Use of a substance inhibiting the PI3K / Akt / mTOR signaling pathway and a substance inhibiting CHI3L1 in the preparation of a product for inhibiting MDSC-mediated metastasis of tumor cells

Abstract

The application discloses application of substances inhibiting PI3K / Akt / mTOR signal paths and substances inhibiting CHI3L1 in preparation of products inhibiting MDSC-mediated tumor cell metastasis. The application finds that CHI3L1 can control cytokine VEGF, active oxygen reaction products and proliferation level of CHI3L1 positive MDSC and immunosuppressive function of T cells through PI3K / Akt / mTOR signal paths, and that blocking CHI3L1 and / or inhibiting PI3K / Akt / mTOR signal paths can reduce cytokine VEGF secretion amount of CHI3L1 positive MDSC, active oxygen reaction products, immunosuppressive function of T cells and CHI3L1 positive MDSC proliferation, and further inhibit CHI3L1 positive MDSC-mediated tumor cell metastasis, thereby realizing CHI3L1 positive tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of substances that inhibit the PI3K / Akt / mTOR signaling pathway and substances that inhibit CHI3L1 in the preparation of products that inhibit MDSC-mediated tumor cell metastasis. Background Technology

[0002] Chitinase 3-Like 1 (CHI3L1), also known as human cartilage glycoprotein 39 or chitosan-like protein YKL-40, is a member of the evolutionarily conserved glycosylhydrolase 18 (GH18) family in mammals. As a chitosan-like protein, it lacks enzymatic activity but retains chitosan-binding activity. It is mainly distributed in the extracellular matrix of multicellular organisms. The CHI3L1 gene is located on human chromosome 1 q32. Its amino acid sequence shares high homology with mammalian chitosan-like proteins, belonging to the glycosylhydrolase 18 family. It resembles chitosan enzymes but lacks their activity. CHI3L1 plays an important role in the migration of vascular endothelial cells and is also involved in growth, proliferation, differentiation, and tissue remodeling.

[0003] As research progresses, the relationship between CHI3L1 and tumors has received increasing attention. Studies have shown that CHI3L1 is a poor prognostic factor for malignant tumors such as breast cancer, colon cancer, glioma, and small cell lung cancer. Recent research has found that CHI3L1 can promote angiogenesis and matrix remodeling, and its tumor-promoting mechanism is complex. CHI3L1 promotes tumor angiogenesis, thereby contributing to the proliferation and metastasis of colon cancer tumors; CHI3L1 promotes the expression of vascular endothelial growth factor in tumor cells, thereby promoting the formation of new blood vessels in gliomas. In astrocytomas, CHI3L1 can enhance their anti-apoptotic effect and is closely related to tumor anti-apoptosis.

[0004] Recent studies have found that CHI3L1 is an anti-apoptotic protein that promotes the growth of various tumor cells, making it a marker for multiple cancers. In patients with advanced lung adenocarcinoma, pre-treatment serum CHI3L1 levels are an independent prognostic factor for lung adenocarcinoma with distant metastases. However, the role of CHI3L1 in different diseases, especially in tumors, and the origin of CHI3L1, its function in the tumor microenvironment, and its regulatory mechanisms remain unclear. Summary of the Invention

[0005] One object of the present invention is to provide novel uses for substances that inhibit the PI3K / Akt / mTOR signaling pathway and / or substances that inhibit CHI3L1.

[0006] This invention provides the use of substances that inhibit the PI3K / Akt / mTOR signaling pathway and / or substances that inhibit CHI3L1 in any of the following: A1) Application in the preparation of products that inhibit tumor cell metastasis and / or invasion; A2) Application in the preparation of products for the prevention or treatment of CHI3L1-positive tumors; A3) Application in the preparation of products that inhibit MDSC proliferation and / or promote MDSC apoptosis.

[0007] In the above applications, in A1), the tumor cell metastasis and / or invasion is MDSC-mediated tumor cell metastasis and / or invasion.

[0008] In some embodiments, the MDSC-mediated tumor cell metastasis and / or invasion is MDSC-mediated lung cancer cell metastasis and / or invasion.

[0009] In some embodiments, the MDSC-mediated tumor cell metastasis and / or invasion is MDSC-mediated breast cancer cell metastasis and / or invasion.

[0010] In the above applications, in A2), the CHI3L1 positive tumor includes CHI3L1 positive lung cancer or CHI3L1 positive breast cancer.

[0011] In the above application, in A3), the MDSC is a CHI3L1 positive MDSC.

[0012] In some implementations, the CHI3L1-positive MDSCs are CHI3L1-positive MDSCs isolated from lung cancer tissue.

[0013] In some implementations, the CHI3L1-positive MDSC is a CHI3L1-positive MDSC isolated from breast cancer tissue.

[0014] Another objective of this invention is to provide a method for in vitro regulation or alteration of MDSC function.

[0015] The method for in vitro regulation or alteration of MDSC function provided by the present invention includes the following steps: treating isolated MDSCs with substances that inhibit the PI3K / Akt / mTOR pathway and / or substances that inhibit CHI3L1 to obtain MDSCs with altered function; wherein the MDSCs are CHI3L1-positive MDSCs isolated from tumor tissue.

[0016] In the above method, the MDSC is a CHI3L1-positive MDSC isolated from tumor tissue.

[0017] In some implementations, the MDSC is a CHI3L1-positive MDSC isolated from lung cancer tissue.

[0018] In some implementations, the MDSC is a CHI3L1-positive MDSC isolated from breast cancer tissue.

[0019] In the above method, the change in function is manifested in any of the following ways: B1) Decreased secretion of the cytokine VEGF; B2) The reactive oxygen species reaction products decrease; B3) The proliferation of MDSCs is inhibited and / or the MDSCs undergo apoptosis; B4) Reduced immunosuppressive function of T cells.

[0020] Another object of the present invention is to provide a pharmaceutical composition for regulating MDSC function.

[0021] The pharmaceutical composition for regulating MDSC function provided by the present invention comprises substances that inhibit the PI3K / Akt / mTOR pathway and / or substances that inhibit CHI3L1; wherein the MDSC is a CHI3L1-positive MDSC isolated from tumor tissue.

[0022] In the above pharmaceutical composition, the MDSC is a CHI3L1-positive MDSC isolated from tumor tissue.

[0023] In some implementations, the MDSC is a CHI3L1-positive MDSC isolated from lung cancer tissue.

[0024] In some implementations, the MDSC is a CHI3L1-positive MDSC isolated from breast cancer tissue.

[0025] In the above-mentioned pharmaceutical composition, the regulation of MDSC function is manifested in any of the following ways: C1) Reduces the secretion of the MDSC cytokine VEGF; C2) Reduces reactive oxygen species reaction products of MDSC; C3) Inhibits MDSC proliferation and / or promotes MDSC apoptosis; C4) Reduces the immunosuppressive function of MDSCs on T cells.

