A set of extracellular vesicle-derived gastrointestinal malignancy markers and applications thereof

Abstract

This invention relates to a group of extracellular vesicle-derived biomarkers for gastrointestinal malignancies and their applications, belonging to the field of molecular biology. The biomarkers of this invention are derived from extracellular vesicles and include proteins CALR, SPP1, OLFM4, and OIT3. When used for the diagnosis of gastrointestinal cancers, these biomarkers have shown good diagnostic efficacy, superior to existing serum biomarkers. Furthermore, the biomarkers of this invention show significant elevations in the early stages of gastrointestinal cancer, which is beneficial for early screening and diagnosis of gastrointestinal cancers.

Description

Technical Field

[0001] This invention relates to a group of gastrointestinal malignant tumor markers derived from extracellular vesicles and their applications, belonging to the field of molecular biology technology. Background Technology

[0002] Gastrointestinal malignancies, or gastrointestinal cancers (GICs), are common malignant tumors of the digestive system, primarily including gastric cancer (GC) and colorectal cancer (CRC). Statistics show that in 2022, new cases and deaths from GICs accounted for 17.9% and 28.3% of all malignant tumor cases, respectively. With advancements in medical technology, clinical treatment outcomes for early-stage GICs have become quite good. The 5-year survival rates for early-stage GC and CRC can reach 69.5% and 90.2%, respectively. However, once distant metastasis occurs, the 5-year survival rate drops sharply to below 10%. Early diagnosis and treatment are crucial for improving the survival rate of patients with GICs and reducing the medical burden.

[0003] Currently, the main diagnostic method for gastrointestinal malignancies is gastrointestinal endoscopy combined with tissue biopsy. This method is invasive, has low comfort levels, is time-consuming, and has low patient compliance. Furthermore, the heterogeneity of tumors means that a single tissue biopsy is insufficient to accurately reflect the overall condition of the patient. Serum tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) have advantages such as simple and minimally invasive testing methods, making them the most widely used diagnostic methods for gastrointestinal inflammatory disease (GIC). However, due to their low sensitivity and specificity, their clinical diagnostic value for GIC remains relatively limited, and in recent years, expert guidelines have increasingly recommended them as indicators for monitoring GIC recurrence.

[0004] Therefore, seeking new non-invasive, highly sensitive, and specific diagnostic biomarkers to improve the diagnostic level of gastrointestinal malignancies, especially the early diagnosis rate of gastrointestinal malignancies, is of great significance for improving patient prognosis. Summary of the Invention

[0005] [Technical Issues]

[0006] The technical problem to be solved by the present invention is to provide a set of non-invasive, highly sensitive and specific diagnostic biomarkers and their application in early screening, diagnosis, staging, monitoring, efficacy evaluation or prognostic evaluation of gastrointestinal cancer.

[0007] [Technical Solution]

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] This invention first provides the application of a biomarker in the preparation of products for early screening, diagnosis, staging, monitoring, efficacy evaluation, or prognostic evaluation of gastrointestinal cancer, characterized in that the biomarker is derived from extracellular vesicles, and the biomarker includes at least one of the proteins CALR, SPP1, OLFM4, and OIT3.

[0010] In one embodiment, the markers include any two, three, or a combination of four of the proteins CALR, SPP1, OLFM4, and OIT3.

[0011] In one embodiment, the gastrointestinal cancer includes, but is not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, gastrointestinal lymphoma, pancreatic cancer, small intestinal tumor, or neuroendocrine tumor. Gastric cancer or colorectal cancer may be selected.

[0012] In one embodiment, the extracellular vesicles include at least one of small extracellular vesicles, exosomes, and extracellular vesicle heteroplasts.

[0013] In one embodiment, the product includes a biochip, a reagent kit, or a device.

[0014] The present invention also provides the application of reagents for detecting the content of biomarkers in samples in the preparation of products for early screening, diagnosis, staging, monitoring, efficacy evaluation or prognosis evaluation of gastrointestinal cancer, wherein the biomarkers are derived from extracellular vesicles and include at least one of the proteins CALR, SPP1, OLFM4, and OIT3.

