Use of a TROP2-FN1 interaction pathway inhibitor in the preparation of a product for diagnosing, preventing, and treating bone marrow metastasis of colorectal cancer

Abstract

The present invention relates to the field of biomedicine, and provides the use of a TROP2-FN1 interaction pathway inhibitor in the preparation of products for diagnosing, preventing, and treating bone marrow metastasis of colorectal cancer. The use of an FN1 inhibitor in the preparation of products for diagnosing, preventing, and treating bone marrow metastasis of colorectal cancer. Advantages: The inventors of the present invention found that TROP2 has the highest expression level in bone metastasis CRC patients. Knocking down the expression of TROP2 in CRC cells can significantly inhibit the proliferation, migration ability, and stemness level of CRC cells themselves; and CRC cells bind to the Fibronectin fiber network derived from bone marrow mesenchymal stem cells (BMSCs) through high expression of TROP2, increasing cell adhesion and bone marrow colonization ability, and promoting bone marrow metastasis of CRC cells. After inhibiting the expression of FN1 or downstream integrin, the adhesion ability of CRC cells is also significantly inhibited. Thus, it provides a new treatment strategy for bone marrow metastasis of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to the use of a TROP2-FN1 interaction pathway inhibitor in the preparation of products for diagnosing, preventing, and treating bone marrow metastasis of colorectal cancer. Background Art

[0002] Colorectal cancer-bone marrow metastasis (CRC-BMM) has a dangerous clinical biological behavior and extremely poor prognosis. Due to the extensive dissemination and proliferation of tumor cells in the bone marrow cavity, resulting in bone marrow hematopoietic dysfunction, the vast majority of patients are accompanied by systemic blood diseases, such as disseminated intravascular coagulation (DIC), prolonged clotting time accompanied by a significant increase in D-dimer, obvious bleeding tendency or pancytopenia, and obvious pain, especially back pain; without effective treatment, the median survival time is about 2 weeks; for patients who have received active treatment, the median survival is only about 8 months. Once bone marrow metastasis occurs, the prognosis and quality of life of CRC patients will decline significantly, and there is an urgent need to develop new treatment methods. The team where the applicant is located focuses on the clinical treatment and basic research of CRC-BMM, and has collected tumor specimens from some CRC-BMM patients for multi-omics analysis, and preliminary research results have been obtained.

[0003] Reviewing the previous literature, in China, two cases of rectal cancer bone marrow metastasis were reported as early as 1985, and only 31 cases have been reported abroad so far. In 1986, Weiss et al. reported a study on the autopsy of colorectal cancer patients to explore their metastasis patterns, and the results showed that when combined with other site metastases, about 27% of patients would have bone marrow metastasis. These reports suggest that although CRC-BMM is rare, with the extension of the survival time of advanced colorectal cancer patients, its incidence may be underestimated clinically.

[0004] In recent years, with the development of surgical and comprehensive treatment techniques, the overall survival of patients with colorectal cancer, even those with advanced metastatic colorectal cancer, has been significantly improved, and the incidence of the previously rare phenomenon of bone marrow metastasis has increased significantly. CRC-BMM is somewhat different from bone metastasis of colorectal cancer. An important difference is that patients with CRC-BMM have hematopoietic system abnormalities and do not necessarily experience skeletal related events (SREs) caused by bone destruction or hyperplasia, suggesting that tumor cells act more on the bone marrow cavity rather than the bone cortex. It is reported that systemic chemotherapy based on XELOX or FOLFOX can effectively prolong the median survival time of patients with CRC-BMM. In addition, Artac et al. found that in patients with breast cancer bone marrow metastasis, the application of targeted HER2 drugs and estrogen therapy can achieve complete remission of the tumor and long-term survival of the patients. Due to the bleeding tendency, the use of targeted drugs (such as bevacizumab) in patients with CRC-BMM is somewhat restricted. Currently, neither domestic nor foreign guidelines mention relevant treatment recommendations for CRC-BMM, making it extremely difficult for clinicians to diagnose and treat corresponding patients. Summary of the Invention

[0005] The present invention aims to overcome at least one defect (shortcoming) of the above-mentioned prior art, and provides the use of a TROP2-FN1 interaction pathway inhibitor in the preparation of products for diagnosing, preventing and treating bone marrow metastasis of colorectal cancer, so as to provide a new treatment strategy for bone marrow metastasis of colorectal cancer.

