Biomarker for detecting intestinal cancer or evaluating lung metastasis risk of intestinal cancer and application of biomarker

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a biomarker for detecting intestinal cancer or evaluating lung metastasis risk of intestinal cancer and application of the biomarker. Based on the biomarker serum SEMA3C, the AUC of intestinal cancer pulmonary metastasis evaluation reaches 0.867, the sensitivity is 90.2%, the specificity is 70.7%, and compared with a traditional tumor marker, the biomarker is better, and can effectively perform early diagnosis on intestinal cancer pulmonary metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to biomarkers for detecting colorectal cancer or assessing the risk of colorectal cancer lung metastasis and their applications. Background Technology

[0002] Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, with metastatic progression being the core reason for over 50% of deaths and a critical clinical challenge in current cancer treatment. The liver is the most common target organ for CRC metastasis, but approximately 15% of CRC patients develop lung metastases, two-thirds of which originate in the rectum. Compared to liver metastases, CRC patients with lung metastases generally have a better prognosis due to a higher proportion of isolated metastases; early detection and surgical intervention are key to prolonging their survival. However, while research on the mechanisms of CRC liver metastasis is extensive, the investment in systematic research on lung metastases is significantly disproportionate to its clinical importance. The lack of in-depth analysis of the specific molecular characteristics of lung metastases leads to significant shortcomings in early clinical diagnostic methods.

[0003] Currently, the clinical diagnosis of CRC lung metastases mainly relies on low-dose computed tomography (LDCT) and serum tumor marker detection. While LDCT is the primary method, its specificity is poor, failing to effectively distinguish early metastatic lesions from benign nodules, potentially leading to unnecessary surgical interventions and psychological burden for benign patients. Commonly used serum markers such as CEA and CA199 have diagnostic sensitivity and specificity both below 50%, making it difficult to differentiate metastatic nodules from granulomatous lesions. Although machine learning models combining imaging features and serum markers have improved detection rates to some extent, their generalization ability is severely limited due to cohort bias and lack of standardized procedures. Existing technologies lack novel biomarkers with organ specificity and ease of detection, failing to meet the clinical needs for sensitivity and specificity in CRC lung metastasis diagnosis.

[0004] Histone methylation imbalance is a crucial mechanism in the development and progression of CRC, leading to the silencing of tumor suppressor genes or the activation of oncogenes. Related methyltransferases have become a hot topic in drug target research. The semaphore (SEMA) family, as the initially discovered axonal guide molecules, has recently been shown to participate in the regulation of malignant biological characteristics of tumors, playing a key role, especially in maintaining the stemness of cancer stem cells. However, current research has not sufficiently explored the specific functions of SEMA family members in CRC lung metastases, and no clinically applicable serum-specific biomarkers for CRC lung metastases have been discovered. This research gap directly results in a lack of convenient and accurate diagnostic methods to differentiate CRC lung metastasis patients from primary CRC patients and patients with benign lung diseases, urgently requiring targeted research to fill this gap. Summary of the Invention

[0005] Based on this, the present invention found that SEMA3C is a specific biomarker for patients with colorectal cancer lung metastases. By detecting the level of SEMA3C in serum, patients with colorectal cancer lung metastases can be distinguished from patients with primary colorectal cancer and other patients with benign lung diseases.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In a first aspect, the present invention provides a biomarker for detecting colorectal cancer or assessing the risk of colorectal cancer lung metastasis, the biomarker being a combination of serum SEMA3C and tissue SMYD3.

[0007] Secondly, the present invention provides the application of a reagent for detecting the above-mentioned biomarkers in the preparation of products for detecting colorectal cancer.

[0008] Preferably, in the above application, the intestinal cancer is colon cancer.

[0009] Preferably, in the above applications, the reagents for detecting the biomarkers include reagents for IHC detection and / or reagents for ELISA detection.

[0010] Preferably, in the above applications, the product for detecting colorectal cancer or assessing the risk of colorectal cancer lung metastasis is a detection reagent or detection kit.

[0011] Thirdly, the present invention provides the application of a reagent for detecting the above-mentioned biomarkers in the preparation of products for assessing the risk of colorectal cancer lung metastasis.

[0012] Preferably, in the above application, the intestinal cancer is colon cancer.

[0013] Preferably, in the above applications, the reagents for detecting the biomarkers include reagents for IHC detection and / or reagents for ELISA detection.

[0014] Preferably, in the above applications, the product for detecting colorectal cancer or assessing the risk of colorectal cancer lung metastasis is a detection reagent or detection kit.

[0015] The beneficial effects of this invention include: (1) Based on the biomarker serum SEMA3C provided by the present invention, the AUC for assessing lung metastasis of colorectal cancer is 0.867, the sensitivity is 90.2% and the specificity is 70.7%, which is better than traditional tumor markers and can effectively diagnose lung metastasis of colorectal cancer at an early stage.

[0016] (2) Compared with some RNA-based diagnostic models, the combination of biomarkers of serum and tissue protein levels in this invention has higher stability and is more stable after three freeze-thaw cycles.

[0017] (3) Biomarker tests based on plasma proteins have higher accessibility, share the same detection procedures as routine biochemical tests, are simple to operate, and have promotional value for predicting postoperative recurrence of locally advanced colorectal cancer.

