Application of LOX in plasma exosomes as an early warning marker for lung adenocarcinoma brain metastasis

Abstract

The application discloses application of LOX in plasma exosomes as a warning marker for lung adenocarcinoma brain metastasis, verifies the content difference of LOX in plasma exosome samples of lung adenocarcinoma brain metastasis and non-brain metastasis patients, and proves that the change of the LOX content in the plasma exosomes can be detected to early warn or diagnose lung adenocarcinoma brain metastasis with high specificity and accuracy, and it is determined that LOX can be used as a marker to provide new guidance for early warning or diagnosis of lung adenocarcinoma brain metastasis, and it provides convenience for clinical diagnosis through liquid biopsy, and has significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of lung adenocarcinoma metastasis diagnosis technology, specifically the application of LOX in plasma exosomes as an early warning biomarker for lung adenocarcinoma brain metastasis. Background Technology

[0002] Lung cancer is a leading cause of cancer-related death worldwide and the most common type of malignant tumor. Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for approximately 85% of all cases. Among these, lung adenocarcinoma (LUAD) exhibits a higher tendency for brain metastasis (BrM) compared to squamous cell carcinoma, with a metastasis rate as high as 30–50%. This unique metastatic tendency may be closely related to LUAD's distinctive driver gene mutation profile and hematogenous metastasis characteristics. Once brain metastasis occurs, patients not only face severe neurological dysfunction but also experience a significant decline in quality of life. More critically, current treatment options have limited efficacy, with a median overall survival typically less than 12 months. In recent years, the widespread use of high-resolution magnetic resonance imaging (MRI) and positron emission tomography-computed tomography (PET-CT) technologies has significantly improved the detection rate of asymptomatic brain metastases. However, frequent imaging monitoring not only increases the economic burden on patients but also has significant limitations in distinguishing tumor progression from treatment-related changes such as pseudoprogression or radiation necrosis. Therefore, identifying non-invasive biomarkers that can predict the early occurrence of LUAD-BrM and elucidating their molecular mechanisms is of great clinical significance for establishing effective intervention strategies and improving patient prognosis.

[0003] Given the specific location and potential risks of neurosurgical biopsies, there are significant gaps in our understanding of the mechanisms of brain metastases and their biological changes over time. Liquid biopsies address many limitations of traditional tissue biopsies by providing a more comprehensive characterization of tumor heterogeneity, facilitating repeat sampling, and enabling non-invasive assessment of tumor biology. Key targets of liquid biopsies include circulating tumor cells, circulating nucleic acids, extracellular vesicles, tumor-educational platelets, proteins, and metabolites present in various body fluids. Despite advancements in various technologies, the application of liquid biopsies in brain metastases lags behind that in other solid tumors and metastases to other organs. Among these liquid biopsy analytes, exosomes, a subset of extracellular vesicles with diameters of 30–150 nm, have been found to be more widely distributed in various body fluids, including blood and urine. They also carry a richer array of biological components (including proteins, nucleic acids, and lipids) and exhibit excellent biostability and long-term storage properties. Tumor-derived exosomes (TDEs) have been identified as a key component of the tumor secretome, playing a crucial role in the bidirectional interaction between tumor and host cells, as is the case in lung cancer. TDEs remodel the tumor microenvironment through multiple mechanisms, including promoting epithelial-mesenchymal transition, enhancing cell proliferation, inhibiting apoptosis, exerting immunosuppressive effects, and promoting hematogenous metastasis and angiogenesis, thereby promoting the invasion and metastasis of lung cancer.