[0015] In one embodiment, the markers include any two, three, or a combination of four of the proteins CALR, SPP1, OLFM4, and OIT3.

[0016] In one embodiment, the sample comprises an ex vivo serum sample.

[0017] In one embodiment, the reagent contains reagents for detecting the expression level of extracellular vesicle proteins.

[0018] In one embodiment, the gastrointestinal cancer includes, but is not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, gastrointestinal lymphoma, pancreatic cancer, small intestinal tumor, or neuroendocrine tumor. Gastric cancer or colorectal cancer may be selected.

[0019] In one embodiment, the extracellular vesicles include at least one of small extracellular vesicles, exosomes, and extracellular vesicle heteroplasts.

[0020] In one embodiment, the product includes a biochip, a reagent kit, or a device.

[0021] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The biomarker proteins CALR, SPP1, OLFM4 and OIT3 of the present invention are derived from extracellular vesicles of blood cells in vitro. Because the proteins are wrapped in a lipid bilayer, they are more stable than free proteins and do not require the separation of gastrointestinal tissue samples from the subjects for further testing. The proteins of the present invention have better compliance when used for gastrointestinal cancer detection and are simpler and more efficient.

[0024] (2) The biomarker proteins CALR, SPP1, OLFM4, and OIT3 of the present invention, when used for the detection of gastrointestinal cancer, exhibit diagnostic efficiencies of 0.8813, 0.9156, 0.7813, and 0.8438, respectively, which are significantly higher than the existing serum biomarkers CEA and CA19-9 for gastrointestinal cancer (0.6125 and 0.6438, respectively). Furthermore, the biomarkers provided by the present invention show a significant increase in early-stage gastrointestinal cancer compared to healthy individuals, while the existing serum biomarkers CEA and CA19-9 only show an increase in late-stage gastrointestinal cancer. Therefore, the biomarkers of the present invention are significantly superior to CEA and CA19-9 in the early screening and diagnosis of gastrointestinal cancer. Attached Figure Description

[0025] Figure 1 The flowchart of this invention is as follows: Left: Screening and confirmation process of biomarker proteins; Right: Diagnostic verification process of biomarker proteins.

[0026] Figure 2 Identification of serum-derived small extracellular vesicles (sEVs). A: The particle size distribution and particle concentration of sEVs were measured using a nanoparticle tracking analysis system; B: The morphology of serum sEVs was examined using transmission electron microscopy, revealing the cup-shaped morphology of sEVs; C: Western blot analysis of serum-derived sEVs showed the presence of the sEV marker (CD9) without cell contamination (calnexin), with cell lysates used as a control.

[0027] Figure 3This is a 4D-LFQ proteomic analysis of 6 groups of sEVs. A: SDS-PAGE of serum sEV proteins in the 6 groups; where M is the protein marker, HC, CRC, and GC are serum sEV proteins from healthy individuals, colorectal cancer patients, and gastric cancer patients, respectively, and HeLa cell lysates are used as controls. B: The number of sEV proteins identified and quantified by 4D-LFQ.

[0028] Figure 4 This is the peptide ion peak area distribution of the sEV protein CALR as confirmed by 4D-PRM. The study included 8 healthy individuals and 20 patients with gastric cancer (GIC): C1-C10 were colorectal cancer patients, G1-G10 were gastric cancer patients, and H1-H8 were healthy controls.

[0029] Figure 5 This is the peptide ion peak area distribution of the sEV protein SPP1 as confirmed by 4D-PRM. The study included 8 healthy individuals and 20 patients with gastric cancer (GIC): C1-C10 were colorectal cancer patients, G1-G10 were gastric cancer patients, and H1-H8 were healthy controls.

[0030] Figure 6 This is the peptide ion peak area distribution of the sEV protein OLFM4 as confirmed by 4D-PRM. The study included 8 healthy individuals and 20 patients with gastric cancer (GIC): C1-C10 were colorectal cancer patients, G1-G10 were gastric cancer patients, and H1-H8 were healthy controls.

[0031] Figure 7 This is the peptide ion peak area distribution of the sEV protein OIT3 as confirmed by 4D-PRM. The study included 8 healthy individuals and 20 patients with gastric cancer (GIC): C1-C10 were colorectal cancer patients, G1-G10 were gastric cancer patients, and H1-H8 were healthy controls.

