Specific M2-TAMs immunodiagnosis marker related to lung adenocarcinoma and application thereof

Abstract

The invention provides a specific M2-TAMs immunodiagnosis marker related to lung adenocarcinoma and application thereof. The technical key point is that SPAG4 is applied to preparation of a tumor diagnosis product as a diagnosis marker. In the early stage, a WGCNA algorithm is adopted, transcriptome data of lung adenocarcinoma patients in TCGA and GEO databases and M2 type macrophage distribution proportion data analyzed by CIBERSORT are integrated, and an M2 type macrophage polarization correlation module is successfully constructed. Through differential gene screening, Cox regression analysis and Kaplan-Meier survival analysis, the SPAG4 is finally determined as the core regulatory gene. Experimental results show that M2 type macrophage polarization and angiogenesis can be remarkably reduced by inhibiting expression of SPAG4, so that tumor growth is inhibited. On the basis, the advantages of an unmarked metabolism living body imaging technology, a living body blood vessel imaging technology, a living body immunostaining technology and molecular epidemiological research are integrated, the regulation and control mechanism of the SPAG4 gene in the lung adenocarcinoma progress and the influence of the SPAG4 gene on macrophage polarization and blood vessel generation are focused, and a novel tumor diagnosis technology and a kit are developed; the accuracy of early screening of the lung cancer is improved, and a new target spot is provided for precise treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a specific M2-TAMs immunodiagnostic biomarker associated with lung adenocarcinoma and its application. Background Technology

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality rate in my country. Non-small cell lung cancer accounts for approximately 85% of all lung cancer cases, with lung adenocarcinoma and squamous cell carcinoma being the main pathological types. In recent years, the age-standardized incidence rate of lung squamous cell carcinoma in my country has fallen below 10 per 100,000 person-years, while the age-standardized incidence rate of lung adenocarcinoma has exceeded 20 per 100,000 person-years. Lung adenocarcinoma has become the main histological subtype of lung cancer in my country (approximately 50% in men and approximately 75% in women). Due to numerous risk factors and a poor prognosis, lung adenocarcinoma is threatening the lives and health of a large portion of the Chinese population, becoming one of the increasingly serious public health problems in my country. Accurately identifying high-risk groups for lung adenocarcinoma and implementing targeted preventive measures is of great public health significance for reducing the incidence and mortality of lung adenocarcinoma.

[0003] Precision oncology is a crucial development direction in the global medical field. With the continuous advancement of molecular imaging and biomarker screening technologies, the demand for precision diagnosis and treatment of tumors based on molecular mechanisms is growing. However, early screening and precision treatment of lung cancer still face many challenges. Traditional tumor metabolic assessment tools include magnetic resonance imaging (MRI), fluorodeoxyglucose-positron emission tomography (FDG-PET), and immunohistochemistry (IHC). These methods cannot fully capture changes in cellular metabolism and reflect the efficacy of drug treatment. While IHC is effective, the process is cumbersome, requiring multiple steps such as tissue biopsy, fixation, and staining, and is only applicable to patients with tumor tissue, making it difficult to provide detailed subcellular information. For example, 18FDG-PET has been widely used in clinical tumor metabolic imaging and is more helpful in assessing efficacy than radiological imaging based on morphological changes, but it is limited by single label, ionizing fluorescence intensity, and low resolution. MRI based on 13C-lactate or 13C-pyruvate has high sensitivity, but its spatial resolution is low, and it still faces challenges in single-cell tracking. In summary, existing tumor metabolic assessment tools still have certain shortcomings and are insufficient to meet the needs of accurate efficacy prediction. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies in early lung cancer screening, which makes it difficult to accurately predict treatment efficacy.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of SPAG4 as a diagnostic biomarker in the preparation of tumor diagnostic products.

[0007] Preferably, the tumor is lung adenocarcinoma.

[0008] Preferably, the diagnostic product is a diagnostic kit or diagnostic reagent.

[0009] This application also provides a lung adenocarcinoma diagnostic kit for early screening of lung adenocarcinoma, wherein the diagnostic kit uses SPAG4 as a diagnostic target.

[0010] This application also provides the application of SPAG4 as a therapeutic target in the preparation of tumor therapeutic drugs, wherein the tumor is lung adenocarcinoma.

[0011] Preferably, the drug inhibits tumor growth by suppressing SPAG4 expression, thereby reducing M2 macrophage polarization and angiogenesis.

