Application of SRPX2 in preparation of product for predicting solid tumor immunotherapy effect, improving immunotherapy effect and treating solid tumor

Abstract

The invention belongs to the technical field of biological medicine or immunotherapy, and particularly relates to application of SRPX2 in preparation of a product for predicting the solid tumor immunotherapy effect and improving the solid tumor immunotherapy effect. According to the invention, it is found for the first time that SRPX2 is highly expressed in various tumor cells and cancer-related fibroblasts (CAF), and both SRPX2 and CAF can secrete SRPX2; meanwhile, it is elaborated that tumor cells and CAF promote M2-like polarization of macrophages by secreting SRPX2 and can induce expression of PD-L1 in the M2-like macrophages; it is proposed that SRPX2 in CAF can cause extracellular matrix remodeling; it is proved that high expression of SRPX2 can predict solid tumor immunotherapy resistance. Based on the principle, the SRPX2 possibly becomes a new marker for screening solid tumor immunotherapy people, and meanwhile, the SRPX2 is used as a potential treatment resistance new target, so that the solid tumor immunotherapy curative effect is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine or immunotherapy technology, and specifically relates to the application of SRPX2 in the preparation of products for predicting the efficacy of immunotherapy for solid tumors, improving the effect of immunotherapy and treating solid tumors. Background Art

[0002] Breast cancer is one of the most common cancers in the world. Triple Negative Breast Cancer (TNBC) is the subtype of breast cancer that is currently the most concerned in breast cancer treatment research, accounting for 15% to 20% of all breast cancer types. Because triple negative breast cancer lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), there is a lack of effective treatments targeting TNBC, making the treatment of triple negative breast cancer an important challenge in clinical practice. With the publication of the results of the phase III clinical trial IMpassion130, immunotherapy has received increasing attention in TNBC and has promoted more and more clinical trials in early triple negative breast cancer. However, whether it is early or advanced TNBC, the efficacy of immunosuppressant monotherapy is low, and the objective response rate (ORR) ranges from 5% in the unscreened population to 23% in the first-line PD-L1 positive population. Several clinical trials on immunotherapy combined with neoadjuvant chemotherapy for early triple-negative breast cancer have been published, and the results show that neoadjuvant immunotherapy combined with neoadjuvant chemotherapy for TNBC can increase the pathologic complete response (pCR) rate by 13.3% to 19.5%. However, immunotherapy is only effective for a small number of patients, and most patients still cannot benefit, which greatly limits the clinical application of immune checkpoint inhibitors (ICIs) in TNBC patients.

[0003] In recent years, immune checkpoint blockade has also made some progress in the treatment of solid tumors such as gastric cancer and pancreatic cancer. Several ICIs such as pembrolizumab, avelumab, sintilimab, tislelizumab and ipilimumab have been approved for clinical use in combination with targeted therapy for the treatment of advanced gastric tumors. Compared with other cancers, the response to immune checkpoint blockade in pancreatic cancer is relatively limited. Only in a very small proportion (~1%) of PDA patients with high microsatellite instability burden (MSI-high), PD-1 blockade in MSI-high patients is effective, and it has recently been approved by the US Food and Drug Administration. In addition, pancreatic cancer has a unique tumor microenvironment, including low immunogenicity, high matrix components and relatively few immune cell infiltration, which also increases the difficulty of immunotherapy.

[0004] Although ICI has become a new treatment for solid tumors, the effective response rate of a single drug is not high, and it is necessary to find a biomarker that predicts drug efficacy. At present, PD-L1 expression is used clinically to predict drug efficacy, but the predictive value of PD-L1 still has many limitations. The overall efficacy of immunotherapy in tumor patients with elevated PD-L1 expression is good. However, it has been observed in many clinical studies that there is no correlation between PD-L1 expression and the benefit of ICIs. PD-L1-negative patients respond to ICIs, and immunotherapy is ineffective when PD-L1 is highly expressed. Therefore, the development of new molecules that can predict sensitivity or resistance to immunotherapy can not only become a biomarker for predicting the efficacy of immunotherapy, but also a new therapeutic target for improving immune response. The combined use with checkpoint blockade is more likely to further improve the efficacy of immunotherapy, and has relatively important clinical value and application prospects.

[0005] SRPX2 (Sushi repeat-containing protein) is a protein containing sushi repeat sequences, encoded by the gene SRPX2. It is mainly distributed in the synapses of the entire cerebral cortex and hippocampus, and is involved in synapse formation. It also plays a role in the peripheral area and is essential for language and cognitive development. In addition, studies have found that extracellular SRPX2 can bind to urokinase-type plasminogen activator receptor (uPAR) and interact in vascular endothelial remodeling, indicating that SRPX2 protein promotes angiogenesis through its receptor pathway. However, the role of SRPX2 in the field of tumor immunotherapy has not been reported. Summary of the invention

[0006] The purpose of the present invention is to provide the use of SRPX2 in the preparation of products for predicting the efficacy of immunotherapy for solid tumors, improving the effect of immunotherapy and treating solid tumors, and to increase new medical uses of SRPX2.

[0007] The present invention provides the use of SRPX2 protein or SRPX2 gene in preparing a product for predicting the efficacy of solid tumor immunotherapy.

[0008] Preferably, the immunotherapy efficacy includes immunotherapy resistance.

[0009] Preferably, the product includes a product for detecting the expression level of the SRPX2 protein or SRPX2 gene.

[0010] Preferably, expression of the SRPX2 protein or SRPX2 gene is upregulated.

[0011] Preferably, the reagents for detecting the expression level of SRPX2 protein or SRPX2 gene include real-time fluorescence quantitative PCR related reagents and / or protein immunoblotting related reagents.

[0012] The present invention also provides the use of SRPX2 protein or SRPX2 gene in preparing products for improving the efficacy of solid tumor immunotherapy and / or in preparing products for treating solid tumors.

[0013] Preferably, the product comprises biological material in which the SRPX2 gene and / or SRPX2 protein is knocked out or knocked down.

[0014] Preferably, the biological material for knocking out or knocking down the SRPX2 gene comprises shRNA and / or siRNA for knocking out or knocking down the SRPX2 gene, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.1; the siRNA comprises siRNA-1 or siRNA-2; the nucleotide sequences of the siRNA-1 or siRNA-2 are as shown in SEQ ID NO.6 and SEQ ID NO.7, respectively;

[0015] The biological material for knocking out or knocking down the SRPX2 protein includes drugs and / or small molecule inhibitors targeting the SRPX2 protein.