[0004] Studies have shown that cancer cells can utilize TDE-mediated regulatory mechanisms to remodel the organ-specific microenvironment, thereby directing metastasis to different organs. Specifically, TDEs exhibit varying affinities for specific receptor cells, leading to differences in metastatic efficiency across different target organs and mediating non-random metastatic spread patterns. In recent years, increasing evidence suggests that TDE-related proteins and nucleic acids play a crucial role in promoting communication between tumor cells and brain microenvironment cells, thus facilitating the formation of metastatic brain tumors. Hoshino et al. discovered that exosomal integrins determine organ-specific metastasis. Specifically, exosomal integrins α6β4 and α6β1 are associated with lung metastasis, while αvβ5 is associated with liver metastasis. Subsequently, their team found elevated levels of cell migration-inducing proteins and hyaluronic acid-binding proteins in tumor tissues and exosomes of patients with brain metastases (BrM), while these were not observed in lung or bone metastases. Advances in proteomics have revealed the molecular characteristics of organ-specific metastatic tumors mediated by TDE-derived proteins, making them a compass encoding organ-specific information in tumor metastasis. Therefore, plasma exosomal proteins are promising candidates for non-invasive prediction of LUAD-BrM. Summary of the Invention

[0005] This invention provides a liquid biomarker for early warning or diagnosis of brain metastases from lung adenocarcinoma. The biomarker is lysine oxidase (LOX) in plasma exosomes, providing clinical guidance for non-invasive prediction of LUAD-BrM.

[0006] In view of this, the solution of the present invention is as follows: The first aspect of this invention is to propose the application of LOX in plasma exosomes as a biomarker in the preparation of a kit for early warning or diagnosis of lung adenocarcinoma brain metastasis.

[0007] A second aspect of the present invention is to propose an application of a LOX content detection product for the preparation of a lung adenocarcinoma brain metastasis early warning or diagnostic kit, wherein the detection product is used for the quantitative detection of LOX in plasma exosomes.

[0008] Furthermore, the kit is used for at least one of the following purposes: a) Early diagnosis of brain metastases from lung adenocarcinoma; b) Screening for brain metastases from lung adenocarcinoma; c) Risk assessment of brain metastasis in lung adenocarcinoma; d) Prognostic assessment of brain metastases from lung adenocarcinoma.

[0009] Furthermore, the detection products include chips, reagent kits, test strips, or analysis platforms.

[0010] Furthermore, the detection product includes at least one of an antibody against exosomal proteins, a reagent for mass spectrometry analysis, or a reagent for chromatographic analysis.

[0011] Preferably, the detection product is an ELISA kit.

[0012] Furthermore, the kit includes plasma exosome separation reagents and / or lysis reagents.

[0013] Furthermore, the kit also includes a reference line, which is the plasma exosomal protein LOX content at any time when the subject has not experienced brain metastasis, or the average plasma exosomal protein LOX content at any time.

[0014] A third aspect of the invention is to provide an apparatus for early warning or diagnosis of brain metastases in lung adenocarcinoma, comprising: The detection module is used to detect the content of exosomal protein LOX in patient plasma samples; The input module is used to obtain the detection results from the detection module; The judgment module is used to compare the test results with the plasma exosomal protein LOX content of the patient before brain metastasis, and to determine whether brain metastasis has occurred or to give the risk of brain metastasis based on the degree of decline. The output module is used to output the diagnostic results.

[0015] Furthermore, the judgment criteria of the judgment module include: if the LOX content in the sample to be tested is significantly lower than the average content during the period before brain metastasis, then it is judged that brain metastasis has occurred.

[0016] Furthermore, the judgment criteria of the judgment module include: if the LOX content in the sample to be tested decreases by ≥30% compared with the average value before brain metastasis, then it is judged that brain metastasis has occurred.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The LOX protein in plasma exosomes provided by this invention serves as a biomarker for predicting brain metastasis of lung adenocarcinoma, offering new guidance for early warning or diagnosis of brain metastasis of lung adenocarcinoma. It also facilitates clinical diagnosis through liquid biopsy.

[0018] The LOX protein described in this application, when used as a biomarker for early warning or diagnosis of brain metastasis in lung adenocarcinoma, is determined by detecting changes in the LOX protein content in plasma exosomes. It has high specificity and accuracy and has significant clinical application prospects. Attached Figure Description

[0019] Figure 1 The results of the enrichment analysis of all identified plasma exosome proteins based on Gene Ontology (GO) in Example 1 of this invention are shown.

[0020] Figure 2 The results of plasma exosomal proteomics analysis of 59 patients in the cohort were obtained in Example 1 of this invention.

