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Apelin-driven endothelial cell migration sustains intestinal progenitor cells and tumor growth

Nature Cardiovascular Research volume 1, pages 476–490 (2022)Cite this article

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Abstract

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. Vascular endothelial growth factor A (VEGFA) is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation-induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells toward progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.

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Fig. 1: Low VEGFA signaling and normoxia are hallmarks of Wnthigh CRC.
Fig. 2: Normal and transformed stem/progenitor cells are maintained in a normoxic VEGFA-independent niche.
Fig. 3: Wnthigh CRC stem/progenitor cells are maintained in a normoxic VEGFA-independent niche.
Fig. 4: Acute progenitor expansion induces endothelial apelin expression.
Fig. 5: Apelin-mediated cryptward endothelial migration maintains crypt venous capillary patency.
Fig. 6: Apelin is necessary for crypt perfusion and progenitor cell maintenance.
Fig. 7: APLN promotes Wnthigh CRC growth by ensuring progenitor cell normoxia.

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Data availability

All data generated or analyzed are included in the main article and associated files. Gene expression profiling data were deposited in a Super Series (GSE178118) including both the intestinal blood endothelial (GSE111783) and epithelial cell data (GSE178110). Source data are provided with this paper.

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Acknowledgements

We thank N. Piazzon (Institute of Pathology Biobank, CHUV and University of Lausanne, Lausanne, Switzerland) for providing human tumor samples, M. Lutolf for providing Rspo1-Fc, A. Zuba, M. Bruand and M. LeCoultre for participation in the initial stages of this work, C. Beauverd for mouse genotyping and colony maintenance and S. Robine and L. Claesson-Welsh for providing Villin-CreERT2 and Kdrfl/fl; Cdh5-CreERT2 mice. The UNIL Animal, Cellular Imaging, Genomic Technologies, Mouse Pathology and Flow Cytometry Facilities and EPFL Histology Core and Protein Production facilities are gratefully acknowledged. We apologize to colleagues whose work we could not reference due to size constraints.

This work was supported by grants from the Swiss Cancer League (KLS 3406-02-2016 and KFS-4895-08-2019), Foundation MEDIC, the Emma Muschamp Foundation, the Swiss National Science Foundation (31003A-156266 to T.V.P., 31003A-166161 to S.A.L.), the European Research Council (ERC EVOLVE-725051 to M.D.P.) and the Gabriela Kummer MD-PhD fellowship and Alfred and Anneliese Sutter-Stöttner private fellowships (to C.C.).

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Author notes

  1. These authors contributed equally: Jeremiah Bernier-Latmani, Christophe Cisarovsky.

Authors and Affiliations

  1. Department of Oncology, Ludwig Center for Cancer Research Lausanne and University of Lausanne, Lausanne, Switzerland

    Jeremiah Bernier-Latmani, Christophe Cisarovsky, Samantha Mahfoud, Simone Ragusa, David Barras, Suzel Davanture, Alejandra González-Loyola, Mauro Delorenzi & Tatiana V. Petrova

  2. Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland

    Isabelle Dupanloup, David Barras, Sina Nassiri, Pascale Anderle, Nadine Fournier & Mauro Delorenzi

  3. Department of Biochemistry, University of Lausanne, Lausanne, Switzerland

    François Renevey, Stefanie Siegert & Sanjiv A. Luther

  4. Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, EPFL, Lausanne, Switzerland

    Sina Nassiri, Mario Leonardo Squadrito, Michele De Palma & Tatiana V. Petrova

  5. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

    Rui Benedito

  6. Institut RESTORE, UMR 1301-INSERM, 5070-CNRS, Université Paul Sabatier, Université de Toulouse, Toulouse, France

    Philippe Valet

  7. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

    Bin Zhou

  8. Institute of Pathology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland

    Christine Sempoux

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  1. Jeremiah Bernier-Latmani
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Contributions

J.B.L. and T.V.P. designed the study; J.B.L., C.C., S.M., S.R., I.D., D.B., F.R., S.N., P.A., M.L.S., S.S., S.D., A.G.L. and N.F. conducted experiments and acquired and analyzed data; S.A.L., R.B., P.V., B.Z., M.D.P., M.D. and C.S. provided reagents and samples; J.B.L. and T.V.P. wrote the manuscript. All authors discussed the results.

Corresponding author

Correspondence to Tatiana V. Petrova.

