tumour (T) areas are indicated wherever applicable. Images shown are representative of specimens obtained and analysed from n = 16 patients with the types of colorectal tumours indicated. Patient characteristics and the type of colorectal tumour per individual are described in the Supplementary Table 3. c, Schematic representation of the mechanism proposed in the present study. TISC, tumour-initiating stem cell.

**Fig. 1 |. Single-cell analyses of the intestinal mesenchyme reveal a rare fibroblast population that expresses *Ptgs2* and its protein product Cox-2, located under the crypts.**
a–c, Single-cell RNA-seq of 3,179 mesenchymal cells from the normal mouse colon. a, t-distributed stochastic neighbour embedding (t-SNE) plot with clustering results. F, fibroblasts. MF, myofibroblasts. b, Violin plots showing the entire range of mesenchymal marker gene expression levels per single cell in each cluster. SMC, smooth muscle cells. c, *Ptgs2* expression levels per single cell visualized by t-SNE plot. d, Immunostaining for Cox-2 and Vimentin in the normal mouse ileum and colon. Cox-2-expressing fibroblasts are located under the crypt (C) epithelium (white arrowheads) and in the muscularis propria (MP). Results are representative of three independent experiments. Scale bars, 20 μm.

**Fig. 2 |. *Ptgs2*-expressing fibroblasts drive tumour initiation by secreting PGE₂ in the crypt microenvironment.**
a, Number of β-catenin⁺ microadenomas in two sections of the small intestine (SI) of 5-week-old *Apc*^Min/+*Ptgs2*^f/f (n = 6) and *Apc*^Min/+*Ptgs2*^ΔFibr (n = 6) mice. Two-tailed t-test. b, Number of macroscopic adenomas in 5.5-month-old *Apc*^Min/+*Ptgs2*^f/f (n = 16) and *Apc*^Min/+*Ptgs2*^ΔFibr (n = 23) mice. Two-tailed Mann–Whitney test. c, Number of macroscopic adenomas in 5.5-month-old *Apc*^Min/+*Ptgs2*^OFF (n = 7) mice in which *Ptgs2* expression is blocked, and *Apc*^Min/+*Ptgs2*^FibrON (n = 11) littermates in which *Ptgs2* is exclusively expressed in fibroblasts (Extended Data Fig. 3i). Two-tailed Mann–Whitney test (duodenum), t-test (jejunum and colon), Welch’s t-test (ileum and total small intestine). d, Incidence of dysplasia and microadenoma development in the colon of *Ptgs2*^f/f (n = 30) and *Ptgs2*^ΔFibr (n = 24) mice treated with 10 weekly injections of azoxymethane (AOM). Two-sided Fisher’s exact test. e, HPLC–MS/MS analysis of prostanoids in the ileum of littermate *Ptgs2*^f/f (n = 9) and *Ptgs2*^ΔFibr (n = 7) mice. IS, internal standard. Two-tailed t-test (PGE₂ and PGF_2a) and Mann–Whitney test (PGI₂ and PGD₂). Data are mean ± s.e.m. NS, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

**Fig. 3 |. Fibroblast-derived PGE₂ drives the expansion of RSCs with a Yap-driven pro-tumorigenic program via receptor Ptger4.**
a, Indicative 3D cultures of crypts isolated from *Ptger4*^f/f and *Ptger4*^ΔIEC mice grown with OGM or OGM supplemented with 0.1 μM dmPGE₂. Scale bar, 100 μm. b, Normal crypts were cultured as 3D organoids in OGM (n = 2) or in co-cultures with primary mouse intestinal fibroblasts (n = 3). The absolute numbers of organoids or spheroids per 3D culture are shown. Results are representative of five independent experiments. Scale bar, 200 μm. c, Crypts isolated from *Ptger4*^f/f and *Ptger4*^ΔIEC mice were grown in co-cultures with wild-type (WT) primary mouse intestinal fibroblasts (n = 3 per genotype). The percentage of organoids and spheroids grown per 3D culture is shown. Results are representative of two independent experiments. Scale bar, 100 μm. In b, c, data are mean ± s.e.m.; two-way ANOVA, ****P < 0.0001. d–l, Single-cell RNA-seq of intestinal crypt–fibroblast co-cultures grown in OGM with 10 μM Ptger4 inhibitor (Ptger4-OFF) or DMSO (Ptger4-ON). Analyses are shown for 1,585 epithelial cells. d, t-SNE plot indicating epithelial cells from co-cultures with Ptger4-ON or Ptger4-OFF. e, t-SNE plot with clustering results. f, Proportion of each epithelial cluster among total epithelial cells in co-cultures with Ptger4-ON or Ptger4-OFF. g–l, Violin plots showing the entire range of metagene expression levels per single cell per cluster for the signatures/transcriptional programs of stem cells (g), tuft cells (h), RSCs (i), β-catenin (j), Yap (k) and early (non-tumour) *Apc*^min/+ tumorigenesis (l).