[0012] Compared with existing technologies, this application has the following beneficial effects: The invention initially employed the WGCNA algorithm, integrating transcriptome data from lung adenocarcinoma patients in the TCGA and GEO databases with M2 macrophage distribution data analyzed by CIBERSORT, successfully constructing an M2 macrophage polarization-related module. Through differential gene screening, Cox regression analysis, and Kaplan-Meier survival analysis, SPAG4 was ultimately identified as the core regulatory gene. Experimental results showed that inhibiting SPAG4 expression significantly reduced M2 macrophage polarization and angiogenesis, thereby inhibiting tumor growth. Based on this, this invention integrates the advantages of label-free metabolic in vivo imaging technology, in vivo vascular imaging technology, in vivo immunostaining technology, and molecular epidemiological research, focusing on the regulatory mechanism of the SPAG4 gene in lung adenocarcinoma progression and its impact on macrophage polarization and angiogenesis, developing novel tumor diagnostic technologies and kits, improving the accuracy of early lung cancer screening, and providing new targets for precision treatment. Attached Figure Description

[0013] Figure 1The expression profile of SPAG4 and its regulatory role in the prognosis of LUAD were investigated. (ab) The KaPlan-Meier (KM) survival curve was generated based on the integration of tam phenotype data and patient OS status in the TCGA-LUAD dataset. M1 and M2 macrophage infiltration were statistically significant with LUAD prognosis (M1, P = 0.008; M2, P = 0.039); (cf) The WGCNA algorithm was used to associate TCGA-LUAD gene expression with M2 macrophage phenotype, and the green module was selected according to the weighted co-expression network (r = 0.2, P < 0.001); (g) Six target genes were obtained by intersecting the three databases (TCGA-LUAD and GTEx differentially expressed genes, green module genes, GSE115002 differentially expressed genes, P < 0.001). FDR <0.05); (h) Cox univariate analysis of the risk scores of 6 genes in LUAD patients (SPAG4: HR = 1.72, P = 0.045). (ij) Pathway analysis showed that SPAG4 mRNA expression in LUAD was highly correlated with immune response; (km) Compared with adjacent non-tumor tissues (normal: n = 347, n = 52, n = 83), SPAG4 mRNA expression in LUAD tumor tissues (LUAD: n = 513, n = 52, n = 83) was significantly increased (P < 0.0001). Significance level: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Results are expressed as mean ± standard deviation.

[0014] Figure 2SPAG4 promotes the development and progression of lung adenocarcinoma. (ac) KM survival curves of LUAD patients based on SPAG4 expression on multiple datasets (P = 0.043, P < 0.0001, P = 0.025, n = 513, n = 226, n = 443); (de) Western blot analysis and quantification of proteins extracted from human LUAD tissue and paired adjacent normal tissues (n = 24, P = 0.0016); (fg) Representative Western blot analysis showing the knockdown efficiency of SPAG4 in SPCA1 cells after transfection with si-SPAG4 plasmid (n = 3, P = 0.0114); (hj) Representative images and quantitative data of scratch healing assay in SPCA1 cells after transfection with si-SPAG4 plasmid (n = 3, P = 0.0328); (ki) Transwell assay images and quantitative data of SPCA1 cells after transfection with si-SPAG4 plasmid. (n=13, P<0.0001); (m) Representative image of colony formation after transfection of SPCA1 cells with si-SPAG4 plasmid. (no) Representative Western Blot analysis showing the knockdown efficiency of SPAG4 in SPCA1 cells after transfection with lv-SPAG4 plasmid; (p) Establishment of a tumor-bearing model (created in BioRender http: / / BioRender.com / z47q423r); (qt) Nude mice with tumors and tumor images and quantitative results (n=16, P=0.0067). Significance level: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Results are expressed as mean ± standard deviation.