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T.V.P. receives research funding from Roche for an unrelated project. The authors declare no other competing interests.

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Nature Cardiovascular Research thanks Hyung Chun, Gou Young Koh and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Reduced VEGFA signaling near Wnthigh progenitors in human CRC.

(a) Wnthigh and Wntlow human CRC tumors are in CMS2 and CMS1, respectively. Distribution of CMS assignment for Wnthigh and Wntlow human CRC tumors, n = 469. (b) Angiogenic gene expression is decreased in CMS2 human CRC tumors compared to CMS1. Experimentally-derived angiogenesis signature score based on Masiero et al. 103 classified by CMS assignment, n = 2,275. p values from one-ANOVA with post-hoc Tukey test are listed. (c) VEGF-dependent angiogenesis gene expression103 is highest in Wntlow CRC tumors, n = 469 tumors. For boxplots, minimum (Q1) and maximum (Q3) of the box are the 25th and 75th percentile, the center is the median, the whiskers minima and maxima highlight the Q1-1.5xIQR and Q3 + 1.5xIQR, where IQR is the interquartile range (Q3-Q1). Points outside of these regions are outliers. (d-h) VEGFA signaling is compartmentalized into low Wnt signaling areas of human CRC tumors. Serial paraffin section immunostaining of high (H) or low (L) areas of PROX1 staining within the same Wnthigh CRC tumors for (d) PROX1, (e) PECAM1 and (f) ESM1. (g-h) Quantification of percentage (g) vessel area (left, p = 0.0401) or (h) ESM1+ vessels (left, p = 0.0008) in Wnthigh-Progenitor (Pro) or Wnthigh-Differentiated (Diff) areas or luminal/bulk or invasive (L/B or Inv) areas of Wntlow CRC tumors (n = 11 Wnthigh tumors and n = 8 Wntlow tumors). Scale bars: 100 μm. All data are shown as mean ± SD. *P < 0.05, ***P < 0.001, 2-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 2 Compartmentalized VEGFA in the intestinal vascular unit.

(a) Filopodia are restricted to villus vessels in the small intestine. White, VEGFR2; arrowheads indicate perinuclear VEGFR2. Quantification of the percentage of filopodia observed in villi or crypts (p < 0.0001, n = 3 mice). (b-c) VEGFA expression and signaling are restricted to the villus. (b) Whole-mount immunostaining for VEGFA (red) in adult intestinal villi is restricted to villus blood vessels (green, PECAM1). (c) ESM1 staining (red) is restricted to villus vessels (green, podocalyxin). Cryosection immunostaining, DNA (blue). (d) Crypt progenitor epithelial cells are normoxic while differentiated epithelial cells are hypoxic. Small intestine and colon paraffin section immunostaining for hypoxia markers pimonidazole (Pimo) and HIF1α (red). Both hypoxia markers co-stain with the differentiated epithelial cell marker Keratin-20 (green, KRT20), DNA (blue). (e) Vessels associated with progenitor and differentiated colon epithelial cells are compartmentalized into normal and angiogenic vessels, respectively, as in the small intestine. Whole-mount immunostaining of colon blood capillaries (red, VEGFR2) with a side- and top-view. (f-h) Villus blood vessel branching is reduced in mice treated with DC101, while crypt vessel length and branching are unaffected. (f) Whole-mount immunostaining for intestinal vessels (red, VEGFR2) and quantification of the length (x100 μm) of crypt vessels and number of branchpoints per 100 μm vessel length in (g) villus (p = 0.0002) and (h) crypt vessels in mice treated with either IgG or DC101 (n = 6 mice). (i) Villus vessel filopodia are dependent on active VEGFA signaling. The number of villus vessel (black, VEGFR2/mTomato) filopodia (arrowheads) is significantly reduced in mice treated with DC101. Quantification of the number of filopodia per 100μm of villus vessel length (n = 4 IgG-treated mice and n = 5 DC101-treated mice; p < 0.0001). (j-k) VEGFA signaling blockade decreases intestinal ESM1 expression. (j) Whole-mount immunostaining for ESM1 (red) on adult intestinal villi blood vessels (green, PECAM1). (k) Total intestine Esm1 expression from adult control antibody- or DC101-treated C57BL/6 mice analyzed by RT–qPCR; Esm1 expression normalized by Cdh5 (n = 6 mice; p = 0.0224). (l) Scheme of intestinal vasculature and VEGFA-dependence. Villus blood vessel are VEGFA-dependent and associated with differentiated epithelial cells while crypt vessels are VEGFA-independent and associated with crypt cells, including stem and proliferating progenitor cells. VEGFA blockade reduces vascular density in the villi but has no impact on crypt vessels or epithelial cell proliferation. Scale bars: 50 μm: b, d, f; 20 μm: a, c, e, i, j. All data are shown as mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, 2-tailed unpaired Student’s t-test. Images in b–e are representative of at least 3 independent experiments.