[0006] One object of the present invention is to provide the use of a TROP2-FN1 interaction pathway inhibitor in the preparation of products for diagnosing, preventing and treating bone marrow metastasis of colorectal cancer.

[0007] The team where the applicant is located referred to the first-line treatment plan for advanced colorectal cancer, and applied a chemotherapy combined with or without a targeted drug plus an anti-DIC treatment plan, which can effectively improve the survival rate of patients with CRC-BMM and relieve clinical symptoms. These results suggest that by deeply exploring the pathogenesis of CRC-BMM and finding appropriate treatment targets, it is possible to improve the current clinical benefits of patients.

[0008] In one or more embodiments of the present invention, it was found that there was significant co-localization between TROP2 and FN1. The interaction between TROP2 and FN1 promoted the bone marrow colonization of CRC cells. TROP2 is tumor-associated calcium signal transducer 2 (also known as trophoblast cell-surface antigen 2), and FN1 is Fibronectin. In the tumor microenvironment, the signal transduction mechanism mediated by Fibronectin and integrin Integrin α / β complex is closely related to the maintenance of proliferation signals, promotion of angiogenesis, promotion of invasion and metastasis, and regulation of tumor immunity in tumor cells. According to the GTEx cell lineage expression data, Fibronectin has extremely high expression levels in BMSCs. In our preliminary experiments, CRC cells with high expression of TROP2 were co-cultured with BMSCs, and then protein purification and tandem mass spectrometry analysis were performed. Compared with the negative control, TROP2 was able to specifically bind to Fibronectin, and confocal microscopy also showed co-localization of TROP2 and Fibronectin on the cell surface. Overexpression of TROP2 significantly increased the number of adherent CRC cells and BMSCs, while the addition of Fibronectin neutralizing antibody weakened the adhesion ability of tumor cells.

[0009] Based on this and combined with more experiments, the inventors found that CRC cells bind to the Fibronectin fiber network derived from bone marrow mesenchymal stem cells (BMSCs) by highly expressing TROP2, increasing cell adhesion and bone marrow colonization ability, and promoting the bone marrow metastasis of CRC cells.

[0010] Another object of the present invention is to provide the use of an FN1 inhibitor in the preparation of products for the diagnosis, prevention, and treatment of colorectal cancer bone marrow metastasis.

[0011] Furthermore, the FN1 inhibitor is FN1-specific siRNA, and the FN1-specific siRNA is any one of the following two siRNAs:

[0012] siFN1#1: The sense strand is: 5’-GGAAAACACTATCAGATAA-3’ (as shown in SEQ ID NO:1), and the antisense strand is: 5’-TTATCTGATAGTGTTTTCC-3’ (as shown in SEQ ID NO:2);

[0013] siFN1#2: The sense strand is: 5’-CTGCGAGAGTAAACCTGAA-3’ (as shown in SEQ ID NO:3), and the antisense strand is: 5’-TTCAGGTTTACTCTCGCAG-3’ (as shown in SEQ ID NO:4).

[0014] Furthermore, the FN1 inhibitor is an FN1 neutralizing antibody.

[0015] Another object of the present invention is to provide the use of a TROP2 inhibitor in the preparation of products for diagnosing, preventing and treating bone marrow metastasis of colorectal cancer.

[0016] Furthermore, the TROP2-FN1 interaction pathway inhibitor is used to inhibit downstream integrin.

[0017] Furthermore, the TROP2-FN1 interaction pathway inhibitor is an Integrin αv / β5 neutralizing antibody, or siRNA that inhibits the expression of Integrinα5, αv, and β3.

