Fig 2: ECs release EVs with dysregulated miRNA levels when cultured in the presence of cancer cells, in particular miR‐142‐5p, miR‐183‐5p and miR‐222‐3p. (a), Overview of the procedure of endothelial EV isolation and small RNA profile analysis. b‐e, Characterization of HUVEC EVs by cryo‐transmission electron microscopy analysis bar represents 100 nm (b), dynamic light scattering analysis (c), and western blotting of the exosomal markers CD9, CD81, CD63 and syntenin in lysates from purified HUVEC EVs and cells from mono‐ and coculture conditions (d). EV lysates: 1: mono‐HUVEC EVs, 2: co‐HUVEC EVs, 3: MDA‐MB‐231‐EVs; cell lysates: 4: mono‐HUVECs, 5: co‐HUVECs, 6: MDA‐MB‐231. (e), Western blotting of endothelial marker CD31 and tumour marker EpCAM in lysates from purified HUVEC EVs and cells from mono‐ and coculture conditions. EV lysates: 1: mono‐HUVEC EVs, 2: co‐HUVEC EVs, 3: MDA‐MB‐231‐EVs; cell lysates: 4: mono‐HUVECs, 5: co‐HUVECs, 6: MDA‐MB‐231. (f), Heatmap of miRNAs with differential abundance between mono‐HUVEC EVs and co‐HUVEC EVs. (g), RT‐qPCR analysis of the miRNAs dysregulated in co‐HUVEC EVs. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 calculated by One‐sample t‐test (n = 3). (h), Gene targets shared by miRNAs with increased abundance in co‐EVs (nodes). Edge thickness is scaled by the number of genes targeted in common by the connected nodes. Node size is scaled by number of predicted targets. i, j, Top canonical pathways (i) and top diseases and disorders (j) by IPA analysis of genes targeted by at least five miRNAs upregulated in co‐EVs. k, RT‐qPCR analysis of miRNAs dysregulated in EVs from MS1 cells in coculture (with 4T1 mouse breast cancer cell line) versus mono‐culture conditions. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 calculated by One‐sample t‐test (n = 3). l, Relative levels of miR‐142‐5p, miR‐183‐5p and miR‐222‐3p in endothelial EVs isolated from plasma of mouse model of 4T1 breast cancer compared to those from control, uninjected BALB/c mice. Data are presented as median ± IQR. **P < 0.01 calculated by unpaired two‐tailed Mann‐Whitney test. ‐4T1 (empty circle) n = 14; +4T1 (black circle) n = 15

Fig 3: Histological detection in EC‐ and LEC‐ specific Foxc KO mouse intestines A–G(A and D) Cre recombination efficiency detection in EC‐Foxc‐DKO and LEC‐Foxc‐DKO mouse strains. (A) Mice (control: mTmG/+;Foxc1 f/f ;Foxc2 f/f , mTmG/EC‐Foxc‐DKO: mTmG/+;Cdh5‐Cre ERT2 ;Foxc1 f/f ;Foxc2 f/f ) were treated with 150 mg/kg Tm by oral gavage for 5 days. Seven days after Tm treatment, the distal jejunum was collected and the frozen sections (15 μm) were stained with GFP/CD31/LYVE1 for the detection of GFP signal in blood vessels (B, CD31+LYVE1−, blue) and lacteals (L, CD31+LYVE1+, purple). Scale bars = 200 μm. (D) Mice (control: mTmG/+;Foxc1 f/f ;Foxc2 f/f , mTmG/LEC‐Foxc‐DKO: mTmG/+;Vegfr3‐Cre ERT2 ;Foxc1 f/f ;Foxc2 f/f ) were treated with 150 mg/kg tamoxifen by oral gavage for 5 days. Twelve days after Tm dose, the distal jejunum was collected and the frozen sections (15 μm) were stained with GFP and LYVE1 antibodies. Confocal images show the VEGFR3‐GFP is expressed in the LYVE1+ lymphatic vessels (L). Scale bars = 200 μm. (B, C, E–G) Representative H&E staining images of the distal jejuna 24 h after I/R in different mouse strains: (B) EC‐Foxc1‐KO, (C) EC‐Foxc2‐KO, (E) LEC‐Foxc‐DKO, (F) LEC‐Foxc1‐KO, (G) LEC‐Foxc2‐KO and their control mice. The intestinal ischemic injury grading in the Chiu scoring system is indicated by red numbers (0 ~ 5). Scale bars = 100 μm. The quantification of Chiu Score for these mouse strains is shown in Figs 2D and E, and 3B–D, respectively.HRepresentative immunostaining images of intestinal crypts labeled with BrdU (proliferative marker, injection performed 2 h before tissue collection) and EpCAM (epithelial marker) show the proliferative epithelial cells in crypts. Paraffin sections (4 μm), scale bars = 20 μm. The quantification of the number of BrdU+ epithelial cells per crypt is shown in Fig 3E.IRepresentative images of intestinal mucosa labeled with Cyclin D1 (CCND1) in LEC‐Foxc‐DKO mice compared with the control group 24 h after I/R. Scale bars = 100 μm. Quantification data for CCND1+ epithelial cells per crypt are shown in Fig 3I. Source data are available online for this figure.

Fig 4: Ectopic expression of EpCAM and Sox9 rescues the self-renewal ability of JMJD2D-knockdown LCSCs.A, Ectopic expression of EpCAM partly rescued the tumorsphere formation ability of JMJD2D-knockdown cells. B, Ectopic expression of Sox9 partly rescued the tumorsphere formation ability of JMJD2D-knockdown cells. C, ectopic expression of both EpCAM and Sox9 significantly rescued the tumorsphere formation ability of JMJD2D-knockdown cells. D, ectopic expression of both EpCAM and Sox9 in JMJD2D-knockdown cells partly rescued subcutaneous tumor growth and tumor weight in vivo. These experiments were performed at least twice with similar results. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Fig 5: Claudin-2 is enriched in extracellular vesicles isolated from patients with replacement-type colorectal cancer liver metastases.a Nanoparticle tracking analysis of EVs detected in plasma derived from either a RHGP liver lesion bearing patient or a DHGP liver lesion bearing patient. b Representative immunoblot analysis of Claudin-2, Claudin-4, CD81, EpCAM, and TSG101 using lysates prepared from EVs isolated from patients with the indicated lesions. c Claudin-2 levels in EVs concentrated from plasma of liver-metastatic CRC patients with the indicated lesions were normalized to TSG101 levels. d Immunoblot analysis of Claudin-2 and TSG101 using lysates prepared from EVs concentrated from patients, pre- and post-resection, with the indicated lesions. e Claudin-2 levels in EVs concentrated from plasma, pre- and post-resection, of liver-metastatic CRC patients with the indicated lesions were normalized to TSG101 levels.