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Extended Data Fig. 3 Stem/progenitor cells are VEGFA-independent and normoxic in mouse adenomas.

(a) Villus vessel length (green, PECAM1) is significantly increased in ApcΔIEC intestine while vessel branching is decreased. (b-c) Quantification of (b) total vessel length (x100μm; p = 0.0019) and (c) total branchpoints per 100μm of vessel length (p = 0.0003) in control or ApcΔIEC intestine (n = 6 mice). (d) Progenitor cell expansion maintains crypt/villus vessel compartmentalization. Whole-mount immunostaining in Apcmin/+; Lgr5-EGFP-CreERT2 adenomas. Blood vessel (red, VEGFR2) density and branching is higher in the luminal part of adenomas (Panel 1) compared to expanded epithelial crypts (Panel 2, Lgr5-GFP, green). Representative of 2 independent experiments. (e) VEGFA expression is not induced during progenitor expansion. qPCR for expression of Vegfa from total intestine of control and ApcΔIEC mice, n = 3 mice. (f-g) VEGFR2 blockade decreases ESM1 expression during progenitor expansion. (f) Whole-mount immunostaining of ESM1 (red) on villus blood vessels (green, PECAM1) in control antibody- or DC101-treated ApcΔIEC mice. (g) ESM1 (red) is reduced in DC101-treated Apcfl/fl; Lgr5-EGFP-CreERT2 adenoma blood vessels (green, MECA32). Paraffin section immunostaining; DNA, blue. (h) Quantification of percentage vessel area in adenomas of control antibody or DC101-treated Apcfl/fl; Lgr5-EGFP-CreERT2 mice, n = 4 mice. (i) Paraffin section immunostaining of GFP+ (green) stem cells in control antibody or DC101-treated Apcfl/fl; Lgr5-EGFP-CreERT2 adenomas (white outlines). (j–l) Quantification of control IgG or DC101-treated Apcfl/fl; Lgr5-EGFP-CreERT2 (j) adenoma area, (k) number of adenomas and (l) percentage tumor area GFP+, n = 4 tumors. (m) KRT20-negative Apcmin/+ adenoma crypts are Pimo-negative while differentiated KRT20+ lumen-adjacent epithelial cells (green) are Pimo+ (red). (n) Apcmin/+ adenoma crypt cells are HIF1α-negative (arrow) while lumen-adjacent epithelial cells are HIF1α+ (red). Scale bars: 200 μm: d, i; 50 μm: a, d (insets), f, g, m, n. All data are shown as mean ± SD, **P < 0.01, ***P < 0.0 1, 2-tailed unpaired Student’s t-test. Images in m and n are representative of 2 independent experiments.

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Extended Data Fig. 4 Stem/progenitor cells are VEGFA-independent and normoxic in models of advanced CRC.

