Application of IGFBP7 in colorectal cancer diagnosis and treatment

Abstract

The invention relates to application of IGFBP7 in clinical diagnosis of colorectal cancer and application of IGFBP7 as a drug target. The research shows that the expression quantity of IGFBP7 secreted by tumor endothelial cells is continuously reduced in the colorectal tumor formation process related to inflammation. Functional analysis proves that the endothelial-derived IGFBP7 is secreted into a tumor microenvironment and plays a role in resisting angiogenesis. In addition, the CRC cells can actively ingest the vesicles containing the IGFBP7. The IGFBP7 reduces the activity of a TGF-beta1 signal channel by inhibiting the expression of EGR1, thereby inhibiting the proliferation and migration of tumor cells. What is worthy of attention is that VAPA (Vesicle-associated Membrane Protein Associated Protein A) is identified to be a key factor for mediating IGFBP7 vesicles to be transferred to lysosome, and VAPA promotes the degradation of IGFBP7, so that the cancer suppression function of the IGFBP7 is weakened. The endothelial cell-derived IGFBP7 inhibits the progress of CRC (cyclic redundancy check) through an EGR1 / TGF-beta 1 pathway, and VAPA-mediated lysosome degradation limits the cancer inhibition effect of the endothelial cell-derived IGFBP7. The discovery emphasizes that the endothelial cell-derived IGFBP7 is a promising therapeutic target in the field of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis and drug target, specifically involving the application of IGFBP7 in the clinical diagnosis of colorectal cancer and as a drug target. Background Technology

[0002] Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths, and the tumor microenvironment (TME) significantly influences disease progression and treatment response. Tumor endothelial cells play a crucial role in the initiation, progression, and metastasis of CRC, but their specific mechanisms of action have not been fully elucidated.

[0003] The IGFBP family comprises a variety of structurally similar proteins that regulate biological processes such as cell proliferation and growth by binding to insulin-like growth factors (IGFs) and modulating their bioavailability. As a key member of the IGFBP family, insulin-like growth factor binding protein 7 (IGFBP7) has been shown to participate in various physiological and pathological processes. It has not only been identified as a urinary biomarker for acute kidney injury (PMID: 35752325), but its downregulation has also been reported to improve symptoms of heart failure (PMID: 38991046). Furthermore, IGFBP7 regulates the interaction between immune cells and endothelial cells in inflammatory diseases such as psoriasis (PMID: 36917196).

[0004] However, there are currently no reports on the relationship between IGFBP7 and colorectal cancer. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of IGFBP7 in the clinical diagnosis of colorectal cancer and as a drug target.

[0006] In a first aspect, the present invention provides the application of IGFBP7 as a biomarker in the preparation of colorectal cancer diagnostic kits.

[0007] Secondly, this invention provides the application of IGFBP7 as a biomarker in the preparation of colorectal cancer prognosis and staging kits.

[0008] Thirdly, this invention provides the application of reagents for detecting the content of IGFBP7 gene or protein in the preparation of colorectal cancer diagnostic kits.

[0009] Fourthly, this invention provides the application of reagents for detecting the content of IGFBP7 gene or protein in the preparation of colorectal cancer prognosis and staging kits.

[0010] Fifthly, the present invention provides the use of IGFBP7 or IGFBP7 promoters in the preparation of drugs for treating colorectal cancer.

[0011] In a sixth aspect, the present invention provides the use of IGFBP7 or IGFBP7 promoters in the preparation of medicaments for preventing recurrence and metastasis of colorectal cancer and treatment resistance.

[0012] In a seventh aspect, the present invention provides a pharmaceutical composition for treating colorectal cancer, the pharmaceutical composition comprising IGFBP7 and an anti-VEGF antibody.

[0013] Eighthly, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating colorectal cancer.

[0014] The advantages of this invention are as follows:

[0015] 1. Endothelial cell-derived IGFBP7 as a biomarker for prognosis and staging of colorectal cancer: Based on a multidimensional chain of evidence including scRNA-seq, NuGEN sequencing after CD31⁺ endothelial cell sorting, and RNAscope / IHC / flow cytometry, studies have shown that IGFBP7 is highly expressed in normal vascular endothelial cells, but significantly decreased in tumor-associated endothelial cells, and progressively decreased from adenoma to adenocarcinoma. This indicates that the change is not a generalized tumor expression difference, but rather a cell type-specific tumor microenvironment endothelial remodeling event, forming a new understanding framework that is different from "tumor cell-derived IGFBP7".

[0016] 2: Revealing the stage-dependent dynamic regulation of endothelial IGFBP7 in the process of inflammation-driven carcinogenesis: In the AOM / DSS colitis-related CRC model, the study subdivided the disease process into acute inflammation (T1), chronic inflammation (T2), early tumorigenesis (T3), and tumor progression (T4), and demonstrated that endothelial IGFBP7 is elevated in the inflammatory stage, downregulated from T3, and significantly reduced in T4. At the same time, it shows an opposing change to the upregulation of pro-angiogenic and TGF-β pathway genes. This "time axis + stage-specific" expression pattern provides new molecular evidence for understanding the key window of inflammation-carcinogenesis transformation.

[0017] 3: Establishing the paracrine transport mechanism of IGFBP7 from endothelial secretion to tumor cell endocytosis: The study approached the problem by examining the paradoxical phenomenon of "almost zero transcriptional level but detectable intracellular protein". Combined with evidence such as enrichment of endothelial cell culture supernatant, sensitivity to Golgi inhibitors but insensitivity to exosome inhibitors, increased tumor cell protein and decreased mRNA after co-culture, and specific recognition of His-tagged recombinant proteins in tumor cells, it was clearly demonstrated that IGFBP7 in tumor cells mainly comes from exogenous uptake from the microenvironment rather than endogenous synthesis, and a new transcellular protein transport mechanism in the tumor microenvironment was proposed.

[0018] 4: Elucidating the axis of IGFBP7 inhibiting CRC migration and proliferation through "inhibition of EGR1 → downregulation of TGF-β1": After identifying core migration-related genes such as TGFB1 through transcriptome enrichment and intersection analysis, the study further verified the downregulation of TGF-β1, Vimentin, and PCNA and the upregulation of E-cadherin in a co-culture system. The EGR1 promoter dual-luciferase assay and the "partial rescue" of EGR1 overexpression confirmed that the inhibition of TGF-β1 by IGFBP7 originates from the transcriptional regulation mediated by EGR1, thus establishing a verifiable causal chain between "endocytosed IGFBP7" and the classic pro-tumor TGF-β1 pathway.

[0019] 5: Demonstrating that IGFBP7 has an autocrine anti-angiogenic effect in endothelial cells and is an endogenous negative regulator of angiogenesis: WGCNA showed that the IGFBP7 co-expression network is enriched in angiogenesis-related pathways, and knockdown of IGFBP7 upregulated pro-angiogenic genes (such as VEGFA, VEGFC, PECAM1, CD34, etc.) and was verified by RT-qPCR in two types of endothelial cells (HMEC-1 / HUVEC). At the same time, in vitro lumen formation experiments directly confirmed that the vascular network structure was more complex and the number of branch points increased, indicating that IGFBP7 not only affects tumor cell behavior, but can also reversely restrict angiogenesis at the endothelial cell level.

[0020] 6: Discovery of VAPA-mediated lysosome-dependent degradation of IGFBP7, proposing a new regulatory axis of "VAPA–lysosome–IGFBP7": After screening VAPA as a conserved interacting protein by IP-MS and verifying it by Co-IP, the study showed that VAPA knockdown did not affect IGFBP7 uptake (no difference in ELISA), but significantly led to intracellular IGFBP7 accumulation, and Bafilomycin A1 further enhanced accumulation. Combined with TEM and LAMP1 immunofluorescence results, VAPA is mainly involved in the transport / activation of IGFBP7 to the lysosomal degradation pathway after endocytosis, which explains for the first time the key node of IGFBP7 fate control in tumor cells.