[0021] Figure 3 The results show the expression abundance of CD9, Alix, and FLOT1 in plasma exosomes in both brain metastases and non-brain metastases in Example 1 of this invention.

[0022] Figure 4 The results of this embodiment of the invention use five different network topology algorithms to identify core proteins from a candidate protein pool.

[0023] Figure 5 This is the result of enhancing the screening of pivot protein interactions (PPIs) using three machine learning algorithms in Embodiment 1 of the present invention.

[0024] Figure 6 This is the integrated analysis result of formalin-fixed paraffin-embedded (FFPE) tissue proteomics and plasma exosome proteomics in Example 1 of the present invention.

[0025] Figure 7 This is the functional association network of 16 core proteins generated based on the GeneMANIA database in Embodiment 1 of the present invention.

[0026] Figure 8 This is the result of detecting the LOX expression level in plasma exosomes and whole plasma samples by ELISA in Example 2 of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] In this invention, the applicant collected peripheral blood plasma samples from patients with stage IV lung adenocarcinoma (LUAD) and categorized them according to the presence of brain metastases (BrM). Exosomes were isolated from the samples in both groups using size exclusion chromatography (SEC). Three core proteins associated with LUAD-BrM were screened using DIA-MS-based proteomics combined with PPI analysis and three machine learning algorithms (LASSO / RF / SVM). Then, by integrating proteomics analysis of a small cohort of FFPE tissue samples, the key role of the LOX family in LUAD-BrM was discovered. Finally, LOX in plasma exosomes from an independent validation cohort was detected by ELISA, and its diagnostic performance for predicting brain metastases was evaluated. The analytical results collectively indicate that LOX proteins in plasma exosomes can serve as liquid biopsy biomarkers for predicting LUAD-BrM progression. This invention aims to provide a liquid biopsy tool for precise monitoring of high-risk populations for LUAD-BrM and lays the foundation for further exploration of the molecular basis of LUAD-BrM pathogenesis and the development of exosome-targeted therapies for LUAD-BrM.

[0029] Therefore, detecting LOX levels can be applied to the early warning or diagnosis of brain metastasis in lung adenocarcinoma. Specifically, a kit is prepared using a LOX level detection product, which is used to quantitatively detect LOX in plasma exosomes.

[0030] It is understood that the kit is intended for at least one of the following purposes: a) Early diagnosis of brain metastases from lung adenocarcinoma; b) Screening for brain metastases from lung adenocarcinoma; c) Risk assessment of brain metastasis in lung adenocarcinoma; d) Prognostic assessment of brain metastases from lung adenocarcinoma.

[0031] In a preferred embodiment, the detection product includes, but is not limited to, chips, kits, test strips, or analysis platforms; specifically, it may include at least one of the following detection or analysis reagents: antibodies using exosomal proteins, reagents for mass spectrometry analysis, or reagents for chromatographic analysis.

[0032] In one specific embodiment, ELISA verification of LOX expression levels confirmed that the plasma exosomal protein LOX levels in patients with brain metastases were significantly lower than those in patients without brain metastases, with a decrease exceeding 30% relative to the mean in patients without brain metastases. Therefore, a diagnostic reference line can be established based on this. The reference line can be the plasma exosomal protein LOX level at any time before brain metastases, or the mean plasma exosomal protein LOX level at any time, or the mean plasma exosomal protein LOX level in a general sample at a time before brain metastases. Optional judgment criteria include: if the LOX level in the test sample is significantly lower than the reference line, then brain metastases are considered to have occurred. Specifically, when the LOX level in the test sample decreases by ≥30% compared to the reference line, brain metastases are considered to have occurred.