(a-b) MC38 tumor cells have low Wnt signaling. (a) qPCR for expression of Wnt target genes Axin2, Lgr5 and Prox1 in AKP organoid and MC38 tumors, n = 3 independent samples. (b) Paraffin section immunostaining for β-CATENIN in AKP organoid and MC38 tumors. DNA, blue. (c-d) VEGFR2 blockade inhibits MC38 tumor growth and proliferation. Quantification of subcutaneous MC38 tumor organoid (c) tumor volume (left, IgG vs DC101, p value: 0.0264, day 10; 0.0093, day 11; 0.0061, day 12; 0.0046, day 13; 0.0035, day 14) and weight (right, p = 0.0002; n = 7 tumors in IgG-treated mice and n = 8 tumors in DC101-treated mice). (d) Percentage of EdU+ MC38 tumor cells in control antibody- and DC101-treated mice (p < 0.0001, n = 6 tumors in IgG-treated mice and n = 7 tumors in DC101-treated mice, data from two independent experiments). (e-f) AKP organoid tumor vessels are less sensitive to VEGFR2 blockade than vessels from MC38 tumors. Quantification of tumor vessel area as a percentage of tumor area of control IgG antibody- or DC101-treated NSG mice with (e) AKP organoid tumors (p = 0.0056, n = 9 tumors) or (f) MC38 tumors (p < 0.0001, n = 14 tumors). (g-h) AKP organoid tumors have fewer vessels expressing the VEGFA signaling-dependent ESM1 than MC38 tumors. Quantification of percentage of ESM1+vessels from control antibody- or DC101-treated mice with (g) AKP organoid tumors (p = 0.0378, n = 6 tumors) or (h) MC38 tumors (p < 0.0001, n = 10 tumors). (i-j) AKP tumor blood endothelial cells (BECs) proliferation is resistant to VEGFR2 blockade. Quantification of % Ki67+ BECs from control antibody- or DC101-treated mice with (i) AKP organoid tumors (n = 9 tumors) or (j) MC38 tumors (p = 0.0039, n = 5 IgG-treated tumors and n = 7 DC101-treated tumors). (k) Endothelial-specific ablation of VEGFR2 does not alter AKP organoid tumor growth. Experimental plan for AKP organoid tumor subcutaneous injection into Kdrfl/fl and Kdrfl/fl; Cdh5-CreERT2 mice; tumor volume (mean ± SEM), dotted line indicates start of tamoxifen injections n = 6 tumors; final tumor weight (mean ± SD, n = 5 tumors in Kdrfl/fl and n = 4 tumors in Kdrfl/fl; Cdh5-CreERT2 mice). (l) VEGFR2 is efficiently depleted in Kdrfl/fl; Cdh5-CreERT2 mice without altering blood vessel density. Staining for PECAM1 (green), VEGFR2 (red) and DAPI (blue). Quantification of vessel density in AKP organoid tumors injected into Kdrfl/fl and Kdrfl/fl; Cdh5-CreERT2 mice (n = 5 tumors in Kdrfl/fl and n = 4 tumors in Kdrfl/fl; Cdh5-CreERT2 mice). (m) Endothelial-specific ablation of VEGFR2 arrests growth of MC38 tumors. Left, experimental plan for MC38 tumor subcutaneous injection into Kdrfl/fl and Kdrfl/fl; Cdh5-CreERT2 mice. Middle, tumor volume (p value: 0.0038, day 22; 0.0011, day 25; dotted line indicates start of tamoxifen injections). Right, final tumor weight (p = 0.0055, n = 4 tumors in Kdrfl/fl and n = 3 tumors in Kdrfl/fl; Cdh5-CreERT2 mice). (n) Increased endothelial VEGFA signaling in tumors with decreased Wnt signaling. Staining for ESM1 (green), PECAM1 (red) and DAPI (blue). Quantification of percentage of ESM1+ vessels in AP and APP organoid tumor vessels (p = 0.0034, n = 7 tumors). Scale bars: 50 μm: b (AKP organoids), l, n; 20 μm: b (MC38). All data (except k, middle) shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t-test.

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Extended Data Fig. 5 Confirmation of Apln expression in villus endothelial cells.

