Fig 2: IGFBP7 inhibits tumor growth and angiogenesis in mouse models. A Schematic diagram illustrating the experimental design of intraperitoneal injection of saline (100 µl/mouse), recombinant IGFBP7 protein (10 µg/mouse), IgG (100 µg/mouse), or anti-mouse IGFBP7 antibody (100 µg/mouse) in the subcutaneous tumor mouse model. Created with BioRender.com. B, C Representative tumor images of subcutaneous tumors from mice in different treatment groups. D Quantification of tumor weight in different treatment groups. E, F Tumor growth curves showing tumor volume in mice from different treatment groups. G Representative immunohistochemistry (IHC) staining images showing the expression of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) in mouse tumor tissues. H Bar graph displaying the optical density values of CD31-positive areas in tumor tissues from different treatment groups, reflecting changes in angiogenesis. I Bar graph displaying the optical density values of TGF-β1-positive areas in tumor tissues from different treatment groups, reflecting TGF-β1 expression levels. J Schematic diagram illustrating the experimental design of intraperitoneal injection of saline (100 µl/mouse), recombinant IGFBP7 protein (10 µg/mouse), IgG (100 µg/mouse), or anti-mouse IGFBP7 antibody (100 µg/mouse) in the AOM/DSS-induced colitis-associated colorectal cancer model. Created with BioRender.com. K, L Representative images of colorectal tumors in AOM/DSS-induced colitis-associated CRC model from different treatment groups. M Quantification of tumor number in AOM/DSS-induced colitis-associated CRC model from different treatment groups. N Representative immunohistochemistry (IHC) staining images showing the expression of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) in colorectal tumor tissues from the AOM/DSS-induced colitis-associated CRC model. O Bar graph displaying the vascular density of CD31-positive areas in tumor tissues from different groups. P Bar graph displaying the optical density values of TGF-β1-positive areas in tumor tissues from different groups. Q Schematic diagram illustrating the experimental design in the AOM/DSS-induced colitis-associated colorectal cancer model. Mice were administered intraperitoneal injections of saline (100 µl/mouse) or recombinant IGFBP7 protein (10 µg/mouse) once per week, as well as mouse IgG2a Kappa antibody (5 mg/kg) or the anti-VEGF monoclonal antibody B20-4.1.1.1 (5 mg/kg) twice per week. Created with BioRender.com. R, S Representative images and quantitative analysis of colon length in mice. T, U Representative images and quantitative analysis of the number of colonic tumors in mice

Fig 3: Endothelial-derived IGFBP7 suppresses colorectal cancer cell migration and proliferation via TGF-β1 signaling. A Venn diagram showing the intersection of enriched GSEA pathways in transcriptome sequencing results of SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h. B GSEA enrichment plot of the “GO_BP_CELL_MIGRATION” pathway in tumor cells after co-culture with recombinant IGFBP7 protein (100 ng/ml) for 48 h. C GSEA enrichment plot of the “GO_BP_BLOOD_VESSEL_MORPHOGENESIS” pathway in tumor cells after co-culture with recombinant IGFBP7 protein (100 ng/ml) for 48 h. D Venn diagram showing the intersection of downregulated genes enriched in the “GO_BP_CELL_MIGRATION” pathway (fc < 0, p < 0.05) identified from transcriptome sequencing of SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h. E Heatmap showing the common downregulated genes in tumor cell lines, with data sourced from SW480. F, G The mRNA expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA in tumor cell lines after co-culture with endothelial cells for 48 h. H The protein expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA in tumor cell lines after co-culturing with endothelial cells for 48 h. I SW480 cells were transfected with plasmids and co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 24 h, followed by measurement of luciferase activity. Data were normalized to Renilla luciferase activity (firefly/Renilla) and are presented as fold change relative to the control group (control = 1). J SW480 cells were transfected with an EGR1 overexpression plasmid and co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h, followed by analysis of EGR1, TGF-β1, and IGFBP7 expression levels. K, L, M Cell migration assays were performed to assess the migration of SW480 and HCT116 tumor cells treated for 48 h with the culture medium supernatant from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA (scale bar, 200 μm). N, O, P Scratch wound healing assays were performed to evaluate the healing of SW480 and HCT116 tumor cells treated for 24 h with the culture medium supernatant from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA (scale bar, 100 μm). Q, R CCK-8 cell proliferation assays were performed to assess the proliferation of SW480 and HCT116 tumor cells treated for 24, 48, 72, and 96 h with the culture medium supernatant from HMEC-1 cells transiently transfected with IGFBP7 siRNA. S, T CCK-8 cell proliferation assays were performed to assess the proliferation of SW480 and HCT116 tumor cells treated for 24, 48, 72, and 96 h with the culture medium supernatant from HUVEC cells transiently transfected with IGFBP7 siRNA