[0033] The following are preferred embodiments of the present invention, illustrating the discovery process of plasma exosome protein biomarkers and their effectiveness in predicting and diagnosing brain metastases. The specific materials, methods, and results involved are as follows: Example 1: Discovery of plasma exosomal protein biomarkers 1) Sample pretreatment and plasma exosome proteomics analysis Sample source and processing for plasma proteomics analysis: A total of 59 patients pathologically diagnosed with LUAD and TNM stage IV were included. Based on the presence or absence of brain metastases, patients were divided into two groups: the experimental group (brain metastases, BrM, n=30) and the control group (no brain metastases, non-BrM, n=29). The inclusion criteria for the BrM group required that brain metastases were detected for the first time by MRI and that the patient had not previously received any brain treatment. For the non-BrM group, the inclusion criteria were that no brain metastases were detected by MRI before blood sample collection. For plasma sampling, 5 mL of fasting upper limb venous blood was collected from each participant into an EDTA anticoagulant tube and centrifuged at 3000×g for 10 minutes at 4°C. The supernatant plasma layer was carefully aspirated and centrifuged again under the same conditions for 10 minutes. The supernatant was stored at -80°C for subsequent exosome extraction. 0.5 mL of the collected plasma sample was used for exosome separation using a size exclusion chromatography kit (DL210340, Duolaimi Biotech Co., Ltd.).

[0034] 200 μL of plasma-derived exosome sample was mixed with an equal volume of BeyoEx Lysis Buffer for Exosome (C3632, Beyotime). The mixture was lysed on ice for 10 minutes to extract proteins. Fragmentation was removed by centrifugation at 14000×g at 4°C for 20 minutes, and the supernatant was collected. Protein concentration was determined using the Bradford method. 10 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) was added to the obtained protein sample, and the mixture was incubated at 37°C for 1 hour to achieve protein reduction. Subsequently, alkylation was carried out by adding 50 μL of 0.2 M iodoacetamide (IAM) at room temperature in the dark for 30 minutes. Next, a dual-enzyme digestion was initiated to obtain peptides: Lys-C (EV01-02-01, EVLiXiR; enzyme-to-substrate ratio 1:100) was added sequentially and incubated at 37°C for 2 hours, followed by trypsin (EV01-01-01, EVLiXiR; enzyme-to-substrate ratio 1:50) digestion overnight at 37°C. The enzyme reaction was terminated by acidification with 50 μL of 0.1% formic acid (FA). Finally, the digested peptide solution was loaded onto a pre-activated peptide desalting column (EV01-04-01, EVLiXiR), and salts were removed with 0.1% formic acid. The bound peptides were eluted sequentially with 50% acetonitrile (ACN) containing 0.1% formic acid and 80% ACN containing 0.1% formic acid. The eluates were combined, lyophilized, and stored at -80°C. Before LC-MS / MS analysis, the peptides were reconstituted in 0.1% FA, and the OD was measured. 280 The peptide concentration was adjusted to approximately 0.5-1.0 µg / μL.

[0035] The obtained peptides were identified using an EASY nLC 1200 UHPLC system (Thermo-Fisher, USA) coupled with a Q Exactive HF-X quadrupole-Orbitrap mass spectrometer (Thermo-Fisher, USA) via a nanoelectrospray ionization source. The chromatographic conditions were as follows: C18 capillary column (75 μm × 20 cm, EV01-06-01, EVLiXiR); column temperature: 60℃; mobile phase A: 0.1% formic acid (FA); mobile phase B: 80% ACN containing 0.1% FA; flow rate: 600 nL / min; gradient program: 0–0.5 min, 8%–10% B; 0.5–52.5 min, 10%–32% B; 52.5–53 min, 32%–95% B; 53–60 min, 95% B. Acquisition methods included a single MS1 scan ranging from 350 to 1500 m / z (resolution: 60,000, maximum injection time: 50 ms, automatic gain control (AGC) target: 3 × 10^6), and segments of 42 different isolation windows ranging from 14 to 312 m / z (resolution: 30,000, maximum injection time: 54 ms, AGC target: 1 × 10^6). Step-normalized collision energies were set to 25, 27.5, and 30, respectively, while the default charge state for MS2 was specified as 3. MS data obtained from the fractionation pool facilitated the creation of a direct-DIA library, which was subsequently merged into a hybrid library using Spectronaut software (Biognosys, version 15.7.220308.50606). This hybrid spectral library was then used to analyze MS data from single-injection samples in Spectronaut software for final protein identification and quantification. All searches were performed on the Uniprot Human Proteome Database, which contains 26,766 target sequences (downloaded on December 29, 2023), with peptide identification filters set to a false positive rate (FDR) of <1%. Protein strength was normalized using the Local Normalization algorithm in the Spectronaut software.