[0026] The substances that inhibit the PI3K / Akt / mTOR signaling pathway mentioned above include substances that inhibit PI3K, substances that inhibit Akt, and / or substances that inhibit mTOR.

[0027] In some implementations, the substance that inhibits the PI3K / Akt / mTOR signaling pathway is a substance that inhibits PI3K.

[0028] In some implementations, the substance that inhibits the PI3K / Akt / mTOR signaling pathway is a substance that inhibits Akt.

[0029] In some implementations, the substance that inhibits the PI3K / Akt / mTOR signaling pathway is a substance that inhibits mTOR.

[0030] In some preferred embodiments, the substance that inhibits the PI3K / Akt / mTOR signaling pathway is a combination of a substance that inhibits PI3K, a substance that inhibits Akt, and a substance that inhibits mTOR.

[0031] Furthermore, the substances that inhibit PI3K include nucleic acid molecules that inhibit PI3K gene expression and PI3K inhibitors. The nucleic acid molecules that inhibit PI3K gene expression include gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc., that inhibit PI3K gene expression; the PI3K inhibitors include proteins, peptides, or small molecule compounds that inhibit the activity or function of the PI3K protein.

[0032] The substances that inhibit Akt include nucleic acid molecules that inhibit AKT gene expression and Akt inhibitors. The nucleic acid molecules that inhibit AKT gene expression include gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc., that inhibit AKT gene expression; the Akt inhibitors include proteins, peptides, or small molecule compounds that inhibit the activity or function of Akt protein.

[0033] The substances that inhibit mTOR include nucleic acid molecules that inhibit mTOR gene expression and mTOR inhibitors. The nucleic acid molecules that inhibit mTOR gene expression include gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc., that inhibit mTOR gene expression; the mTOR inhibitors include proteins, peptides, or small molecule compounds that inhibit the activity or function of the mTOR protein.

[0034] The substances that inhibit CHI3L1 include nucleic acid molecules that inhibit CHI3L1 gene expression and CHI3L1 inhibitors. The nucleic acid molecules that inhibit CHI3L1 gene expression include gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc., that inhibit CHI3L1 gene expression; the CHI3L1 inhibitors include proteins, peptides, or small molecule compounds that inhibit the activity or function of the CHI3L1 protein.

[0035] Furthermore, the nucleic acid molecule that inhibits PI3K gene expression is siRNA that inhibits PIK3CA gene expression.

[0036] The PI3K inhibitor may be selected from any of the following compounds: Alpelisib, Taselisib, Inavolisib, Idelalisib, Umbralisib, Buparlisib, Copanlisib, and Gedatolisib.

[0037] In some embodiments, the sense strand sequence of the siRNA that inhibits PIK3CA gene expression is shown in Sequence 1, and the antisense strand sequence is shown in Sequence 2.

[0038] The nucleic acid molecules that inhibit AKT gene expression include siRNA that inhibits AKT1 gene expression.

[0039] The Akt inhibitor may be selected from any of the following compounds: Capivasertib, Ipatasertib.

[0040] In some embodiments, the sense strand sequence of the siRNA that inhibits AKT1 gene expression is shown in Sequence 3, and the antisense strand sequence is shown in Sequence 4.

[0041] The nucleic acid molecules that inhibit mTOR gene expression include siRNA that inhibits mTOR gene expression.

[0042] The mTOR inhibitor may be selected from any of the following compounds: Rapamycin, Sirolimus, Everolimus, Temsirolimus, Ridaforolimus, Rapalogs, and Sapanisertib.

[0043] In some embodiments, the sense strand sequence of the siRNA that inhibits mTOR gene expression is shown in Sequence 5, and the antisense strand sequence is shown in Sequence 6.

[0044] In some implementations, the mTOR inhibitor is Rapamycin.

[0045] The protein that inhibits the activity or function of CHI3L1 protein is an anti-CHI3L1 antibody.

[0046] In some embodiments, the anti-CHI3L1 antibody is a rabbit anti-human CHI3L1 antibody.

[0047] The tumors mentioned above include lung cancer and breast cancer.

[0048] In some implementations, the lung cancer is lung adenocarcinoma.

[0049] In some implementations, the lung adenocarcinoma is clinical stage III or IV.

[0050] In some implementations, the lung adenocarcinoma is poorly differentiated lung adenocarcinoma.

[0051] In some implementations, the lung adenocarcinoma has lymph node metastasis.

[0052] The tumor cells mentioned above include lung cancer cells and breast cancer cells.

[0053] In some implementations, the lung cancer cells are the mouse Lewis lung cancer cell line LLC.

[0054] In some embodiments, the breast cancer cells are the mouse breast cancer cell line 4T1.

[0055] The PI3K / Akt / mTOR signaling pathway described above includes PI3K (phosphatidylinositol-3-kinase), Akt (protein kinase B / serine-threonine kinase), and mTOR (mammalian target of rapamycin).

[0056] The PI3K mentioned above includes the PIK3CA (Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha) protein, the amino acid sequence (size 1068aa) of which is registered in NCBI with accession number NP_006209.2.

[0057] The Akt mentioned above includes the amino acid sequence (480 aa) of Akt1 (RAC-alpha serine / threonine-protein kinase), the Akt1 protein having accession number NP_005154.2 in NCBI.

[0058] The amino acid sequence (size 2549aa) of any of the above-mentioned mTOR (Mechanistic target of rapamycin kinase) has the accession number NP_004949.1 in NCBI.

[0059] The amino acid sequence (383aa) of any of the above-mentioned CHI3L1 (Chitinase-3-like protein 1) has the accession number NP_001267.2 in NCBI.

[0060] This invention reveals that CHI3L1 can control the levels of cytokine VEGF, reactive oxygen species (ROS) products, and proliferation in CHI3L1-positive MDSCs via the PI3K / Akt / mTOR signaling pathway, as well as their immunosuppressive function on T cells. Blocking CHI3L1 and / or inhibiting the PI3K / Akt / mTOR signaling pathway can reduce the secretion of VEGF, ROS products, and immunosuppressive function on T cells in CHI3L1-positive MDSCs, and inhibit their proliferation, thereby inhibiting CHI3L1-positive MDSC-mediated tumor cell metastasis and achieving the treatment of CHI3L1-positive tumors. Attached Figure Description

[0061] Figure 1 To perform immunofluorescence double staining of the lungs of c-fms-rtTA / (TetO)7-CMV-Api6 transgenic mice with CHI3L1 and F4 / 80 antibodies, CHI3L1 expression was observed on increased F4 / 80 positive macrophages after tetracycline induction.

[0062] Figure 2 To analyze lung MDSCs in c-fms-rtTA / (TetO)7-CMV-Api6 transgenic mice induced by tetracycline for 1 month, 2 months, 4 months, and after induction of lung cancer, using flow cytometry combined with marker antibodies. A represents CD11b+LY6GLY6Chi M-MDSCs and CD11b+LY6GhiLY6C PMN-MDSCs. B represents the expression of CHI3L1 in the lungs of transgenic mice at different tetracycline induction times and after lung cancer. The shaded area represents the IgG antibody control; the dashed line represents PMN-MDSCs, and the solid line represents M-MDSCs.