**Fig. 4 |. Epithelial Ptger4 induces Yap nuclear translocation, mediates RSC mobilization and drives tumour initiation.**
a, b, Wild-type organoids pretreated with or without 10 μM Ptger4 inhibitor were stimulated with 0.1 μM dmPGE₂.Western blots for phosphorylated Yap Ser127 (pYap) and total Yap in cytoplasmic lysates (a), Yap and TBP in nuclear lysates (b). Results are representative of two experiments. c, d, Relative expression of Yap target genes. c, Wild-type organoids treated with 10 μM Ptger4 inhibitor and 0.1 μM dmPGE₂ for 13 h. n = 3 to 4 cultures per condition. One-way ANOVA. d, *Yap1*^ΔIEC and *Yap1*^f/f organoids treated with 0.1 μM dmPGE₂ for 13 h. Three cultures per genotype per condition. Two-tailed t-test. e, f, *Ptger4*^f/f (n = 3) and *Ptger4*^ΔIEC (n = 3) mice received 14 Gy of abdominal irradiation. On day 3, the ileum was analysed by haematoxylin and eosin staining (e) and immunostaining for lysozyme (f). Results are representative of three independent experiments. Scale bars, 50 μm. g, Immunostaining for Yap in the small intestine of five-week-old wild-type and *Apc*^Min/+ mice. Nuclear Yap is evaluated on the basis of colocalization with DAPI. Results are indicative of at least eight microadenomas. h, Immunostaining for Sca-1 in the small intestine of five-week-old wild-type and *Apc*^Min/+ mice. Results are representative of two independent experiments. i, k, Quantification of areas of the small intestine with expansion of Sca-1⁺ epithelial cells in *Apc*^Min/+*Yap1*^f/f (n = 3) and *Apc*^Min/+*Yap1*^ΔIEC (n = 5) mice (i), and *Apc*^Min/+*Ptger4*^f/f (n = 7), *Apc*^Min/+*Ptger4*^ΔIEC (n = 5) mice (k). Two-tailed t-test. j, l, Number of BrdU⁺ microadenomas per small-intestinal section of *Apc*^Min/+*Yap1*^f/f (n = 4) and *Apc*^Min/+*Yap1*^ΔIEC (n = 5) mice (j), and *Apc*^Min/+*Ptger4*^f/f (n = 5) and *Apc*^Min/+*Ptger4*^ΔIEC (n = 7) mice (l). Two-tailed Mann–Whitney test (j); two-tailed t-test (l). m, Number of macroscopic adenomas in 5.5-month-old *Apc*^Min/+*Ptger4*^f/f (n = 9) and *Apc*^Min/+*Ptger4*^ΔIEC (n = 8) mice. Two-tailed t-test (duodenum and colon); Welch’s t-test (jejunum and total small intestine); Mann–Whitney test (ileum). Data are mean ± s.e.m.

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Fibroblasts orchestrate tumour initiation.
Seton-Rogers S. Seton-Rogers S. Nat Rev Cancer. 2020 Jun;20(6):301. doi: 10.1038/s41568-020-0264-z. Nat Rev Cancer. 2020. PMID: 32317743 No abstract available.
Fibroblasts fuel intestinal tumorigenesis.
Wang D, DuBois RN. Wang D, et al. Cell Res. 2020 Aug;30(8):635-636. doi: 10.1038/s41422-020-0340-7. Cell Res. 2020. PMID: 32494022 Free PMC article. No abstract available.

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References

1. Vermeulen L & Snippert HJ Stem cell dynamics in homeostasis and cancer of the intestine. Nat. Rev. Cancer 14, 468–480 (2014). - PubMed
1. Powell DW, Pinchuk IV, Saada JI, Chen X & Mifflin RC Mesenchymal cells of the intestinal lamina propria. Annu. Rev. Physiol. 73, 213–237 (2011). - PMC - PubMed
1. Kinchen J et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386 (2018). - PMC - PubMed
1. Hamilton TG, Klinghoffer RA, Corrin PD & Soriano P Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol. Cell. Biol. 23, 4013–4025 (2003). - PMC - PubMed
1. Molotkov A, Mazot P, Brewer JR, Cinalli RM & Soriano P Distinct requirements for FGFR1 and FGFR2 in primitive endoderm development and exit from pluripotency. Dev. Cell. 41, 511–526 (2017). - PMC - PubMed

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[1] Vermeulen L & Snippert HJ Stem cell dynamics in homeostasis and cancer of the intestine. Nat. Rev. Cancer 14, 468–480 (2014). - PubMed

[2] Vermeulen L & Snippert HJ Stem cell dynamics in homeostasis and cancer of the intestine. Nat. Rev. Cancer 14, 468–480 (2014). - PubMed

[3] Powell DW, Pinchuk IV, Saada JI, Chen X & Mifflin RC Mesenchymal cells of the intestinal lamina propria. Annu. Rev. Physiol. 73, 213–237 (2011). - PMC - PubMed

[4] Powell DW, Pinchuk IV, Saada JI, Chen X & Mifflin RC Mesenchymal cells of the intestinal lamina propria. Annu. Rev. Physiol. 73, 213–237 (2011). - PMC - PubMed

[5] Kinchen J et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386 (2018). - PMC - PubMed

[6] Kinchen J et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386 (2018). - PMC - PubMed

[7] Hamilton TG, Klinghoffer RA, Corrin PD & Soriano P Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol. Cell. Biol. 23, 4013–4025 (2003). - PMC - PubMed

[8] Hamilton TG, Klinghoffer RA, Corrin PD & Soriano P Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol. Cell. Biol. 23, 4013–4025 (2003). - PMC - PubMed

[9] Molotkov A, Mazot P, Brewer JR, Cinalli RM & Soriano P Distinct requirements for FGFR1 and FGFR2 in primitive endoderm development and exit from pluripotency. Dev. Cell. 41, 511–526 (2017). - PMC - PubMed

[10] Molotkov A, Mazot P, Brewer JR, Cinalli RM & Soriano P Distinct requirements for FGFR1 and FGFR2 in primitive endoderm development and exit from pluripotency. Dev. Cell. 41, 511–526 (2017). - PMC - PubMed

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Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche

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