[0036] Based on the above experimental process, a total of 2,230 proteins were identified in the discovery cohort. Gene Ontology (GO) enrichment analysis of these proteins is as follows: Figure 1 As shown, they are mainly enriched in secretory vesicle cavities, cytoplasmic vesicle cavities, and blood granules, confirming the purity of the isolated exosome samples and their plasma origin. When the identified proteins were compared with the Exocarta and Vesiclepedia databases, it was found that more than 80% of the proteins were present in both databases. Figure 2A), and more than 90% of the top 100 proteins in each database were identified in the samples. Figure 2 B). Furthermore... Figure 2 C shows the positive expression rates of the top 10 proteins from the two databases in both groups. Except for CD63 and TSG101, the positive expression rates of almost all proteins were close to 100%, further emphasizing the high purity of exosomes isolated using SEC. Of the 2,230 proteins, 2,146 (approximately 96.2%) were detected in both groups. This was achieved by applying a |log2 fold change| ≥ 1 and... P Based on a threshold of <0.05, we identified a total of 35 differentially expressed proteins between the two groups. Compared to the non-BrM group, the BrM group showed 26 upregulated proteins and 9 downregulated proteins. Figure 2 D), a detailed list is shown in Table 1, and the corresponding volcano map is shown below. Figure 2 As shown in E. Heatmap ( Figure 2 F) shows the expression patterns of the top five upregulated and downregulated proteins in the two groups after Z-score normalization. Notably, the expression levels of CD9, Alix, and FLOT1 did not show significant differences between the two groups. Figure 3 This indicates that these protein differences do not originate from the number or composition of exosomes.

[0037] Table 1: 35 significantly differentially expressed (|log2 fold change| ≥ 1 and ...) P Protein details (<0.05) KLK11 Q9UBX7 2.74121 0.00000132 IGHV1OR15-1 A0A075B7D0 1.89822 0.00000870 IGHV1-69 P01742 1.71575 0.00119682 TRANK1 O15050 1.68526 0.00052843 IGKV1D-16 P01601 1.43099 0.00037469 RPRD1A Q96P16 1.38054 0.00053942 TADA2B Q86TJ2 1.35784 0.00025959 KRT73 Q86Y46 1.26332 0.00027092 ABCB9 Q9NP78 1.22955 0.00064711 IGLV2-23 P01705 1.21954 0.02278143 IGHV2-70 P01814 1.19756 0.00429797 IGLV3-21 P80748 1.16341 0.00512628 LEFTY2 O00292 1.15290 0.01327820 IGLV9-49 A0A0B4J1Y8 1.14186 0.01762827 ACTB P60709 1.13732 0.00000003 PDGFC Q9NRA1 1.10933 0.00013228 SVIP Q8NHG7 1.10685 0.00003307 CD63 P08962 1.10273 0.00022635 IGHV5-10-1 A0A0J9YXX1 1.10066 0.03902793 IGHV1-8 P0DP01 1.07527 0.00390087 NDUFS7 O75251 1.04575 0.00070706 DEFA1 P59665 1.01256 0.00073929 DEFA1B P59665 1.01256 0.00073929 C5AR1 P21730 1.01253 0.00585618 PLSCR1 O15162 1.00882 0.00076288 IGHV2-5 P01817 1.00661 0.00032151 LTBP2 Q14767 -1.66919 0.00020864 LOX P28300 -1.49975 0.00004307 LMTK2 Q8IWU2 -1.44099 0.02950704 PPBP P02775 -1.39661 0.00101982 NPW Q8N729 -1.32544 0.00087181 CRB2 Q5IJ48 -1.15751 0.04885077 GPSM3 Q9Y4H4 -1.10579 0.00064621 LTBP1 Q14766 -1.03728 0.00249134 FPR1 P21462 -1.02864 0.00011468 2) Screening of differentially expressed proteins at the hub of plasma exosome proteomics Based on the identified differentially expressed proteins (DEEPs) associated with LUAD-BrM, we first constructed a protein-protein interaction (PPI) network using the STRING database (version 12.0) and identified 22 interacting proteins using Cytoscape software. Subsequently, we performed hub analysis on the candidate proteins using five network topology algorithms: DMNC, MCC, MNC, Degree, and EPC. Finally, we identified 16 common core proteins: LOX, NPW, PLSCR1, C5AR1, FPR1, GPSM3, DEFA1, DEFA1B, PPBP, CRB2, LTBP2, LTBP1, CD63, PDGFC, LEFTY2, and ACTB. Figure 4Subsequently, three machine learning algorithms were used for enhanced screening: LASSO regression, through L1 regularization, effectively reduced redundant variables and identified 13 key proteins with the strongest biological interpretability: ACTB, C5AR1, CD63, DEFA1, DEFA1B, FPR1, GPSM3, LEFTY2, LOX, NPW, PDGFC, PLSCR1, and PPBP. Figure 5 A). Random Forest (RF) assessed feature contributions based on the average reduction in Gini coefficient, retaining eight high-influence proteins with Gini values ​​exceeding 1.5: ACTB, PLSCR1, PDGFC, FPR1, PPBP, LOX, LTBP2, and LTBP1 ( Figure 5 B). SVM-RFE optimizes the hyperplane by maximizing the classification margin, identifying 14 proteins with the highest classification discriminative power: ACTB, C5AR1, CD63, CRB2, DEFA1, DEFA1B, FPR1, GPSM3, LEFTY2, LOX, LTBP1, LTBP2, NPW, and PDGFC. Figure 5 C). Intersection analysis of the screening results of three machine learning algorithms using Venn diagrams revealed three common candidate proteins: LOX, FPR1, and ACTB. Figure 5 (D). This finding indicates that these three proteins are consistently considered to be highly associated with brain metastasis in predictive models.