[0018] Another object of the present invention is to provide the application of TROP2 as a diagnostic and therapeutic marker for bone marrow metastasis of colorectal cancer.

[0019] Another object of the present invention is to provide a kit for diagnosing bone marrow metastasis of colorectal cancer, comprising a reagent for detecting the expression level of TROP2 or a reagent for detecting the expression levels of Integrin α5, αv, and β3 proteins.

[0020] Another object of the present invention is to provide a pharmaceutical composition for treating bone marrow metastasis of colorectal cancer, comprising an FN1 inhibitor.

[0021] Furthermore, the FN1 inhibitor is FN1-specific siRNA, or the FN1 inhibitor is an FN1 neutralizing antibody.

[0022] Furthermore, the FN1-specific siRNA is any one of the following two siRNAs:

[0023] siFN1#1: The sense strand is: 5’-GGAAAACACTATCAGATAA-3’ (as shown in SEQ ID NO:1), and the antisense strand is: 5’-TTATCTGATAGTGTTTTCC-3’ (as shown in SEQ ID NO:2);

[0024] siFN1#2: The sense strand is: 5’-CTGCGAGAGTAAACCTGAA-3 (as shown in SEQ ID NO:3), and the antisense strand is: 5’-TTCAGGTTTACTCTCGCAG-3’ (as shown in SEQ ID NO:4).

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The inventors of the present invention found that TROP2 has the highest expression level in CRC patients with bone metastasis. Knockdown of TROP2 expression in CRC cells can significantly inhibit the proliferation, migration ability and stemness level of CRC cells; and CRC cells bind to the fibronectin fiber network derived from bone marrow mesenchymal stem cells (BMSCs) through high expression of TROP2, increasing cell adhesion and bone marrow colonization ability, promoting bone marrow metastasis of CRC cells. After inhibiting FN1 expression or downstream integrin expression, the adhesion ability of CRC cells is also significantly inhibited. Thus, it provides a new treatment strategy for bone marrow metastasis of colorectal cancer. Brief Description of the Drawings

[0027] Figure 1 Shown: TROP2 is more highly expressed in CRC patients with bone metastasis (BMM) than in patients without metastasis (Met. Free). The adjacent normal intestinal epithelial tissue in the section shows negative for TROP2.

[0028] Figure 2 Shown: After co-culturing CRC cells with BMSCs, it was identified that TROP2 and FN1 are co-localized on the cell surface.

[0029] Figure 3 Shown: The interaction between TROP2 and FN1 promotes the adhesion ability of CRC cells.

[0030] Figure 4 Shown: TROP2 is significantly elevated in the cancer tissues of CRC-BMM patients. (A) Heat map of differential gene expression analyzed by whole transcriptome sequencing; (B) Volcano plot of gene expression analyzed by whole transcriptome sequencing; (C) TROP2 expression results in cancer and adjacent tissues of 3 CRC-BMM patients; (D) Cancer and adjacent expression of TROP2 in TCGA-COAD data.

[0031] Figure 5 Shown: TROP2 is more highly expressed in CRC patients with bone metastasis (BMM) than in patients without metastasis (Met. Free). (Left) Representative figure; (Right) Statistical chart. It can be seen in the figure that the adjacent normal intestinal epithelial tissue is negative for TROP2.

[0032] Figure 6 Shown: TROP2 promotes the malignant phenotype of CRC cells. (A-B) Verification of overexpression and knockdown of TROP2; (C) Monitoring of cell proliferation; (D) Determination of colony formation ability; (E) Transwell assay for cell migration; (F) Cell adhesion assay.

[0033] Figure 7 Shown: FN1 protein expression abundance in cells from different sources (the red arrow indicates BMSCs).

[0034] Figure 8Show: There is an interaction between TROP2 and FN1. (A) Silver staining detection after purification of TROP2-Flag tag; (B) Mass spectrometry map of specific peptide of FN1; (C-D) Immunoprecipitation verification of TROP2-FN1 interaction.

[0035] Figure 9 Show: Co-localization of TROP2 and FN1 was observed by confocal microscopy.

