Application of TROP2 inhibitor in preparation of product for diagnosing, preventing and treating colorectal cancer bone marrow metastasis

Abstract

The invention relates to the field of biological medicine, and provides application of a TROP2 inhibitor in preparation of a product for diagnosing, preventing and treating colorectal cancer bone marrow metastasis. The application has the advantages that the inventor finds that the expression quantity of the TROP2 in bone metastasis CRC patients is the highest, and the proliferation, migration ability and dryness level of CRC cells can be remarkably inhibited by knocking down the expression of the TROP2 in the CRC cells; and the CRC cells are combined with Fibronectin fiber webs derived from bone marrow mesenchymal stem cells (BMSCs) through high expression of TROP2, so that the cell adhesion and bone marrow colonization capacities are increased, the bone marrow metastasis of the CRC cells is promoted, the expression of FN1 or the expression of downstream integrin is inhibited, and the adhesion capacity of the CRC cells is also remarkably inhibited, thereby providing a new treatment strategy for the bone marrow metastasis of colorectal cancer.

Description

[0001] This application is a divisional application. The application date of the original application is August 6, 2024, the application number is 2024110697773, and the name of the invention is: Use of TROP2-FN1 interaction pathway inhibitors in the preparation of products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis. Technical Field

[0002] The present invention relates to the field of biomedicine, and more specifically, to the use of a TROP2 inhibitor in preparing products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis. Background Art

[0003] Colorectal cancer-bone marrow metastasis (CRC-BMM) presents a dangerous clinical and biological condition with an extremely poor prognosis. Due to the widespread dissemination and proliferation of tumor cells in the bone marrow cavity, bone marrow hematopoietic dysfunction occurs, leading to the majority of patients developing concurrent systemic hematologic disorders, such as disseminated intravascular coagulation (DIC), prolonged coagulation time with significantly elevated D-dimers, a pronounced bleeding tendency or pancytopenia, and significant pain, particularly back pain. Without effective treatment, the median survival is approximately two weeks; even with active treatment, the median survival is only about eight months. Once bone marrow metastasis occurs, the prognosis and quality of life of CRC patients are significantly reduced, necessitating the development of novel treatments. The applicant's team 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, yielding preliminary results.

[0004] A review of previous literature reveals that the earliest cases of bone marrow metastasis in rectal cancer were reported in my country in 1985, with two cases reported internationally to date. In 1986, Weiss et al. reported a study exploring the metastatic patterns of colorectal cancer patients through autopsy. Their results showed that approximately 27% of patients with concurrent metastases to other sites developed bone marrow metastases. These reports suggest that, although CRC-BMM is rare, its incidence may be clinically underestimated as survival rates for patients with advanced colorectal cancer increase.

[0005] In recent years, advances in surgical and combined treatment techniques have significantly improved the overall survival of patients with colorectal cancer, even those with advanced metastatic colorectal cancer. However, the incidence of bone marrow metastasis, previously rare, has increased significantly. CRC-BMM differs from colorectal cancer bone metastasis in certain ways. A key difference is that patients with CRC-BMM have hematopoietic abnormalities and do not necessarily experience skeletal-related events (SREs) resulting from bone destruction or proliferation, suggesting that tumor cells primarily target the bone marrow cavity rather than the cortical bone. Systemic chemotherapy based on XELOX or FOLFOX has been reported to effectively prolong the median survival of patients with CRC-BMM. Furthermore, Artac et al. demonstrated that the use of HER2-targeted drugs and estrogen therapy in patients with breast cancer bone marrow metastasis resulted in complete tumor remission and long-term survival. The use of targeted agents (such as bevacizumab) in patients with CRC-BMM is limited by the presence of a bleeding tendency. Currently, no domestic or international guidelines provide treatment recommendations for CRC-BMM, making it challenging for clinicians to diagnose and treat these patients. Summary of the Invention

[0006] The present invention aims to overcome at least one defect (shortcoming) of the above-mentioned prior art, provide a use of a TROP2 inhibitor in the preparation of products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis, and provide a new treatment strategy for colorectal cancer bone marrow metastasis.

[0007] One object of the present invention is to provide a use of a TROP2 inhibitor in the preparation of products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis.

[0008] Another object of the present invention is to provide a use of a TROP2-FN1 interaction pathway inhibitor in the preparation of products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis.

[0009] The applicant's team, drawing on first-line treatment options for advanced colorectal cancer, has demonstrated that chemotherapy with or without targeted drugs, combined with anti-DIC therapy, can effectively improve the survival rate and alleviate clinical symptoms of CRC-BMM patients. These results suggest that further research into the pathogenesis of CRC-BMM and the identification of appropriate therapeutic targets may improve the clinical benefits currently available to patients.