[0015] Figure 3SPAG4 promoted the polarization of m2-like macrophages in vitro. (a) Western blot analysis showed that the SPAG4low group had lower expression levels of CD206 and IL10 proteins and higher expression levels of CD86 and INOS proteins. (bc) Western blot analysis showed that the SPAG4low group of mice had lower expression levels of CD206 and IL10 proteins and higher expression levels of CD86 and INOS proteins in tumor tissue (P = 0.0303, P = 0.0135, P = 0.0091, P = 0.0305, n = 16). P-values ​​were expressed using one-way ANOVA with Tukey multiple comparison test. (dg) Transwell indirect co-culture model diagram; (e) Representative results of indirect co-culture immunofluorescence; (fg) Single-cell metabolic kinetics of CD68 and CD206 signaling. Each colored square represents the visible pixel intensity of CD68 and CD206 in each macrophage (n=15, P<0.0001 for all); (hk)(h) Schematic diagram of co-culture model in conditioned medium; (i) Representative results of immunofluorescence in co-culture in conditioned medium; (jk) Single-cell metabolic kinetics under CD68 and CD206 signals. Each colored square represents the visible pixel intensity of CD68 and CD206 in each macrophage (n=15, P<0.0001 for all). Significance level: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Results are expressed as mean ± standard deviation.

[0016] Figure 4SPAG4 affects the polarization of M2-like macrophages in vivo. (ab) In nude mice bearing tumors, dextran (a marker for M2-like macrophages) was injected into the tail vein for 2.5 h, followed by in vivo and in vitro imaging to observe the differences in the distribution of M2-like macrophages between the SPAG4WT and SPAG4Low groups; (cd) Quantitative results of in vivo mean fluorescence intensity efficiency (n=7, P<0.0001) and in vitro mean fluorescence intensity efficiency (n=6, P=0.0219); (ef) Flow cytometry was performed on some tumors, and CD206-labeled M2-like macrophages were used to explore the differences between the SPAG4WT and SPAG4Low groups. Flow cytometry data showed that the number of CD206+ cells decreased after transfection with SPAG4Low (n=12, P=0.0095); (g) Immunofluorescence staining showed the precise cellular localization of dextran targeting M2 macrophages (red, scale bar = 50 μm). (hk) Compared with the SPAG4wt group, the SPAG4low group showed a significant reduction in the infiltration of CD68+ (green) and CD206+ (red) macrophages (P<0.0001, P=0.0066). Unpaired t-tests were used to evaluate the p-values. Significance levels: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Results are expressed as mean ± standard deviation.

[0017] Figure 5 SPAG4 modulates the vascular network of LUAD (lumbar vascular angiogenesis). (ac) Multi-label immunofluorescence staining and quantification of SPAG4, CD31, and CD206 in human LUAD tumor tissue (P = 0.0069, P < 0.0001, n = 3 cases). P-values ​​were analyzed using an unpaired t-test. (d) Immunofluorescence staining showed that the vascular structure of human LUAD tumor tissue was more disordered, complex, and unstructured than that of normal tissue from patients. (ei) AngioTool analysis of the vascular network in human LUAD tumor tissue, including total vessel length, average vessel length, vessel area, and total number of connections (P < 0.0001, n = 3 cases). P-values ​​were analyzed using an unpaired t-test. (j) Intravenous injection of 2MDa dextran Tritc (red) was used for in vivo visualization of tumor vessels. Tumor angiogenesis in SPAG4wt mice was highly leaky, disordered, and branched; AngioTool analysis of the vascular network in (kn)LUAD skinfold mice, including total vessel length, mean length, vessel area, and total number of connections (P<0.0001, P=0.0148, P=0.0434, P=0.0012, P=0.0004, n=9). P values ​​were expressed as a one-way ANOVA using Tukey's multiple comparison test. Significance levels: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Results are expressed as mean ± standard deviation. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments.

[0019] The application of SPAG4 as a diagnostic biomarker in the preparation of tumor diagnostic kits, wherein the tumor is lung adenocarcinoma, and the SPAG4 sequence is as shown in SEQ ID NO:01.

[0020] Based on the above-mentioned application of SPAG4, this application also provides a diagnostic kit for early screening of lung cancer, wherein the diagnostic kit uses SPAG4 as a diagnostic target.

[0021] In addition, this application also provides the application of SPAG4 as a therapeutic target in the preparation of tumor therapeutic drugs, wherein the drugs inhibit tumor growth by inhibiting the expression of SPAG4 to reduce M2 macrophage polarization and angiogenesis.

[0022] The above content will be explained below with reference to specific verification experiments:

[0023] I. Experimental Materials and Sources

[0024]

[0025]

[0026] II. Implementation Examples

[0027] Example 1 (Bioinformatics): Screening of Tissue-Specific Diagnostic Markers for Lung Adenocarcinoma M2-TAMs

[0028] First, infiltration data of 22 immune cell types were obtained using CIBERSORT for all genes in TCGA-LUAD. Based on the research objective, Kaplan-Meier (KM) survival analysis was performed on the infiltration status of M1 and M2 macrophages combined with the survival information from TCGA-LUAD. The results showed that only the KM curve for M2 met the logical requirements.