[0038] 3) Integrated analysis of proteomics in small sample sizes of paraffin-embedded tissues We included formalin-fixed, paraffin-embedded (FFPE) tissue samples from 13 patients, including surgically resected brain tissue samples from 5 BrM patients and lung biopsy samples from 8 non-BrM patients. FFPE specimens used for proteomics analysis were obtained from our institution's pathology department and cut into 8-micrometer-thick sections. To extract proteins, the sections were first dewaxed with xylene and rehydrated via a gradient of ethanol, followed by rinsing with deionized water. The rehydrated sections were incubated at 95°C for 20 min in lysis buffer (8 M urea, 1% SDS, 50 mM Tris-HCl pH 8.5, and a mixture of 1× protease inhibitors) to reverse cross-linking and promote protein dissolution. After cooling to room temperature, the samples were sonicated to break down tissue fragments, then centrifuged at 14,000×g for 20 min at 4°C to remove insoluble particles, and the supernatant was collected. Then, the same digestion and desalting process as in 1) is performed, and finally, LC-MS / MS analysis is conducted, with the final identification and quantification of proteins achieved using Spectronaut software.

[0039] FFPE tissue proteomics results showed that lysine oxidase-like 1 (LOXL1) was significantly downregulated in brain metastases (log2 fold change = -1.43). P = 0.005), which is consistent with the downregulation of LOX in plasma exosomes ( Figure 6 A). Since both LOXL1 and LOX are members of the lysine oxidase family, they catalyze the oxidation and deamination of lysine residues in collagen and elastin to enhance the mechanical stability of the extracellular matrix (ECM). These two proteins share a domain in the protein-protein interaction (PPI) network. Figure 7 Furthermore, PPI analysis combining FFPE and plasma exosome proteomics further confirmed the direct interaction between LOX and LOXL1. Figure 6 B). Furthermore, data from the Cancer Genome Atlas (TCGA) support a positive correlation between LOXL1 and LOX expression (r = 0.43, p < 0.001). Figure 6 C). In summary, the cross-dimensional consistency between LOXL1 downregulation in tissues and LOX downregulation in plasma exosomes suggests that the LOX family may promote brain metastasis and colonization of lung adenocarcinoma cells by jointly disrupting extracellular matrix (ECM) homeostasis. Compared with traditional tissue biopsy, LOX in plasma exosomes represents a more suitable non-invasive liquid biopsy target for early warning and efficacy monitoring, thus providing a new strategy for the clinical management of LUAD-BrM.

