A biomarker of fecal exosome protein for colorectal cancer diagnosis and its application

Abstract

This invention relates to a fecal exosomal protein biomarker for the diagnosis of colorectal cancer and its application, said biomarker comprising exosomal proteins CD147 and A33. This biomarker set can be used accurately, efficiently, and conveniently in the diagnosis and staging of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biomarkers, and in particular to a fecal exosome protein biomarker for colorectal cancer diagnosis and its application. Background Technology

[0002] According to the "Guidelines for Colorectal Cancer Screening and Early Diagnosis and Treatment in China" published in 2020 by the Expert Group for the Development of the Guidelines for Colorectal Cancer Screening and Early Diagnosis and Treatment in China by the National Cancer Center of my country, there are currently five main methods for colorectal cancer screening and early diagnosis: (1) colonoscopy; (2) fecal occult blood test (FIT); (3) sigmoidoscopy; (4) colon CT imaging technology; and (5) multi-target fecal FIT-DNA detection.

[0003] Colonoscopy is the core component of the entire colorectal cancer screening process, and pathological examination of endoscopic biopsies or resected specimens is the gold standard for diagnosing colorectal cancer. Colonoscopy allows direct visualization of the colorectal lumen wall, making it the most sensitive method for detecting intestinal tumors. However, colonoscopy requires rigorous bowel preparation beforehand and is highly invasive, causing significant discomfort to patients, resulting in low patient compliance. Considering my country's large population and the scarcity and uneven distribution of colonoscopy resources, colonoscopy screening can be promoted as an important means of personalized screening, but it is not suitable for large-scale population-wide screening.

[0004] Fibroin irradiation (FIT) uses the principle of hemoglobin antigen-antibody reaction to detect intestinal bleeding. As one of the early signs of colorectal cancer, it is currently the most widely used colorectal cancer screening technology. However, FIT has low sensitivity for early colorectal cancer and is prone to false positives (other intestinal bleeding diseases such as ulcerative colitis, Crohn's disease, ischemic enteritis, and hemorrhoids can also cause a positive FIT result).

[0005] Similar to colonoscopy, sigmoidoscopy primarily examines the descending colon, sigmoid colon, and rectum. However, due to its inherent limitations, sigmoidoscopy does not significantly reduce the incidence of proximal colonic tumors. Statistics show that 38% of colonic adenomas and 42% of colorectal cancers in Chinese patients are located in the proximal colon, indicating that sigmoidoscopy misses a large number of colonic lesions. Therefore, sigmoidoscopy is currently not recommended for colorectal cancer screening. Colonic CT imaging, also known as CT-simulated colonoscopy, involves obtaining three-dimensional images of the colon and rectum using high-resolution abdominal CT scans after bowel preparation, thereby diagnosing intestinal tumors. This method requires bowel preparation, is relatively complex to perform, and is expensive. It also suffers from false positives, radiation hazards, and low public acceptance. Currently, it is not recommended for general screening and is only suitable for cases where a full colonoscopy is not feasible.

[0006] Multi-target fecal FIT-DNA testing utilizes DNA technology to detect abnormal methylation and gene mutations in the DNA of cancer cells detached from intestinal tumors in feces. Combined with FIT, this improves the sensitivity and specificity for screening advanced adenocarcinoma of colorectal cancer. However, its sensitivity for early-stage colorectal cancer remains unsatisfactory, and the cost is high. Therefore, there is an urgent need to develop more effective, simple, and convenient colorectal cancer screening methods.

[0007] Exosomes carry proteins, lipids, nucleic acids, and other bioactive molecules derived from blastocysts, serving as mediators of intercellular communication and participating in various physiological and pathological processes such as cell proliferation, cell differentiation, angiogenesis, immune regulation, and tumor invasion. Exosomes secreted by different cells (or the same cells under different physiological and pathological conditions) possess specific molecular biological information. Therefore, screening for exosomal biomarkers secreted by tumor cells has become one of the most important directions in cancer research, holding significant clinical importance for early cancer diagnosis, molecular subtyping, efficacy evaluation, and prognostic assessment. Studies have shown that miRNA dysregulation is closely related to the occurrence and development of colorectal cancer, potentially involving various stages of colon cancer transformation. miRNAs isolated from blood can serve as potential biomarkers for early diagnosis and prognostic assessment of colorectal cancer. For example, CN109439749B discloses exosomal miRNA biomarkers and diagnostic kits for colorectal cancer diagnosis, wherein the miRNA biomarkers are let-7b-3p, miR-139-3p, and miR-145-3p. CN114672560A discloses a detection kit and method for identifying colorectal cancer status using exosomal miRNA biomarkers. The kit detects the levels of miRNA biomarkers in the serum or plasma of the subject. The miRNA biomarkers are selected from hsa-miR-320a-3p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3p, and any combination thereof. However, the detection specificity of colorectal cancer screening using peripheral blood miRNAs is not high, and it cannot accurately distinguish colorectal cancer from other cancers. Therefore, how to more accurately screen for colorectal cancer remains an urgent problem to be solved. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a colorectal cancer diagnostic biomarker and kit that is highly specific, accurate, and stable.