[0029] Next, the RNA-seq data in TCGA-LUAD was cleaned through a series of processes, including transposition, deduplication, and retention of parts with significant survival value. The remaining RNA-seq data of all genes were then combined with M2 macrophage infiltration data using the WGCNA algorithm, and the green module with the highest logical correlation r was selected.

[0030] Subsequently, the intersection of the module genes with the differentially expressed genes in the TCGA-LUAD database (which integrates the GTEx database) and the external dataset GSE115002 was taken to obtain 6 genes that met the criteria. Then, a univariate Cox regression was performed on these 6 genes (P<0.05, HR>1) to finally obtain the target gene SPAG4.

[0031] Simultaneously, the GEPIA2 website was used to obtain the top 300 genes related to SPAG4. GSEA pathway analysis was performed using their TCGA-LUAD expression data. In this application, the top 10 related pathways were selected, with the immune response pathway ranking eighth and P... FDR <0.05. In this application, multiple GEO databases (GSE115002, GSE75037, GSE31210, GSE68465) were used to verify the expression and prognosis of SPAG4, and the results were all logically consistent with P<0.05. Figure 1 )

[0032] Example 2 (Molecular Epidemiology) ： Validation of tissue-specific M2-TAMs diagnostic markers for lung adenocarcinoma

[0033] To preliminarily verify the biomarker's ability to differentiate between lung adenocarcinoma tissue and adjacent normal tissue, Western blotting was used to examine the differential expression of SPAG4 in 24 pairs of human lung adenocarcinoma tissue samples. After knocking down SPAG4 in tumor cells using interfering plasmids, a series of experiments were conducted to demonstrate the pro-cancer effect of SPAG4, including proliferation, migration, scratch assays, transwell invasion, and tumor bearing. Figure 2 )

[0034] (I) Example 3: Sample collection experiment verification

[0035] The first phase included tumor tissue from 8 nude mice and 3 case studies from the preliminary experiments. Baseline information for the cases is shown in Table 1. All three lung cancer cases were newly diagnosed lung adenocarcinoma cases at the First People's Hospital of Nantong City, Jiangsu Province, between October 2024 and May 2025. All cases were confirmed by pathological histology, excluding patients with a history of malignant tumors, metastases to other organs, or who had received chemotherapy or radiotherapy prior to blood collection. The adjacent tissue from the three cases served as controls.

[0036] Table 1. Characteristics of the subjects included in this study

[0037]

[0038] Tumor tissue proteins were extracted from eight nude mice, and Western blot analysis was performed to detect markers of M1 and M2 macrophages. It was found that injection of SPAG4... WTThe expression levels of markers for M1 macrophages were higher than those for SPAG4. Low In the group, the expression levels of M2 macrophage markers were opposite. Furthermore, frozen sections of matched cancer and adjacent normal tissues from three patients with early-stage lung adenocarcinoma were stained, and the results were consistent with those in mouse tumor tissues, showing high expression levels of CD206 (a marker for M2 macrophages) in cancer tissues. Figure 3 )

[0039] (II) Example 4: In vivo imaging in mice to verify the polarization of M2 macrophages in vivo

[0040] The second stage involved constructing M2 macrophage-labeled mice in vivo, initially consistent with the construction of tumor-bearing mouse models. Until in vivo IVIS (label-free metabolic in vivo imaging) was performed, both groups of mice were injected intravenously with the M2 macrophage labeling probe Dextran. Two hours later, in vivo IVIS observation of the fluorescence intensity in both groups revealed that the injection of SPAG4... WT The fluorescence intensity of group Dextran was higher than that of SPAG4. Low Group. Subsequently, half of the tumor was extracted for flow cytometry, and the other half was subjected to immunofluorescence staining. The results were consistent with previous experiments, demonstrating that SPAG4 promotes M2 macrophage polarization in vivo. Figure 4 )

[0041] (III) Example 5: In vivo imaging in mice to verify that SPAG4 promotes M2 macrophage polarization and affects angiogenesis (in vivo vascular imaging technology, in vivo immunostaining technology)