[0063] Figure 3 The images show the inhibitory effects of different subsets of MDSCs on T cells. A shows that CHI3L1+ MDSCs inhibit T cell proliferation more strongly than CHI3L1-MDSCs. B shows that CHI3L1+ MDSCs inhibit T cell activity more strongly than CHI3L1-MDSCs. C shows that CHI3L1+ MDSCs promote the generation of more regulatory T cells than CHI3L1-MDSCs.

[0064] Figure 4 The CHI3L1 protein reduces MDSC apoptosis via mTOR, and mTOR siRNA inhibits mTOR expression in MDSCs, thus promoting MDSC apoptosis. Here, CHI3L1 represents the CHI3L1 protein.

[0065] Figure 5The CHI3L1 protein induces AKT / mTOR protein expression in MDSCs. mTOR siRNA inhibits the promoting function of CHI3L1 on the mTOR pathway, but has little effect on AKT expression in MDSCs, demonstrating that CHI3L1 induces mTOR expression in MDSCs through AKT. Here, CHI3L1 represents the CHI3L1 protein.

[0066] Figure 6 The CHI3L1 protein induces AKT / mTOR protein expression in MDSCs, while AKT siRNA inhibits the promoting function of CHI3L1 in the AKT / mTOR pathway. Here, CHI3L1 represents the CHI3L1 protein.

[0067] Figure 7 The CHI3L1 protein promotes the production of more reactive oxygen species (ROS) by MDSCs through mTOR, thereby inhibiting T cell activity. mTOR siRNA blocks mTOR expression in MDSCs, reducing ROS production. Here, CHI3L1 represents the CHI3L1 protein.

[0068] Figure 8 In 4T1 highly metastatic breast cancer tumor tissue, MDSCs purified using immunomagnetic beads were cultured in vitro under different conditions, and VEGF content was detected. A shows the mTOR blocking assay of CHI3L1 positive MDSCs. B shows the mTOR blocking assay of CHI3L1 negative MDSCs.

[0069] Figure 9 In 4T1 highly metastatic breast cancer tumor tissue, MDSCs purified using immunomagnetic beads were cultured in vitro under different conditions, and VEGF content was detected. A shows the PI3K siRNA and AKT siRNA blocking assays of CHI3L1 positive MDSCs. B shows the PI3K siRNA and AKT siRNA blocking assays of CHI3L1 negative MDSCs.

[0070] Figure 10 To purify MDSCs from tumor tissue, they were pre-cultured in the lower chamber of a Transwell chamber under different conditions, and then mixed cells were added to the upper chamber and cultured for 12 hours. The number of cells that migrated through the polycarbonate membrane was counted (mean number of migrating cells, in units). A is the mTOR blocking assay for CHI3L1-positive MDSCs. B is the mTOR blocking assay for CHI3L1-negative MDSCs.

[0071] Figure 11To purify MDSCs from tumor tissue, they were pre-cultured in the lower chamber of a Transwell chamber under different conditions, and then mixed cells were added to the upper chamber and cultured for 12 hours. The number of cells that migrated through the polycarbonate membrane was counted (mean number of cells, in units). A shows the PI3K siRNA and AKT siRNA blocking assays for CHI3L1-positive MDSCs. B shows the PI3K siRNA and AKT siRNA blocking assays for CHI3L1-negative MDSCs.

[0072] Figure 12 Immunohistochemical staining of lung adenocarcinoma tissue. A shows CHI3L1 distributed in macrophages, type II alveolar cells, and cancer cells, with significant enhancement (X20). B shows strong expression of mTOR in the cytoplasm and nucleus of lung adenocarcinoma cells. C shows strong expression of PI3K in the cytoplasm and nucleus of lung adenocarcinoma cells. D shows weak expression of AKT in the cytoplasm and nucleus of lung adenocarcinoma cells.

[0073] Figure 13 This study analyzed the expression of CHI3L1 in lung cancer tissues and adjacent normal tissues. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0076] The biological materials involved in the following examples and their sources are as follows: The mouse Lewis lung cancer cell line LLC is a product of Shanghai Boson Biotechnology Co., Ltd., with the product code BES-20596CL.

[0077] The mouse breast cancer cell line 4T1 is a product of Shanghai Boson Biotechnology Co., Ltd., with the catalog number BES-2845HC.

[0078] The vascular endothelial cell line SVEC is a product of Shanghai Yingwan Biotechnology Co., Ltd., with the catalog number C2028.

[0079] The anti-IgG antibody is an Abcam product, catalog number ab771.

[0080] Rabbit anti-human CHI3L1 antibody is a product of Abcam, catalog number ab77528.

[0081] The rabbit anti-human Akt polyclonal antibody is from Abcam, catalog number ab227385.

[0082] Rabbit anti-mTOR (7C10) antibody is a product of Cell Signaling Technology, catalog number #2983.

[0083] Rabbit anti-PI3K antibody is a product of Abcam, catalog number ab191606.

[0084] Rapamycin (mTOR blocking compound) is a product of Yisheng Biotechnology (Shanghai) Co., Ltd., with catalog number 52404ES10 (10mg).

[0085] CHI3L1 protein is a product of Shanghai Enzyme-Link Biotechnology Co., Ltd., with the product code P36222.

[0086] The ELISA kit for detecting the pro-angiogenic cytokine VEGF is a product of Wuhan E-EL-H0111, manufactured by Wuhan E-EL-H0111.

[0087] The siRNAs and their sense and anti-sense sequences involved in the following examples are as follows: The sense and antisense strand sequences of PI3CA siRNA control are as follows: Sense: 5′-CCCUAAGAUCCGAUGGUAAUAAUUA-3′; Anti-sense: 5′-UAAUUAUUACCAUCGGAUCUUAGGG-3′.

[0088] The sense and antisense sequences of PI3CA siRNA (PI3K siRNA) are as follows: Sense: 5′-CCCAAGAAUCCUAGUAGAAUGUUUA-3′ (Sequence 1); Anti-sense: 5′-UAAACAUUCUACUAGGAUUCUUGGG-3′ (sequence 2).

[0089] The sense and antisense strand sequences of the AKT1 siRNA control are as follows: Sense: 5′-CGUACGCGGAAUACUUCGA-3′; Anti-sense: 5′-UCGAAGUAUUCCGCGUACG-3′.

[0090] The sense and antisense sequences of AKT1 siRNA (AKT siRNA) are as follows: Sense: 5′-GCACCUUCAUUGGCUACAA-3′ (Sequence 3); Anti-sense: 5′-UUGUAGCCAAUGAAGGUGC-3′ (sequence 4).

[0091] The sense and antisense strand sequences of mTOR siRNA control are as follows: Sense: 5′-GGUGUAUCUUCGAUCCUGAUUCGGGUC-3′; Anti-sense: 5′-CAGGCCUAAGACUAGAGCAGACAUCCA-3′.