[0009] This invention is achieved through the following technical solution:

[0010] This invention provides a biomarker for the diagnosis, grading, and / or prognosis of colorectal cancer, the biomarker comprising exosomal proteins CD147 and / or A33. Preferably, it is a fecal exosomal protein biomarker for the diagnosis of colorectal cancer. The colorectal cancer is colorectal cancer, preferably colorectal cancer. The exosomal proteins CD147 and / or A33 are exosomal membrane proteins. Preferably, it is a biomarker group of exosomal proteins CD147 and A33, or a biomarker group of CEA and exosomal proteins CD147 and A33.

[0011] The exosomes mentioned are fecal exosomes and are not derived from exosomes in peripheral blood, plasma, serum, etc.

[0012] Furthermore, the biomarkers of the present invention may further include CEA (carcinoembryonic antigen).

[0013] The markers used in this invention are preferably exosome marker groups.

[0014] The present invention also provides the use of the above-mentioned biomarkers in the preparation of diagnostic reagents or kits for colorectal cancer diagnosis, grading and / or prognosis.

[0015] The present invention further provides a detection reagent or kit for detecting the above-mentioned markers.

[0016] The detection reagent or kit preferably includes an exosome extraction reagent and a detection reagent for exosome proteins CD147 and / or A33.

[0017] The present invention also provides a system for the diagnosis, grading, and / or prognosis of colorectal cancer, the system comprising a patient exosome biomarker information acquisition module and an analysis and evaluation module, wherein: the patient exosome information acquisition module is used to perform the step of acquiring detection information of the biomarker; the evaluation module is used to perform the step of diagnosing, grading, and / or prognostically determining the patient based on the detection information of the biomarker.

[0018] This invention utilizes proteomics, bioinformatics, transcriptomics, and other methods, combined with clinical practice, to obtain fecal exosome biomarkers for colorectal cancer screening.

[0019] In a specific embodiment, the exosomes of the present invention are obtained by extraction and separation using one or more of the following methods: ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, size exclusion chromatography, and immunoaffinity assay.

[0020] The present invention preferably uses high-speed centrifugation to extract exosomes from fecal samples of colorectal cancer patients, which are then fully dissolved in PBS buffer and repeatedly centrifuged at high speed for 4-8 times at 2-6℃.

[0021] This invention aims to provide fecal exosome membrane protein biomarkers for the diagnosis of colorectal cancer, thereby improving the speed, accuracy, and convenience of diagnosis. This invention describes the use of ELISA to examine the expression of CD147 and A33 in fecal exosomes from healthy donors (n=14) and colorectal cancer patients (n=48). Binary logistic regression was used to combine the two biomarkers (CD147 & A33), and p-values ​​were obtained through an unpaired t-test. In distinguishing between healthy donors and colorectal cancer patients, the p-values ​​for CD147 and A33 were 0.0006 and 0.0001, respectively, indicating that both are significantly associated with colorectal cancer. Furthermore, the AUC values ​​were 0.903 and 0.904, respectively. The p-value for the combined CD147 & A33 was less than 0.0001, and the AUC value was 0.913, demonstrating better diagnostic efficacy.

[0022] Meanwhile, in comparing the diagnostic efficacy of fecal exosomes CD147, A33, and CEA, this invention also found that the clinical cut-off value for CEA is 5 ng / mL. The cut-off values ​​for CD147, A33, and CD147&A33 were obtained by ROC analysis and set to 0.192, 0.277, and 0.625, respectively. The comparison revealed that CD147, A33, and CD147&A33 can effectively distinguish between colorectal cancer patients and healthy donors, with a clinical sensitivity of 89%; while the sensitivity of CEA is only 40%. These results indicate that CD147 and / or A33 in fecal exosomes have better diagnostic efficacy for colorectal cancer than conventional CEA markers. Studies have found that CEA, previously used as a specific marker for early diagnosis of colon and rectal cancer, has been found through extensive clinical practice to be elevated not only in gastrointestinal malignancies but also in the serum of breast cancer, lung cancer, and other malignancies.