[0036] Figure 10 Show: FN1 promotes the migration and adhesion ability of CRC cells. (A) 3D migration experiment to determine cell migration ability; (B) Knockdown of FN1 inhibits the adhesion ability of CRC cells to BMSCs.

[0037] Figure 11 Show: The interaction between TROP2 and FN1 depends on the participation of integrin. (A) Detection of the expression of some Integrin molecules in TROP2-overexpressing CRC cells; (B) The interaction between FN1 and TROP2 depends on Integrin expression; (C) TROP2-promoted adhesion of CRC cells depends on Integrin expression.

[0038] Figure 12 Show: Integrin is involved in the regulation of TROP2-FN1 interaction and the adhesion of CRC cells. (A) Integrin neutralizing antibody inhibits the TROP2-FN1 interaction; (B) FN1 or Integrin neutralizing antibody inhibits the adhesion of TROP2-high-expressing CRC cells.

[0039] Figure 13 Show: TROP2-high-expressing CRC cells can achieve tibial colonization and growth in nude mouse models faster. Detailed implementation manners

[0040] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0041] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0042] The present invention will be further described in conjunction with specific examples. The following examples are only for explaining the present invention and do not constitute a limitation to the present invention. The test samples and test procedures used in the following examples include the following content (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources).

[0043] Example 1

[0044] Expression study of TROP2 in CRC - BMM:

[0045] Our team was the first to apply multi - omics high - throughput sequencing methods to conduct systematic basic research on CRC - BMM. Previously, by obtaining in - situ cancer and adjacent tissue samples from 3 patients for RNA sequencing analysis, it was found that multiple proteins related to colorectal cancer metastasis and stemness regulation were highly expressed in the tumor tissues of CRC - BMM patients, including TACSTD2 (log2(fc)=6.67, FDR = 3.39E - 17). TACSTD2 (Tumor - associated calcium signal transducer 2, tumor - associated calcium signal transducer 2; also known as TROP2, trophoblast cell - surface antigen 2, trophoblast cell - surface antigen 2) belongs to a single - pass transmembrane glycoprotein, which consists of an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Our previous results showed that TROP2 had the highest expression level in CRC patients with bone metastasis, followed by CRC patients without metastasis, and had a very low expression level in normal intestinal epithelium. Knocking down the expression of TROP2 in CRC cells could significantly inhibit the self - proliferation, migration ability, and stemness level of CRC cells (as Figure 1 shown).

[0046] Example 2

[0047] Previous research basis:

[0048] According to the GTEx cell lineage expression data, Fibronectin has an extremely high expression level in BMSCs. In the inventor's preliminary experiment, CRC cells with high expression of TROP2 were co - cultured with BMSCs, and then protein purification tandem mass spectrometry analysis was performed. Compared with the negative control, TROP2 could specifically bind to Fibronectin, and confocal microscopy also showed the co - localization of TROP2 and Fibronectin on the cell surface (as Figure 2 、 Figure 8 and Figure 9). Overexpression of TROP2 can significantly increase the number of CRC cells adhering to BMSCs, while the addition of Fibronectin neutralizing antibody weakens the adhesion ability of tumor cells (as Figure 3 , Figure 10 , Figure 11 and Figure 12 ). These results suggest that Fibronectin may play an important role in mediating the bone marrow colonization of CRC cells.

[0049] Example 3

[0050] I. Clinical characteristics and treatment of CRC-BMM patients

[0051] Table 1 shows 8 CRC-BMM patients diagnosed and treated in our system. All patients had diffuse bone marrow invasion

[0052] signs. Most of these 8 patients received chemotherapy combined with targeted therapy, and tumor control and DIC status were improved in some patients. Among them, 5 patients had died and 3 were still under observation.