[0032] Figure 8 The expression levels of four proteins (8A: CALR, 8B: SPP1, 8C: OLFM4 and 8D: OIT3) in serum sEVs of healthy individuals and patients with GIC were detected using 4D-PRM.

[0033] Figure 9 The expression levels of four proteins (9A: CALR, 9B: SPP1, 9C: OLFM4 and 6D: OIT3) in serum sEVs of healthy individuals, patients with early-stage GIC (GIC I / II), and patients with late-stage GIC (GIC III / IV) were measured using 4D-PRM. Serum CEA (9E) and CA19-9 (9F) levels were used as controls.

[0034] Figure 10To analyze the diagnostic ability of sEV proteins (10A: CALR, 10B: SPP1, 10C: OLFM4 and 10D: OIT3) for GIC using ROC curve analysis, the ROC analysis results of serum CEA (10E) and CA19-9 (10F) were used as controls. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Example 1: 4D-LFQ proteomics screening for GIC-related sEV proteins

[0038] This embodiment screened GIC-related sEV proteins; the specific process is detailed in the appendix. Figure 1 .

[0039] (1) Clinical serum specimen collection: This study was approved by the Ethics Committee of the Affiliated Hospital of Jiangnan University, and informed consent was obtained from patients and their families. All patients were pathologically confirmed to have colorectal cancer or gastric cancer and met the following inclusion criteria: ① no chemotherapy or radiotherapy; ② no malignant diseases; ③ no diabetes or immune diseases. Healthy controls (HC) had normal blood biochemical indicators and tumor markers, and other related diseases were excluded. Before treatment, whole blood samples were collected from GIC patients (including GC and CRC patients) and healthy controls using vacuum coagulation tubes. After standing at room temperature for 20 minutes, the serum was extracted by centrifugation at 2000 rpm for 10 minutes and stored in [location missing]. It was stored in an 80°C refrigerator for subsequent serum sEV separation.

[0040] (2) Isolation of sEVs from clinical serum: During the 4D-LFQ proteomics analysis, sEVs were isolated from serum samples using ultracentrifugation. The serum samples were mixed into 6 groups as follows: HC1 (15 HC cases), HC2 (15 HC cases), CRC1 (15 CRC cases), CRC2 (15 CRC cases), GC1 (15 GC cases), and GC2 (15 GC cases). The specific sEV isolation experimental steps are as follows:

[0041] ① Sample preparation before centrifugation: Take 15 mL of human serum and dilute it 5 times with 1×PBS; 500× g Centrifuge at 4°C for 5 minutes to remove cells; ② Transfer the supernatant to a new centrifuge tube and centrifuge at 2000× g Centrifuge at 4℃ for 10 min to remove cell debris; ③ Transfer the supernatant to a new centrifuge tube and centrifuge at 10000× g ④ Centrifuge at 4℃ for 30 min to remove large cell vesicles; ⑤ Filter the supernatant using a 0.22 μm filter to remove any large particles that may have been introduced during the process; ⑥ Transfer the filtered supernatant to an ultracentrifuge tube, add 1×PBS buffer to make up the remaining volume, weigh precisely to balance, and place on the rotor of an ultracentrifuge (model: P70AT). Centrifuge at 130,000 × 10⁻⁶. g Centrifuge at 4°C for 80 min; ⑥ After centrifugation, discard the supernatant. A translucent sediment will be visible on the bottom side of the tube. Resuspend the sediment in 1×PBS buffer and centrifuge at 130000× g Centrifuge at 4°C for 80 min. Repeat this step once to wash the sEV; ⑦ Resuspend the sEV precipitate in 150 μL of 1×PBS buffer, then transfer it to a 1.5 mL EP tube. Depending on the requirements of subsequent experiments, it can be directly used for downstream experiments or stored at -80°C.