[0092] The sense and antisense strand sequences of mTOR siRNA are as follows: Sense: 5′-ACAUCGAAGCUAGGACUAAGCCCAG-3′ (Sequence 5); Anti-sense: 5′-GGUGUAUCUUCGAUCCUGAUUCGGGUC-3′ (sequence 6).

[0093] The method for purifying CHI3L1-positive and CHI3L1-negative MDSCs from lung cancer tissue using immunomagnetic beads in the following examples is described in the literature "Qu P, Yan C, Du H. Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis. Blood. 2011 Apr 28;117(17):4476-89."

[0094] The transgenic mouse lung cancer models c-fms-rtTA / (TetO)7-CMV-Api6 and c-fms-rtTA / (TetO)7-CMV-MMP12 described in the following examples are described in the literature “Qu P, Du H, Li Y, Yan C. Myeloid-specific expression of Api6 / AIM / Sp alpha induces systemic inflammation and adenocarcinoma in the lung. J Immunol. 2009 Feb 1;182(3):1648-59.” and “Qu P,Yan C,Du H. Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis. Blood. 2011 Apr 28;117(17):4476-89.”.

[0095] Example 1: CHI3L1 regulates the proliferation and immunosuppressive function of tumor myeloid immunosuppressive cells (MDSCs) through the PI3K / Akt / mTOR pathway. I. Construction of the mouse model Transgenic mouse lung cancer models c-fms-rtTA / (TetO)7-CMV-Api6 and c-fms-rtTA / (TetO)7-CMV-MMP12 were constructed according to the methods described in the literature “Qu P, Du H, Li Y, Yan C. Myeloid-specific expression of Api6 / AIM / Sp alpha induces systemic inflammation and adenocarcinoma in the lung. J Immunol. 2009 Feb 1;182(3):1648-59.” and “Qu P, Yan C, Du H. Matrixmetalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis. Blood. 2011 Apr 28;117(17):4476-89.”, respectively.

[0096] The c-fms-rtTA / (TetO)7-CMV-Api6 and c-fms-rtTA / (TetO)7-CMV-MMP12 transgenic mouse lung cancer models, after tetracycline induction, exhibited overexpression of Api6 and MMP12, resulting in chronic obstructive pulmonary disease (COPD) after three months and tumors appearing after six months. 30%–40% of the transgenic mice developed lung adenocarcinoma after induction. Mice with lung adenocarcinoma obtained from the transgenic mouse lung cancer model after tetracycline induction were designated as the tetracycline-induced group mice.

[0097] Following the method described in the literature "Mackert JR, Qu P, Min Y, Johnson PF, Yang L, Lin PC. Dualnegative roles of C / EBPα in the expansion and pro-tumor functions of MDSCs. Sci Rep. 2017. 7(1):14048.", normal mice were intraperitoneally injected with mouse Lewis lung cancer cell line LLC and mouse breast cancer cell line 4T1 to establish a tumor-bearing mouse model.

[0098] II. The role and regulatory mechanism of CHI3L1 in MDSCs within the tumor microenvironment 1. A study on CHI3L1 expression in a mouse lung cancer model (c-fms-rtTA / (TetO)7-CMV-Api6 and c-fms-rtTA / (TetO)7-CMV-MMP12 transgenic mice induced with tetracycline to obtain lung adenocarcinoma mice) revealed that CHI3L1 was expressed in lung cancer cells and surrounding blood vessels; it was also highly expressed on infiltrating cells within the tumor region, but not on benign cells. This indicates that the elevated serum CHI3L1 originates from cancer cells and surrounding macrophages. Furthermore, compared to normal mice, transgenic mice with chronic obstructive pulmonary disease induced by overexpression of Api6 and MMP12 showed significantly elevated levels of CHI3L1 in BALF (bronchial lavage fluid) and serum, and increased expression of CHI3L1 in alveolar macrophages. Figure 1 Its level is related to airflow limitation and impaired lung diffusion capacity.

[0099] 2. Flow cytometry was used in conjunction with the marker antibodies GR-1, CD11b, Ly6C, and Ly6G to analyze lung MDSCs in c-fms-rtTA / (TetO)7-CMV-Api6 transgenic mice induced with tetracycline for 1 month, 2 months, and 4 months, as well as after induction to obtain lung adenocarcinoma. Results showed that in c-fms-rtTA / (TetO)7-CMV-Api6 transgenic mice, inflammation increased with age, and the number of CHI3L1-positive CD11b+LY6GLY6Chi M-MDSCs in the blood and lungs increased, while CD11b+LY6GhiLY6CPMN-MDSCs did not express CHI3L1. After tumor formation, 40%–60% of M-MDSCs in the blood, lungs, and surrounding tumors expressed CHI3L1. Only 10% of CHI3L1-positive PMN-MDSCs in the blood (…) Figure 2 ).

[0100] 3. CHI3L1+ / - M-MDSCs or CHI3L1+ / - PMN-MDSCs were purified from lung tissue of transgenic mice (c-fms-rtTA / (TetO)7-CMV-Api6) induced for 4 months and co-cultured with CFSE-labeled CD4+CD25-T cells. T cell proliferation in all groups was stimulated with anti-CD3 / CD28. A control group without anti-CD3 / CD28 was used. Results showed that CHI3L1 plays an important regulatory role in the growth and immunosuppressive function of chronic inflammatory cells, primarily MDSCs. CHI3L1-positive MDSCs more strongly inhibited T cell proliferation and activity than CHI3L1-negative MDSCs. Specifically, in the lung cancer mouse model, CHI3L1-positive MDSCs had a stronger immunosuppressive ability than CHI3L1-negative MDSCs, while simultaneously promoting the generation of more regulatory T cells to suppress T cell function. Figure 3 ).

[0101] III. The role and regulatory mechanism of CHI3L1 in MDSCs within the tumor microenvironment 1. CHI3L1-positive MDSCs were purified from lung cancer tissue of tumor-bearing mice injected with Lewis lung cancer cell line LLC using immunomagnetic beads.

[0102] 2. The obtained CHI3L1-positive MDSCs were cultured in RPMI medium (Thermo Fisher Scientific) containing 10% serum at 37°C and 5% CO2 to obtain the CHI3L1-positive MDSCs culture system (the concentration of CHI3L1-positive MDSCs was 4 × 10⁻⁶). 5 (number / mL), and then divided into the following 4 groups according to different in vitro culture conditions: Unblocked group / anti-IgG control group: siRNA control (concentration of siRNA control in the culture system was 50 nM) was added to the CHI3L1 positive MDSCs culture system, and after 48 hours of culture, anti-IgG antibody (concentration of anti-IgG antibody in the culture system was 0.5 μg / mL) was added and cultured for 72 hours.

[0103] Unblocked group / CHI3L1 treatment group: siRNA control (concentration of siRNA control in the culture system is 50 nM) was added to the CHI3L1 positive MDSCs culture system, and after 48 hours of culture, CHI3L1 protein (concentration of CHI3L1 protein in the culture system is 20 μg / mL) was added and cultured for 72 hours.

