(A) Tumor cell medium–stimulated HUVECs were treated with PBS or OX40L protein (100 ng/mL) for 48 hours and then subjected to transcriptome sequencing. Enriched pathways were pooled using differentially expressed genes after OX40L treatment (left). The heatmap displays the expression of genes associated with endothelial-mesenchymal transition (right). (B and C) Genes encoding mesenchymal markers and adhesive molecules were evaluated in OX40^– or OX40⁺ tumor ECs using scRNA-Seq data. (D) PBS- or OX40L-treated HUVECs were fractionated into cytoplasmic (Cyto) and nuclear (Nuc) fractions and subjected to protein mass spectrometry. (E) PBS- or OX40L-treated HUVECs were immunostained with YAP antibody. Scale bars: 5 μm. (F and G) Expression of YAP downstream genes was measured in sorted CD31⁺ cells from CRC and NT tissues by qRT-PCR (F, n = 3) and in OX40^– and OX40⁺ ECs from CRC tissues using scRNA-Seq data (G, n = 5). (H) Representative images of CRC tissues immunostained using the indicated antibodies. The white boxes indicate OX40⁺ or OX40^– ECs (n = 162). Scale bar: 5 μm. (I) YAP⁺ percentages in OX40^– ECs and OX40⁺ ECs from tumor tissues (n = 162). (J) Subcutaneous tumors were established in mice with conditional knockin of OX40 in ECs or in control mice. The mice were treated with verteporfin for 3 weeks (n = 10). (K–M) Subcutaneous tumor and pulmonary metastasis models were established using MC38 cells in BALB/c nude mice. The mice were treated with OX40L or a combination of mouse OX40L protein and verteporfin. (K) Tumor volume (n = 8). (L and M) Metastatic nodules in the lung (L) and mouse survival (M) (n = 6). Two-tailed Student’s t test (F and I), 2-way ANOVA (J and K), 1-way ANOVA (L), or log-rank test (M) was used for statistical analysis.