[0040] Example 2: ELISA verification of LOX expression levels The validation cohort consisted of 158 patients, including 79 patients with BrM and 79 patients with non-BrM. First, plasma samples were collected and exosomes were extracted according to the method described in Example 1. Subsequently, exosome lysates were obtained using lysis buffer, and the supernatant was collected after centrifugation at 14000 × g for 20 min. Protein concentrations in the samples were normalized to similar levels using a BCA assay kit. Finally, LOX expression levels in both groups were measured using the Human LOX ELISA Kit (HM10997, Bioswamp). In addition, LOX concentrations in plasma samples were also directly measured for comparison. Receiver operating characteristic (ROC) curves of the biomarkers were plotted, and diagnostic performance was compared using the area under the curve (AUC). All other statistical analyses and graphing were performed using GraphPad Prism software (version 10.4.2), IBM SPSS Statistics software (version 23.0), or R packages (version 4.3.1). Differences between groups were analyzed using unpaired Student's t-tests or ANOVA, with statistical significance set at 0.5%. P <0.05.

[0041] The results showed that the expression of LOX in plasma exosomes and total plasma was significantly downregulated in the BrM group. Figure 8 A, Figure 8 B). The areas under the curve (AUC) for LOX in plasma exosomes and total plasma samples were 0.786 (95% CI: 0.713–0.859) and 0.665 (95% CI: 0.579–0.750), respectively, indicating that separating exosomes from plasma first and then detecting LOX within them provides higher predictive performance for LUAD-BrM prediction compared to directly detecting LOX in total plasma (Delong test). P The AUC value increased to 0.798 (95% CI: 0.729–0.866) when both plasma exosomal LOX and total plasma LOX were included in the model. Figure 8 C). However, the addition of total plasma LOX resulted in only a slight increase in AUC (ΔAUC = 0.012), according to the Delong test. P The value was 0.561, indicating no significant difference in diagnostic performance between the two models. Therefore, directly using LOX from patient serum exosomes as a single predictive biomarker for LUAD-BrM offers greater practical convenience and cost-effectiveness.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a product for detecting LOX protein content in plasma exosomes, characterized in that, The application is to prepare a kit for early warning or diagnosis of lung adenocarcinoma brain metastasis, and the detection product is used for quantitative detection of LOX protein.

2. The application according to claim 1, characterized in that, The kit is used for at least one of the following purposes: a) Early diagnosis of brain metastases from lung adenocarcinoma; b) Screening for brain metastases from lung adenocarcinoma.

3. The application according to claim 1, characterized in that, The detection product includes antibodies against the LOX protein in exosomes.

4. The application according to claim 3, characterized in that, The detection product is an ELISA kit.

5. The application according to claim 1, characterized in that, The kit includes plasma exosome separation reagents and / or lysis reagents.

6. The application according to claim 1, characterized in that, The kit also includes a reference line, which is the content of protein LOX in plasma exosomes of the subject at any time when no brain metastasis has occurred, or the average content of protein LOX in plasma exosomes at any time.

7. A device for early warning or diagnosis of brain metastasis in lung adenocarcinoma, characterized in that, include: The detection module is used to detect the content of exosomal protein LOX in plasma samples from patients with lung adenocarcinoma; The input module is used to obtain the detection results from the detection module; The judgment module is used to compare the test results with the LOX protein content in the plasma exosomes of patients before brain metastasis occurred, and to determine whether brain metastasis has occurred based on the degree of decrease. The output module is used to output the diagnostic results.

8. The apparatus according to claim 7, characterized in that, The judgment criteria of the judgment module include: if the LOX content in the sample to be tested is significantly lower than the average content in the period before brain metastasis, then it is judged that brain metastasis has occurred.

9. The apparatus according to claim 8, characterized in that, The judgment criteria of the judgment module include: if the LOX content in the sample to be tested decreases by ≥30% compared with the average value before brain metastasis, then it is judged that brain metastasis has occurred.

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