[0104] Blocking group / anti-IgG control group: AKT siRNA or mTOR siRNA (50 nM concentration in the culture system) was added to the CHI3L1 positive MDSCs culture system. After blocking for 48 hours, anti-IgG antibody (20 μg / mL concentration in the culture system) was added and cultured for 72 hours.

[0105] Blocking group / CHI3L1 treatment group: AKT siRNA or mTOR siRNA (concentration of AKT siRNA or mTOR siRNA in the culture system is 50 nM) was added to the CHI3L1 positive MDSCs culture system. After blocking for 48 hours, CHI3L1 protein (concentration of CHI3L1 protein in the culture system is 20 μg / mL) was added and cultured for 72 hours.

[0106] 3. Flow cytometry was used to detect the proliferation and apoptosis of MDSCs in each treatment group. Flow cytometry results showed that in vitro, CHI3L1 protein promoted the expression of MDSCs in tumor-bearing mice, inhibited T cell proliferation, and increased T cell apoptosis. This effect was regulated by CHI3L1 protein through mTOR; when mTOR siRNA was used to block the mTOR pathway, CHI3L1 protein could not promote MDSC expression. Figure 4 ).

[0107] 4. Western blotting was used to detect the protein expression levels of Akt / mTOR in MDSCs cells of each treatment group. Western blotting results showed that CHI3L1 protein can activate AKT / mTOR signaling molecules in MDSCs; in vitro, AKT siRNA or mTOR siRNA mediating AKT or mTOR expression can inhibit the function of CHI3L1 in MDSCs. Figure 5 and Figure 6 ).

[0108] Furthermore, by detecting the reactive oxygen species (ROS) products produced by MDSCs in each treatment group, it was found that MDSCs treated with CHI3L1 protein had a significantly stronger immunosuppressive effect on T cells than MDSCs treated with IgG. This is mainly because CHI3L1 regulates ROS products through the mTOR signaling pathway. When the mTOR pathway is blocked by mTOR siRNA, CHI3L1 protein cannot promote the production of more ROS products by MDSCs. Figure 7 ).

[0109] Example 2: CHI3L1 regulates MDSC-mediated tumor cell metastasis and / or invasion via the PI3K / Akt / mTOR pathway. I. CHI3L1 activates genes such as PI3K / AKT / mTOR to regulate MDSCs-mediated tumor cell metastasis CHI3L1-positive and CHI3L1-negative MDSCs were purified from tumor tissues of a 4T1 highly metastatic breast cancer model using immunomagnetic beads. The obtained CHI3L1-positive and CHI3L1-negative MDSCs were cultured in RPMI medium (Thermo Fisher Scientific) containing 10% serum at 37°C and 5% CO2, respectively, to obtain CHI3L1-positive and CHI3L1-negative MDSC culture systems.

[0110] 1. mTOR blockade experiment Based on different in vitro culture conditions, CHI3L1-positive MDSCs were divided into the following 4 groups for treatment: Unblocked / Anti-IgG control group (Anti-IgG+Alc): Anti-IgG antibody (concentration of anti-IgG antibody in CHI3L1 positive MDSCs culture system is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after culturing for 72 hours, 10 μL Alc (solvent) was added and cultured for 48 hours.

[0111] Blocking group / anti-IgG control group (Anti-IgG+Rap): Anti-IgG antibody (concentration of anti-IgG antibody in CHI3L1 positive MDSCs culture system was 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after culturing for 72 hours, Rapamycin (mTOR blocking compound, concentration of Rapamycin in CHI3L1 positive MDSCs culture system was 50 nM) was added to block for 48 hours.

[0112] Unblocked / Anti-CHI3L1 antibody treatment group (Anti-CHI3L1+Alc): Anti-CHI3L1 antibody (concentration of anti-CHI3L1 antibody in the CHI3L1 positive MDSCs culture system is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after culturing for 72 hours, 10 μL Alc (solvent) was added and cultured for 48 hours.

[0113] Blocking group / Anti-CHI3L1 antibody treatment group (Anti-CHI3L1+Rap): Anti-CHI3L1 antibody (concentration of anti-CHI3L1 antibody in the culture system of CHI3L1 positive MDSCs is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system. After culturing for 72 hours, Rapamycin (mTOR blocking compound, concentration of Rapamycin in the culture system of CHI3L1 positive MDSCs is 50 nM) was added for 48 hours of blocking.

[0114] Based on different in vitro culture conditions, CHI3L1 negative MDSCs were divided into the following 4 groups for treatment: Unblocked group / CHI3L1 protein uncultured group (Alc+PBS): 10 μL Alc (solvent) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 10 μL PBS solution was added and cultured for 72 hours.

[0115] Unblocked group / CHI3L1 protein culture group (Alc+CHI3L1): 10 μL of Alc (solvent) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL of CHI3L1 protein solution (solvent was PBS solution, and the concentration of CHI3L1 protein in the CHI3L1 negative MDSCs culture system was 10 μg / mL) was added and cultured for 72 hours.

[0116] Blocking group / CHI3L1 protein uncultured group (Rap+PBS): Rapamycin (mTOR blocking compound, solvent Alc) was added to the CHI3L1 negative MDSCs culture system and its concentration in the CHI3L1 negative MDSCs culture system was 50 nM. After blocking for 48 hours, 10 μL of PBS solution was added and cultured for 72 hours.

[0117] Blocking group / CHI3L protein culture group (Rap+CHI3L1): Rapamycin (mTOR blocking compound, solvent Alc) was added to the CHI3L1 negative MDSCs culture system and its concentration in the CHI3L1 negative MDSCs culture system was 50 nM. After blocking for 48 hours, 20 μL of CHI3L1 protein solution (solvent PBS solution, CHI3L1 protein concentration in the CHI3L1 negative MDSCs culture system was 10 μg / mL) was added and cultured for 72 hours.

[0118] 2. PI3K siRNA or AKT siRNA blocking experiment Based on different in vitro culture conditions, CHI3L1-positive MDSCs were divided into the following 6 groups for treatment: Unblocked / Anti-IgG control group (Anti-IgG+control siRNA): Anti-IgG antibody (concentration of anti-IgG antibody in CHI3L1 positive MDSCs culture system was 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after 72 hours of culture, siRNA control (concentration of siRNA control in CHI3L1 positive MDSCs culture system was 70 nM) was added and cultured for 48 hours.

[0119] Unblocked / Anti-CHI3L1 antibody treatment group (Anti-CHI3L1+control siRNA): Anti-CHI3L1 antibody (concentration of anti-CHI3L1 antibody in the culture system of CHI3L1 positive MDSCs is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after culturing for 72 hours, siRNA control (concentration of siRNA control in the culture system of CHI3L1 positive MDSCs is 70 nM) was added and cultured for 48 hours.