[0016] Preferably, the drug includes a degradation drug targeting the SRPX2 protein and / or a drug targeting the receptor uPAR of the SRPX2 protein.

[0017] Preferably, the solid tumor includes one or more of breast cancer, pancreatic cancer and gastric cancer.

[0018] Beneficial effects:

[0019] The present invention provides an application of SRPX2 protein or SRPX2 gene in the preparation of a solid tumor immunotherapy efficacy prediction product. The present invention first discovered that SRPX2 is highly expressed in a variety of tumor cells and cancer-associated fibroblasts (CAFs), and both can secrete SRPX2; at the same time, it is explained that tumor cells and CAFs promote macrophage M2-like polarization by secreting SRPX2, and can induce the expression of PD-L1 in M2-like macrophages; it is proposed that SRPX2 in CAFs can cause extracellular matrix remodeling; it is proved that high expression of SRPX2 can predict solid tumor immunotherapy resistance. Based on the above principle, the SRPX2 protein or SRPX2 gene may become a new marker for screening solid tumor immunotherapy populations, and at the same time as a potential new target for treatment resistance, improve the efficacy of solid tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1This is a heat map of differentially expressed genes obtained by screening based on the data sets GSE173839 and GSE176580 in Example 1;

[0022] Figure 2 This is a Venn diagram of differentially expressed genes obtained by screening based on the data sets GSE173839 and GSE176580 in Example 1;

[0023] Figure 3 The 14 differentially expressed genes screened based on the datasets GSE173839 and GSE176580 in Example 1;

[0024] Figure 4 The fluorescence quantitative expression result diagram of SRPX2 gene in various breast cancer cells in Example 2;

[0025] Figure 5 The Western Blot expression results of SRPX2 in various breast cancer cells in Example 2 are shown;

[0026] Figure 6 The expression of SRPX2 in various cell types in the pancreatic cancer single-cell sequencing dataset in Example 3.

[0027] Figure 7 The expression of SRPX2 in the human pancreatic cancer cell line provided by CCLE in Example 3.

[0028] Figure 8 This is a graph showing the Western Blot expression results of SRPX2 in various pancreatic cancer cells in Example 3.

[0029] Fig. 9 This is the correlation analysis between SRPX2 expression and survival of pancreatic cancer patients in Example 3.

[0030] Fig.10 This is the expression of the downstream molecule COL1A after knocking down SRPX2 in pancreatic cancer CAFs in Example 3.

[0031] Fig.11 The expression of SRPX2 in various types of cells in the gastric cancer single-cell sequencing data set in Example 4.

[0032] Fig.12 The expression of SRPX2 in the human gastric cancer cell line provided by CCLE in Example 4.

[0033] Fig.13 This is a graph showing the Western Blot expression results of SRPX2 in various gastric cancer cells in Example 4.

[0034] Fig.14This is the correlation analysis between SRPX2 expression and survival of gastric cancer patients in Example 4.

[0035] Fig.15 This is the expression of the downstream molecule COL1A after knocking down SRPX2 in gastric cancer CAFs in Example 4.

[0036] Fig.16 This is a graph showing the content of type I collagen in the extracellular matrix after knocking down SRPX2 in CAF cells in Example 5;

[0037] Figures 17-18 This is the collagen contraction after SRPX2 was knocked down in CAF cells in Example 5;

[0038] Fig.19 The expression of CD163 membrane protein on the surface of macrophages after adding SRPX2 recombinant protein (rhSRPX2) in Example 6;

[0039] Fig. 20 The expression of CD86 membrane protein in macrophages in the rhSRPX2 combined with Anti-uPAR group in Example 6;

[0040] Figures 21-22 The changes in tumor volume and weight of 4T1 cells overexpressing SRPX2 and control 4T1 cells after injection of Anti-PD-L1 in Example 7;

[0041] Fig.23 The expression of SRPX2 in breast tumor tissues of mice in the SRPX2 overexpression group and the control group in Example 7;

[0042] Fig.24 A technical roadmap for clinical samples in Example 8 to verify that high expression of SRPX2 promotes TNBC immunotherapy resistance;

[0043] Fig.25 The expression of SRPX2 protein and CD163 protein in the immunotherapy non-response group and immunotherapy response group in Example 8.

[0044] Fig.26 The mRNA levels of CD163, CCL17, CCL22, PPARγ, CD86, iNOS and CXCL9 in macrophages cultured with the conditioned medium of TNBC cells with SRPX2 knocked down in Example 9, and the expression of macrophage surface membrane proteins CD163 and CD86.

[0045] Fig. 27The mRNA levels of CD163, CCL17, CCL22, PPARγ, CD86, iNOS and CXCL9 in macrophages cultured with the conditioned medium of TNBC cells overexpressing SRPX2 in Example 9, and the expression of macrophage surface membrane proteins CD163 and CD86.

[0046] Figures 28 to 31 This is the situation in Example 10 where SRPX2 induces M2-like macrophages to express PD-L1. DETAILED DESCRIPTION

[0047] The present invention provides an application of SRPX2 protein or SRPX2 gene in preparing a solid tumor immunotherapy efficacy prediction product. The accession number of the SRPX2 gene in the NCBI database is Gene ID: 27286.

[0048] As an embodiment, the solid tumor of the present invention may be one or more of breast cancer, pancreatic cancer and gastric cancer; as another embodiment, the breast cancer may be triple-negative breast cancer. As an embodiment, the immunotherapy efficacy of the present invention may be immunotherapy resistance; the immunotherapy method is not particularly limited, and conventional immunotherapy methods in the art may be used. As an embodiment, the product of the present invention may be a product for detecting the expression level of the SRPX2 protein or SRPX2 gene; as another embodiment, the reagent for detecting the expression level of the SRPX2 protein or SRPX2 gene of the present invention may be a real-time fluorescence quantitative PCR-related reagent and / or a protein immunoblotting-related reagent; the specific types of the real-time fluorescence quantitative PCR-related reagent and the protein immunoblotting-related reagent are not particularly limited, and conventional real-time fluorescence quantitative PCR-related reagents and protein immunoblotting-related reagents in the art may be used.