[0042] The third stage involved constructing a mouse dorsal lateral visual window model. Visual windows were installed on the dorsal side of 8-week-old nude mice, and SPCA1-SPAG4 was injected into the visual window area of ​​both groups of nude mice. WT With SPCA1-SPAG4 Low Cells were injected with the vascular labeling probe 2MDa Dextran via the tail vein three days later. After 30 minutes, in vivo imaging of angiogenesis in mice was performed using a two-photon focusing microscope. Data analysis showed that SPAG4 promoted M2 macrophage polarization and affected angiogenesis. Figure 5 )

[0043] III. Some of the experiments and procedures used in this application

[0044] 1. Immune infiltration analysis

[0045] CIBERSORT (https: / / cibersortx.stanford.edu / ) is a computational tool widely used in tumor immunology research. It facilitates the quantitative assessment of different immune cell subsets within tumor tissues by analyzing gene expression data. This method relies on a linear regression model that predicts the relative abundance of specific immune cell types in a mixed cell sample. CIBERSORT achieves this by utilizing known gene expression profiles characteristic of various immune cell subsets. In the study presented in this application, CIBERSORT was used to estimate the proportions of 22 different tumor-infiltrating immune cell types in the TCGA-LUAD cohort. To ensure the robustness of the results, all data were normalized and 1000 replicate analyses were performed.

[0046] 2. Construction of co-expression networks

[0047] Expression data from the TCGA-LUAD database were analyzed using the Weighted Gene Co-expression Network Analysis (WGCNA) software package, with a focus on the expression levels of m2-like macrophages within the samples. Following a series of preprocessing steps, including translocation, duplication removal, and preservation of relevant survival information, gene expression data from 459 individuals were merged with M2 macrophage phenotypic data obtained through CIBERSORT. Subsequently, gene clusters showing the highest positive correlation with M2 macrophage abundance were identified. Specifically, the modules with the highest correlation coefficients (r>0) were selected.

[0048] 3. Western blot analysis

[0049] Cells were first washed with pre-cooled PBS, then lysed with RIPA lysis buffer (Beyotime) and a protease inhibitor (Roche, Shanghai, China) to extract total protein. Protein concentration was determined by bicinchonic acid (BCA) protein quantification, and corresponding samples were prepared. Subsequently, proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk powder and incubated overnight at 4°C with RNF213 antibody (1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA), ZBTB20 antibody (1:2000, Proteintech, Wuhan, China), and microtubule antibody (1:1000, Beyotime). Finally, the membranes were incubated with goat anti-mouse secondary antibody (1:1000, Beyotime) and goat anti-rabbit secondary antibody according to specifications, followed by color development. The relative expression of the target protein against Tubulin was normalized, and the relative quantification of the immunoblot images was performed using ImageJ software (version 154d).

[0050] 4. Cell culture and transfection

[0051] Human LUAD cell line SPCA1 was purchased from ATCC (Gaithersburg, MD, USA) and stored in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. The commercially available SPAG4 plasmid was obtained and si-SPAG4 was designed and synthesized by Nanjing Keruis Biotechnology Co., Ltd. The construction of the rs191959201G>T overexpression plasmid was performed by Shanghai Jikai Co., Ltd. Lipo8000 was used. TM The DNA plasmid or siRNA was transfected into SPCA1 cells according to the manufacturer's instructions using the transfection reagent (Beyotime, Shanghai, China). Western blot was used to determine the success of plasmid transfection.

[0052] 5. Cell proliferation assay

[0053] In the cell proliferation experiment, the SPCA1 cell line was first placed in a large culture dish. After the dish was filled with cells, cell counting was performed after 2 days. 1000 LUAD cells were seeded into 6-well plates, and the designed plasmids were transfected into the corresponding sites in an incubator at 37°C. Two weeks later, the cells were fixed with methanol for half an hour, stained with crystal violet (Beyotime, Shanghai, China) for 2 hours, and observed under natural light.

[0054] 6. Scratch healing test

[0055] A scratch healing assay was performed on the back of a six-well plate by drawing six parallel lines 0.5 cm apart using a ruler. After transfection, 10,000 lung adenocarcinoma cells were evenly seeded onto the six-well plate. Cells were allowed to proliferate until they covered 80-90% of the plate surface. Then, a scratch perpendicular to the parallel lines was created using a pipette tip. The culture dishes were incubated at 37°C in a cell culture incubator. Cells were observed and photographed under a microscope at 0, 48, and 96 hours.