[0120] Blocking group / anti-IgG control group (Anti-IgG + PI3K siRNA): Anti-IgG antibody (concentration of anti-IgG antibody in CHI3L1 positive MDSCs culture system was 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after 72 hours of culture, PI3K siRNA (concentration of PI3K siRNA in CHI3L1 positive MDSCs culture system was added to block for 48 hours.

[0121] Blocking group / Anti-CHI3L1 control group (Anti-CHI3L1+PI3K siRNA): Anti-CHI3L1 antibody (concentration of anti-CHI3L1 antibody in the culture system of CHI3L1 positive MDSCs is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and PI3K siRNA (concentration of PI3K siRNA in the culture system of CHI3L1 positive MDSCs is 70 nM) was added to block for 48 hours.

[0122] Blocking group / anti-IgG control group (Anti-IgG+AKT siRNA): Anti-IgG antibody (concentration of anti-IgG antibody in CHI3L1 positive MDSCs culture system was 0.5 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after 72 hours of culture, AKT siRNA (concentration of AKT siRNA in CHI3L1 positive MDSCs culture system was 70 nM) was added to block for 48 hours.

[0123] Blocking group / Anti-CHI3L1 control group (Anti-CHI3L1+AKT siRNA): Anti-CHI3L1 antibody (concentration of anti-CHI3L1 antibody in the culture system of CHI3L1 positive MDSCs is 20 μg / mL) was added to the CHI3L1 positive MDSCs culture system, and after 72 hours of culture, AKT siRNA (concentration of AKT siRNA in the culture system of CHI3L1 positive MDSCs is 70 nM) was added to block for 48 hours.

[0124] Based on different in vitro culture conditions, CHI3L1 negative MDSCs were divided into the following 6 groups for treatment: Unblocked group / CHI3L1 protein uncultured group (control siRNA+PBS): siRNA control (the concentration of siRNA control in the CHI3L1 negative MDSCs culture system is 70 nM) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL of PBS solution was added and cultured for another 72 hours.

[0125] Unblocked group / CHI3L1 protein culture group (control siRNA+CHI3L1): siRNA control (the concentration of siRNA control in the CHI3L1 negative MDSCs culture system is 70 nM) was added to the CHI3L1 negative MDSCs culture system. After culturing for 48 hours, 20 μL of CHI3L1 protein solution (the concentration of CHI3L1 protein solution in the CHI3L1 negative MDSCs culture system is 10 μg / mL) was added and cultured for 72 hours.

[0126] Blocking group / CHI3L1 protein uncultured group (PI3K siRNA+PBS): PI3K siRNA (concentration of PI3K siRNA in CHI3L1 negative MDSCs culture system is 70 nM) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL PBS solution was added and cultured for 72 hours.

[0127] Blocking group / CHI3L1 protein culture group (PI3K siRNA+CHI3L1): PI3K siRNA (concentration of PI3K siRNA in CHI3L1 negative MDSCs culture system is 70 nM) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL of CHI3L1 protein solution (concentration of CHI3L1 protein solution in CHI3L1 negative MDSCs culture system is 10 μg / mL) was added and cultured for 72 hours.

[0128] Blocking group / CHI3L1 protein uncultured group (AKT siRNA+PBS): AKT siRNA (70 nM concentration of AKT siRNA in CHI3L1 negative MDSCs culture system) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL of PBS solution was added and cultured for 72 hours.

[0129] Blocking group / CHI3L1 protein culture group (AKT siRNA+CHI3L1): AKT siRNA (70 nM concentration of AKT siRNA in CHI3L1 negative MDSCs culture system) was added to the CHI3L1 negative MDSCs culture system, and after culturing for 48 hours, 20 μL of CHI3L1 protein solution (10 μg / mL concentration of CHI3L1 protein solution in CHI3L1 negative MDSCs culture system) was added and cultured for 72 hours.

[0130] 3. Detection of pro-angiogenic cytokines After the MDSCs culture medium of each group was collected in step 2 above, the content of the pro-angiogenic cytokine VEGF in the MDSCs culture medium was measured using an ELISA kit.

[0131] In the mTOR blockade assay, for CHI3L1-positive MDSCs, blocking mTOR or CHI3L1 inhibited VEGF levels ( Figure 8 A). For CHI3L1-negative MDSCs, treatment with CHI3L1 protein promotes VEGF secretion, while blocking mTOR does not promote VEGF secretion even with CHI3L1 protein treatment. Figure 8 B). Therefore, CHI3L1 controls the secretion of the pro-angiogenic cytokine VEGF by MDSCs through mTOR.

[0132] In PI3K siRNA or AKT siRNA blocking experiments, for CHI3L1-positive MDSCs, blocking AKT or PI3K or CHI3L1 inhibited VEGF levels ( Figure 9 A). For CHI3L1-negative MDSCs, treatment with CHI3L1 protein promotes VEGF secretion. However, if AKT or PI3K is blocked, even treatment with CHI3L1 protein will not promote VEGF secretion. Figure 9 B). Therefore, CHI3L1 controls the secretion of the pro-angiogenic cytokine VEGF by MDSCs through Akt and PI3K.

[0133] II. CHI3L1 regulates the secretion of pro-angiogenic factors by MDSCs through the PI3K / Akt / mTOR pathway to promote tumor cell vascular metastasis. To investigate the role of CHI3L1 in regulating the secretion of pro-angiogenic factors by MDSCs to promote tumor vascular metastasis, an in vitro tumor cell invasion assay was conducted using a Transwell chamber. The specific experimental method is as follows: The upper and lower chambers of the Transwell chamber were separated by a 3 mm diameter polycarbonate membrane (8 µm pore size), and Matrigel was evenly spread on the membrane. 1 × 10⁶ vascular endothelial cell line (SVEC) was added to the upper chamber. 5 Cells / mL, add 100µL of mixed cell solution to the lower chamber (the cells in the mixed cell solution are from 2×10⁶ cells cultured in vitro in step one). 5 Each group of MDSCs cells and 1×10 5 The cell line consisted of 4T1 breast cancer cells, with a control group consisting only of 4T1 breast cancer cells. After 12 hours of culture, the number of cells that crossed the polycarbonate membrane was counted, and the number of invading cells was used to represent the invasive ability of tumor cells.

[0134] In mTOR blockade experiments, for CHI3L1-positive MDSCs, blocking mTOR or CHI3L1 reduced the promoting effect of MDSCs on tumor cell migration. Figure 10 A). For CHI3L1-negative MDSCs, treatment with CHI3L1 protein enhances the promoting effect of MDSCs on tumor cell migration. However, if mTOR is blocked, even with CHI3L1 protein treatment, the promoting effect of MDSCs on tumor cell migration cannot be enhanced. Figure 10 B). Therefore, CHI3L1 controls the promoting effect of MDSCs on tumor cell migration through mTOR.