[0049] The present invention finds that SRPX2 protein or SRPX2 gene is highly expressed in solid tumor tumor cells and cancer-associated fibroblasts (CAFs), and both can secrete SRPX2; at the same time, it is explained that tumor cells and CAFs promote macrophage M2-like polarization by secreting SRPX2, and can induce the expression of PD-L1 in M2-like macrophages; it is proposed that SRPX2 in CAFs can cause extracellular matrix remodeling, and it is proved that high expression of SRPX2 can predict resistance to immunotherapy of solid tumors, that is, when SRPX2 is highly expressed in the body, it indicates that the body is resistant to immunotherapy, and thus the SRPX2 may become a new marker for screening solid tumor immunotherapy populations or predicting the effect of immunotherapy.

[0050] The present invention also provides the use of SRPX2 in the preparation of products for improving the efficacy of solid tumor immunotherapy and / or for the preparation of solid tumor treatment products. As an embodiment, the solid tumor of the present invention may be one or more of breast cancer, pancreatic cancer and gastric cancer; as another embodiment, the breast cancer may be triple-negative breast cancer. As an embodiment, the product of the present invention may be a drug. As an embodiment, the product of the present invention may be a biological material for knocking out or knocking down the SRPX2 gene and / or SRPX2 protein. As an embodiment, the biological material for knocking out or knocking down the SRPX2 gene of the present invention can be shRNA and / or siRNA for knocking out or knocking down the SRPX2 gene, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.1, specifically 5'-CCAGCGAAGATGCGAAGATAT-3'; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the siRNA-1 or siRNA-2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively, specifically 5'-GAGAGCA TGTGATTCGTTTA-3' and 5'-CCACCAATCTGTGCTCCTA-3', respectively; in a specific embodiment, the siRNA-1 and siRNA-2 are siG000027286B and siG000027286C purchased from Ruibo Bio, respectively.

[0051] As one embodiment, the biological material for knocking out or knocking down the SRPX2 protein of the present invention includes drugs and / or small molecule inhibitors targeting the SRPX2 protein; as another embodiment, the drugs include degradation drugs targeting the SRPX2 protein and / or drugs targeting the receptor uPAR of the SRPX2 protein; as another embodiment, the drug of the present invention targeting the receptor uPAR of the SRPX2 protein can be a uPAR neutralizing antibody.

[0052] uPAR, as a receptor for SRPX2, is anchored to the cell membrane surface through C-terminal glycosylated phosphatidylinositol (GPI). uPAR is mainly expressed on the surface of immune cells, especially macrophages, and can mediate a variety of biological activities, including plasminogen activation, extracellular matrix (ECM) remodeling, cell adhesion and migration, and intracellular signal transduction. Therefore, uPAR neutralizing antibodies (Anti-uPAR) can be used to block the immune resistance mediated by SRPX2 protein.

[0053] The present invention has no particular limitation on the specific process of the knockout and knockdown, and conventional knockout or knockdown steps in the art may be used.

[0054] The present invention discovered through experiments that high expression of SRPX2 promotes resistance to immunotherapy of solid tumors and can serve as a potential therapeutic target for resistance to immunotherapy.

[0055] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] The data sources and method steps used in the following examples are:

[0057] 1. Data Collection and Processing

[0058] The transcriptome dataset GSE173839 of breast cancer patients receiving neoadjuvant anti-PD-L1 therapy (including 12 immunotherapy non-responders and 9 immunotherapy responders) and the transcriptome dataset GSE176580 of breast cancer mice receiving anti-PD-L1 therapy (including 2 immunotherapy-resistant tumor cells and 2 immunotherapy-responsive tumor cells) were downloaded from the Gene Expression Omnibus Data Base (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ).

[0059] The differentially expressed genes (DEGs) between non-responders (NR) and responders (pCR) in GSE173839 and GSE176580 datasets were screened using the "limma" package in R language with a threshold of false discovery rate (FDR) < 0.05 or FDR < 0.01 and fold change (FC) log value |logFC|> 1. For the DEGs obtained from the GSE176580 dataset, the "biomaRt" package in R language was used to convert mouse genes into homologous human genes. Then, the "ggvenn" package in R software was used to obtain the common differentially expressed genes by taking the intersection of the DEGs obtained by differential analysis of the two datasets. The key genes with survival differences in TNBC were screened by Kaplan-Meier analysis of the TCGA-TNBC dataset.

[0060] 2. Cell sources and cell culture

[0061] Human breast cancer cell lines MDA-MB-231, MDA-MB-468, BT549, MCF7, T47D, human pancreatic cancer cell lines BXPC3, PANC1, SW1990, human gastric cancer cell lines MKN45, AGS, MKN74, HGC27, SNU216, MKN7, and human THP-1 monocytic cell line were purchased from the Chinese Academy of Sciences Cell Bank, and the mouse breast cancer cell line 4T1 carrying a luciferase (luc) marker was provided by the Department of Cell Biology, China Medical University. MDA-MB-231, T47D, MCF7, 4T1, PANC1, and SW1990 cell lines were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and passaged every 2-3 days. MDA-MB-468 was cultured in L-15 medium containing 10% fetal bovine serum, 1% penicillin (100 U / mL) and 1% streptomycin (100 μg / mL), and passaged every 2-3 days. BT549, BXPC3, MKN45, AGS, MKN74, HGC27, SNU216 and MKN7 cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 10% fetal bovine serum, 1% penicillin (100 U / mL) and 1% streptomycin (100 μg / mL), and passaged every 2-3 days. THP-1 cell line was cultured in RPMI-1640 medium containing 15% fetal bovine serum, 1% penicillin (100 U / mL) and 1% streptomycin (100 μg / mL), and passaged every 2-3 days. All cell lines were grown in an incubator at 37°C and saturated humidity, and all cell lines except MDA-MB-468 cells were supplied with 5% CO2. All cells used in the experiment were in the logarithmic growth phase and mycoplasma-negative cells. Breast cancer, pancreatic cancer, and gastric cancer CAF cells were derived from primary cancer-associated fibroblasts extracted from human breast cancer, pancreatic cancer, and gastric cancer tumors. They were cultured in DMEM / F12 medium containing 20% ​​fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin, and passaged every 3 days.