(a) Density plots showing the gating strategy for flow cytometric sorting of intestinal blood and lymphatic endothelial cell (BEC and LEC) sorting. Single, live, CD45neg, EpCAMneg cells were selected (upper plot). Among those, BEC were identified and collected as CD31+, gp38neg cells and LEC as CD31+, gp38+ cells. (b) Intestinal LEC and BEC quality control qPCR for Flt1 (p = 0.0059), Nrp1 (p = 0.0036), Lyve1 (p = 0.0051) and Prox1 (p < 0.0001, n = 3 sorted cell samples from individual mice). (c-d) Apelin expression is induced during progenitor cell expansion. qPCR for Apln expression in (c) sorted intestinal BECs (p = 0.0071, n = 3 sorted cell samples from individual mice) and (d) total intestine from control and ApcΔIEC mice (p = 0.0051, n = 4 WT and n = 3 ApcΔIEC mice). (e) Paraffin section immunostaining for blood vessels (red, PECAM1) and in situ hybridization (ISH) for Apln (green). Zoomed areas marked by 1 + 2. (f-h) The majority of apelin-expressing endothelial cells are in or near the villus venule. (f) Whole-mount immunostaining of GFP expression (green) and either all intestinal vessels (red, VEGFR2/mTomato) or MADCAM1+ vessels (blue) in Apln-CreERT2; mTmG mice 24 hours post 4-OHT injection. (g) Quantification of the percentage of GFP+ ECs that are in the MADCAM1+ villus venule or endothelial cells directly in contact with the villus venule (MADCAM1-adjacent) or crypt vessels in Apln-CreERT2; mTmG mice 24 hours post 4-OHT injection (n = 3 mice). (h) Quantification of percentage of GFP+ endothelial cells in the villus or crypt (n = 3 mice). (i) Aplnr is widely expressed in intestinal blood vessels. Paraffin section immunostaining/ISH for Aplnr (green) expressed on intestinal endothelial cells (red, PECAM1) of wild-type and Apcfl/fl; p53fl/fl Vil-CreERT2 intestine; blue, DAPI. (j) Apln expression is maintained after VEGFR2 signaling blockade. Experimental setup for Apln-CreERT2; mTmG lineage tracing combined with DC101 treatment. Whole-mount immunostaining for intestinal vessels (white, PECAM1) and GFP+ endothelial cells (green) after 24 hours of treatment with control or DC101 blocking antibodies. Red, ESM1; arrowheads, villus venule; arrows, capillary endothelial cells; asterisk, ESM1 staining comparison. Quantification of number of GFP+ endothelial cells in the villus or crypt, n = 4 IgG-treated and n = 5 DC101-treated mice). Scale bars: 50 μm: e, f, j; 20 μm: b (insets), i. All data are shown as mean ± SD. **P < 0.01, ***P < 0.001 2-tailed unpaired Student’s t-test. Images in e and i representative of 2 independent experiments.

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Extended Data Fig. 6 Apelin maintains crypt venous capillary patency at steady state and during crypt expansion.

(a-b) Neither vessel density nor blood endothelial cell (BEC) proliferation are decreased in Apln-/- mice. (a) Quantification of crypt vessel density in control and Apln-/- mice (p = 0.0368, n = 3 WT mice and n = 4 Apln-/- mice). (b) Quantification of percentage of villus and crypt Ki67+ BECs in control and Apln-/- mice (n = 8 WT mice and n = 6 Apln-/- mice). (c) Quantification of the number of empty collagen sleeves / villus area of wild-type and Apln-/- mice (n = 4 WT mice and n = 3 Apln-/- mice). (d) Venous identity of regressing crypt vessels in Apln-/- mice. Whole-mount immunostaining for VEGFR2 (green), venous marker endomucin (red) and collagen IV (white). Arrowheads, empty collagen sleeves between endomucin+ vessels; arrow, regressing vessel. Quantification of number of regressed crypt vessels in Apln-/- mice sorted by endomucin status of each end of regressed vessel (n = 3 mice). (e) Quantification of crypt vessel density in ApcΔIEC and ApcΔIEC; Apln-/- mice (n = 3 ApcΔIEC and n = 4 ApcΔIEC; Apln-/- mice). (f) Quantification of percentage of villus and crypt Ki67+ BECs in ApcΔIEC and ApcΔIEC; Apln-/- mice (n = 4 mice). (g) Increased number of blind-ended crypt blood vessels in ApcΔIEC; Apln-/- mice. Whole-mount immunostaining of intestinal vessels (red, VEGFR2) from ApcΔIEC and ApcΔIEC; Apln-/- mice. Insets: blind-ended crypt vessels (arrowheads). (h) Increased empty collagen sleeves (green, collagen IV) in crypt vessels (red, VEGFR2) of ApcΔIEC; Apln-/- compared to ApcΔIEC mice. Quantification of number of empty collagen sleeves per crypt vessel length (p = 0.0002, n = 5 ApcΔIEC and n = 4 ApcΔIEC; Apln-/- mice). (i) Venous identity of regressing crypt vessels in ApcΔIEC; Apln-/- mice. Whole-mount immunostaining of crypt vessels (green, VEGFR2) and venous identity marker endomucin (red) from ApcΔIEC and ApcΔIEC; Apln-/- mice. Quantification of number of regressed crypt vessels in ApcΔIEC; Apln-/- mice sorted by endomucin status of each end of regressed vessel (n = 4 mice). Scale bars: 50 μm: g-i; 20 μm: d, g + h (insets). All data are shown as mean ± SD. *P < 0.05, ***P < 0.001, 2-tailed unpaired Student’s t-test.