[0053] Table 1: Summary of treatment data of 8 CRC-BMM patients

[0054] Patient ID Age / Gender Primary Tumor Location Metastatic Site Blood Abnormality Bone Marrow Infiltration Treatment Outcome Survival Period (days) 1 67 / M Proximal Ascending Colon Near Ileocecal Junction Lymph Node, Liver, Bone Marrow, Peritoneum DIC + Cetuximab + mXeliri Survived 2 64 / M Ascending Colon Bone Marrow DIC + Bevacizumab + mFolfiri Died 105 3 47 / M Ileocecal Junction Lymph Node, Bone Marrow DIC + mXeliri Survived 4 64 / M Hepatic Flexure of Colon Lymph Node, Liver, Bone Marrow DIC + mXeliri Survived 5 58 / F Rectum Bone Marrow DIC + Cetuximab + mXeliri Died 60 6 70 / M Ileocecal Junction Lymph Node, Liver, Bone Marrow, Peritoneum DIC + No Treatment Died 3 7 44 / F Transverse Colon Bone Marrow, Peritoneum DIC + FOLFIRI Died 18 8 63 / M Sigmoid Colon Lymph Node, Liver, Bone Marrow DIC + FOLFIRI Died 18

[0055] In Table 1, mXeliri is capecitabine combined with irinotecan, and FOLFIRI is leucovorin, 5-FU and irinotecan combined chemotherapy.

[0056] II. High expression of TROP2 in CRC-BMM patients

[0057] We obtained the in-situ carcinoma and adjacent tissues of 3 CRC-BMM patients for whole transcriptome sequencing analysis. The results showed that there were 51 co-upregulated genes and 6 co-downregulated genes. We drew expression heatmaps and volcano plots for the differentially expressed genes ( Figure 4 A and Figure 4 B). According to the fold change of expression and P value screening, we selected TACSTD2 (i.e., TROP2) as the focus of subsequent research. TROP2 ranked the top in the list of differentially expressed genes in CRC-BMM sequencing (log2(fc)=6.67, FDR=3.39E-17) ( Figure 4 C), and this gene was also significantly upregulated in the cancer tissues of the TCGA colorectal cancer dataset (COAD) ( Figure 4 D).

[0058] We then detected the expression of TROP2 in in-situ tumor tissue sections of 50 CRC patients with bone metastases (BMM) and 50 CRC patients without metastases (Met.Free) in our hospital, and found that the expression of TROP2 was significantly higher in CRC patients with bone metastases ( Figure 5 ). These results suggest that the increased expression of TROP2 in CRC-BMM patients may have an important impact on the CRC tumor metastasis pattern.

[0059] III. High expression of TROP2 promotes the malignant phenotype of CRC cells

[0060] We first constructed CRC cell lines (HCT-116) with stable overexpression and knockdown of TROP2. Western Blotting and qPCR results showed high efficiency of TROP2 overexpression and knockdown ( Figure 6 A and 6B). Subsequently, we used a live cell real-time monitoring system to measure cell proliferation. Overexpression of TROP2 promoted the proliferation of CRC cells, while knockdown inhibited their growth ( Figure 6 C); Enhanced colony formation ability was also observed by the plate colony formation assay, and the opposite result was obtained after knockdown ( Figure 6 D). To explore the interaction factors between CRC and bone marrow colonization, we selected BMSCs and CRC cells for co-culture experiments. The results showed that after co-culture with BMSCs, the migration ability of TROP2 overexpressing cells was significantly enhanced compared with the control group ( Figure 6 E); Similarly, to observe the difference in cell adhesion ability, we seeded fluorescently labeled CRC cells onto a well plate covered with a monolayer of BMSCs, and washed away the non-adherent cells 30 minutes after seeding. The results showed that overexpression of TROP2 significantly enhanced the adhesion of CRC cells to BMSCs, while knockdown of TROP2 weakened this effect ( Figure 6 F).

[0061] In this example, the siRNA used for knocking down TROP2 is any one of the following two:

[0062] siTROP2#2: The sense strand is: 5’-CGTGTCCCACCAACAAGAT-3’ (as shown in SEQ ID NO:5), and the antisense strand is: 5’-ATCTTGTTGGTGGGACACG-3’ (as shown in SEQ ID NO:6);

[0063] siTROP2#3: The sense strand is: 5’-TCAAGGGCGAGTCTCTATT-3’ (as shown in SEQ ID NO:7), and the antisense strand is: 5’-AATAGAGACTCGCCCTTGA-3’ (as shown in SEQ ID NO:8).