[0062] 3. RNA Extraction and Real-time Quantitative PCR (RT-qPCR)

[0063] The cell samples treated with different conditions were collected, the supernatant was discarded after centrifugation, and the remaining steps were performed according to the recommended method of EastepSuper total RNA extraction kit (Promega, LS1040) to extract total RNA. The extracted RNA was quantified by measuring the absorbance at 260 nm using a NanoDrop ND-100 spectrophotometer. The extracted RNA was reverse transcribed into cDNA using the GoScript reverse transcription kit (Promega, A2790). Quantitative real-time PCR was performed using SYBR Premix ExTaq II and detected on Applied Biosystems 7500 Real-Time PCR Systems. A total of 45 cycles were performed at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, followed by one cycle at 95°C for 15 seconds, 60°C for 1 minute, 95°C for 30 seconds, and 60°C for 15 seconds. Use 2 -ΔΔCt The relative expression level was calculated by the 18S method, with 18S as the internal reference.

[0064] 4. Protein Extraction and Western Blot

[0065] Collect cells treated with different conditions, discard the culture medium, rinse with pre-cooled PBS solution, add appropriate amount of lysis buffer (1% TritonX-100, 50mM Tris-HCl pH 7.4, 150mMNaCl, 10mM EDTA, 100mMNaF, 1mM Na3VO4, 1mM PMSF, 2μg / mL Aprotinin) and mix well, and place on ice for lysis for 20 minutes. After ultrasonic lysis of the cell lysate in an ice-water mixture, centrifuge the cell lysate at 4°C at 13000rpm for 25 minutes, and absorb the supernatant to obtain cell protein. Then use the Coomassie brilliant blue method to quantify protein, mix the quantified protein sample with 3× loading buffer, separate instantly, boil the sample in a 95°C thermostat metal device for 5-10 minutes to denature the protein, and then cool to room temperature. The cooled protein sample is subjected to protein electrophoresis by SDS-PAGE. According to the molecular weight of the protein, the SDS-polyacrylamide separation gel of the corresponding concentration is configured to condense for 20-30 minutes, and then the concentrated gel is configured. After inserting the comb, it is condensed at room temperature for more than 40 minutes, and the gel is removed and placed in distilled water for standby. Install the electrophoresis device, fill it with electrophoresis buffer, unplug the comb, and load the samples in sequence according to the loading order. During electrophoresis, the concentrated gel is at a constant voltage of 80V, and the constant voltage is adjusted to 130V after the protein sample is electrophoresed to the separation gel. After the electrophoresis is completed, the separation gel is placed on the filter paper in the transfer folder prepared in advance, and then the activated PVDF membrane (Merke Millipore, IPVH00010) is placed on the separation gel, and enough transfer fluid is added. The protein is transferred to the PVDF membrane at a constant voltage of 80V for 120 minutes. After the transfer is completed, the PVDF membrane is soaked in TTBS buffer containing 5% skim milk and sealed at room temperature for 40 minutes. The PVDF membrane was cut according to the pre-designed method, incubated in the primary antibody diluted in TTBS buffer containing 5% skim milk at an appropriate ratio, and incubated at room temperature for more than 6 hours or at 4°C overnight. After the primary antibody is blocked, it is thoroughly washed with 1×TTBS buffer (10 minutes / time, 4 times in total). The PVDF membrane was then placed in the secondary antibody labeled with horseradish peroxidase of the corresponding property diluted in TTBS buffer at 1:2000, incubated at room temperature for 4 minutes, and then thoroughly washed again with 1×TTBS buffer (10 minutes / time, 4 times in total). Finally, the ECL method was used for color development. After the ECL luminometer was precooled, the developer was prepared and evenly added to the surface of the PVDF membrane. After development, the original image was analyzed and stored using GelCapture software.

[0066] 5. Detection of cell surface markers by flow cytometry

[0067] Antibody single staining method was used to detect changes in cell surface proteins by flow cytometry. Cell samples of the blank control group, control group and treatment group were collected, and 0.25% trypsin was added to the culture dish to be collected to digest the cells. After adding culture medium to terminate digestion, centrifuge at 4°C and 1000rpm for 5 minutes, discard the supernatant, resuspend and wash the cells with pre-cooled PBS and centrifuge again, and discard the supernatant. A total volume of 50μL PBS solution containing 10μCD86 or CD163 flow antibodies was added to the cell pellet and incubated in a 4°C refrigerator for 30 minutes. After the incubation, resuspend with 1mL PBS and centrifuge. After carefully aspirating the supernatant, add 500μL PBS and resuspend again. Flow cytometry was used to detect changes in cell surface protein expression, and FlowJo software was used to analyze the results.

[0068] 6. Decellularized Extracellular Matrix Preparation and Immunofluorescence

[0069] A certain number of CAF cells were inoculated on a glass slide in a 12-well plate and allowed to grow for 72 hours. An initial concentration of 250 μg / mL sodium ascorbate was added to promote cell secretion of extracellular matrix, and then the sodium ascorbate concentration was maintained at 50 μg / mL. After growing, wait for 3 days to secrete extracellular matrix. Lyse the cells with 0.5% X-triton for 15 minutes. Wash with PBS three times to remove X-triton. Perform immunofluorescence: (1) Fixation: Fix with 500 μL 4% paraformaldehyde (PFA) and let stand at room temperature for 30 minutes. Wash with PBS three times. Wash once with 3% BSA. (2) Blocking: Block with 500 μL 3% BSA and let stand for 30 minutes. (3) Block with primary antibody: dilute 200-300 μL of primary antibody (COL1 Asanta: 59772) with 3% BSA, shake slowly for a while, and let stand at 4°C overnight. (4) Wash with 1 mL PBS, 10 minutes / time, for a total of 3 times. (5) Fluorescent secondary antibody: dilute the secondary antibody with 200-300 μL of 3% BSA, shake slowly at room temperature for 1 hour (avoid light). (6) Wash with 1 mL of PBS, 10 minutes / time, 3 times in total (avoid light). (7) Sealing: place 30-40 μL of anti-fluorescence quenching sealing solution on the slide, and put the cover slip on the slide. Observe under a fluorescence microscope.