[0135] In PI3K siRNA or AKT siRNA blocking experiments, for CHI3L1-positive MDSCs, blocking AKT or PI3K or CHI3L1 reduced the promoting effect of MDSCs on tumor cell migration. Figure 11 A). For CHI3L1-negative MDSCs, treatment with CHI3L1 protein enhances the promoting effect of MDSCs on tumor cell migration. However, if AKT or PI3K is blocked, even with CHI3L1 protein treatment, the promoting effect of MDSCs on tumor cell migration cannot be enhanced. Figure 11 B). Therefore, CHI3L1 controls the promoting effect of MDSC on tumor cell migration through Akt and PI3K.

[0136] In summary, CHI3L1 activates genes such as PI3K / AKT / mTOR to regulate MDSCs-mediated tumor cell metastasis; at the same time, CHI3L1 regulates the secretion of pro-angiogenic cytokines VEGF by MDSCs through PI3K / AKT / mTOR to promote vascular metastasis of tumor cells.

[0137] Example 3: Multivariate correlation statistical analysis of the relationship between CHI3L1 protein distribution and various pathological factors in clinical cancer patients. Currently, there is a lack of systematic studies on the abnormalities of the CHI3L1 and PI3K / Akt / mTOR signaling pathways in lung adenocarcinoma. This application investigates the expression of CHI3L1 and PI3K / Akt / mTOR signaling pathways in lung adenocarcinoma cells and adjacent normal tissue cells, and their correlation with imaging findings. It explores the roles of CHI3L1 and PI3K / Akt / mTOR signaling pathways in the pathogenesis of lung adenocarcinoma, and the correlation between CHI3L1 and PI3K / Akt / mTOR signaling pathways and imaging findings in lung adenocarcinoma cells, providing clinical evidence for the early diagnosis and targeted therapy of lung adenocarcinoma. The specific steps are as follows: I. Clinical Trials and Multi-Data Statistical Analysis This study included lung adenocarcinoma tissue specimens collected from 80 patients with primary lung adenocarcinoma (diagnosed according to the 2014 NCCN Guidelines) between October 2012 and October 2014. Simultaneously, relatively normal lung tissue specimens (near-cancerous normal tissue specimens) were selected as controls. Patient inclusion criteria were as follows: (1) pathologically confirmed lung adenocarcinoma; (2) no history of other tumors; (3) no history of radiotherapy, chemotherapy, targeted therapy, or other treatments; and (4) no history of using immune enhancers, immune system diseases, tuberculosis, or other serious infections. All specimens were fixed in 10% formalin, then routinely embedded in paraffin and sectioned.

[0138] All patients ranged in age from 38 to 78 years, with a mean age of 62.39 ± 9.38 years. Among them, 47 patients were ≥60 years old, and 33 were <60 years old. There were 59 males and 21 females. 43 patients had a history of smoking, and 37 had no smoking history. According to WHO classification standards: 32 cases were adenocarcinoma, 31 cases were squamous cell carcinoma, and 17 cases were other types. Differentiation grade: 24 cases were moderately or poorly differentiated, 45 cases were moderately differentiated, and 11 cases were well differentiated. TNM staging: 5 cases were stage I, 47 cases were stage II, 16 cases were stage III, and 12 cases were stage IV.

[0139] II. Expression of CHI3L1 in Lung Adenocarcinoma and its Relationship with Clinicopathological Features Immunohistochemical staining was performed to detect the expression of CHI3L1 and PI3K / Akt / mTOR proteins in lung adenocarcinoma tissues and adjacent normal tissues. The specific steps included: 1. Preparation of paraffin slices Postoperatively, lung cancer tissue and tissue with relatively normal margins are rapidly harvested and immediately immersed in a 4% formaldehyde solution. Tissue blocks are generally no larger than 1.2cm × 0.5cm × 0.5cm. After fixation for 24 hours, the tissue is thoroughly washed with water, followed by dehydration using a gradient of 70%, 80%, 90%, 95%, and 100% alcohol. The dehydrated tissue is then cleared with xylene for 40 minutes. The cleared tissue blocks are then immersed in molten paraffin wax with a melting point of 58℃–60℃, allowing the liquid wax to penetrate the tissue cells for 2 hours. After this treatment, the tissue blocks are placed in an embedding cassette containing molten paraffin wax and allowed to cool.

[0140] 2. Slicing and mounting Before sectioning, trim the wax block to the desired shape, fix it on the microtome, and cut it into wax sections 5–6 μm thick. Spread the connected wax sections in warm water and attach them to poly-L-lysine-coated glass slides. Dry them in a 37°C oven for later use.

[0141] 3. Immunohistochemical SP method was used to detect the expression of CHI3L1 protein and PI3K / Akt / mTOR protein in lung cancer and adjacent normal tissues. (1) Select spare paraffin sections, dewax with xylene for 10 min × 2 times; hydrate with gradient alcohol (100%, 95%, 90%, 80%, 70%) for 2 minutes each time. Rinse with tap water for 5 min, rinse with distilled water twice, and place in PBS buffer with pH 7.2-7.4.

[0142] (2) Antigen retrieval using citric acid retrieval solution under high pressure. The specific steps are as follows: Boil 0.1M citric acid retrieval solution with pH 6 in the pressure cooker. After the antigen retrieval solution boils, put the slide in to completely immerse it. Cover the pot and continue heating. Start timing after the pressure valve releases steam. Stop heating after 2 minutes. After naturally cooling to room temperature, remove the slide from the retrieval solution and wash it with PBS buffer with pH 7.2-7.4 for 3 minutes × 3 times. Shake off excess water.

[0143] (3) Add 100 μL of 3% H2O2 solution to each slide and incubate in a humidified chamber at room temperature for 10 min to eliminate endogenous peroxidase activity. Wash with PBS buffer (pH 7.2-7.4) for 3 min × 3 times, discarding excess water; add normal sheep serum (secondary antibody animal allogeneic serum) and incubate at room temperature for 10-15 minutes, then discard the solution without washing. Add 100 μL of CHI3L1 or PI3K / Akt / mTOR primary antibody working solution to each slide and incubate overnight at 4°C. Wash again with PBS buffer (pH 7.2-7.4) for 3 min × 3 times, discarding excess water.

[0144] (4) Add the second antibody and incubate at room temperature for 15 min. After washing, add horseradish enzyme-labeled streptavidin working solution (SA / HRP) and incubate at room temperature or 37°C for 10-15 minutes. Finally, develop the color with DAB chromogenic solution at room temperature and observe the color development under a microscope. Control the reaction time, and after about 30 s to 10 min of color development, place it under a microscope for observation. If necessary, rinse with tap water to stop the color development reaction.

[0145] (5) Counterstaining and mounting: counterstain with hematoxylin for 5 seconds, rinse with tap water for 5 minutes; differentiate with 1% hydrochloric acid alcohol for 3 seconds, rinse with tap water; return to blue with 0.5% ammonia water for 5 seconds, rinse with tap water for 5 minutes. Dehydrate with graded alcohols (70%, 80%, 90%, 100%); clear with xylene, mount with neutral resin, and observe under a microscope.