[0064] IV. Interaction between TROP2 and Fibronectin Promotes CRC Cell Colonization in Bone Marrow

[0065] Considering that a large number of cancer cells colonize and grow in the bone marrow cavity of CRC-BMM patients, we hypothesized that CRC cells might communicate with BMSCs through cell-cell interaction to construct a microenvironment suitable for tumor survival. As a cell membrane protein, TROP2 can transduce signals through protein-protein interactions. Therefore, we performed tag protein purification followed by mass spectrometry analysis in a co-culture system of TROP2-Flag overexpressing CRC cells and BMSCs, aiming to explore the protein profile derived from BMSCs that can interact with TROP2. After screening the background proteins of the control groups (a total of two control groups: ① TROP2-CRC cells alone; ② co-culture of Vector-CRC cells and BMSCs), we obtained a series of candidate proteins (as shown in Table 2).

[0066] Table 2: The top ten candidate proteins derived from BMSCs that specifically bind to TROP2 with the highest mass spectrometry scores after screening against the background proteins of the control groups.

[0067] Gene Name Total PEP Score Coverage Rate [%] # Specific Peptide Bait TACSTD2 147.752 51 18 Prey FN1 366.342 38 62 MYH10 295.11 32 34 MYO6 216.601 45 45 MYH14 147.432 20 21 TUBA1C 135.253 57 2 KRT8 129.883 57 22 MYO1C 120.953 33 27 MYO1B 111.682 27 26 TUBB4A 104.558 59 3 KRT19 96.231 55 12

[0068] We focused on the FN1 protein. By searching the ProteomicsDB database, we learned that FN1 has a relatively high protein expression level in BMSCs, suggesting its enrichment in the bone marrow cavity ( Figure 7 ). We performed silver staining detection and found a specific band around 220kD in the purification system of co-culture of TROP2 overexpressing CRC cells and BMSCs (the molecular weight of FN1 in SDS-PAGE is approximately 220kD). Mass spectrometry identification confirmed that it was the FN1 protein ( Figure 8 A and 8B). Subsequently, we performed co-immunoprecipitation experiments. Through co-expression in 293T cells or positive and negative interaction verification in the co-culture system of CRC cells and BMSCs, we confirmed the interaction between TROP2 and FN1 ( Figure 8 C and 8D); confocal results also showed that compared with the TROP2 homologous protein EPCAM, TROP2 and FN1 showed significant co-localization, while no co-localization was found between EPCAM and FN1 ( Figure 9 ).

[0069] Next, we performed functional verification. In the 3D migration experiment, when exogenous FN1 protein was added, the migration circles of CRC cells, especially TROP2-overexpressing cells, were significantly enlarged ( Figure 10 A); in the adhesion experiment, when co-cultured with BMSCs treated with FN1-specific siRNA, the adhesion ability of TROP2-high-expressing CRC cells was significantly reduced, showing no significant difference from the control group ( Figure 10 B). These results suggest that the TROP2-FN1 interaction signal plays an important role in promoting the bone marrow colonization of CRC cells. The FN1-specific siRNA used in this example is any one of the following two siRNAs:

[0070] siFN1#1: The sense strand is: 5’-GGAAAACACTATCAGATAA-3’ (as shown in SEQ ID NO:1), and the antisense strand is: 5’-TTATCTGATAGTGTTTTCC-3’ (as shown in SEQ ID NO:2);

[0071] siFN1#2: The sense strand is: 5’-CTGCGAGAGTAAACCTGAA-3 (as shown in SEQ ID NO:3), and the antisense strand is: 5’-TTCAGGTTTACTCTCGCAG-3’ (as shown in SEQ ID NO:4).