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Extended Data Fig. 7 Clonal analysis of Apln-CreERT2: mTmG lineage tracing.

(a) Clonal expansion of GFP+ cells in the villus, but not crypt vessels of Apln-CreERT2; mTmG mice. Analysis of venule and venule adjacent veinous GFP+ cell cluster size in the villus and crypt after 24 hr and 3 weeks of lineage tracing after 4-OHT injection, (mean ± SD, n = 4 Apln-CreERT2; mTmG 24 hr mice and n = 3 Apln-CreERT2; mTmG 3w mice). (b) Lack of sex-specific differences in crypt vessel phenotype in Apln-deficient mice. Representative whole-mount immunostainings for crypt vessels (white, VEGFR2) in male and female mice heterozygous or null for Apln. (c) Paraffin section immunostaining for crypt proliferation (green, Ki67) in mice treated with PBS or Rspo1-FC for 4 days; blue, DAPI. (d) Model for APLN-mediated control of crypt vessel patency. APLN+ endothelial cells migrate during steady state and expansion to the crypts in an APLN-dependent fashion. Scale bars: 50 μm: b; 20 μm: c. Images in a and c representative of 2 independent experiments.

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Extended Data Fig. 8 Apelin promotes crypt perfusion and epithelial cell fitness.

(a) Villi are smaller in Apln-/- mice compared to controls. Whole-mount immunostaining for villus vessels (red, VEGFR2) in control and Apln-/- mice. Quantification of villus area between control and Apln-/- mice (p = 0.0221, n = 4 mice). (b) Crypt length is smaller in Apln-/- mice compared to controls. Paraffin section immunostaining; red, EdU; blue, DNA. Quantification of crypt length (p = 0.0004, n = 4 mice). (c) Decreased cypt epithelial cell proliferation in Apln-/- mice. Quantification of the number of Ki67+ crypt epithelial cells in control and Apln-/- mice (p = 0.0004, n = 4 WT mice and n = 3 Apln-/- mice). (d) Epithelial cell migration is slowed in Apln-/- mice. Paraffin immunostaining for EdU (red) after 72 h pulse; blue, DNA. Quantification of position of EdU+ epithelial cells 72 h after pulse as a percentage of villus height (p = 0.0028, n = 3 mice). (e) Increased number of EdUneg phospho-γH2A.X+ crypt epithelial cells in Apln-/- mice. Paraffin section immunostaining; green, EdU; red, phospho-γH2A.X; blue, DNA. Quantification of percentage of crypt EdUneg phospho-γH2A.X+ epithelial cells (p = 0.032, n = 3 mice). (f) Definition of crypt epithelial proliferation polarization index. EdU+ crypt intestinal epithelial cells were annotated as either on the left or right side and the percentage of each to a side was used to generate a score where 0 indicates exactly the same number of EdU+ epithelial cells on each side of the crypt and 1 indicates all EdU+ epithelial cells are on one side of the crypt. (g) Crypt EdU+ epithelial cells are polarized on one side of the crypt in Apln-/- mice. Polarization index of EdU+ crypt epithelial cells in control and Apln-/- mice (p < 0.0001, n = 4 mice). (h) Pulsed EdU+ IECs are polarized on one side of the villus in Apln-/- mice. Polarization index of EdU+ IECs after 72 h pulse in control and Apln-/- mice (p = 0.0214, n = 3 mice). (i) Decreased perfusion of crypt vessels in Apln-/- mice. Whole-mount immunostaining for VEGFR2 (green) after i.v. injection of PE-conjugated PECAM1 antibody (red). Arrowheads: non-perfused VEGFR2+PECAM1-PEneg vessels. (j) Density plots depicting the gating strategy for sorting CD44+ and CD44neg epithelial cells using flow cytometry. Single, live CD45neg CD31neg cells expressing EPCAM were selected (left plot) and then collected as either CD44+ or CD44neg epithelial cell subsets. (k) Apelin receptor is not expressed on intestinal epithelial cells (IECs). qPCR for Lgr5, Cdh5 and Aplnr expression in CD44+ IECs (progenitor cells), CD44neg IECs (differentiated IECs) and BECs (n = 2 CD44+ sorted IECs, n = 2 CD44neg sorted IECs and n = 5 sorted BECs from individual animals). (l, m) Defect of crypt epithelial progenitor survival in Apln-/- mice is cell extrinsic. Images and quantification of organoid area from either wild-type or Apln-/- mice after (l) first plating (p = 0.0013) or (m) re-plating (n = 4 organoid populations isolated from individual mice). Scale bars: 50 μm: a, b, d, i, l, m; 20 μm: e. All data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t-test.