[0021] 7: In vivo studies have demonstrated that IGFBP7 possesses potential for tumor suppression and anti-angiogenic therapy, and can enhance the efficacy of anti-VEGF therapy: In CT26 subcutaneous tumor and AOM / DSS models, exogenous supplementation with rmIGFBP7 reduced tumor burden, decreased CD31-positive vascular invasion, and downregulated TGF-β1, while neutralization of IGFBP7 had the opposite effect; more importantly, the combination of rmIGFBP7 and anti-VEGF antibody significantly improved colon shortening and reduced tumor number and burden compared to monotherapy, indicating that IGFBP7 is not only a mechanistic molecule, but also has the availability and feasible pathway to be transformed into a "combined anti-angiogenic strategy". Attached Figure Description

[0022] Appendix Figure 1 IGFBP7 is lowly expressed in vascular endothelial cells of colorectal cancer (CRC) tumors and is associated with poor prognosis. (A) Schematic diagram of experimental design for single-cell RNA sequencing (scRNA-seq) and NuGEN RNA sequencing (drawn by BioRender.com); (B) UMAP map of cell sources from two different tissue types; (C) Cell-annotated UMAP map of the two tissues; (D) Marker genes for each cell type; (E) Expression level of IGFBP7 in each cell type; (F) Expression level of IGFBP7 in endothelial cells of adenomas and adenocarcinomas; (G) NuGEN RNA sequencing results showing the expression level of endothelial IGFBP7 in CD31⁺ cells; (H) Representative RNAscope images showing IGFBP7 expression in endothelial cells of three pairs of colorectal cancer tumors and adjacent normal tissues (scale bar, 50 μm); (I) Expression level of IGFBP7 in endothelial cells of paired colorectal cancer tumors and adjacent normal tissues; (J) Kaplan-Meier analysis showing the relationship between endothelial cell IGFBP7 expression level and overall survival of colorectal cancer patients; (K) Kaplan-Meier analysis showed the relationship between endothelial cell IGFBP7 expression level and disease-free survival in colorectal cancer patients; (L) Cox regression analysis showed the correlation between endothelial cell IGFBP7 expression level and disease-free survival in colorectal cancer patients; (M) The proportion of endothelial cell IGFBP7 and representative flow cytometry images in paired colorectal cancer tumor tissues and adjacent normal tissues; (N) Serum IGFBP7 expression levels in colorectal cancer patients and healthy controls;

[0023] Appendix Figure 2During inflammation-driven carcinogenesis, the expression of IGFBP7 in endothelial cells is downregulated in stages. (A) Schematic diagram of the timeline of mice sacrificed at different time points in an AOM / DSS-induced colitis-associated colorectal cancer mouse model (drawn by BioRender.com); (B) PCA analysis of mouse colon tissue transcriptome sequencing; (C) Representative immunofluorescence images of IGFBP7 expression in mouse endothelial cells at different time points (scale bar, 50 μm); (D, E) Statistical analysis of fluorescence intensity of IGFBP7 expression in mouse endothelial cells at different time points; (F, G) Statistical analysis of IGFBP7 expression in mouse endothelial cells at different time points by flow cytometry; (H) Pseudo-temporal analysis of colon tissue from T1 to T4 stages in an AOM / DSS-based AOM / DSS model mouse based on Mfuzz; (I) Enrichment analysis of genes in Cluster 7; (J) Schematic diagram of an AOM / DSS-induced colitis-associated colorectal cancer model in Igfbp7^fl / fl; Cdh5-Cre^+ / - mice (drawn by BioRender.com); (K) Colon length (L) of mice treated with AOM / DSS and Igfbp7^fl / fl; Cdh5-Cre^+ / -; Number of tumors in the colon of mice treated with AOM / DSS and Igfbp7^fl / fl; Cdh5-Cre^+ / -.

[0024] Appendix Figure 3IGFBP7, secreted by endothelial cells, can be endocytosed by tumor cells. (A) Expression levels of IGFBP7 mRNA in endothelial cell lines, colorectal cancer (CRC) cell lines, and FHC; (B) Secretion levels of IGFBP7 in the culture supernatant of endothelial cell lines, CRC cell lines, and FHC; (C) Expression levels of IGFBP7 protein in endothelial cell lines, CRC cell lines, and FHC; (D) Changes in IGFBP7 secretion levels in HMEC-1 cells after treatment with Golgi Stop (1 μl / ml) or GW4869 (10 μM) for 6 hours; (E) Schematic diagram of endothelial cells and CRC cells co-cultured for 48 hours (drawn by BioRender.com); (F) Changes in IGFBP7 protein levels in CRC cells and HMEC-1 cells co-cultured for 48 hours; (G) Changes in IGFBP7 mRNA levels in CRC cells and HMEC-1 cells co-cultured for 48 hours; (H, I) Changes in IGFBP7 secretion levels in the culture supernatant of CRC cells and HMEC-1 cells co-cultured for 48 hours; (J) The levels of IGFBP7 protein in CRC cells were detected after co-culturing with HMEC-1 for 48 hours and adding MG132 (20 μM) or DMSO in the last 5 hours; (K, L) The levels of IGFBP7 mRNA expression in CRC tumor cells were detected after treatment with recombinant IGFBP7 protein (100, 300 or 500 ng / ml) for 48 hours; (M) The levels of IGFBP7 and 6×His tag protein expression in CRC tumor cells were detected after treatment with recombinant IGFBP7 protein (100, 300 or 500 ng / ml) for 48 hours.

[0025] Appendix Figure 4IGFBP7, derived from endothelial cells, inhibits the migration and proliferation of colorectal cancer cells through the TGF-β1 signaling pathway. (A) Venn plot showing the intersection of GSEA pathway enrichment in transcriptome sequencing results after SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 48 hours; (B) GSEA enrichment curve of the "GO_BP_CELL_MIGRATION" pathway after SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 48 hours; (C) GSEA enrichment curve of the "GO_BP_BLOOD_VESSEL_MORPHOGENESIS" pathway after SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 48 hours; (D) Venn plot showing genes enriched in the "GO_BP_CELL_MIGRATION" pathway and downregulated (fc < 0, p < 0) in transcriptome sequencing results after SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 48 hours.(E) Heatmap showing genes commonly downregulated in tumor cell lines, data from SW480; (F, G) mRNA expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA after 48 hours of co-culture of tumor cells and endothelial cells; (H) Protein expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA after 48 hours of co-culture of tumor cells and endothelial cells; (I) Luciferase activity was detected after SW480 cells were transfected with plasmid and co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 24 hours. Data were normalized by Renilla luciferase activity (firefly / Renilla), and the control group was used as 1 to represent fold change; (J) SW480 cells were transfected with EGR1 overexpression plasmid and co-cultured with recombinant IGFBP7 protein (100 ng / ml) for 24 hours. After co-culturing for 48 hours (ng / ml), the expression levels of EGR1, TGF-β1, and IGFBP7 were detected; (K, L, M) Cell migration assays were used to assess the migration ability of SW480 and HCT116 tumor cells after treatment with culture supernatants obtained from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA for 48 hours (scale bar, 200 μm); (N, O, P) Scratch healing assays were used to assess the healing of SW480 and HCT116 tumor cells after treatment with culture supernatants obtained from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA for 24 hours (scale bar, 100 μm); (Q, R) CCK-8 cell proliferation assays were used to assess the proliferation of SW480 and HCT116 tumor cells after treatment with culture supernatants obtained from HMEC-1 cells transiently transfected with IGFBP7 siRNA for 24, 48, 72, and 96 hours; (S, T) The CCK-8 cell proliferation assay was used to evaluate the proliferation of SW480 and HCT116 tumor cells after treatment with culture supernatants obtained from HUVEC cells transiently transfected with IGFBP7 siRNA for 24, 48, 72, and 96 hours.

[0026] Appendix Figure 5IGFBP7 deficiency enhances the angiogenesis potential of endothelial cells. (A) A hierarchical clustering dendrogram of co-expression modules obtained by WGCNA analysis based on NuGEN RNA sequencing performed after endothelial cell sorting; (B) A dot plot showing the pathways enriched by the network containing IGFBP7; (C) GSEA enrichment curve of the "GOBP_REGULATION_OF_VASCULATURE_DEVELOPMENT" pathway after transient transfection of endothelial cells with IGFBP7 siRNA; (D) A heatmap of transcriptome data after transient transfection of endothelial cells with IGFBP7 siRNA, with red indicating upregulated genes and blue indicating downregulated genes, and the color intensity reflecting the degree of expression change; (E, F) Verification of mRNA levels of angiogenesis-related genes after transient transfection of endothelial cells with IGFBP7 siRNA; (G, H, I) Angiogenesis experiments were used to evaluate the angiogenesis capacity of endothelial cells after transient transfection with IGFBP7 siRNA, using HMEC-1 cells (2 × 10⁻⁶) transfected with siRNA. 4 HUVEC cells (5 × 10³ cells / well) or HUVEC cells (5 × 10³ cells / well) were seeded on Matrigel and incubated at 37°C for 6 hours (scale bar, 100 μm).