[0146] 4. Interpretation criteria The score is determined based on the staining intensity of the protein in the cell membrane and cytoplasm, and the area covered by positive cells. Staining intensity is divided into four levels (no staining = 0 points, pale yellow = 1 point, brownish yellow = 2 points, and light brown = 3 points); the area covered by positive cells is divided into five levels (<5% = 0 points, 5%-25% = 1 point, 26%-50% = 2 points, 51%-75% = 3 points, and >75% = 4 points). The final score is the product of the staining intensity of the cell membrane and cytoplasm and the area covered by positive cells, and is ultimately divided into negative (0 points), moderate expression (1 and 2 points), and high expression (3 and 4 points).

[0147] 5. Image Analysis Evaluation method: (1) Region of interest (ROI) selection: Measurements are taken away from blood vessels, calcifications and cavities. To avoid the influence of partial volume effect, measurements are not taken at the upper and lower edge levels. The ROI area is ≥1cm². 2 (2) Three slides were selected from each lung cancer tumor tissue and adjacent normal tissue, and five fields of view were selected from each slide for immunohistochemical image analysis.

[0148] 6. Statistical Analysis Data analysis was performed using SPSS 19.0. Quantitative data were described using mean ± standard deviation (x ± s) and analyzed using t-tests (or ANOVA). Categorical data were expressed as rates and analyzed using chi-square tests (or Fisher's exact test). The significance level was set at α = 0.05, and p < 0.05 was considered statistically significant.

[0149] Immunohistochemical results showed that CHI3L1 protein positivity in adjacent normal tissues was mainly distributed in macrophages, with very few type II cells showing weak positivity; mTOR was strongly expressed in the cytoplasm and nucleus of lung adenocarcinoma cells; PI3K was strongly expressed in the cytoplasm and nucleus of lung adenocarcinoma cells; Akt was weakly expressed in the cytoplasm and nucleus of lung adenocarcinoma cells. Figure 12 ).

[0150] The expression rate of CHI3L1 in adjacent normal tissues of lung adenocarcinoma was 27.5% (22 / 80), while the expression rate in lung adenocarcinoma tissues was 80.0% (64 / 80), with a statistically significant difference (P < 0.05). This indicates that the expression rate of CHI3L1 protein gradually increases between adjacent normal tissues and lung adenocarcinoma tissues (Table 1). Figure 13 ).

[0151] Table 1. Expression of CHI3L1 in different tissues

[0152] In 80 cases of lung adenocarcinoma, the positive expression rate of CHI3L1 was 69.44% in clinical stages I and II and 88.64% in stages III and IV. The positive rates in the lymph node metastasis group and the non-metastasis group were 90.91% and 68.09%, respectively. The positive rates of CHI3L1 in the high, intermediate, and low differentiation groups were 72.73%, 68.89%, and 91.67%, respectively. Statistical analysis showed that the positive expression of CHI3L1 was significantly correlated with the clinicopathological stage of the tumor, the presence or absence of lymph node metastasis, and the degree of tumor differentiation (P < 0.05), but not significantly different from tumor size, patient age, or gender (P > 0.05) (Table 2).

[0153] Table 2. Relationship between CHI3L1 expression and clinicopathological features

[0154] The correlation between the expression of each protein was analyzed by Spearman rank correlation analysis. The results showed that the expression levels of CHI3L1 were significantly correlated with those of Akt, PI3K and mTOR (r=0.342, 0.320, 0.281, all P<0.01), indicating that Akt, PI3K and mTOR are co-expressed (Table 3).

[0155] Table 3. Correlation analysis of various proteins in lung adenocarcinoma expression

[0156] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of substances that inhibit the PI3K / Akt / mTOR signaling pathway and / or substances that inhibit CHI3L1 in any of the following: A1) Application in the preparation of products that inhibit tumor cell metastasis and / or invasion; A2) Application in the preparation of products for the prevention or treatment of CHI3L1-positive tumors; A3) Application in the preparation of products that inhibit MDSC proliferation and / or promote MDSC apoptosis.

2. Use according to claim 1, characterized in that: The tumor cell metastasis and / or invasion are MDSC-mediated tumor cell metastasis and / or invasion.

3. The application according to claim 1 or 2, characterized in that: The MDSCs were CHI3L1 positive MDSCs.

4. A method for in vitro regulation or alteration of MDSC function, comprising the following steps: treating isolated MDSCs with substances that inhibit the PI3K / Akt / mTOR pathway and / or substances that inhibit CHI3L1 to obtain functionally altered MDSCs; wherein the MDSCs are CHI3L1-positive MDSCs isolated from tumor tissue.

5. The method according to claim 4, characterized in that: The change in functionality is manifested in any of the following ways: B1) Decreased secretion of the cytokine VEGF; B2) The reactive oxygen species reaction products decrease; B3) The proliferation of MDSCs is inhibited and / or the MDSCs undergo apoptosis; B4) Reduced immunosuppressive function of T cells.

6. A pharmaceutical composition for regulating MDSC function, comprising a substance that inhibits the PI3K / Akt / mTOR pathway and / or a substance that inhibits CHI3L1; wherein the MDSC is a CHI3L1-positive MDSC isolated from tumor tissue.

7. The composition according to claim 6, characterized in that: The aforementioned MDSC regulation function is manifested in any of the following ways: C1) Reduces the secretion of the MDSC cytokine VEGF; C2) Reduces reactive oxygen species reaction products of MDSC; C3) Inhibits MDSC proliferation and / or promotes MDSC apoptosis; C4) Reduces the immunosuppressive function of MDSCs on T cells.

8. The application according to any one of claims 1-3, the method according to claim 4 or 5, or the pharmaceutical composition according to claim 6 or 7, characterized in that: The substances that inhibit the PI3K / Akt / mTOR signaling pathway include substances that inhibit PI3K, substances that inhibit Akt, and / or substances that inhibit mTOR.

9. The application, method, or pharmaceutical composition according to claim 8, characterized in that: The substances that inhibit PI3K include nucleic acid molecules that inhibit PI3K gene expression and PI3K inhibitors; Alternatively, the substance that inhibits Akt may include nucleic acid molecules that inhibit AKT gene expression and Akt inhibitors; Alternatively, the substance that inhibits mTOR may include nucleic acid molecules that inhibit mTOR gene expression and mTOR inhibitors; Alternatively, the substances that inhibit CHI3L1 include nucleic acid molecules that inhibit CHI3L1 gene expression and CHI3L1 inhibitors; Alternatively, the nucleic acid molecule that inhibits PI3K gene expression includes siRNA that inhibits PIK3CA gene expression; Alternatively, the nucleic acid molecule that inhibits AKT gene expression may include siRNA that inhibits AKT1 gene expression; Alternatively, the nucleic acid molecule that inhibits mTOR gene expression may include siRNA that inhibits mTOR gene expression.

10. The application according to any one of claims 1-3, the method according to claim 4 or 5, or the pharmaceutical composition according to claim 6 or 7, characterized in that: The tumors include lung cancer and breast cancer.

Images