[0010] In more than one embodiment of the present invention, it was found that TROP2 and FN1 were significantly co-localized. TROP2 and FN1 interacted to promote the bone marrow colonization of CRC cells. TROP2 is a tumor-associated calcium signal transduction factor 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 the integrin α / β complex is closely related to maintaining proliferation signals of tumor cells, promoting angiogenesis, promoting invasion and metastasis, and regulating tumor immunity. According to GTEx cell lineage expression data, Fibronectin has extremely high expression levels in BMSCs. In our preliminary experiments, CRC cells with high TROP2 expression 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 that TROP2 and Fibronectin co-localized on the cell surface. TROP2 overexpression 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.

[0011] Based on this, combined with more experiments, the inventors found that CRC cells highly expressed TROP2 and combined with the Fibronectin fiber network derived from bone marrow mesenchymal stem cells (BMSCs), increasing cell adhesion and bone marrow colonization ability, and promoting CRC cell bone marrow metastasis.

[0012] Another object of the present invention is to provide a use of an FN1 inhibitor in the preparation of products for diagnosing, preventing and treating colorectal cancer bone marrow metastasis.

[0013] Furthermore, the FN1 inhibitor is an FN1-specific siRNA, and the FN1-specific siRNA is any one of the following two siRNAs:

[0014] siFN1#1: sense strand: 5'-GGAAAACACTATCAGATAA-3' (as shown in SEQ ID NO: 1), antisense strand: 5'-TTATCTGATAGTGTTTTCC-3' (as shown in SEQ ID NO: 2);

[0015] 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).

[0016] Furthermore, the FN1 inhibitor is an FN1 neutralizing antibody.

[0017] Furthermore, the TROP2-FN1 interaction pathway inhibitor is used to inhibit downstream integrins.

[0018] Furthermore, the TROP2-FN1 interaction pathway inhibitor is an Integrin αv / β5 neutralizing antibody, or an siRNA that inhibits the expression of Integrin α5, αv, and β3.

[0019] Another object of the present invention is to provide a use of TROP2 as a diagnostic and therapeutic marker for colorectal cancer bone marrow metastasis.

[0020] Another object of the present invention is to provide a kit for diagnosing colorectal cancer bone marrow metastasis, comprising a reagent for detecting the expression level of TROP2 or a reagent for detecting the expression level of Integrin α5, αv, and β3 proteins.

[0021] Another object of the present invention is to provide a pharmaceutical composition for treating colorectal cancer bone marrow metastasis, comprising an FN1 inhibitor.

[0022] Furthermore, the FN1 inhibitor is an FN1-specific siRNA, or the FN1 inhibitor is an FN1 neutralizing antibody.

[0023] Furthermore, the FN1-specific siRNA is any one of the following two siRNAs:

[0024] siFN1#1: sense strand: 5'-GGAAAACACTATCAGATAA-3' (as shown in SEQ ID NO: 1), antisense strand: 5'-TTATCTGATAGTGTTTTCC-3' (as shown in SEQ ID NO: 2);

[0025] 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).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The inventors of the present invention found that TROP2 is most highly expressed in patients with CRC bone metastasis. Knocking down TROP2 expression in CRC cells can significantly inhibit the proliferation, migration ability and stemness level of CRC cells themselves. Moreover, CRC cells highly express TROP2 and bind to the Fibronectin fiber network derived from bone marrow mesenchymal stem cells (BMSCs), increasing cell adhesion and bone marrow colonization ability, thereby promoting CRC cell bone marrow metastasis. After inhibiting FN1 expression or downstream integrin expression, the adhesion ability of CRC cells is also significantly inhibited, thus providing a new treatment strategy for colorectal cancer bone marrow metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results showed that TROP2 was more highly expressed in CRC patients with bone metastasis (BMM) than in patients without metastasis (Met.Free), and the normal intestinal epithelial tissue adjacent to the cancer was TROP2 negative in the sections.

[0029] Figure 2 Shown: After CRC cells were co-cultured with BMSCs, TROP2 and FN1 were identified to co-localize on the cell surface.

[0030] Figure 3 Shown: TROP2-FN1 interaction promotes CRC cell adhesion ability.

[0031] Figure 4 TROP2 expression is significantly elevated in cancerous tissues of CRC-BMM patients. (A) Heat map of differential gene expression from whole-transcriptome sequencing analysis; (B) Volcano plot of gene expression from whole-transcriptome sequencing analysis; (C) TROP2 expression in cancerous and adjacent tissues from three CRC-BMM patients; (D) TROP2 expression in cancerous and adjacent tissues from TCGA-COAD datasets.

[0032] Figure 5 TROP2 expression is higher in CRC patients with bone metastasis (BMM) than in patients without metastasis (Met-Free). (Left) Representative images; (Right) Statistical graph. The images show that normal intestinal epithelial tissue adjacent to the tumor is negative for TROP2.