[0027] Appendix Figure 6IGFBP7 inhibits tumor growth and angiogenesis in a mouse model. (A) Schematic diagram of the experimental design for intraperitoneal injection of physiological saline (100 μl / mouse), recombinant IGFBP7 protein (10 μg / mouse), IgG (100 μg / mouse), or anti-mouse IGFBP7 antibody (100 μg / mouse) in a subcutaneous tumor mouse model (drawn by BioRender.com); (B, C) Representative tumor images of subcutaneous tumors in mice of different treatment groups; (D) Quantitative analysis of tumor weight in different treatment groups; (E, F) Tumor growth curves showing changes in tumor volume in mice of different treatment groups; (G) Representative immunohistochemical (IHC) staining images of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) expression in mouse tumor tissue; (H) Bar chart of optical density values ​​of CD31-positive regions in tumor tissue of different treatment groups, used to reflect changes in angiogenesis; (I) Bar chart of optical density values ​​of TGF-β1-positive regions in tumor tissue of different treatment groups, used to reflect the expression level of TGF-β1; (J) Schematic diagram of the experimental design for 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 an AOM / DSS-induced colitis-associated colorectal cancer model (illustrated by BioRender.com); (K, L) Representative images of colorectal tumors in AOM / DSS-induced colitis-associated CRC models in different treatment groups; (M) Quantitative analysis of tumor number in AOM / DSS-induced colitis-associated CRC models in different treatment groups; (N) Representative immunohistochemical (IHC) staining images of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) expression in colorectal tumor tissue in the AOM / DSS-induced colitis-associated CRC model; (O) Bar chart of vascular density in CD31-positive areas of tumor tissue in different treatment groups; (P) (Q) Bar chart of optical density values ​​of TGF-β1 positive regions in tumor tissues of different treatment groups; (Q) Schematic diagram of experimental design of AOM / DSS-induced colitis-related colorectal cancer model: mice were intraperitoneally injected weekly with physiological saline (100 μl / mouse) or recombinant IGFBP7 protein (10 μg / mouse), and intraperitoneally injected twice weekly with mouse IgG2a Kappa antibody (5 mg / kg) or anti-VEGF monoclonal antibody B20-4.1.1.1 (5 mg / kg) (drawn by BioRender.com); (R, S) Representative images and quantitative analysis of mouse colon length; (T, U) Representative images and quantitative analysis of mouse colon tumor number.

[0028] Appendix Figure 7VAPA promotes lysosomal degradation of IGFBP7 and regulates the TGF-β1 signaling pathway. (A) Immunoprecipitation-mass spectrometry (IP-MS) results showing proteins interacting with IGFBP7; (B) Immunoprecipitation analysis confirming the interaction between endogenous IGFBP7 and VAPA in HCT116 cells; (C) Immunoprecipitation analysis confirming the interaction between endogenous VAPA and IGFBP7 in CaCO2 cells; (D, E) Verification of VAPA knockdown protein levels in tumor cells; (F) IGFBP7 protein expression level after co-culturing VAPA-knockdown tumor cells with endothelial cells for 48 hours; (G) Changes in IGFBP7 concentration in the culture medium after co-culturing VAPA-knockdown tumor cells with recombinant IGFBP7 protein (100 ng / ml) for 48 hours; (H) Detection of IGFBP7 protein expression level in VAPA-knockdown SW480 cells under co-culturing conditions with and without recombinant IGFBP7 (100 ng / ml) for 48 hours, with Bafilomycin A1 (20 μM) added during the last 6 hours; (I) (J) Determination of lysosome count in VAPA-knockdown SW480 cells after treatment with recombinant IGFBP7 protein (100 ng / ml) for 48 hours; (K) Representative immunofluorescence staining images of IGFBP7 and LAMP1 in HCT116 cells after VAPA knockdown or co-culture with recombinant IGFBP7 protein (100 ng / ml) for 48 hours, with green representing IGFBP7, red representing LAMP1, and blue representing cell nuclei (scale bar, 50 μm); (L) Quantitative analysis of relative fluorescence intensity of IGFBP7 in each group; (M) Quantitative analysis of relative fluorescence intensity of LAMP1 in each group; (M) Expression levels of IGFBP7, TGF-β1 signaling pathway and EMT pathway-related marker proteins in VAPA-knockdown SW480 tumor cells after co-culture with HMEC-1 endothelial cells for 48 hours. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] It should be noted that, due to formatting issues, the XML file submitted with the sequence listings involved in the embodiments requires all "U" characters in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 to be replaced with "T". The specific details are as shown in the embodiments.

[0031] Example 1

[0032] I. Materials and Methods

[0033] Cell lines: Colorectal cancer (CRC) cell lines RKO, SW620, SW480, SW1116, HCT116, HT29, Caco-2, LoVo, and FHC were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and confirmed by STR. All cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin / streptomycin (HyClone). HMEC-1 (iCell, Shanghai, China; catalog number iCell-h375) and HUVEC (iCell, Shanghai, China; catalog number iCell-h110) cells were cultured using the manufacturer's dedicated endothelial cell culture medium (iCell-h375-001b and iCell-h110-001, respectively). All cells were cultured in a 37°C, 5% CO2 incubator.

[0034] Cell treatment and inhibitor treatment: HMEC-1 cells were treated with either a Golgi transport inhibitor (BDBiosciences, catalog number 554724, 1 μl / ml) or GW4869 (GLPBIO, catalog number GC19186, 10 μM) for 6 hours. After treatment, the culture supernatant was collected for ELISA detection. Tumor cells were treated with either MG132 (20 μM, MedChem Express, USA, catalog number HY-13259) for 5 hours or Bafilomycin A1 (20 μM) for 6 hours, and then collected for Western blot analysis. Tumor cells were also treated with recombinant IGFBP7 protein (R&D Systems, catalog number 1334-B7) for 48 hours. After treatment, the culture supernatant and cells were collected for subsequent experiments.

[0035] siRNA and plasmid transfection: IGFBP7 siRNA and negative control (NC) siRNA were purchased from ObioTechnology (Shanghai, China). siRNA transfection of HMEC-1 and HUVEC cells was performed according to the manufacturer's instructions. siRNA sequences are shown in Table 1. shVAPA and NC plasmids were designed and synthesized by ObioTechnology (Shanghai, China). Tumor cells were transfected with either the NC or shVAPA plasmid using Lipofectamine™ 8000 transfection reagent (Beyotime, China, catalog number C0533), and selected for 7 days using puromycin (5 μg / mL) to obtain stable cell lines. Transfection efficiency was verified by real-time quantitative reverse transcription PCR (RT-qPCR) and Western blot. shRNA sequences are listed in Table 2.

[0036]

[0037] Cell co-culture: Colorectal cancer (CRC) cells and endothelial cells were co-cultured in Transwell chambers (Corning, New York, USA). CRC cells were cultured at 5 × 10⁶ cells / year. 5 Endothelial cells were seeded at a density of 10 cells / well in the lower chamber, with endothelial cells at a density of 2 × 10⁶ cells / well. 5 Cells were seeded at a density of 10 cells / well in the upper chamber and cultured for 48 hours. The cells and culture supernatant were then collected for subsequent experiments.

[0038] Quantitative reverse transcription PCR (RT-qPCR): Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen, catalog number 15596026). The RNA was reverse transcribed into complementary DNA (cDNA) using the HyperScript one-strand cDNA synthesis kit (NovaBio), followed by RT-qPCR detection using SYBR Green PCR Mix (NovaBio). Primer sequences are shown in Table 3.

[0039]

[0040]

[0041] Western blotting: Total cell protein was lysed at 4°C for 30 min using RIPA lysis buffer (Beyotime, catalog number P0013B) containing an InStab™ protease inhibitor mixture (EDTA-free, Yeasen, catalog number 20124ES03) and an InStab™ phosphatase inhibitor mixture (Yeasen, catalog number 20109ES05). The lysis buffer was centrifuged at 12,000 × g at 4°C for 10 min, and protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher, catalog number 23227). Western blot experiments were performed according to standard procedures.

[0042] Immunofluorescence staining: Cells or tumor tissues were treated according to standard methods and fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with phosphate-buffered saline (PBS) containing 0.2–0.5% Triton X-100 for 10 minutes. Samples were blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature to reduce nonspecific binding. Primary antibodies were diluted with blocking buffer and incubated overnight at 4°C. After washing with PBS, samples were incubated with the corresponding fluorescently labeled secondary antibody at room temperature for 1 hour in the dark. Cell nuclei were then counterstained with DAPI for 5 minutes, mounted with anti-quenching mounting medium, and imaged under a fluorescence microscope. Images were processed and analyzed using ImageJ software.

[0043] Immunohistochemistry (IHC): Tissue samples were rapidly fixed in 10% formalin and sectioned using standard paraffin embedding procedures. Subsequently, standard immunohistochemical staining procedures were performed, including antibody incubation and colorimetric reactions, to detect the expression of the target protein. The stained sections were imaged using an optical microscope, and quantitative analysis was performed using ImageJ software.

[0044] Co-immunoprecipitation (Co-IP) assay: Lysate of colorectal cancer (CRC) cells containing the target protein was incubated overnight at 4°C with a specific antibody, followed by immunoprecipitation using Protein G agarose beads (MedChemExpress, catalog number HY-K0204). After thorough washing, the protein complex bound to the agarose beads was eluted with SDS loading buffer and analyzed by Western blot.