[0042] (3) Identification of serum sEVs:

[0043] 1) Transmission Electron Microscopy (TEM): The morphology of sEVs was observed using negative staining. Specifically, 10 μL of sEVs was added to a copper grid using a pipette, allowed to stand for 1 min, and then excess exosome suspension was carefully aspirated with filter paper. The grid was stained with 2% uranyl acetate for 1 min, and then excess liquid was aspirated with filter paper again. The copper grid with the sEVs attached was then transferred to a lamp to dry, and the morphology of the sEVs was observed under a transmission electron microscope.

[0044] 2) Nanoparticle Tracking Analysis (NTA): The sEV suspension was pipetted to homogenize it and diluted with 0.22 μm filtered 1×PBS buffer to the instrument's optimal detection concentration (20-100 particles per field of view). 1 mL was injected into the instrument. Following the manufacturer's recommendations, the sample was irradiated with a laser (blue 488) and the movement of nanoparticles due to Brownian motion was recorded at an average frame rate of 20 frames per second for 60 seconds. Finally, the data was output and analyzed using NTA 3.3 software to obtain the particle size distribution and particle concentration of the sEVs.

[0045] 3) sEV marker protein detection: Add an equal volume of RIPA protein lysis buffer containing protease inhibitors to the sEV suspension, lyse on ice for 30 min, shaking for 20 s every 10 min. Finally, 13000× g Centrifuge at 4°C for 15 min, and then transfer the protein-containing supernatant to a new EP tube. After protein quantification using the BCA method, add an equal volume of 2×SDS-PAGE loading buffer, mix thoroughly, and then denature. Western blot is used to detect the sEV positive marker CD9 and the negative marker calnexin.

[0046] (4) 4D-LFQ proteomics analysis (4D - Label-Free Quantitation):

[0047] Protein lysis buffer (a mixture of 8 M urea and 1% protease inhibitor) was added to the sEV and shaken for 1 h. After complete lysis, protein concentration was determined using a BCA kit. 5 mM dithiothreitol was added to the protein solution (5 μg), and the mixture was incubated at 56°C for 30 min to reduce disulfide bonds. Then, 11 mM iodoacetamide was added, and the mixture was incubated at room temperature in the dark for 15 min to alkylate the protein and maintain its reduced state. Finally, the urea concentration in the sample was diluted with triethylammonium bicarbonate to below 2 M. Trypsin was added at a 1:50 (w / w) ratio to protein, and the mixture was incubated overnight at 37°C. Finally, trypsin was added at a 1:100 (w / w) ratio to protein, and the mixture was incubated for another 4 h.

[0048] The peptide fragments obtained from enzymatic hydrolysis were dissolved in liquid chromatography-mobile phase A and separated using a NanoElute UPLC system. Mobile phase A was an aqueous solution consisting of 0.1% formic acid and 2% acetonitrile. Mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were: 0–68 min, 6%–23% B; 68.0–82.0 min, 23%–32% B; 82.0–86.0 min, 32%–80% B; 86.0–90.0 min, 80% B, maintaining a flow rate of 500 nL / min. The separated peptides were injected into a capillary ion source for ionization and then analyzed by timsTOF Pro mass spectrometry. The electrospray voltage was 1.6 kV. Precursor ions and secondary fragments were analyzed on a TOF detector. The primary mass spectrometry scan range was set to 100–1700 m / z, and the scan resolution was set to 60,000. Data acquisition mode was set to Parallel Accumulation Serial Segmentation (PASEF). After acquiring a single mass spectrometer, a secondary spectrum was obtained in PASEF mode, specifically for precursor ions with charges between 0 and 5. This process was repeated ten times. The dynamic exclusion time for tandem mass spectrometry scans was set to 30 seconds to avoid duplicate scans of precursor ions. The raw data was then processed using ProteomeDiscoverer (version 2.4.1.15). The enzyme digestion method was set to trypsin (Full). The number of missing sites was set to 2. The minimum peptide fragment length was set to 6 amino acid residues. The maximum number of peptide modifications was set to 3; the mass error tolerance for precursor ions was 10 ppm, and for fragment ions, it was 0.02 Da. The FDR for protein, peptide, and PSM identification was 1%. The differential expression threshold was set to a 1.5-fold change.