[0033] Figure 6 Shown: TROP2 promotes the malignant phenotype of CRC cells. (AB) TROP2 overexpression and knockdown validation; (C) Cell proliferation monitoring; (D) Clonogenicity assay; (E) Cell migration Transwell assay; (F) Cell adhesion assay.

[0034] Figure 7 Shown: the expression abundance of FN1 protein in cells from different sources (the red arrows indicate BMSCs).

[0035] Figure 8Showing the interaction between TROP2 and FN1. (A) Silver staining of TROP2 after purification with the Flag tag; (B) Mass spectrometry of FN1-specific peptides; (C) Immunoprecipitation verification of the TROP2-FN1 interaction.

[0036] Figure 9 Display: Confocal microscopy revealed co-localization of TROP2 and FN1.

[0037] Figure 10 Figure: FN1 promotes CRC cell migration and adhesion. (A) 3D migration assay to measure cell migration; (B) FN1 knockdown inhibits CRC cell adhesion to BMSCs.

[0038] Figure 11 The interaction between TROP2 and FN1 is dependent on integrin. (A) Integrin expression in selected TROP2-overexpressing CRC cells; (B) The interaction between FN1 and TROP2 is dependent on integrin expression; (C) TROP2-promoted CRC cell adhesion is dependent on integrin expression.

[0039] Figure 12 Integrin is involved in the regulation of TROP2-FN1 interaction and CRC cell adhesion. (A) Integrin neutralizing antibodies inhibit TROP2-FN1 interaction; (B) FN1 or integrin neutralizing antibodies inhibit the adhesion of CRC cells with high TROP2 expression.

[0040] Figure 13 It shows that TROP2-high-expressing CRC cells can achieve faster colonization and growth in the tibia of nude mice model. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and 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 skilled in the art to which the present application belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments 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 "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] The present invention will now be further described in conjunction with specific examples. The following examples are only intended to explain the present invention but do not limit the present invention. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources).

[0044] Example 1

[0045] TROP2 expression in CRC-BMM:

[0046] Our team was the first to use multi-omics high-throughput sequencing methods to conduct systematic basic research on CRC-BMM. In the early stage, RNA sequencing analysis was performed on the carcinoma in situ and adjacent tissues of three patients. 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-related calcium signal transducer 2; also known as TROP2, trophoblast cell-surface antigen 2, trophoblast cell surface antigen 2) is a single transmembrane glycoprotein composed of an extracellular structural region, a transmembrane region, and an intracellular signaling region. Our early results showed that TROP2 was expressed most highly in patients with bone metastasis CRC, followed by patients with CRC without metastasis, while its expression was very low in normal intestinal epithelium. Knocking down TROP2 expression in CRC cells can significantly inhibit the proliferation, migration ability and stemness level of CRC cells themselves (such as Figure 1 shown).

[0047] Example 2

[0048] Preliminary research basis:

[0049] According to GTEx cell lineage expression data, Fibronectin has extremely high expression levels in BMSCs. In the inventors' preliminary experiments, CRC cells that highly express 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 that TROP2 and Fibronectin co-localized on the cell surface (such as Figure 2 、 Figure 8 and Figure 9TROP2 overexpression can significantly increase the number of CRC cells adhering to BMSCs, while the addition of Fibronectin neutralizing antibodies weakens the adhesion ability of tumor cells (e.g. Figure 3 、 Figure 10 、 Figure 11 and Figure 12 These results suggest that Fibronectin may play an important role in mediating CRC cell bone marrow colonization.

[0050] Example 3

[0051] 1. Clinical Characteristics and Treatment of CRC-BMM Patients

[0052] Table 1 shows the eight patients with CRC-BMM that we systematically diagnosed and treated. All patients had signs of diffuse bone marrow invasion. Most of these eight patients received chemotherapy combined with targeted therapy, and tumor control and DIC status improved in some patients. Among them, five patients have died, and three are still under observation.

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

[0054]

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

[0056] TROP2 is highly expressed in CRC-BMM patients

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

[0058] We then performed TROP2 expression detection on in situ tumor tissue sections of 50 CRC patients with bone metastasis (BMM) and 50 CRC patients without metastasis (Met.Free) in our hospital. The results showed that TROP2 expression was significantly higher in CRC patients with bone metastasis ( Figure 5 These results suggest that TROP2 expression is elevated in CRC-BMM patients and may have an important impact on the metastatic pattern of CRC tumors.