[0056] 7. Transwell test

[0057] Cell migration ability was measured in a Transwell chamber (pore size 8 μm; 3472, Corning, New York, USA). The day before the experiment, a layer of fibronectin (F8180, Solarbio, Beijing, China) was coated on the bottom of a Transwell culture dish, which was then placed in a cell culture incubator. On the day of the experiment, 30,000 treated SPCA1 cells were seeded in serum-free medium in the upper chamber and medium containing 10% fetal bovine serum was added to the lower chamber. After 24 hours of culture, SPCA1 cells were collected and fixed with methanol for 1 hour. They were then stained with crystal violet dye (C0121, Beyotime, Shanghai, China) for 1 hour and photographed for analysis.

[0058] 8. Co-culture experiment

[0059] 1) Co-culture based on conditioned medium (CM)

[0060] CM harvested from SPCA1 cells (cultured for 48 hours) was applied to M0 macrophages and then incubated for 48 hours before the end of treatment.

[0061] 2) Transwell indirect co-culture

[0062] Indirect co-culture was performed using a Transwell system (24-well plate; 0.4 μm pore size, 353095, Corning, New York, USA). SPCA1 cells (5 × 10⁶ cells / well) were used. 5 ) Inoculated into the superior vena cava, M0 macrophages (5×10 5 Inoculated into the lower cavity.

[0063] 9. Macrophage polarization

[0064] THP-1 cells were added at a rate of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 100 ng / mL in 24-well plates with PMA (100 ng / mL; MCE, Shanghai, China). Cells were cultured at 37°C and 5% CO2 for 48 hours, then cultured for another 24 hours after removing the PMA to induce differentiation into M0 adherent macrophages. Subsequently, M0 macrophages were treated with 30 ng / mL IL-4 (novoProtein, Suzhou, China) for 72 hours to promote M2 phenotype polarization.

[0065] 10. Tumor formation experiment

[0066] Animal experiments were approved by the Institutional Animal Welfare and Utilization Committee of Nantong University (Approval No.: S20220224-006). Male BALB / c mice (5 weeks old) were purchased from the SLAC Laboratory Animal Center (Shanghai, China) and housed in an SPF facility. Mice were randomly divided into two groups of 8 mice each. 5 × 10⁶ / 100 μL of SPAG4WT or SPAG4Low SPCA1 cells were subcutaneously injected into the right axilla of each BALB / c mouse. Tumor volume was measured using calipers at specified time points and calculated using the formula: L (length) × W (width)² × 2. -1 At the end of the experiment, all mice were euthanized, and the tumors were photographed, weighed, and then frozen for further analysis. According to the humane endpoint assessment criteria established by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animals, Nantong University, tumor weight should not exceed 10% of the animal's body weight, corresponding to a maximum subcutaneous tumor diameter of 20 mm in a 25g mouse. In this study, the tumor burden of all animals did not exceed these limits.

[0067] 11. Immunofluorescence staining

[0068] Nude mouse tumor tissue was embedded in an optimal cutting temperature (OCT) compound, frozen overnight at -80°C, and then sectioned (8-10 μm thickness) using a cryostat (CM3050 S, LEICA, Germany). Human LUAD specimens from the First People's Hospital of Nantong, Jiangsu Province, China, were sectioned using the same method. After fixation and infiltration, sections were blocked with 10% normal goat serum at room temperature for 2 hours, incubated overnight at 4°C with primary antibodies (CD206: AF2534-SP, R&D Systems, MN, USA; CD68: ab303565, Abcam, Cambridge, UK; CD31: 14-0319-82, eBioscience, CA, USA), and reverse stained with DAPI (C1002, Beyotime, Beijing, China). Fluorescence images were captured using a confocal microscope (Olympus FV3000) for subsequent analysis.

[0069] 12. In Vitro Imaging System (IVIS)

[0070] Two hours prior to imaging, tumor-bearing mice were anesthetized with 2% isoflurane and injected via the tail vein with 110 mg / kg Dextran-AF680 (10,000 MW, D34680, Invitrogen, CA, USA). Bioluminescence signals were acquired using an IVIS spectral system (PerkinElmer, MA, USA) with an exposure time of 1 min, binning of 4, f / stop of 1, and wavelengths of 560-660 nm. Quantitative analysis was performed using the built-in software. All procedures followed institutional guidelines for humane animal imaging.