Fig 4: Loss of IGFBP7 enhances angiogenic potential of endothelial cells. A Hierarchical clustering dendrogram of coexpression modules identified using WGCNA following NuGEN RNA sequencing of endothelial cell sorting. B Dot plot illustrating the enriched pathway of IGFBP7 containing network. C GSEA enrichment plot of the “GOBP_REGULATION_OF_VASCULATURE_DEVELOPMENT” pathway in endothelial cells following transient transfection with IGFBP7 siRNA. D Heatmap of transcriptome data following transient transfection of IGFBP7 siRNA in endothelial cells. Red indicates upregulated genes, blue indicates downregulated genes, and the intensity of the color reflects the degree of expression change. E, F mRNA level validation of angiogenesis-related genes following transient transfection of IGFBP7 siRNA in endothelial cells. G, H, I Angiogenesis assays were performed to assess the angiogenic ability of endothelial cells following transient transfection with IGFBP7 siRNA. HMEC-1 cells (2 × 10⁴ cells/well) or HUVECs (5 × 10³ cells/well) transfected with siRNA were seeded onto Matrigel and incubated at 37 °C for 6 h (scale bar, 100 μm)

Fig 5: VAPA facilitates lysosomal degradation of IGFBP7 and modulates TGF-β1 signaling. A Immunoprecipitation-mass spectrometry (IP-MS) results showing the proteins that interact with IGFBP7. B Immunoprecipitation analysis demonstrating the interaction between endogenous IGFBP7 and VAPA in HCT116cells. C Immunoprecipitation analysis demonstrating the interaction between endogenous VAPA and IGFBP7 in CaCO2cells. D, E Validation of protein levels after VAPA knockdown in tumor cells. F Protein expression levels of IGFBP7 after co-culturing VAPA knockdown tumor cells with endothelial cells for 48 h. G Changes in the concentration of IGFBP7 in the culture medium after co-culturing VAPA knockdown tumor cells with recombinant IGFBP7 protein (100 ng/ml) for 48 h. H Measurement of IGFBP7 protein expression levels in VAPA -knockdown SW480 cells with or without co-culture with recombinant IGFBP7 (100 ng/ml) for 48 h, followed by treatment with Bafilomycin A1 (20 µM) during the final 6 h. I Measurement of lysosome counts in VAPA knockdown SW480 cells after treatment with recombinant IGFBP7 protein (100 ng/ml) for 48 h. J Representative immunofluorescence staining of IGFBP7 and LAMP1 in HCT116 after VAPA knockdown or co-culturing with recombinant IGFBP7 protein (100 ng/ml) for 48 h. Green, IGFBP7; Red, LAMP1; Blue, nucleus (scale bars, 50 μm). K Relative fluorescence intensities of IGFBP7 from the indicated groups were quantified. L Relative fluorescence intensities of LAMP1 from the indicated groups were quantified. M Protein expression levels of IGFBP7, TGF-β1 signaling pathway, and EMT pathway markers after co-culturing VAPA knockdown SW480 tumor cells with HMEC-1 endothelial cells for 48 h