[0049] Experimental results:

[0050] The results of serum sEV identification are as follows: Figure 2 As shown, the peak particle size of serum sEVs is 122 nm. Figure 2 A), consistent with the classic size range of sEVs. TEM observation of serum sEVs showed a typical cup-shaped morphology, with a diameter of approximately 100 nm (A). Figure 2 B). Western blot analysis showed that the CD9 band, a marker of sEV, was detected in serum sEVs, and the sEV-negative marker calnexin was hardly observed in sEV samples. Figure 2 C). The above characterization confirmed the successful isolation of sEVs from clinical serum samples and their suitability for subsequent 4D-LFQ proteomics analysis.

[0051] First, the quality of sEV protein was assessed by SDS-PAGE electrophoresis. 1 μg of sEV protein was loaded, and 1 μg of HeLa protein was loaded as a control. Results showed clear protein bands, normal band distribution, no protein degradation, and good parallelism within each lane. Figure 3 A). Next, the protein expression profiles of serum sEV samples (HC1, HC2, CRC1, CRC2, GC1, GC2) were analyzed using 4D-LFQ proteomics technology. A total of 3046 proteins were identified in the serum sEV samples, of which 2728 were quantifiable, with each group containing approximately 2250 quantifiable proteins. Figure 3 B). This invention uses a 1.5-fold change threshold to select four sEV proteins (CALR, SPP1, OLFM4, and OIT3) that are upregulated in the serum of CRC and GC patients compared with healthy controls as potential biomarkers for GIC in subsequent experiments (Table 1).

[0052] Table 1 Potential biomarkers of GIC

[0053]

[0054] Example 2: Detection and evaluation of GIC-related sEV proteins

[0055] This embodiment verifies the diagnostic efficacy of the sEV protein screened in Example 1 for GIC. The specific procedure is detailed in the appendix. Figure 1 .

[0056] (1) Collection of clinical serum specimens:

[0057] Example 2: Serum samples were collected again from GIC patients and healthy individuals. The inclusion criteria and serum collection methods for patients and healthy individuals were the same as in Example 1. The specific groups were as follows: 8 healthy individuals and 20 GIC patients (including 5 cases of stage I / II CRC, 5 cases of stage I / II GC, 5 cases of stage III / IV CRC and 5 cases of stage III / IV GC).

[0058] (2) In order to be suitable for clinical use during the 4D-PRM validation phase, the more convenient column membrane adsorption method (exoEasy kit) was used to separate sEVs from a single serum sample.

[0059] The specific operating steps are as follows: ① Take 2 mL of human serum, 500× g Centrifuge at 4℃ for 5 min, and collect the supernatant into a new centrifuge tube; ② 2000× g Centrifuge at 4℃ for 10 min. After centrifugation, collect the supernatant into another clean centrifuge tube; ③ 16000× gCentrifuge at 4°C for 10 min to remove cell debris and large cell vesicles; ④ Transfer the supernatant to a new centrifuge tube, then add an equal volume of XBP buffer, invert 5 times to mix thoroughly; ⑤ Add the supernatant and XBP mixture to an exoEasy filter column equipped with a collection tube, centrifuge at 500× g Centrifuge at 4℃ for 1 min to allow the SEVs to bind to the membrane in the filter column; ⑥ Discard the waste liquid in the collection tube, return the exoEasy filter column to the collection tube, and centrifuge again at 5000× g Centrifuge at 4℃ for 1 min to remove residual liquid from the column membrane; ⑦ Discard the small amount of waste liquid in the collection tube, put the exoEasy filter column back into the collection tube, and add 3.5 mL of XWP buffer, 5000× g Centrifuge at 4℃ for 5 min to wash away sEVs; ⑧ Then transfer the exoEasy filter column to a new collection tube, add 200 μL of XE buffer, cover the column membrane, incubate at room temperature for 1 min, and centrifuge at 5000× g Centrifuge at 4℃ for 5 min, and collect the eluent containing sEVs; ⑨ Add the eluent back to the centrifuge column, incubate at room temperature for 1 min, and centrifuge at 5000× g Centrifuge at 4°C for 5 min to fully elute residual sEVs on the column membrane; collect the sEV eluent and dispense it into new EP tubes.