[0070] 7. Rat Tail Collagen Extraction

[0071] Take 4 rat tails and put them at -80℃ when not in use. Thaw them for 30 minutes in advance to soften and restore elasticity, and soak them in alcohol for 30 minutes to disinfect. Put the soft and sterilized rat tail in a large dish, insert the scissors from the broken end to cut the skin, and then cut it again from the symmetry, and peel off the skin from the cut. Rotate it to break the bones, and further rotate it to break the skin connection, exposing the silvery-white tail tendon. Pull out the tail tendon from the short end, and then pull out the tail tendon from the long end first. Put the tail tendon in an Ep tube, peel it, weigh it, and dissolve it in 0.02N acetic acid solution. Stir it at 4-8℃ for more than 48h, and shake it every 1h. Transfer the solution to a high-speed centrifuge tube under sterile conditions. Centrifuge it at 10000g, 4℃ for 1h, transfer the upper solution to a sterile Ep tube, and store it at -20℃.

[0072] 8. Collagen gel contraction experiment

[0073] Take a sample containing 2×10 5 Count the number of CAF cells and place them in 100 μL of phenol red culture medium. Take the collagen solution extracted in step 7 out of the refrigerator, melt it, and place it on ice. Take 400 μL of collagen solution and use 0.1M sodium hydroxide solution for acid-base titration, about 50-75 μL is required. Titrate to about pH 7. Mix 400 μL of collagen solution and 100 μL of phenol red culture medium containing cells evenly, quickly add to the sodium hydroxide required for titration, and mix quickly. Aspirate 500 μL of the mixed solution into one well of a 24-well plate. Place it in a 37°C incubator for 1 hour to allow it to fully coagulate. After 1 hour, the collagen gel was observed to gel. At this time, a 20 μL pipette tip was used to separate the gel from the outer wall and add 500 μL of double-free culture medium. Observe and take pictures at 0h, 24h, 48h, and 72h.

[0074] 9. Immunohistochemistry

[0075] The tissue was fixed with 10% neutral formalin, embedded in paraffin and then sliced ​​continuously. (1) Dewaxing and hydrating: Use a 70°C oven to bake the slices for 1 hour. After the baking is completed, wait for the temperature to drop slightly before starting dewaxing and hydrating. After xylene and alcohol 100%, 95%, and 75% in sequence, put the slice rack in for 3 minutes and rinse with distilled water. (2) Sodium citrate high-pressure antigen repair: Fill the slice box with 1× sodium citrate repair solution, use high pressure method to perform antigen repair, cool naturally to room temperature and put in distilled water. (3) Blocking: After rinsing with PBS twice, use solution A in the immunohistochemistry kit for 10 minutes, rinse with PBS three times, and then treat with solution B for 1 hour. (4) Incubation: Add 200μL of primary antibody diluted in PBS to each slice and incubate overnight at 4°C. The next day, after rinsing with PBS three times, add solution C for 10 minutes. (5) Color development: After rinsing with PBS three times, use solution D for 20 minutes. After rinsing with PBS twice, add 200 μL DAB to each slide and develop color under a microscope. When the color development is appropriate, rinse with tap water for 10 minutes and place in a slice box filled with distilled water. (6) Nuclear counterstaining: After the slides are rinsed dry, they are stained with hematoxylin for 1 minute for nuclear counterstaining and then rinsed with tap water for 10 minutes. Finally, dehydrate, make transparent, and seal the slides. For each pathological section, three random fields of view were selected for immunohistochemical scoring (H-score), H-score = staining intensity score × positive cell frequency score, where the staining intensity score is: negative 0 point, weak 1 point, medium 2 points, strong positive 3 points; positive cell frequency score is: <5% is 0 point, 5%-25% is 1 point, 25%-50% is 2 points, 50%-75% is 3 points, >75% is 4 points. Immunohistochemical interpretation was completed by two independent pathologists.

[0076] 10. Preparation of Tumor Cell Conditioned Medium (CM)

[0077] Well-growing MDA-MB-231 and BT549 cells were digested with trypsin, centrifuged at 1000 rpm for 3-5 minutes, and plated in 25 cm 2 In a cell culture flask. When the cells adhere to the wall and grow to about 70%-75% fusion, wash the cells twice with PBS, add 10mL of DMEM or RPMI-1640 culture medium containing 0.5% fetal bovine serum to each cell culture flask, and continue to culture for 48 hours. Collect the cell culture medium, centrifuge at 1000 rpm for 10 minutes, take the supernatant, and be careful to avoid inhaling cells or cell debris. Ultrafilter the cell supernatant through a 0.45μm filter (Millipore). Combine the same cell culture supernatant samples and put them into sterile glass bottles. They can be stored at 4°C for a short period of time (1-2 days) and frozen in a -80°C refrigerator for long-term storage.

[0078] 11. Statistical Analysis

[0079] All statistical analyses of the following embodiments were performed using R4.1.0 software. Kaplan-Meier was used to generate the survival curves of each subgroup in each data set, and the Log-Rank test was used to evaluate statistically significant differences. Statistical analysis was performed using GraphPadPrism software 8.0 and SPSS25.0. Comparisons between two groups and multiple groups were analyzed using Student's-t test and one-wayANOVA single factor analysis of variance, respectively. The difference between the two groups of clinical samples was compared using the chi-square test. P<0.05 was considered to be statistically significant, and P<0.01 was considered to be statistically significant. All data in this experiment were the results of three independent repeated experiments, and the data were expressed as "mean ± standard error", *P<0.05, **P<0.01, ***P<0.001.

[0080] Example 1

[0081] Bioinformatics analysis was used to screen SRPX2 as a key molecule that promotes resistance to immunotherapy in breast cancer patients. The steps are as follows:

[0082] The expression profiles of samples of non-responders (NR) and pathologic complete responses (pCR) in the GSE173839 and GSE176580 datasets were differentially analyzed.

[0083] Using |logFC|>1, P<0.05 as the screening criteria, 67 up-regulated genes and 131 down-regulated genes were found in the NR group compared with the pCR group in the GSE173839 dataset; using |logFC|>1, P<0.01 as the screening criteria, 140 up-regulated genes and 157 down-regulated genes were found in the NR group compared with the pCR group in the GSE176580 dataset ( Figure 1 ).