[0045] Cell migration assay: SW480 or HCT116 cells (5 × 10⁻⁶) were transported to the target cell line. 4Cells (10 cells / well) were seeded in the upper chamber of a Transwell (Corning, New York, USA) containing serum-free medium, while the lower chamber was supplemented with serum-containing medium supplemented with recombinant IGFBP7 protein, or with conditioned medium derived from endothelial cell culture. Cells were incubated at 37°C for 48 hours. After incubation, cells that had migrated to the submembrane surface were fixed with 4% paraformaldehyde for 15 minutes and stained with crystal violet. Three microscopic fields were randomly selected for counting and quantitative analysis.

[0046] Scratch healing assay: SW480 or HCT116 cells were seeded in 6-well plates and cultured to 90%–100% confluence. A linear scratch was then made on the cell monolayer using a sterile 200 μL pipette tip. The cells were then gently washed twice with PBS to remove detached cells, and further cultured in conditioned medium supplemented with recombinant IGFBP7 protein or conditioned medium derived from endothelial cells transfected with IGFBP7 siRNA. Images of the scratched areas were captured using a phase-contrast microscope at 0 and 24 hours, and the degree of scratch healing was quantitatively analyzed using ImageJ software. Results were expressed as a percentage relative to the initial scratch width.

[0047] Cell counting kit-8 (CCK-8) assay: SW480 or HCT116 cells (3 × 10³ cells / well) were seeded in 96-well plates and cultured in conditioned medium derived from endothelial cells transfected with IGFBP7 siRNA. Cell viability was assessed at 0, 24, 48, 72, and 96 hours using the CCK-8 assay kit (MedChemExpress, catalog number HY-K0301). Absorbance was measured at 450 nm using a microplate reader; higher absorbance indicated stronger cell viability.

[0048] Lumen formation assay: Matrigel (BD Biosciences, catalog number 354234) was melted overnight at 4°C. 50 μL was added to each well of a 96-well plate and incubated at 37°C for 30 minutes to solidify. Subsequently, HMEC-1 cells transfected with siRNA (2 × 10⁶ cells / well) were... 4 HUVEC cells (5 × 10³ cells / well) or HUVEC cells (5 × 10³ cells / well) were seeded on Matrigel surfaces and incubated at 37°C for 6 hours. The total vessel length was statistically analyzed using the angiogenesis analysis plugin in ImageJ software to assess lumen-forming capacity.

[0049] Dual-luciferase reporter gene assay: When cell confluence reached approximately 70%–80%, cells were seeded in 24-well plates and co-transfected with the target promoter reporter plasmid and the internal control plasmid. After transfection, recombinant IGFBP7 protein was added to the designated experimental groups and the cells were co-cultured for 24 hours. Cells were then collected and lysed, and the luciferase activities of firefly and renal luciferase were detected sequentially using a dual-luciferase reporter gene assay kit. Firefly luciferase activity was normalized to renal luciferase activity, and results are expressed as relative luciferase activities. Each experiment was performed in at least three biological replicates.

[0050] ELISA assay: Conditioned culture medium from colorectal cancer cells and endothelial cells was collected and centrifuged at 1000 g for 10 minutes to remove cell debris and impurities; serum samples were clarified by centrifugation at 3000 g for 10 minutes. Following the manufacturer's instructions, IGFBP7 levels were quantitatively detected using the human IGFBP7 ELISA kit (Abcam, catalog number ab229894) and the mouse IGFBP7 ELISA kit (Abcam, catalog number ab245712-1), respectively.

[0051] Endothelial cell isolation: Before isolating endothelial cells, the colon was first rinsed with PBS to remove intestinal contents, and the mucus on the surface of the intestinal lumen was gently scraped off with a sterile scalpel. The colonic tissue was then transferred to a culture dish, minced into small pieces, and placed in a 50 mL centrifuge tube. The tissue was then digested for 30 minutes at 37°C in a MACS C-tube (Miltenyi Biotec, catalog number 130-093-237) with agitation in a digestion solution containing collagenase I, collagenase IV, Dispase, and DNase I (Sigma-Aldrich). After adding serum-containing buffer to terminate digestion, the cell suspension was filtered through a 40 μm nylon filter to remove undigested tissue pieces. Finally, endothelial cells were enriched by positive selection using CD31-coupled magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions.

[0052] Flow cytometry: Endothelial cells obtained from isolation were used to detect IGFBP7 expression by flow cytometry. For human samples, cells were first incubated on ice for 10 minutes with an Fc receptor blocking reagent to block Fc receptors. Subsequently, cells were incubated with surface antibodies (including active dyes, anti-CD45 antibody, and anti-CD31 antibody) in FACS buffer at 4°C for 30 minutes. After washing, cells were permeabilized at 4°C for 30 minutes, followed by blocking with buffer containing 4% BSA at room temperature in the dark for 30 minutes. Cells were then divided into two groups: a control group without primary antibody and an experimental group incubated with anti-IGFBP7 primary antibody at 4°C in the dark for 60 minutes. After washing, cells were incubated with fluorescently labeled secondary antibody at 4°C in the dark for 30 minutes. For mouse samples, cells were directly incubated with fluorescently labeled anti-IGFBP7 antibody after permeabilization. Finally, IGFBP7 expression in endothelial cells was detected using BD flow cytometry, and data analysis was performed using FlowJo software.

[0053] Clinical samples: Paired tumor tissues and adjacent normal tissues were obtained from the Department of Pathology, Xiang'an Hospital, Xiamen University. This study protocol was approved by the Ethics Committee of Xiang'an Hospital, Xiamen University (Approval No.: 20200722YJ001). All patients signed informed consent forms and agreed to participate in this study before sample collection.

[0054] Mouse models: Igfbp7-Flox mice (NM-CKO-231807) were purchased from Shanghai Baimo Biotechnology Co., Ltd., and Cdh5-Cre mice were purchased from The Jackson Laboratory (JAX #006137; PMID: 16450386). Igfbp7^fl / fl; Cdh5-Cre^+ / - mice were constructed using Cre recombinase mediated by the Cdh5 (VE-cadherin) promoter to achieve specific knockout of Igfbp7 in endothelial cells. A colitis-associated colorectal cancer model was established using azomethane (AOM) / dextran sulfate sodium (DSS). Male C57BL / 6 mice aged 6–8 weeks were used in the experiment. Mice were given a single intraperitoneal injection of AOM (10 mg / kg body weight) on day 0. From day 8, 2.5% (w / v) DSS was added to the drinking water for 7 consecutive days, followed by normal drinking water for 14 days. This cycle was repeated 3 times. During the experiment, mouse body weight changes, fecal characteristics, and rectal bleeding were monitored regularly. To assess the effect of IGFBP7 on colorectal cancer formation, mice were intraperitoneally injected weekly with IGFBP7 neutralizing antibody (100 μg / mouse, ABclonal, catalog number A4615), control IgG (100 μg / mouse, ABclonal, catalog number AC005), recombinant IGFBP7 protein (10 μg / mouse, Sino Biological, catalog number 51125-M02H), or sterile saline. Mice were sacrificed at the end of the experiment, and colons were collected for gross tumor counting, histological evaluation, and molecular biological analysis. In addition, mouse anti-VEGF monoclonal antibody (SydLabs, clone number B20-4.1.1.1) and isotype control IgG2a kappa antibody (Syd Labs, clone number 12B9) were administered intraperitoneally twice weekly at a dose of 5 mg / kg.

[0055] In a subcutaneous xenograft model, colon cancer cells from CT26 mice (1 × 10⁻⁶) were transplanted. 6 Tumor cells (suspended in 100 μL of sterile PBS) were subcutaneously injected into the right back of 8-week-old female BALB / c mice. Tumor size was measured every 2 days using calipers, and tumor volume was calculated using the formula: Volume (mm³) = (Length × Width²) / 2. When the tumor volume reached approximately 100 mm³, the mice were randomly divided into 4 groups and injected intraperitoneally twice weekly with either IGFBP7 neutralizing antibody (100 μg / mouse), control IgG (100 μg / mouse), recombinant IGFBP7 protein (10 μg / mouse), or sterile saline. Mice were sacrificed at the experimental endpoint, and tumor tissue was collected for subsequent histological and molecular analysis.

[0056] Immunoprecipitation-mass spectrometry (IP-MS): To identify proteins interacting with IGFBP7, SW480 and HCT116 cells were treated with recombinant human IGFBP7 protein (R&D Systems, catalog number 1334-87-025) for 48 hours under standard culture conditions. After treatment, cells were washed with pre-chilled PBS and lysed on ice with IP lysis buffer for 30 minutes. Cell debris was removed by centrifugation at 12,000 × g, 4°C for 10 minutes, and the supernatant was incubated overnight at 4°C with anti-6×His-tagged antibody. The next day, pre-washed Protein G beads were added, and the cells were incubated at 4°C for several hours to capture the antibody-protein complex. After thorough washing to remove non-specifically binding proteins, the bound protein complex was sent for mass spectrometry analysis (Thermo Orbitrap Fusion Lumos).