[0060] (3) 4D-PRM targeted proteomics technology (4D - Parallel Reaction Monitoring):

[0061] Based on the results of the 4D-LFQ proteomics analysis in Example 1, the 4D-PRM targeted proteomics technology was selected to detect the levels of CALR, SPP1, OLFM4, and OIT3 in serum sEVs:

[0062] Trypsin peptides were dissolved in solvent A and separated using a NanoElute UHPLC system. Solvent A consisted of an aqueous solution containing 0.1% formic acid and 2% acetonitrile. Solvent B consisted of 0.1% formic acid in 98% acetonitrile, with the concentration gradually increased: from 6% to 24% over 40 minutes, from 24% to 35% over 54 to 57 minutes, and finally from 35% to 80% over 57 to 60 minutes. The flow rate was maintained at 450 nL / min. The separated peptides were injected into a capillary ion source for ionization and then analyzed using a TIMS-TOF Pro mass spectrometer. The ionization voltage was 1.7 kV, and the parent peptide ion and its secondary fragments were detected and analyzed using a TOF mass spectrometer. The secondary mass spectrometry scan range was set to 100–1700. Secondary spectra of the parent ion charge in the range of 0–5 were acquired using the Parallel Reaction Monitoring-Parametric Accumulation-Tandem Fragmentation (PRM-PASEF) mode. Data was processed using Skyline 21.2 with the following peptide parameters: protease set to trypsin / P, maximum deletion site set to 0, and peptide length ranging from 7 to 25 amino acid residues. The mass error tolerance in ion matching was set to 0.02 Da.

[0063] (4) Detection of serum CEA and CA19-9 levels: The levels of traditional serum tumor markers CEA and CA19-9 were detected using chemiluminescent immunoassay (CLIA) on a Beckman Coulter DXI-800 fully automated chemiluminescence analyzer.

[0064] (5) Statistical analysis: Statistical analysis was performed using GraphPad Prism 9.0 and SPSS 23.0 software. Differences between groups were compared using the Mann-Whitney nonparametric test or one-way ANOVA. A p-value <0.05 was considered statistically significant. The diagnostic efficacy of sEV-derived CALR, SPP1, OLFM4, and OIT3 as GIC markers was assessed using the area under the curve (AUC) method.

[0065] Experimental results:

[0066] 4D-PRM was used to quantify peptides from four proteins that met quality control standards. The results are shown in [Figure number missing]. Figures 4-7 Compared with healthy controls, patients with GIC showed significantly elevated levels of four sEV proteins (CALR, SPP1, OLFM4, and OIT3). Figure 8Furthermore, after dividing the samples into three groups, it was found that the traditional GIC serum markers CEA and CA19-9 were elevated only in a few patients with advanced GIC, while the four sEV proteins (CALR, SPP1, OLFM4, and OIT3) showed an increasing trend in early GIC. Figure 9 ).

[0067] The receiver operating characteristic (ROC) curve analysis results of 4D-PRM data and traditional serum protein biomarker data are as follows: Figure 10 As shown, the areas under the curve (AUC) values ​​for CALR, SPP1, OIT3, and OLFM4 were 0.8813, 0.9156, 0.7813, and 0.8438, respectively. The AUC values ​​for CEA and CA19-9 were 0.6125 and 0.6438, respectively. Further ROC curve analysis of the four sEV protein combinations revealed that the AUC values ​​were all greater than 0.9 (Table 2). This demonstrates that sEV proteins CALR, SPP1, OIT3, and OLFM4, alone or in any combination, exhibit superior diagnostic capabilities. In conclusion, the four serum sEV proteins (CALR, SPP1, OLFM4, and OIT3) screened in this invention have clinical application value as non-invasive biomarkers for GIC.

[0068] Table 2. ROC curve analysis of sEV protein combinations

[0069]

[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The use of a serum small extracellular vesicle marker composition in the preparation of products for the diagnosis or monitoring of gastrointestinal cancer, characterized in that, The biomarker composition is a combination of at least one of the proteins CALR, SPP1, and OLFM4 with protein OIT3, and the gastrointestinal cancer is gastric cancer or colorectal cancer.

2. The application according to claim 1, characterized in that, The products include biochips, reagent kits, or devices.

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