[0023] This invention also describes the efficacy of CD147 and / or A33 in monitoring the prognosis of colorectal cancer. By examining the expression of CD147 and / or A33 in fecal exosomes of colorectal cancer patients and postoperative colorectal cancer patients, the results showed that the expression changes of CD147 and / or A33 before and after surgery were analyzed. The p-values ​​were obtained by unpaired t-test. The p-values ​​for both CD147 and A33 were less than 0.05, indicating that the expression of the two markers was significantly different before and after the patients underwent surgery. Attached Figure Description

[0024] Figure 1 Process for preparing fecal exosomes by ultracentrifugation

[0025] Figure 2 Fecal exosome colorectal cancer marker screening process

[0026] Figure 3Western blot screening for candidate biomarkers

[0027] Figure 4 ELISA detection method diagram

[0028] Figure 5 p-values ​​of CD147 and A33 for diagnosing colorectal cancer

[0029] Figure 6 Sensitivity and specificity of CD147 and A33 in the diagnosis of colorectal cancer

[0030] Figure 7 Comparison of diagnostic efficacy of fecal exosomes CD147, A33, and CEA

[0031] Figure 8 Changes in CD147 and A33 expression before and after surgery

[0032] Figure 9 Changes in the expression of CD147 and A33 in longitudinal samples Detailed Implementation

[0033] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0034] Example 1

[0035] Fecal exosome separation

[0036] Exosomes were isolated from fecal samples using ultracentrifugation. Fecal samples were dissolved by vortexing with PBS buffer; centrifuged twice at 3,000×g for 10 minutes at 4°C; the supernatants were combined and centrifuged again at 3,000×g for 30 minutes at 4°C; the supernatant was filtered through filter paper and the filtrate was collected; the filtrate was centrifuged at 10,000×g for 30 minutes at 4°C and the supernatant was collected; the supernatant was filtered through a 0.22 μm filter membrane and the filtrate was collected; the filtrate was centrifuged at 150,000×g for 2.5 hours at 4°C and the supernatant was discarded; the precipitate was resuspended in PBS buffer and centrifuged again at 150,000×g for 2.5 hours at 4°C; the precipitate was then resuspended with a small amount of PBS buffer and BCA protein quantification was performed. For detailed preparation procedures, please refer to [link to detailed preparation process]. Figure 1 .

[0037] Example 2: Biomarker Screening

[0038] To screen for biomarkers associated with colorectal cancer in fecal exosomes, bioinformatics was used to identify candidate protein biomarkers. These were further screened using the UniProt and ExoCarta databases to identify exosomal membrane proteins, initially yielding 11 candidate biomarkers, including A33, ADAM10, CD147, CDH17, CPNE3, Galectin-4, GPC-1, HSP60, HSP70, KRT19, and SRC. Exosomes were isolated from fecal samples from healthy donors (n=4) and colorectal cancer patients (n=4), and their expression was examined using Western blot.

[0039] Exosomes were treated with RIPA lysis buffer, and protein concentration was determined using the BCA method. 36 μg of protein was loaded and separated by electrophoresis using a 4-20% high-resolution precast gel (HEPES-Tris). The protein was then transferred to a nitrocellulose (NC) membrane, blocked with 5% skim milk for 1 hour, followed by incubation overnight at 4°C with primary antibody (diluted with 5% skim milk, dilution ratio according to antibody instructions). The membrane was washed three times with TBST, incubated for 2 hours with horseradish peroxidase (HRP)-labeled secondary antibody (diluted 1:1000 with 5% skim milk), washed three times with TBST, and then photographed using a gel imaging system after adding ECL chromogenic buffer. The grayscale values ​​of the bands were calculated using ImageJ software. See details... Figure 2 The screening process.

[0040] from Figure 3 The results of Western blot screening for candidate biomarkers showed that, except for CD147 and A33 which were only detected in the colorectal cancer group, the other nine proteins were detected in both the healthy group and the colorectal cancer group. Therefore, CD147 and A33 are initially considered as potential exosomal membrane protein biomarkers for colorectal cancer.