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Extended Data Fig. 9 Apelin maintains distinct populations of crypt epithelial cells.

(a, b) Secretory progenitor- and differentiated secretory-specific gene expression is decreased in Apln-/- crypt epithelial cells. (a) UMAP plots of mouse intestinal epithelial scRNAseq data55. Cell type-specific markers allowed cluster annotation (left). The top 100 cluster-specific genes were used as input for GSEA of differentially expressed genes between wild-type and Apln-/- CD44+ crypt epithelial cells. Clusters enriched for genes increased or decreased in Apln-/- crypt epithelial cells are marked in green and red, respectively (right). (b) GSEA of genes differentially regulated in Apln-/- crypt epithelial cells against top 100 genes of each cluster shown in (a). (c) Annotation of clusters used for GSEA analysis for genes up- and downregulated in wild-type vs Apln-/- crypt epithelial cells. (d) No increase in the number Lgr5-GFP stem cells (green, GFP, p = 0.0266) or their proliferation (red, Ki67, p = 0.0484) in Apln-/- crypts; blue, DAPI (n = 3 mice). (e, f) Decreased number of epithelial cells expressing tuft and enteroendocrine markers in Apln-/- crypts. Paraffin section immunostaining for (e) DCLK1 (green) or (f) CHGA (red) in wild-type and Apln-/- mice; blue, DAPI. Quantification of the number of (e) DCLK1+ (p = 0.0188, WT vs Apln-/- crypt) or (f) CHGA+ (p = 0.0055, WT vs Apln-/- crypt) in crypts and villi of wild-type and Apln-/- mice (n = 3 mice). Scale bars: 50 μm: e, f; 20 μm: d. *P < 0.05, **P < 0.01 2-tailed unpaired Student’s t-test.

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Extended Data Fig. 10 Apelin maintains tumor progenitor cell fitness and proliferation.

(a-d) Apln deficiency reduces crypt expansion (a) Staining for EdU (green), Ki67 (red) and DNA (blue) in ApcΔIEC and ApcΔIEC; Apln-/- mice. (b-d) Quantification for the (b) crypt depth (p = 0.0007) and percentage of (c) Ki67+ (p = 0.0026) and (d) EdU+ crypt IECs (p = 0.0303, n = 4 mice). (e) Increased number of EdUneg phospho-γH2A.X+ crypt epithelial cells in ApcΔIEC; Apln-/- mice. Paraffin section immunostaining; green, EdU; red, phospho-γH2A.X; blue, DNA. Quantification of percentage of crypt EdUneg phospho-γH2A.X+ epithelial cells (p = 0.0004, n = 4 mice). (f) In situ hybridization for Lgr5 (green) in AKP organoid tumors implanted into wild-type or Apln-/- mice; DAPI, blue. Quantification for percentage of tumor area positive for Lgr5 (n = 6 tumors). (g) The majority of DCLK1+ cells in AKP organoid tumors also express PROX1. Quantification of percentage DCLK1+ cells that are also PROX1+ in either control or Apln-/- mice (n = 5 tumors in WT mice and n = 3 tumors in Apln-/- mice). (h) Apelin deficiency reduces vessel density in progenitor cell areas. Staining for progenitor marker PROX1 (green), PECAM1 (red) and DNA (blue) in AKP tumor grown in control and Apln-/- mice. Quantification of tumor vascularization as percentage PECAM1+ area in PROX1+ (p = 0.0082, n = 8 tumors in WT mice and n = 5 tumors in Apln-/- mice) and PROX1neg zones (n = 8 tumors in WT mice and n = 6 tumors in Apln-/- mice) of AKP tumor organoids grown in control and Apln-/- mice. Scale bars: 50 μm: a, f; 20 μm: e. All data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t-test.

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Bernier-Latmani, J., Cisarovsky, C., Mahfoud, S. et al. Apelin-driven endothelial cell migration sustains intestinal progenitor cells and tumor growth. Nat Cardiovasc Res 1, 476–490 (2022). https://doi.org/10.1038/s44161-022-00061-5

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