[0057] Transmission electron microscopy (TEM): SW480 cells stably transduced with control shRNA (SW480-nc) or VAPA-targeting shRNA (SW480-shVAPA) were cultured under standard conditions and treated with or without recombinant human IGFBP7 protein (300 μg / mL) for 6 hours, as per the experimental design. TEM samples were prepared according to standard procedures and imaged using a transmission electron microscope (Hitachi HT-7800). Lysosomes were identified based on their morphological characteristics, and the number of lysosomes in each cell was quantitatively analyzed in randomly selected fields of view.

[0058] RNAscope In Situ Hybridization: Human colorectal cancer (CRC) tissue microarrays (Zhongke Guanghua, China, catalog number D160Co01S) were analyzed using RNAscope® Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, ACD), strictly following the manufacturer's instructions (PMID: 22166544). Tissue sections were first dewaxed, then subjected to heat-induced target repair using RNAscope TargetRetrieval Reagent, and further processed with RNAscope Protease Plus for optimal permeabilization. Specific probes targeting human IGFBP7 (ACD, catalog number 316681) and CD31 (ACD, catalog number 487381-C2) mRNA were incubated in a HybEZ™ hybridization oven at 40°C for 2 hours. After a series of signal amplification steps, signal detection was performed using Opal fluorescent dye (Akoya Biosciences) following a multiplex fluorescence detection protocol, with cell nuclei counterstained with DAPI. Images were acquired using confocal fluorescence microscopy, where RNA signals appeared as punctate fluorescent signals localized in the cytoplasm and / or nucleus. Using ImageJ software, the expression levels of IGFBP7 and CD31 were quantitatively analyzed by calculating the average number of fluorescent spots per cell within representative regions.

[0059] Single-cell RNA sequencing (scRNA-seq): Following the manufacturer's instructions, a single-cell RNA sequencing library was constructed using the 10x Genomics Chromium Single Cell 3′ Library and Gel Bead Kit v3. Nuclear suspensions were stained with 0.4% trypan blue, and cell viability was assessed under a microscope. Samples with greater than 80% viability were selected for subsequent experiments. Samples were generated using gel bead-in-emulsion on a Chromium Controller (10x Genomics), during which individual mRNA molecules were barcoded and reverse transcribed to generate cDNA. After cDNA synthesis, the emulsion was broken and purified, followed by fragmentation, end repair, and A addition treatment. Sequencing adapters were then ligated, and PCR amplification was performed. Library quality was assessed using Qubit and Bioanalyzer. Circularized DNA was replicated using rolling circle replication to generate DNA nanoballs, which were loaded onto a high-density array and sequenced using combinatorial probe anchoring synthesis (cPAS) technology on the MGI sequencing platform.

[0060] Whole RNA sequencing (Bulk RNA-seq): Total RNA was extracted from each sample as the starting material for constructing the whole RNA sequencing library, and the library was constructed according to standard procedures. The final library was quantified and quality-checked using methods such as qPCR or capillary electrophoresis. Qualified libraries were then loaded onto the BGISEQ sequencing platform (MGI Tech Co., Ltd., China) for sequencing.

[0061] NuGEN RNA Sequencing: Total RNA was extracted using the RNeasy Micro Kit (QIAGEN, Germany) according to the manufacturer's instructions, and the procedure was strictly followed. Library preparation was then performed using the Revelo™ RNA-Seq High Sensitivity Library Construction Kit (Tecan, Switzerland). The library was amplified using phi29 DNA polymerase to generate DNA nanospheres (DNBs), which were then loaded onto a high-density DNA nanosphere chip. Sequencing was performed on the DNBSEQ sequencing platform (MGI Tech Co., Ltd., China) using cPAS technology to obtain PE100 paired-end sequencing data.

[0062] Bioinformatics analysis: Differential expression analysis of bulk RNA-seq data was performed using the limma software package. The criteria for screening differentially expressed genes (DEGs) were fold change >1 or <1 and false discovery rate (FDR) <0.01. Gene set enrichment analysis (GSEA) was then performed.

[0063] In the co-expression analysis, a gene co-expression network was constructed using the WGCNA R software package (v1.72-1) (PMID: 19114008). Based on the weighted signed correlation network method, eight endothelial cell samples were analyzed, with a soft threshold parameter set to 15, identifying 24 initial gene modules. The modules were then merged using the "mergeCloseModules" function, with a cutoff height set to 0.25. Functional enrichment analysis of each module was performed using the DAVID database (PMID: 19131956).

[0064] For single-cell RNA sequencing (scRNA-seq) data, cells with fewer than 1000 unique molecular identifiers (UMIs) and fewer than 200 detected genes were excluded. Data standardization, dimensionality reduction, and graph-based clustering analysis were performed using the Seurat software package (version 4.1.1) according to standard procedures.

[0065] II. Statistical Analysis

[0066] All statistical analyses were performed using GraphPad Prism 9.5 (GraphPad, San Diego, CA) and R software (v4.1.2). Data are expressed as mean ± standard deviation (SD). Two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, supplemented by Tukey's post-hoc test. Statistical significance was defined as: *p < 0.05, **p < 0.01, ***p < 0.001, and p > 0.05 was considered not statistically significant (ns).

[0067] Third, the results

[0068] (a) IGFBP7 is expressed at low levels in vascular endothelial cells of colorectal cancer tumors and is associated with poor prognosis.

[0069] To investigate the role of IGFBP7 in the transformation of precancerous adenomas into colorectal cancer (CRC), we collected adenoma and adenocarcinoma tissue samples from CRC patients for single-cell RNA sequencing (scRNA-seq). Furthermore, tumor tissues and their paired adjacent normal tissues were enzymatically digested, and CD31⁺ vascular endothelial cells were sorted and subjected to NuGEN RNA sequencing. Figure 1 A). scRNA-seq analysis identified 15 cell populations that were distinct from each other at the transcriptional level. Figure 1B–C, including naive T cells (CD3D, CD3E, CD3ζ, CCR7, LEF1), helper T17 (Th17) cells (CD3D, CD3E, RORA, IL17A, CCL20), regulatory T (Treg) cells (CD3D, CD3E, TIGIT, CTLA4, FOXP3), CD8⁺ T cells (CD3D, CD3E, CD8A, CD8B, GZMK, KLRD1), natural killer (NK) cells (NKG7, CST7, PRF1, GZMB), natural killer T (NKT) cells (CD3D, CD3E, KLRC2, GZMA), and NCR⁺ type 3 innate lymphocytes (NCR⁺). ILC3s (KIT, IL1R1, AHR, PCDH9), B cells (BANK1, CD79A, CD79B, VPREB3, CD74), plasma cells (JCHAIN, MZB1, XBP1), CD16⁺ monocytes (FCGR3B, CSF3R, S100A8, S100A9), macrophages (CD68, CD163, C1QA, C1QB, CSF1R), mast cells (TPSAB1, CPA3, KIT, IL1RL1), epithelial cells (KRT8, EPCAM, CLDN4, TSPAN8, LCN2), endothelial cells (IGFBP7, FLT1, SPARC, VWF, TCF4), and MKI67⁺ progenitor cells (MKI67, STMN1, TUBB) Figure 1 D). Compared with other cell types, IGFBP7 exhibits specific and significant enrichment expression in vascular endothelial cells. Figure 1 E). Notably, compared with endothelial cells derived from adenomatous tissue, the expression of IGFBP7 in tumor-associated endothelial cells was significantly reduced, suggesting that endothelial IGFBP7 expression is gradually downregulated during malignant transformation. Figure 1 F). Consistent with single-cell results, transcriptomic analysis of purified CD31⁺ cells also showed that IGFBP7 expression in tumor tissue was significantly lower than in paired adjacent normal colorectal tissue. Figure 1 (G). In summary, these unbiased transcriptomic analyses indicate that IGFBP7 is an endothelial cell-enriched gene that is gradually downregulated during the development and progression of CRC.