[0071] 13. Flow cytometry

[0072] Single-cell suspensions were prepared from tumor tissues from nude mice and stained with CD206-AF488 (568806, Biolendy, CA, USA) to quantify M2 macrophage infiltration. Flow cytometry analysis was performed using a Gallios flow cytometer (Beckman Coulter, IN, USA), and data processing software was FlowJo 10.9.0 win.

[0073] 14. In vivo angiography

[0074] Male Balb / c nude mice (8 weeks old) were surgically implanted with a small dorsal implantation kit (SM100; APJTrading, CA, USA) to establish an optical imaging window. One × 10⁶ stable SPAG4WT or SPAG4Low-SPCA1 cells were injected into the chamber-related tissue. After the postoperative recovery period, high molecular weight dextran and a fluorescent tracer (2,000,000 MW; D7139, Invitrogen, CA, USA) were administered intravenously via the tail vein. Vascular dynamics were captured by focusing on the chamber window using a two-photon confocal microscope (FV31S, Olympus, Tokyo, Japan). Quantification of the fluorescence signal was performed using the manufacturer's imaging software (FluoView FV31S-SW, Olympus).

[0075] 15. Enzyme-linked immunosorbent assay (ELISA)

[0076] Target protein levels were quantified according to the manufacturer's instructions using a commercial ELISA kit (IL-8: MM-1558H2, IL-10, MM-0066H2, Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China). Standard curves were generated using GraphPad Prism 9.5 (GraphPad Software, CA, USA), and sample concentrations were interpolated from a linear regression model.

[0077] 16. Data Collection

[0078] GTEx ( https: / / www.gtexportal.org / home / This database supplements the normal tissue sequencing information in the TCGA-LUAD database. Other databases for further validation can be found from GEO (…). Home-GEO-NCBI Download. All SNP information for SPAG4 comes from NCBI (…). National Center for Biotechnology Information The GWAS dataset used for validation is from the GWAS Catalog ( GWASCatalog Search within ).

[0079] 17. Statistical Analysis

[0080] Data analysis was performed using GraphPad Prism 8 and R version 4.3.3 (R Foundation for Statistical Computing, Vienna, Austria). Experimental data are expressed as mean ± standard deviation. Student's t-test was used to analyze differences between two groups, and one-way ANOVA was used to analyze differences among multiple groups. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance).

[0081] Based on the above description, this application previously employed the WGCNA algorithm to integrate transcriptome data from lung adenocarcinoma patients in the TCGA and GEO databases with M2 macrophage distribution data analyzed by CIBERSORT, successfully constructing an M2 macrophage polarization-related module. Through differential gene screening, Cox regression analysis, and Kaplan-Meier survival analysis, SPAG4 was ultimately identified as the core regulatory gene. Experimental results showed that inhibiting SPAG4 expression significantly reduced M2 macrophage polarization and angiogenesis, thereby inhibiting tumor growth. This application utilized a SPAG4 knockdown plasmid to verify that SPAG4 promotes M2 macrophage polarization and angiogenesis. Based on this, this invention integrates the advantages of label-free metabolic in vivo imaging technology, in vivo vascular imaging technology, in vivo immunostaining technology, and molecular epidemiology research, focusing on the regulatory mechanism of the SPAG4 gene in lung adenocarcinoma progression and its impact on macrophage polarization and angiogenesis, developing novel tumor diagnostic technologies and kits, improving the accuracy of early lung cancer screening, and providing new targets for precision treatment.

Claims

1. Application of SPAG4 as a diagnostic biomarker in the preparation of tumor diagnostic products.

2. The application of SPAG4 as a diagnostic biomarker in the preparation of tumor diagnostic kits according to claim 1, characterized in that: The tumor is lung adenocarcinoma.

3. The application of SPAG4 as a diagnostic biomarker in the preparation of tumor diagnostic products according to claim 2, characterized in that: The diagnostic product is a diagnostic kit or diagnostic reagent.

4. A diagnostic kit for lung adenocarcinoma, used for early screening of lung adenocarcinoma, characterized in that: The diagnostic kit uses SPAG4 as the diagnostic target.

5. The application of SPAG4 as a therapeutic target in the preparation of tumor therapeutic drugs, characterized by: The tumor is lung adenocarcinoma.

6. The application of SPAG4 as a therapeutic target in the preparation of tumor therapeutic drugs according to claim 5, characterized in that: The drug inhibits tumor growth by suppressing SPAG4 expression, thereby reducing M2 macrophage polarization and angiogenesis.

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