[0084] Then, the mouse sample genes were converted into human-mouse homologous genes into human differentially expressed genes. Finally, the differentially expressed genes (DEGs) of the above two datasets were intersected to obtain 22 common differentially expressed genes ( Figure 2 ), and further screening revealed 14 differentially expressed genes with consistent expression trends in the immunotherapy non-response group (NR) ( Figure 3 ). Among them, SRPX2 was most correlated with resistance to immunotherapy, HR for breast cancer = 13.361, P = 0.013.

[0085] Example 2

[0086] SRPX2 is highly expressed in breast cancer CAFs and tumor cell lines and in their secretory supernatants

[0087] The heterogeneous interaction between cancer cells and stromal cells leads to the proliferation and metastasis of malignant cells. In addition to the malignant biological behavior of tumor cells themselves, the stromal cell content and proliferation of stromal connective tissue are also significantly associated with the poor prognosis of breast cancer. Due to the extensive proliferation of breast connective tissue, CAFs account for 80% of the tumor mass and are the most common stromal cell component in the breast TME. The complexity of CAFs and the tumor microenvironment interact with each other to worsen tumor progression. The transformation and activation of CAFs is one of the foundations of cancer progression. When fibroblasts are overactivated, they no longer perform tissue repair but become harmful components that promote organ fibrosis or tumor growth. CAFs can achieve tumor survival and continued spread through different mechanisms, mainly by releasing secreted paracrine factors, cytokines, and exocrine vesicles, and also by direct or indirect interactions with other cells to achieve physical remodeling of the extracellular matrix and ultimately enhance motility.

[0088] Breast cancer tumor-associated fibroblasts (CAFs), breast cancer tumor cell lines MDA-MB-468, MDA-MB-231, BT-549, T-47D, and MCF-7 were cultured, and cell samples and supernatant samples were collected. PCR was performed after quantification of each cell sample ( Figure 4 ) Detection of SRPX2 expression The SRPX2 primer sequences are: SRPX2-F: 5'-CGGACTGAGTTCCTCTAATCCC-3' (SEQ ID NO.2), SRPX2-R: 5'-CACTGCCCAGATGTTACGAC-3' (SEQ ID NO.3). At the same time, Western Blot was performed on the supernatant samples according to the cell count ratio ( Figure 5 ) to detect the expression of SRPX2 (SRPX2 antibody (66266-1-1g) was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., China).

[0089] The results showed that SRPX2 was highly expressed in breast cancer CAF cells, tumor cells and their secretory supernatants.

[0090] Example 3

[0091] SRPX2 is highly expressed in human pancreatic cancer primary CAFs and cell lines, and pancreatic cancer patients with high SRPX2 expression have worse survival

[0092] Pancreatic cancer single-cell sequencing data TISCH (http: / / tisch.comp-genomics.org) were used to classify cell types and found that SRPX2 was more highly expressed in CAFs and pancreatic cancer cells compared with other cell types ( Figure 6 ).

[0093] The expression of SRPX2 in human pancreatic cancer cell lines was obtained from Cancer Cell Line Encyclopedia (CCLE) (http: / / broadinstitute.org), and it was found that SRPX2 was highly expressed in human pancreatic cancer cell lines ( Figure 7 ). Pancreatic cancer CAFs and pancreatic cancer cell lines (PANC1, SW1990, BXPC3) were cultured and samples were collected for Western Blot detection of SRPX2 protein expression. It was found that SRPX2 was highly expressed in pancreatic cancer CAFs, PANC1, SW1990 and BXPC3 ( Figure 8 ).

[0094] The survival data of pancreatic cancer patients in the TCGA database were obtained using the online website TIDE (http: / / tide.dfci.harvard.edu / ) to conduct a correlation analysis between SRPX2 expression and pancreatic cancer patient survival. It was found that pancreatic cancer patients with high SRPX2 expression had a worse survival rate ( Fig. 9 ).

[0095] Pancreatic cancer CAFs were transfected with two siRNAs (purchased from Rebo Bio siG000027286B: target sequence 5'-GAGAGCATGTGATTCGTTTA-3', SEQ ID NO.6, Rebo Bio siG000027286C: target sequence 5'-CCACCAATCTGTGCTCCTA-3', SEQ ID NO.7) to knock down SRPX2. Samples were collected 48 hours later and Western Blot was performed to detect the expression of SRPX2 and its downstream type I collagen (COL1A: 59772, purchased from SANTA). It was found that after knocking down SRPX2 in pancreatic cancer CAFs, the expression of the downstream molecule COL1A protein decreased ( Fig.10 ). The above results confirm that SRPX2 promotes the expression of collagen COL1A protein in pancreatic cancer CAFs.

[0096] Example 4

[0097] SRPX2 is highly expressed in primary CAFs and cell lines of human gastric cancer, and high expression of SRPX2 worsens the survival of gastric cancer patients

[0098] The gastric cancer single-cell sequencing dataset was used to classify cell types on the online website TISCH (http: / / tisch.comp-genomics.org). The analysis found that SRPX2 was more highly expressed in gastric cancer CAFs and gastric cancer cells compared with other cell types ( Fig.11 ).

[0099] In the online website Cancer Cell Line Encyclopedia (CCLE) (http: / / broadinstitute.org), the expression of SRPX2 in some human gastric cancer cell lines was obtained, and it was found that SRPX2 was highly expressed in human gastric cancer cell lines ( Fig.12 ). Gastric cancer CAFs and gastric cancer cell lines (MKN45, AGS, MKN74, HGC27, SNU216 and MKN7) were cultured and samples were collected for Western Blot detection of SRPX2 protein expression. It was found that SRPX2 was highly expressed in gastric cancer CAFs, MKN45, AGS, MKN74, HGC27, SNU216 and MKN7 ( Fig.13 ).

[0100] The survival data of gastric cancer patients in the TCGA database were obtained from the online website TIDE (http: / / tide.dfci.harvard.edu / ) to conduct a correlation analysis between SRPX2 expression and gastric cancer patient survival. It was found that gastric cancer patients with high SRPX2 expression had a worse survival ( Fig.14 ).

[0101] Gastric cancer CAFs were transfected with two siRNAs ((purchased from Rebo Bio siG000027286B: target sequence 5'-GAGAGCATGTGATTCGTTTA-3', SEQ ID NO.6, Rebo Bio siG000027286C: target sequence 5'-CCACCAATCTGTGCTCCTA-3', SEQ ID NO.7)) to knock down SRPX2. Samples were collected 48 hours later and Western Blot was performed to detect the expression of SRPX2 and its downstream type I collagen (COL1A: 59772, purchased from SANTA). It was found that after knocking down SRPX2 in gastric cancer CAFs, the expression of the downstream molecule COL1A protein decreased ( Fig.15 ).