[0070] To validate these findings in a larger clinical cohort, we used RNAscope technology to perform dual in situ detection of CD31 and IGFBP7 on a tissue microarray containing 70 pairs of CRC tumors and adjacent normal tissues. Consistent with sequencing results, IGFBP7 expression in tumor-associated endothelial cells was significantly lower than in adjacent normal tissues. Figure 1H–I). Furthermore, in patients with lymph node metastasis, endothelial IGFBP7 expression showed a further decreasing trend. More importantly, survival analysis showed that patients with high IGFBP7 expression in tumor endothelial cells had significantly better overall survival (OS) and disease-free survival (DFS). Figure 1 J–K). Multivariate Cox regression analysis further confirmed that endothelial IGFBP7 expression was an independent prognostic factor for DFS in CRC patients (HR = 0.2, p = 0.05; Figure 1 L). Flow cytometry analysis of endothelial cells isolated from paired tumors and adjacent normal tissues also confirmed a significant decrease in IGFBP7 expression in tumor endothelial cells. Figure 1 M). Given that IGFBP7 is a secreted protein, we further examined circulating IGFBP7 levels, and the results showed that serum IGFBP7 concentrations in CRC patients were significantly lower than those in healthy controls. Figure 1 In summary, these results indicate that IGFBP7 is highly expressed in normal vascular endothelial cells, but is significantly downregulated in tumor-associated endothelial cells during CRC progression, and its absence is closely associated with poor clinical outcomes.

[0071] (ii) During inflammation-driven carcinogenesis, the expression of IGFBP7 in endothelial cells is downregulated in stages.

[0072] To systematically evaluate the potential role of IGFBP7 expression in vascular endothelial cells at different stages of the inflammatory-to-carcinogenic process, we constructed an AOM / DSS-induced colitis-associated colorectal cancer (CRC) mouse model. Figure 2 A), this model is widely regarded as a classic animal model for studying inflammation-related tumorigenesis (PMID: 33160389). In this model, colon tissue was collected at four representative stages, including the acute inflammatory phase (T1), the chronic inflammatory phase (T2), the early tumorigenesis phase (T3), and the tumor progression phase (T4), and the samples were subjected to transcriptome sequencing analysis.

[0073] Principal component analysis (PCA) results showed that the T1 and T2 stage samples were highly clustered at the transcriptomic level, suggesting that they have similar inflammation-related transcriptional features. In contrast, the T3 and T4 stage samples were not only clearly separated from the inflammatory stage, but also showed significant differences between them, indicating that significant transcriptomic remodeling occurred during the transformation of inflammation into tumor and tumor progression. Figure 2 B). The above results suggest that T3 and T4 are key stages in the development and progression of colorectal tumors. Based on this, we further analyzed the expression changes of IGFBP7 in colon tissue at different stages.

[0074] Immunofluorescence analysis showed that, compared with healthy control colon tissue, the expression of IGFBP7 in vascular endothelial cells during the inflammatory phase was significantly increased; however, starting from the early tumorigenesis stage (T3), the expression of IGFBP7 in endothelial cells significantly decreased, and further decreased during the tumor progression stage (T4). Figure 2 C–E). Flow cytometry analysis of the isolated endothelial cells also confirmed that IGFBP7 expression showed a continuous downregulation trend with tumor initiation and progression. Figure 2 (F–G), suggesting that endothelial IGFBP7 has a significant stage-dependent regulatory feature in the development of colorectal cancer.

[0075] To further characterize the dynamic changes of IGFBP7 and its related gene programs over time, we used the Mfuzz algorithm to perform soft clustering analysis on RNA-seq data from stages T1 to T4. Figure 2 H). Mfuzz analysis yielded nine gene clusters, with IGFBP7 primarily enriched in Cluster 7. Within this cluster, IGFBP7 was gradually downregulated during the inflammatory phase, showed some recovery during early tumorigenesis (T3), but decreased sharply during tumorigenesis (T4). Conversely, several key angiogenesis-related genes (including VEGFA, HIF-1α, ANGPT2, MMP9, and VEGFC) and TGF-β signaling pathway-related genes (TGF-β1 and SMAD2) exhibited the opposite expression trend, showing lower expression in T3 and significant upregulation in tumorigenesis (T4). Furthermore, these gene expression changes were further validated by RT-qPCR. Functional enrichment analysis of Cluster 7 genes revealed their primary involvement in biological processes such as apoptosis, cell adhesion, cell migration, signal transduction, angiogenesis, and endosome-lysosome transport. Figure 2 (I) This suggests that the downregulation of endothelial IGFBP7 is closely related to the activation of pro-angiogenic and pro-tumor-related pathways.

[0076] To investigate the functional significance of endothelial cell IGFBP7 in colorectal tumorigenesis, we further constructed a mouse model of endothelial cell-specific IGFBP7 knockout (Igfbp7^fl / fl; Cdh5-Cre^+ / −, see Methods section). Under basal physiological conditions, endothelial cell-specific IGFBP7 deletion did not lead to significant changes in colon length, and histological analysis showed that the colon structure and morphology remained intact, with no obvious pathological changes. Based on this, we established an AOM / DSS-induced colitis-related CRC model (…). Figure 2 J). Compared with control mice, mice lacking IGFBP7 in their endothelial cells exhibited more pronounced colonic shortening (J). Figure 2K) and a significantly increased tumor burden ( Figure 2 L).

[0077] In summary, these results indicate that the expression of IGFBP7 in vascular endothelial cells is dynamically and stage-specifically regulated during the transformation from inflammation to carcinogenesis, and its deficiency exacerbates disease severity in colitis-associated CRC models. These findings highlight the crucial role of endothelial IGFBP7 in maintaining colonic homeostasis and inhibiting tumor progression.

[0078] (iii) IGFBP7 secreted by endothelial cells can be endocytosed by tumor cells.

[0079] To investigate the cellular origin and potential mechanisms of action of IGFBP7, we first systematically analyzed the expression characteristics of IGFBP7 in various cell lines at the transcriptional level, intracellular protein abundance, and secretion level in conditioned media. These included two vascular endothelial cell lines (HMEC-1 and HUVEC), normal intestinal epithelial cells (FHC), and eight colorectal cancer (CRC) cell lines. The results showed that IGFBP7 transcripts were highly expressed in HMEC-1 and HUVEC cells, while they were almost unexpressed or completely undetectable in all the CRC cell lines examined. Figure 3 A). Consistent with this, high levels of IGFBP7 secretion were detected in the culture supernatants of HMEC-1 and HUVEC, while almost no IGFBP7 was detected in the culture supernatants of CRC cell lines. Figure 3 B). These results suggest that IGFBP7 is mainly secreted by endothelial cells, rather than produced by tumor cells themselves.

[0080] It is noteworthy that although IGFBP7 exhibits high transcriptional levels and is secreted in large quantities in HMEC-1 and HUVEC cells, the protein is almost undetectable by Western blot in their intracellular lysates. Figure 3 C). Conversely, although IGFBP7 transcription and secretion were lacking in CRC cell lines, a certain level of intracellular IGFBP7 protein was detectable in all tested CRC cell lines. Figure 3 C). This significant inconsistency between transcriptional and secretion levels and intracellular protein abundance strongly suggests that IGFBP7 in tumor cells is not derived from its endogenous synthesis, but more likely from the extracellular microenvironment.

[0081] To clarify the secretion pathway of IGFBP7, we treated HMEC-1 cells with a Golgi transport inhibitor (Golgi stop) and an exosome secretion inhibitor (GW4869), respectively. The results showed that Golgi stop treatment significantly reduced the level of IGFBP7 in the culture supernatant, while GW4869 treatment had no significant effect on its secretion. Figure 3 (D) indicates that IGFBP7 is mainly released through the classical secretory pathway, rather than relying on exosome-mediated secretion mechanisms.

[0082] Given the extremely low expression of IGFBP7 in tumor cells while endothelial cells can secrete large amounts of IGFBP7, we hypothesized that the IGFBP7 secreted by endothelial cells might be endocytosed by neighboring tumor cells. To verify this hypothesis, we established a co-culture system of colorectal cancer cell lines (SW480 and HCT116) with endothelial cells (HMEC-1 and HUVEC). Figure 3 E). After co-culturing for 48 hours, the level of IGFBP7 protein in tumor cells significantly increased ( Figure 3 F), while its mRNA expression level decreased (F). Figure 3 The presence of G suggests that the increased protein levels are not due to endogenous transcriptional upregulation, but more likely to be from extracellular uptake. Simultaneously, elevated IGFBP7 concentrations were detected in both the upper and lower chambers of the co-culture system. Figure 3 The H–I assay confirmed the efficient diffusion and translocation of IGFBP7 between cells. Similar results were obtained using a co-culture model constructed with HUVEC cells.

[0083] To rule out the possibility that IGFBP7 accumulation in CRC cells stemmed from protein degradation, we treated tumor cells with the proteasome inhibitor MG132 under both endothelial cell co-culture conditions and without endothelial cells. The results showed that MG132 treatment alone did not affect IGFBP7 protein levels in tumor cells, and a significant increase in IGFBP7 was only observed when co-cultured with endothelial cells. Figure 3 J), thus ruling out the possibility that altered proteasome degradation was the main factor, further supporting the paracrine uptake mechanism.