[0041] Example 3: Marker Validation

[0042] Enzyme-linked immunosorbent assay (ELISA) method

[0043] Enzyme-linked immunosorbent assay (ELISA) was used, with mouse anti-human CD63 antibody as the capture antibody and rabbit anti-human CD147 antibody and rabbit anti-human A33 antibody as the detection antibodies, to further verify the performance of CD147 and A33 as markers for colorectal cancer. Dilute the capture antibody to a concentration of 2 μg / mL with coating buffer (carbonate buffer, pH 9.5). Add 100 μL of capture antibody to each well of a microplate and incubate overnight at 4°C. Discard the solution and wash three times with PBST (0.2% Tween-20 in PBS, v / v). Add 100 μL of blocking buffer (1% casein in PBS, w / v) to each well and incubate at 37°C for 1 hour. Discard the solution and add 100 μL of exosome sample (diluted with blocking buffer, 40 μg) to each well and incubate at 37°C for 3 hours. Discard the solution and wash three times with PBST. Add 100 μL of detection antibody (diluted with blocking buffer; rabbit anti-human CD147 antibody final concentration 1 μg / mL, rabbit anti-human A33 antibody final concentration 2 μg / mL) to each well and incubate at 37°C for 1 hour. Discard the solution and wash three times with PBST. Add 100 μL of detection antibody to each well. HRP-labeled goat anti-rabbit antibody (diluted with blocking buffer to a final concentration of 2 μg / mL) was incubated at 37°C for 40 minutes. The solution was discarded, and the cells were washed five times with PBST solution. 100 μL of TMB luminescent reagent was added to each well, and the cells were incubated in the dark for 5 minutes. Then, 100 μL of stop solution was added, and the absorbance (A) at 450 nm was measured using a microplate reader. 450 .

[0044] Validation of CD147 and A33 for the diagnosis of colorectal cancer

[0045] The expression of CD147 and A33 in fecal exosomes from healthy donors (n=14) and colorectal cancer patients (n=48) was examined using ELISA. Binary logistic regression was used to combine the two biomarkers (CD147 & A33), and p-values ​​were obtained using an unpaired t-test. Results are as follows: Figure 5 , 6 As shown, when distinguishing between healthy donors and colorectal cancer patients, the p-values ​​for CD147 and A33 were 0.0006 and 0.0001, respectively, indicating that both were significantly associated with colorectal cancer. Table 1 shows that the AUC values ​​were 0.903 and 0.904, respectively; the p-value for the combined CD147&A33 was less than 0.0001, with an AUC of 0.913, demonstrating good diagnostic efficacy. Meanwhile, the specificities of CD147 and A33 were 0.875 and 0.875, respectively; the specificity of the combined CD147&A33 was significantly improved to 0.937.

[0046] Table 1. Validation results of CD147 and A33 for the diagnosis of colorectal cancer.

[0047]

[0048] Example 4: Comparison of diagnostic efficacy of fecal exosomes CD147, A33 and CEA

[0049] In 35 patients with colorectal cancer, the diagnostic efficacy of CEA (a commonly used marker of colorectal cancer), CD147 and A33 in fecal exosomes, and CD147 & A33 were further compared and contrasted. The results are as follows: Figure 7 As shown, the clinical cut-off value for CEA is 5 ng / mL. The cut-off values ​​for CD147, A33, and CD147&A33 were obtained by ROC analysis and set to 0.192, 0.277, and 0.625, respectively. The comparison revealed that CD147, A33, and CD147&A33 could effectively distinguish between colorectal cancer patients and healthy donors, with a clinical sensitivity of 89%; while the sensitivity of CEA was only 40%. These results indicate that CD147 and A33 in fecal exosomes have good diagnostic efficacy for colorectal cancer.

[0050] Example 5: CD147 and A33 for monitoring colorectal cancer prognosis

[0051] The expression of CD147 and A33 in fecal exosomes of colorectal cancer patients (n=48) and postoperative colorectal cancer patients (n=29) was investigated using ELISA. The results are as follows: Figure 8 The changes in the expression of CD147 and A33 before and after surgery were shown. The p-values ​​were obtained by unpaired t-test. The p-values ​​for both CD147 and A33 were less than 0.05, indicating that the expression of the two markers was significantly different before and after the patient underwent surgery.

[0052] Furthermore, longitudinal stool samples were collected from seven colorectal cancer patients before and after surgery to examine the changes in the expression of CD147 and A33 in fecal exosomes. The results are as follows: Figure 9 As shown in the figure. Paired t-tests yielded p-values; the p-values ​​for CD147 and A33 were both less than 0.05, indicating significant differences in expression before and after individual surgery; while the p-value for CEA was greater than 0.05. These results suggest that CD147 and A33 in fecal exosomes have the potential to monitor the prognosis of colorectal cancer.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of a biomarker combination in the preparation of diagnostic reagents or kits for colorectal cancer diagnosis, grading, and / or prognosis, characterized in that, The biomarker combination is exosomal proteins CD147 and A33, the exosomal is fecal exosomal, and the colorectal cancer is colorectal cancer.

2. The detection reagent or kit according to claim 1, characterized in that, It includes exosome extraction reagents and detection reagents for exosome proteins CD147 and A33.

3. A system for the diagnosis, grading, and / or prognosis of colorectal cancer, characterized in that, The system includes a patient exosome biomarker information acquisition module and an analysis and evaluation module, wherein: the patient exosome biomarker information acquisition module is used to perform the step of acquiring detection information of the biomarker combination of claim 1; the evaluation module is used to perform the step of diagnosing, classifying and / or prognosticating the patient based on the detection information of the biomarker combination.

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