[0102] Example 5

[0103] SRPX2 in CAFs promotes extracellular matrix remodeling

[0104] The pLenti-U6-shRNA-CMV-BSR-WPRE vector carrying SRPX2 short hairpin RNA (shRNA sequence: 5'-CCAGCGAAGATGCGAAGATAT-3') and the corresponding lentivirus carrying an empty vector were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai). CAFs were cultured at 2×10 4The number of cells was inoculated in a six-well plate. After the cells adhered to the wall the next day, shRNA transfection was performed according to the instructions, and the medium was changed 12 hours after transfection. After the number of cells was sufficient and in good condition, puromycin (2 μg / mL) was added to screen the stably transfected cells. After 4-5 days of screening, RT-qPCR, SRPX2 primer sequence SRPX2-F: 5'-CGGACTGAGTTCCTCTAATCCC-3' (SEQ ID NO.2), SRPX2-R: 5'-CACTGCCCAGATGTTACGAC-3' (SEQID NO.3) and Western Blot were used to verify the efficiency of lentiviral transfection, and the established shSRPX2 CAF stable transfection line and CAFshNC negative control cell line were used for subsequent experiments.

[0105] The above shSRPX2 CAF stable transfected cell line and CAFshNC negative control cell line were respectively cultured at 1×10 5 Several cells were seeded on sterile glass slides, and sodium ascorbate (initial concentration 250ng / mL, maintenance concentration 50ng / mL) was added to promote extracellular matrix secretion. After about 2 days of growth, secretion continued for another 3 days. After 3 days, the cells were lysed with 0.5% Triton X-100, and the decellularized extracellular matrix was immunofluorescently stained to observe the expression of type I collagen. The results showed that after knocking down SRPX2 in CAFs, the content of type I collagen in the extracellular matrix decreased ( Fig.16 ).

[0106] 400 μL of the extracted collagen solution was mixed with shSRPX2 CAF stable transfected cell line and CAFshNC negative control cell line in 100 μL of phenol red culture medium containing 2×10 5 The cells were mixed and the total volume of 500 μL of the mixed solution was inoculated in a 24-well plate. The degree of collagen contraction was observed by taking photos at 24 hours, 48 ​​hours and 72 hours. The results showed that after knocking down SRPX2, the ability of CAF to promote collagen contraction was weakened ( Figure 17-18 ). The above results indicate that SRPX2 in CAFs promotes matrix formation and remodeling in the tumor microenvironment.

[0107] Example 6

[0108] SRPX2 recombinant protein induces M2-like polarization of macrophages

[0109] M0-like macrophages, M2-like macrophages induced with IL-4 (50 ng / mL), macrophages cultured with SRPX2 recombinant protein (rhSRPX2H00027286, Abnova, USA, 100 ng / mL), and macrophages cultured with SRPX2 recombinant protein combined with uPAR neutralizing antibody (Anti-uPAR, #946204, BioLegend, USA, 100 ng / mL) were compared after 48 hours of culture.

[0110] Flow cytometry results showed that compared with M0-like macrophages, the expression of CD163 membrane protein on the surface of rhSRPX2-treated macrophages was significantly increased (P<0.001), while the expression of CD163 membrane protein on macrophages in the rhSRPX2 combined with Anti-uPAR group was significantly decreased (P<0.01) ( Fig.19 ). At the same time, this example found that the expression of CD163 membrane protein on the surface of macrophages after adding rhSRPX2 was higher than that of M2-like macrophages induced by adding IL-4 ( Fig. 20 ). However, there was no significant change in the expression of CD86 membrane protein in macrophages in the rhSRPX2 combined with Anti-uPAR group ( Fig.21 ).

[0111] Example 7

[0112] In vivo experiments in mice confirm that high expression of SRPX2 promotes TNBC immunotherapy resistance

[0113] In this example, a TNBC orthotopic transplant tumor model was established by injecting 4T1 cells into the mammary fat pad of wild-type BALB / c mice. The GL186pcSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE 3 vector (upstream and downstream cloning restriction sites: EcoR I and BamH I) carrying the promoter sequence CMV-F of the lentivirus overexpressing (OE) mouse SRPX2: 5'-CGCAAATGGGCGGTAGGC GTG-3' (SEQ ID NO.4); the sequence MSCV-rev related to the Rev protein in the lentiviral vector: 5'-CAGCGGGGCT GCTAAAGCGCATGC-3' (SEQ ID NO.5) and the corresponding lentivirus carrying the empty vector were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai).

[0114] Luc-labeled murine breast cancer 4T1 cells were cultured at 4×10 4The cells were inoculated in 6-well plates. The next day, after the cells adhered to the wall, the overexpression lentivirus and negative photovirus were infected with 4T1 cells at a concentration of MOI = 20 (multiplicity of infection) and the corresponding virus titer. The culture medium was replaced 8 hours later. After 72 hours, puromycin (2μg / mL) was used to select cells expressing the puromycin resistance tag. After 1 week of screening, the efficiency of lentiviral transfection was verified by RT-qPCR and Western Blot experiments. The Luc-labeled 4T1 stable transfection line overexpressing SRPX2 was used for mouse experiments.

[0115] The mammary fat pads of mice were injected with 5×10 5 The cells were injected with 100 μL PBS suspension of 4T1 cells stably overexpressing SRPX2 (OE-SRPX2) and control (Vector) 4T1 cells. After tumor formation, the Vector group and the SRPX2 group were randomly divided into a PBS injection group (control group: 1 mL) and an Anti-PD-L1 injection group (200 μg / 1 mL / each mouse: 1 mL).