[0059] 3. Overexpression of TROP2 promotes the malignant phenotype of CRC cells

[0060] We first constructed a CRC cell line (HCT-116) with stable overexpression and knockdown of TROP2. Western Blotting and qPCR results showed that the efficiency of TROP2 overexpression and knockdown was high ( Figure 6 A and 6B). We then used a live cell real-time monitoring system to measure cell proliferation. TROP2 overexpression promoted CRC cell proliferation, while knockdown inhibited its growth ( Figure 6 C); The plate clone formation experiment also observed that TROP2 overexpression enhanced the clone formation ability, while knockdown had the opposite effect ( 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 filled with a monolayer of BMSCs. After 30 minutes of seeding, we washed away the non-adherent cells. The results showed that TROP2 overexpression significantly enhanced the adhesion of CRC cells to BMSCs, while knocking down TROP2 weakened this effect ( Figure 6 F).

[0061] In this embodiment, siRNA is used to knock down TROP2, and the siRNA is any one of the following two types:

[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] 4. TROP2 interacts with Fibronectin to promote CRC cell bone marrow colonization

[0065] Considering that CRC-BMM patients harbor a large number of cancer cells colonizing and growing in the bone marrow cavity, we hypothesized that CRC cells might engage in cell-cell communication with BMSCs to establish a microenvironment conducive to tumor survival. TROP2, as a membrane protein, can transduce signals through protein-protein interactions. Therefore, we performed tag protein purification and mass spectrometry analysis in a co-culture system of TROP2-Flag-overexpressing CRC cells and BMSCs, aiming to explore the spectrum of BMSC-derived proteins that interact with TROP2. After background protein screening compared to control groups (two control groups: ① TROP2-only CRC cells; ② Vector-CRC cells co-cultured with BMSCs), we obtained a series of candidate proteins (shown in Table 2).

[0066] Table 2: Top ten candidate proteins derived from BMSCs that specifically bind to TROP2 with the highest mass spectrometry scores after screening with background proteins in the control group.

[0067] Gene name Total PEP score Coverage [%] #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 FN1 protein and found that FN1 has a high protein expression level in BMSCs cells by searching the ProteomicsDB database, suggesting that it is enriched in the bone marrow cavity ( Figure 7 We performed silver staining and found a specific band around 220 kD in the co-culture purification system of TROP2-high-expressing CRC cells and BMSCs (the molecular weight of FN1 in SDS-PAGE is approximately 220 kD). Mass spectrometry identification confirmed that it was FN1 protein ( Figure 8 A and 8B). We subsequently performed immunoprecipitation experiments and verified the interaction between TROP2 and FN1 by co-expression in 293T cells or co-culture of CRC cells and BMSCs. Figure 8 C and 8D); The confocal microscopy results also showed that compared with the TROP2 family protein EPCAM, TROP2 and FN1 were significantly co-localized, while EPCAM and FN1 were not co-localized ( Figure 9 ).

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

[0070] siFN1#1: sense strand: 5'-GGAAAACACTATCAGATAA-3' (as shown in SEQ ID NO: 1), antisense strand: 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] 4. TROP2-Fibronectin interaction promotes CRC cell adhesion and depends on downstream integrin signaling

[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 of some integrin molecules in CRC cells. The results showed that the expression levels of Integrinα5, αv, and β3 proteins were significantly increased after TROP2 overexpression ( Figure 11 A); After knocking down the expression of the corresponding integrin using siRNA, immunoprecipitation with FN1 revealed that the interaction between TROP2 and FN1 was significantly weakened ( Figure 11 B), and TROP2-mediated CRC cell adhesion ability was also significantly inhibited ( Figure 11 C). Furthermore, we used neutralizing antibodies to verify that the interaction between TROP2 and FN1 was weakened after the addition of Integrinαv / β5 neutralizing antibody (P1F6) ( Figure 3 .10A), and after adding 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 embodiment,

[0075] The siRNA for knocking down Integrinα5 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 Integrinαv 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 Integrinβ3 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] Establishment of CRC metastasis model by orthotopic injection into the tibial cavity of mice:

[0083] Currently, there is no mature model of colorectal cancer bone marrow metastasis. We used BALB / c nude mice to microinject luciferase-labeled HCT-116 cells into the tibial cavity to successfully establish a mouse tibial cavity CRC metastasis model. Through in vivo imaging, we initially found that compared with the control group, TROP2-overexpressing CRC cells can achieve faster tibial colonization and growth, not only in terms of the number of tumors (control group: 1 / 3; TROP2-overexpressing group: 3 / 3) but also in terms of fluorescence intensity ( Figure 3 .11). We will subsequently use this model to conduct in vivo functional validation of TROP2-targeted drugs and related small molecule inhibitors.

[0084] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of TROP2 inhibitors in the preparation of products for the diagnosis, prevention and treatment of colorectal cancer bone marrow metastasis.

2. The use according to claim 1, characterized in that The TROP2 inhibitor is an siRNA that inhibits TROP2 expression, and the siRNA is any one of the following two types: 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); 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).

3. Application of TROP2 as a diagnostic and therapeutic marker for colorectal cancer bone marrow metastasis.

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