[0084] Furthermore, after co-culturing CRC cells with different concentrations of recombinant IGFBP7 protein for 48 hours, no significant changes in IGFBP7 mRNA expression were observed. Figure 3 K–L), but intracellular IGFBP7 protein levels were significantly elevated. More importantly, the accumulated IGFBP7 protein could be recognized by specific antibodies targeting the recombinant protein tag (K–L). Figure 3 M) clearly confirmed that IGFBP7 in tumor cells originates from exogenous supply rather than endogenous transcription.

[0085] In summary, these results indicate that IGFBP7 is a secretory protein released by vascular endothelial cells via the classical secretory pathway and can be actively endocytosed by neighboring tumor cells, thus potentially playing an important biological role in the tumor microenvironment.

[0086] (iv) Endothelial-derived IGFBP7 inhibits the migration and proliferation of colorectal cancer cells through the TGF-β1 signaling pathway.

[0087] To investigate the biological effects of IGFBP7 after endocytosis by tumor cells, we co-cultured recombinant IGFBP7 protein with three colorectal cancer (CRC) cell lines (SW480, HCT116, and Caco2) for 48 hours, and then performed RNA sequencing analysis on the collected cells. Pathway enrichment analysis of differentially expressed genes (DEGs) in the three cell lines revealed that 60 signaling pathways were consistently altered after IGFBP7 treatment. Figure 4 A). Among them, several pathways related to tumor cell migration were significantly downregulated ( Figure 4 B). At the same time, pathways related to vascular morphogenesis and vascular system development were also significantly suppressed (B). Figure 4 C), suggesting that IGFBP7 can reduce the pro-angiogenic potential of CRC cells.

[0088] To further clarify the key molecular mechanism by which IGFBP7 inhibits cell migration, we performed an intersection analysis of migration-related genes commonly regulated in three CRC cell lines, identifying six genes with consistent regulation, including TGFB1, HDAC5, GRN, PIK3CD, ITGA3, and SEMA4B. Figure 4 (D–E). Among them, the TGF-β1 signaling pathway has been widely confirmed to play a pro-cancer role in tumor growth and progression (PMID: 39061061). Based on the above results, we speculate that IGFBP7 may exert its tumor-suppressive effect by regulating the TGF-β1 signaling pathway.

[0089] To test this hypothesis, we co-cultured CRC cells (SW480 and HCT116) with HMEC-1 endothelial cells for 48 hours. RT-qPCR analysis showed that the mRNA expression of TGF-β1, Vimentin, and PCNA in tumor cells was significantly downregulated, while the expression of E-cadherin was significantly upregulated. Figure 4 F–G). Western blot analysis further confirmed the consistency of the above changes at the protein level (F–G). Figure 4 H). Similar results were observed in the co-culture system with HUVEC cells, indicating that endothelial-derived IGFBP7 can stably regulate TGF-β1-related signaling and epithelial-mesenchymal plasticity.

[0090] To elucidate the molecular mechanism by which IGFBP7 inhibits TGF-β1, we systematically analyzed the expression of key TGF-β1 regulators in transcriptome data. The results showed that under IGFBP7 co-culture conditions, the expression of EGR1, P300, and CREBBP was significantly downregulated in both tumor cell lines. Previous studies have shown that EGR1 can bind to and regulate the TGF-β1 promoter, thereby participating in its transcriptional regulation (PMID: 38385088, 15958557, 10984481). Furthermore, EGR1 can also affect downstream TGF-β1 signaling pathways by regulating the transcriptional coactivators P300 and CREBBP (PMID: 15225550). Based on this, we propose that IGFBP7 may block TGF-β1 expression by inhibiting the transcriptional activity of EGR1.

[0091] To verify this hypothesis, we constructed an EGR1 promoter-driven dual-luciferase reporter plasmid and treated cells with recombinant IGFBP7 protein for 24 hours after successful transfection. The dual-luciferase assay results showed that, compared with the control group, IGFBP7 significantly reduced the transcriptional activity of the EGR1 promoter, while in cells transfected with the empty vector, IGFBP7 had no significant effect on luciferase activity. Figure 4 I). Furthermore, we constructed a stable SW480 cell line overexpressing EGR1 (SW480-OE-EGR1) and performed Western blot analysis 48 hours after treatment with or without recombinant IGFBP7. The results showed that IGFBP7 significantly inhibited TGF-β1 protein expression, while EGR1 overexpression significantly upregulated TGF-β1 levels; notably, EGR1 overexpression could partially offset the inhibitory effect of IGFBP7 on TGF-β1 (…). Figure 4 These results collectively indicate that IGFBP7 downregulates TGF-β1 expression by inhibiting EGR1-mediated transcriptional activation.

[0092] Subsequently, we further evaluated the effect of IGFBP7 on tumor cell migration. In the Transwell assay, the addition of recombinant IGFBP7 protein to the lower chamber significantly inhibited tumor cell migration. The scratch healing assay also showed that CRC cell migration was significantly reduced after 24 hours of IGFBP7 treatment. To elucidate the functional role of endothelial-derived IGFBP7, we constructed an IGFBP7 knockdown endothelial cell line and verified its knockdown efficiency using RT-qPCR and ELISA. Co-culturing CRC cells with IGFBP7-deficient endothelial cells significantly enhanced tumor cell migration. Figure 4 K–M). Furthermore, conditioned medium derived from IGFBP7 knockdown endothelial cells (after centrifugation to remove cell debris) significantly promoted tumor cell migration in the scratch assay. Figure 4 N–P). Meanwhile, CCK-8 cell proliferation assays showed that conditioned medium derived from IGFBP7-deficient endothelial cells significantly enhanced the proliferation of tumor cells. Figure 4 Q–T).

[0093] In summary, these results indicate that IGFBP7 secreted by endothelial cells effectively inhibits the migration and proliferation of colorectal cancer cells by suppressing the TGF-β1-related signaling pathway, thereby limiting the motility and growth potential of tumor cells.

[0094] (v) IGFBP7 deficiency enhances the angiogenic potential of endothelial cells

[0095] To further elucidate the autocrine regulatory role of IGFBP7 in endothelial cells, we conducted weighted gene co-expression network analysis (WGCNA) based on NuGEN RNA sequencing data from tumor endothelial cells to analyze the co-expression relationship between IGFBP7 and other genes in the network. Figure 5 A). The results showed that the co-expression network containing IGFBP7 was significantly enriched in angiogenesis-related pathways (A). Figure 5 B) suggests that IGFBP7 may play a regulatory role in endothelial cell angiogenesis.

[0096] To directly investigate the functional significance of IGFBP7, we constructed an IGFBP7 knockdown HMEC-1 endothelial cell line and performed transcriptome comparison analysis with control cells. Pathway enrichment results showed that IGFBP7 deficiency significantly enhanced the angiogenesis capacity of endothelial cells. Figure 5C), and simultaneously, cellular immune response-related functions are also enhanced. Further analysis of differentially expressed genes related to angiogenesis revealed that several key pro-angiogenic genes were significantly upregulated, including CEACAM1, TGF-β2, KLF2, ADM2, MDK, CD34, PECAM1, VEGFA, and VEGFC. Figure 5 D).

[0097] Subsequently, we validated the expression changes of the above genes in two types of endothelial cells, HMEC-1 and HUVEC, using RT-qPCR. The results consistently showed that IGFBP7 knockdown significantly upregulated the expression levels of these pro-angiogenic genes in both types of endothelial cells. Figure 5 Based on this, we further evaluated the functional impact of IGFBP7 deficiency on endothelial cell angiogenesis capacity using in vitro angiogenesis experiments. Consistent with transcriptomic and gene expression results, HMEC-1 and HUVEC cells lacking IGFBP7 exhibited significantly enhanced angiogenesis capacity, specifically manifested in an increased number of lumen formations and a more complex vascular network structure. Figure 5 G–I).

[0098] In summary, these results indicate that IGFBP7 acts as a negative regulator of angiogenic potential in endothelial cells by inhibiting the expression of pro-angiogenic genes.

[0099] (vi) IGFBP7 inhibits tumor growth and angiogenesis in mouse models

[0100] To further investigate the biological function of IGFBP7 in vivo, we established a subcutaneous xenograft model by subcutaneously inoculating mouse colorectal cancer CT26 cells onto the dorsal side of BALB / c mice. Starting from day 9 post-inoculation, mice were intraperitoneally injected twice weekly with saline, recombinant mouse IGFBP7 protein (rmIGFBP7), IgG isotype control antibody, or anti-IGFBP7 neutralizing antibody, and tumor volume was measured every two days. Figure 6 A). On day 19, the mice were sacrificed, and the tumors were removed for weighing and photographic recording. Figure 6 B–C). The results showed that rmIGFBP7 treatment significantly reduced tumor weight (B–C). Figure 6 D) and inhibited tumor growth ( Figure 6 E), while treatment with anti-IGFBP7 neutralizing antibodies significantly increased tumor weight and accelerated tumor progression (E). Figure 6F). To elucidate its mechanism of action, we performed immunohistochemical analysis on tumor tissue. The results showed that rmIGFBP7 treatment significantly reduced intratumoral vascular density and downregulated TGF-β1 protein expression, while anti-IGFBP7 antibody treatment led to enhanced vascular invasion and increased TGF-β1 expression (F). Figure 6 Furthermore, rmIGFBP7 treatment significantly promoted tumor cell apoptosis and inhibited their proliferation, while anti-IGFBP7 antibody treatment showed the opposite effect.