[0116] By detecting the bioluminescent signal of tumor cells through the in vivo imaging system, it was found that compared with the Vector group, the tumor in the stable overexpression SRPX2 group was significantly enlarged and the fluorescence signal was enhanced. In the SRPX2 overexpression group, there was no significant change in the tumor fluorescence signal of the Anti-PD-L1 group, while the fluorescence signal of the Vector group was significantly weakened after the application of Anti-PD-L1 (P<0.05). The results of mouse tumor analysis showed that compared with the Vector group, the tumor volume and weight of the stable overexpression SRPX2 group were significantly increased; after overexpression of SRPX2, the injection of Anti-PD-L1 did not reduce the tumor volume; and the application of Anti-PD-L1 significantly reduced the volume and weight of the tumor in the Vector group mice, with a significant statistical difference (P<0.01.)( Fig. 22 ). The results of immunohistochemistry showed that compared with the Vector group, the expression of SRPX2 in breast tumor tissues of mice in the OE-SRPX2 group was significantly increased (P<0.01) ( Fig.23 ).

[0117] In summary, in vivo experiments in mice confirmed that TNBC with high expression of SRPX2 is resistant to anti-PD-L1 immunotherapy, and SRPX2 can promote the growth of TNBC tumors in vivo.

[0118] Example 8

[0119] Clinical samples verified that high expression of SRPX2 promotes TNBC immunotherapy resistance and can be used as a marker and therapeutic target for immunotherapy resistance.

[0120] A total of 38 breast tumor puncture specimens were collected from patients with triple-negative breast cancer before neoadjuvant immunotherapy, and immunohistochemical staining was performed on the patients' baseline puncture specimens. According to the Miller-Payne grade of the primary breast tumor of the patients after surgery, the patients were divided into an immunotherapy response group (pCR group) of 25 cases and an immunotherapy non-response group (NR group) of 13 cases ( Fig.24 ).

[0121] The results showed that the expression levels of SRPX2 and CD163 proteins in the non-responder group (NR) were higher than those in the pCR group. The IHC staining results of the baseline breast tumor puncture specimens showed that the expression levels of SRPX2 and CD163 in the NR group of non-responders to immunotherapy were significantly higher than those in the pCR group of immunotherapy responders ( Fig.25 ). Clinical sample analysis confirmed that high expression of SRPX2 and increased M2-like macrophages promoted TNBC immunotherapy resistance. The above results indicate that SRPX2 can be used as a marker and therapeutic target for immunotherapy resistance.

[0122] Example 9

[0123] Conditioned medium of TNBC cells with SRPX2 knockdown / overexpression inhibits / promotes macrophage M2-like polarization

[0124] The conditioned medium of SRPX2-knockdown MDA-MB-231 cells (shSRPX2-CM) and the conditioned medium of SRPX2-overexpressing BT549 cells (OE-SRPX2-CM) were used to culture human THP-1 cell-derived M0-like macrophages.

[0125] After culture with shSRPX2-CM, the mRNA levels of CD163, CCL17, CCL22, and PPARγ in macrophages were significantly downregulated (P<0.001); the surface CD163 membrane protein expressed by macrophages decreased (P<0.05), while the surface CD86 membrane protein expressed by macrophages did not change significantly (P<0.05). Fig.26 ).

[0126] After culturing macrophages with conditioned medium of BT549 cells overexpressing SRPX2 (OE-SRPX2-CM), the mRNA levels of CD163, CCL17, CCL22, and PPARγ in macrophages were significantly upregulated; at the same time, flow cytometry detected a significant increase in the expression of CD163 membrane protein on the surface of macrophages ( Fig. 27 ).

[0127] Example 10

[0128] SRPX2 induces PD-L1 expression in M2-like macrophages

[0129] M0-like macrophages were cultured with conditioned medium from MDA-MB-231 cells or BT549 cells that knocked down or overexpressed SRPX2. Flow cytometry results showed that the expression of PD-L1 membrane protein on the surface of macrophages was significantly reduced after macrophages were cultured with shSRPX2-CM, while the expression of PD-L1 membrane protein on macrophages cultured with OE-SRPX2-CM was significantly increased.

[0130] After adding rhSRPX2 (50ng / mL, 100ng / mL, 200ng / mL), the expression of PD-L1, a surface membrane protein of macrophages, increased. After further adding Anti-uPAR and STAT3 inhibitor Stattic, the expression of PD-L1 membrane protein decreased significantly (P<0.05). Western Blot detection of total PD-L1 protein in macrophages also showed corresponding changes. shSRPX2-CM can reduce the expression level of total PD-L1 protein in macrophages and the phosphorylation level of STAT3 in macrophages. OE-SRPX2-CM and rhSRPX2 increase the expression of PD-L1 protein in macrophages and the phosphorylation level of STAT3. After further adding Anti-uPAR and STAT3 inhibitor Stattic, the expression of PD-L1 protein in macrophages decreased ( Figures 28 to 31 ).

[0131] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of SRPX2 protein or SRPX2 gene in the preparation of products for predicting the efficacy of immunotherapy for solid tumors.

2. The use according to claim 1, characterized in that: The immunotherapy efficacy includes immunotherapy resistance.

3. The use according to claim 1, characterized in that: The products include products for detecting the expression level of the SRPX2 protein or SRPX2 gene.

4. The use according to claim 3, characterized in that: The expression of the SRPX2 protein or SRPX2 gene is upregulated.

5. The use according to claim 4, characterized in that: The reagents for detecting the expression level of SRPX2 protein or SRPX2 gene include real-time fluorescence quantitative PCR related reagents and / or protein immunoblotting related reagents.

6. Use of SRPX2 protein or SRPX2 gene in the preparation of products for improving the efficacy of immunotherapy for solid tumors and / or in the preparation of products for treating solid tumors.

7. The use according to claim 6, characterized in that: The product includes biological materials in which the SRPX2 gene and / or SRPX2 protein is knocked out or knocked down.

8. The use according to claim 7, characterized in that: The biological material for knocking out or knocking down the SRPX2 gene includes shRNA and / or siRNA for knocking out or knocking down the SRPX2 gene, the nucleotide sequence of the shRNA is shown as SEQ ID NO.1; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the siRNA-1 or siRNA-2 are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively; The biological material for knocking out or knocking down the SRPX2 protein includes drugs and / or small molecule inhibitors targeting the SRPX2 protein.

9. The use according to claim 7, characterized in that: The drugs include degradation drugs targeting the SRPX2 protein and / or drugs targeting the receptor uPAR of the SRPX2 protein.

10. The use according to any one of claims 1 to 9, characterized in that: The solid tumor includes one or more of breast cancer, pancreatic cancer and gastric cancer.

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