[0072] IV. The interaction between TROP2 and Fibronectin promotes CRC cell adhesion depending on downstream integrin signal transduction

[0073] Because the integrin family plays an important role in mediating the physiological functions of extracellular matrix proteins such as FN1, we detected the expression levels of some integrin molecules in CRC cells. The results showed that after TROP2 overexpression, the protein expression levels of Integrin α5, αv, and β3 were significantly increased ( Figure 11 A); after knocking down the corresponding integrin expression using siRNA and performing immunoprecipitation with FN1, it was found that the interaction between TROP2 and FN1 was significantly weakened ( Figure 11 B), and at the same time, the adhesion ability of CRC cells mediated by TROP2 was also significantly inhibited ( Figure 11 C). Furthermore, we verified it again using neutralizing antibodies. After adding the Integrin αv / β5 neutralizing antibody (P1F6), the interaction between TROP2 and FN1 was weakened ( Figure 3 .10A), and after adding the FN1 neutralizing antibody (HFN7.1) or P1F6 to the co-culture system of CRC cells and BMSCs, the adhesion ability of CRC cells was also significantly inhibited ( Figure 12B). These results suggest that the interaction between TROP2 and Fibronectin and its mediated CRC cell adhesion function depend on the participation of downstream integrins.

[0074] In this example,

[0075] The siRNA for knocking down Integrin α5 is:

[0076] siITGA5: The sense strand is: 5’-TGGCTCAGACATTCGATCC-3’ (as shown in SEQ ID NO:9), and the antisense strand is: 5’-GGATCGAATGTCTGAGCCA-3’ (as shown in SEQ ID NO:10);

[0077] The siRNA for knocking down Integrin αv is:

[0078] siITGAV: The sense strand is: 5’-GAATATCGGTTGGATTATA-3’ (as shown in SEQ ID NO:11), and the antisense strand is: 5’-TATAATCCAACCGATATTC-3’ (as shown in SEQ ID NO:12);

[0079] The siRNA for knocking down Integrin β3 is:

[0080] siITGB3: The sense strand is: 5’-CCAAGACTCATATAGCATT-3’ (as shown in SEQ ID NO:13), and the antisense strand is: 5’-AATGCTATATGAGTCTTGG-3’ (as shown in SEQ ID NO:14).

[0081] Example 4

[0082] Establish a mouse model of CRC metastasis by orthotopic injection into the tibial cavity:

[0083] Currently, there is no mature model of colorectal cancer bone marrow metastasis. Referring to the bone metastasis of prostate cancer and breast cancer, we used BALB / c nude mice to perform orthotopic microinjection of luciferase-labeled HCT-116 cells into the tibial cavity, and successfully constructed a mouse model of CRC metastasis in the tibial cavity. Through in vivo imaging detection, we initially found that compared with the control group, TROP2-high-expressing CRC cells could achieve tibial colonization and growth faster, not only in terms of the number of tumors formed (control group: 1 / 3; TROP2-high-expressing group: 3 / 3) but also in terms of fluorescence intensity, which was higher than that of the control group ( Figure 3 .11). Subsequently, we will use this model to perform in vivo functional verification of TROP2-targeted drugs and related small molecule inhibitors.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of a TROP2-FN1 interaction pathway inhibitor in the preparation of a product for treating bone marrow metastasis of colorectal cancer, characterized in that, The TROP2-FN1 interaction pathway inhibitor is siRNA that inhibits the expression of Integrin αv and β3; The siRNA that inhibits Integrin αv is siITGAV, The sense strand of siITGAV is: 5'-GAATATCGGTTGGATTATA-3' (as shown in SEQ ID NO: 11), The antisense strand of siITGAV is: 5'-TATAATCCAACCGATATTC-3' (as shown in SEQ ID NO: 12); The siRNA that inhibits Integrin β3 is siITGB3, The sense strand of siITGB3 is: 5'-CCAAGACTCATATAGCATT-3' (as shown in SEQ ID NO: 13), The antisense strand of siITGB3 is: 5'- AATGCTATATGAGTCTTGG-3' (as shown in SEQ ID NO: 14).

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