[0101] Given that the AOM / DSS-induced colitis-associated CRC model better mimics the inflammation-driven pathogenesis of human CRC, we further used this model to validate the in vivo function of IGFBP7. During the first and second DSS treatment cycles, mice were intraperitoneally injected weekly with either IgG control antibody or anti-IGFBP7 antibody; during the second and third DSS cycles, mice were intraperitoneally injected weekly with either saline or rmIGFBP7. Mice were sacrificed after completing all three DSS treatment cycles, and the colonic tumor burden was assessed by counting tumor numbers. Figure 6 J). The results showed that administration of anti-IGFBP7 antibody during the early inflammatory phase did not significantly increase tumor burden (J). Figure 6 K), while supplementation with rmIGFBP7 in the later stages of inflammation-to-carcinogenesis significantly reduced the number of tumors and the overall tumor burden (K). Figure 6 L–M). Immunohistochemical staining further showed that rmIGFBP7 treatment inhibited vascular invasion and TGF-β1 expression in colon tumor tissue, reduced tumor cell proliferation, and promoted apoptosis, while anti-IGFBP7 antibody treatment produced the opposite effect. Figure 6 N–P).

[0102] Subsequently, to evaluate whether IGFBP7 could enhance the efficacy of anti-angiogenic therapy, we combined mouse anti-VEGF monoclonal antibody B20-4.1.1.1 with rmIGFBP7 in the AOM / DSS model and systematically evaluated its anti-tumor effects. Figure 6 Q). The results showed that both rmIGFBP7 and antiVEGF antibody administration alone significantly alleviated inflammation-induced bowel shortening and reduced tumor burden (Q). Figure 6 R–S). Notably, combination therapy showed a more significant effect in inhibiting tumor development and progression than monotherapy, suggesting that IGFBP7 can enhance the anti-tumor effect of anti-VEGF therapy (R–S). Figure 6 T–U).

[0103] In summary, we systematically evaluated the in vivo function of IGFBP7 under different treatment conditions using the CT26 subcutaneous xenograft model and the AOM / DSS-induced colitis-related CRC model. Exogenous supplementation of IGFBP7 can inhibit tumor progression by limiting vascular invasion and suppressing the TGF-β1 signaling pathway in the tumor microenvironment; while neutralizing IGFBP7 promotes angiogenesis and accelerates tumor growth. More importantly, IGFBP7 can enhance the anti-tumor efficacy of anti-VEGF therapy, suggesting its potential therapeutic target value in anti-angiogenic therapy for colorectal cancer.

[0104] (vii) VAPA promotes lysosomal degradation of IGFBP7 and regulates the TGF-β1 signaling pathway.

[0105] To further elucidate the molecular mechanism by which IGFBP7 functions in tumor cells, we used immunoprecipitation-mass spectrometry (IP-MS) to identify proteins that interact with IGFBP7. Colorectal cancer cell lines (SW480 and HCT116) were treated with recombinant IGFBP7 protein carrying a 6×His tag, followed by IP-MS analysis targeting the 6×His tag. A total of 14 co-enriched proteins were identified in both cell lines. Figure 7 The presence of proteins A suggests that these proteins may be conserved IGFBP7 interactors. Among them, vesicle-associated membrane protein-associated protein A (VAPA) is a member of the VAMP-associated protein family and a highly conserved membrane protein located in the endoplasmic reticulum, known to participate in lipid transport and membrane transport processes (PMID: 9657962). To verify the interaction between IGFBP7 and VAPA in tumor cells, we further conducted a co-immunoprecipitation (Co-IP) experiment, using VAPA antibody for immunoprecipitation combined with Western blot analysis. The results confirmed that IGFBP7 and VAPA interact at the protein level. Figure 7 B–C).

[0106] To further explore the functional significance of IGFBP7–VAPA interaction, we constructed a tumor cell line with stable VAPA knockdown and corresponding control cells, and verified the knockdown efficiency of VAPA at the protein level. Figure 7 D–E). Notably, the loss of VAPA did not alter the expression level of endogenous IGFBP7 in tumor cells (D–E). Figure 7D–E). Subsequently, we evaluated whether VAPA affected the intracellular processing of IGFBP7 after tumor cells took up IGFBP7. After co-culturing endothelial cells with VAPA-knockdown tumor cells, the level of IGFBP7 in tumor cells was detected by Western blot. The results showed that VAPA knockdown significantly led to a large accumulation of endocytosed IGFBP7 in tumor cells. Figure 7 F) indicates that the cleaning process is hindered.

[0107] To clarify whether VAPA affects the actual uptake efficiency of exogenous IGFBP7, we further supplemented the culture medium with recombinant IGFBP7 and quantitatively analyzed the intracellular IGFBP7 level by ELISA. The results showed no significant difference in IGFBP7 uptake between VAPA-knockdown cells and control cells. Figure 7 (G) indicates that VAPA does not affect the process of IGFBP7 entering tumor cells, but mainly participates in its intracellular processing after entering the cells.

[0108] Given the important role of VAPA in membrane transport, we hypothesized that VAPA might promote the lysosome-dependent degradation of endocytosed IGFBP7. To test this hypothesis, we combined the lysosomal inhibitor Bafilomycin A1 with recombinant IGFBP7 protein in VAPA-knockdown tumor cells. Western blot results showed that VAPA deficiency itself significantly increased intracellular IGFBP7 protein levels, while lysosomal inhibition further exacerbated IGFBP7 accumulation. Figure 7 The results suggest that IGFBP7 is primarily degraded via the lysosomal pathway, with VAPA playing a promoting role in this process. Consistent results were observed in co-culture systems with HMEC-1 or HUVEC endothelial cells, where lysosomal inhibition further enhanced IGFBP7 accumulation in VAPA-deficient tumor cells.

[0109] To further corroborate this mechanism, we performed transmission electron microscopy analysis. The results showed that recombinant IGFBP7 treatment significantly enhanced lysosomal activity in tumor cells, while under the same conditions, VAPA knockdown significantly reduced lysosomal activation. Figure 7I). Furthermore, we performed immunofluorescence staining analysis of IGFBP7 and the lysosomal marker LAMP1 in VAPA-knockdown HCT116 cells under co-culture conditions with recombinant IGFBP7. The results showed that although VAPA knockdown itself did not change the basal lysosome number, it significantly weakened the IGFBP7-induced increase in lysosomes, leading to further accumulation of intracellular IGFBP7. Figure 7 J–L).

[0110] Finally, to assess the downstream effects of enhanced IGFBP7 stability in the VAPA-deficient context, we examined key molecular changes in the TGF-β1 signaling pathway in VAPA-knockdown tumor cells co-cultured with HMEC-1. The results showed that VAPA deficiency was accompanied by decreased expression of TGF-β1 and the stromal marker N-cadherin, decreased Smad2 phosphorylation levels, and increased expression of the epithelial marker E-cadherin. Figure 7 M).

[0111] In summary, our study shows that IGFBP7 endocytosed by tumor cells undergoes lysosome-dependent degradation via the VAPA-mediated membrane transport pathway; the absence of VAPA weakens lysosomal function, leading to the accumulation of IGFBP7 in cells and inhibiting the activation of the pro-tumor TGF-β1 signaling pathway, thereby enhancing the antitumor effect of IGFBP7.

Claims

1. Application of IGFBP7 as a biomarker in the preparation of colorectal cancer diagnostic kits.

2. Application of IGFBP7 as a biomarker in the preparation of colorectal cancer prognosis and staging kits.

3. Application of reagents for detecting IGFBP7 gene or protein content in the preparation of colorectal cancer diagnostic kits.

4. Application of reagents for detecting IGFBP7 gene or protein content in the preparation of colorectal cancer prognosis and staging kits.

5. Application of IGFBP7 or IGFBP7 promoters in the preparation of drugs for treating colorectal cancer.

6. Application of IGFBP7 or IGFBP7 promoters in the preparation of drugs to prevent recurrence and metastasis of colorectal cancer and treatment resistance.

7. A pharmaceutical composition for treating colorectal cancer, characterized in that, The pharmaceutical composition consists of IGFBP7 and an anti-VEGF antibody.

8. The use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating colorectal cancer.

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