[0018] (4) ELISA-based detection methods are low-cost, easy to standardize, and suitable for large-scale screening. Attached Figure Description

[0019] Figure 1a The distribution of the β-catenin signaling pathway in the integrated, publicly available, and self-tested dimensionality-reduced intestinal epithelial single-cell feature maps; Figure 1b To identify gene modules upregulated in metastatic epithelium using single-cell module scoring; Figure 1c Bubble graph for transcription factor enrichment analysis of normal, primary, and metastatic intestinal epithelial cells; Figure 1d Pie chart showing the correlation analysis between differentially expressed transcriptional regulators and signaling pathways; Figure 2a A schematic diagram of the experimental procedure for verifying in vivo that the SMYD3 gene promotes lung metastasis of colorectal cancer and the survival curves of mice; Figure 2b Changes in mouse body weight after modeling MC38 cell lines overexpressing Smyd3; Figure 2c HE staining results of lungs in mice overexpressing Smyd3 and control group lung transfer mice; Figure 2d A schematic diagram of the experimental procedure and mouse survival curves for in vivo validation of the use of chlorphosphate depletion (CLOD) to promote lung metastasis in alveolar macrophages; Figure 3a Dimensionally reduced clustering diagrams of primary colorectal cancer, lung metastases, and liver metastases in a publicly available dataset; Figure 3b Box plots showing the expression levels of SMYD3 in different tissue samples; Figure 3c A scatter plot showing the correlation between SMYD3 and SEMA3C expression; Figure 3d The relative expression levels of SMYD3 and SEMA3C at different capture sites in spatially recorded genome sequencing of colorectal cancer lung metastases; Figure 4a These are representative SMYD3 immunohistochemical images of recurrent colorectal cancer, early metastatic primary colorectal cancer, and colorectal cancer lung metastases in our center's cohort. Figure 4b Box plots of SMYD3 immunohistochemical scores for primary, metastatic, early metastatic, and non-metastatic colorectal cancer lesions; Figure 4cSurvival curves for colorectal cancer patients grouped according to the SMYD3 immunohistochemical score of the primary lesion; Figure 4d ROC curves for predicting lung metastasis in colorectal cancer patients using serum SEMA3C in our center's colorectal cancer cohort (n=82). Detailed Implementation

[0020] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0022] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0023] Example 1 This invention discloses the screening and validation of serum SEMA3C and / or tissue SMYD3 expression levels as biomarkers for colorectal cancer lung metastasis.

[0024] (a) Screening of biomarkers (1) Data integration and initial screening The "harmony" algorithm was used to integrate single-cell sequencing datasets of primary colorectal cancer tissues from public databases (GEO, EBI) (GSE200997, GSE166555, EMTAB8107) with single-cell sequencing data from 19 resectable colorectal cancer lung metastases and adjacent normal tissues from our center. The "FindAllMarkers" function combined with the "SCENIC" algorithm was used to screen for differentially expressed transcriptional regulatory factors, and the "AddModuleScore" function combined with the "cNMF" algorithm was used to analyze changes in biological pathways. A list of secreted proteins was obtained from the Human Protein Atlas database (www.proteinatlas.org), and potential biomarkers were screened using Pearson correlation.

[0025] The results are as follows Figures 1a to 1d As shown, the results indicate that the Wnt-β-catenin pathway is specifically upregulated in metastatic colorectal epithelium (see [link to study]). Figure 1a and 1b Furthermore, correlation analysis showed that among numerous transcriptional regulators, SMYD3 was most positively correlated with RAD21 and the significantly upregulated MYC and β-catenin pathways (see [link to relevant documentation]). Figure 1c Furthermore, enrichment analysis using the SCENIC algorithm showed that SMYD3 was the most significantly upregulated factor in metastatic lesions. Figure 1d ).

[0026] (2) Mouse experiment The most significantly different molecule (SMYD3) was selected as a candidate biomarker. Stable knockout / overexpression cell lines were constructed by transfecting cells with knockout and overexpression lentiviral vectors (MOI=10). Ten molecule-10 cells were injected via tail vein into 8-week-old BALB / c and C57BL / 6 mice (n=10). 6 A mouse model of lung metastasis was constructed using mouse colon cancer cells (MC38 and CT26). The weight of the mice was recorded every 10 days. After 40 days, the mice were sacrificed by cervical dislocation, and the lung metastases were dissected for subsequent experiments. After HE staining, the slides were scanned with a slide scanner, and the number and size of the lung metastases were counted using QuPath software. Finally, the selected biomarkers were verified as driving factors of colon cancer lung metastasis.

[0027] The results are as follows Figures 2a to 2d As shown, the results indicated that, using a mouse lung metastasis model constructed via tail vein, overexpression of Smyd3 significantly increased tumor-related deaths in mouse colon cancer cell lines MC38 and CT26 (see [link to study]). Figure 2a The mice's body weight decreased significantly 3 weeks after injection (see...). Figure 2b The lung metastases have significantly increased in size (see...). Figure 2c This leads to poor prognosis in mice, and treatment with the macrophage scavenger CLOD also shows the same phenomenon (see...). Figure 2d This provides insights for subsequent drug target development.

[0028] (3) bulk transcriptome sequencing data This integrates bulk transcriptome sequencing data from 524 primary colorectal cancer, normal colorectal tissue, colorectal cancer metastases, and corresponding adjacent normal tissues from publicly available datasets in the GEO database (see [link to GEO database]). Figure 3a SMYD3 was also significantly upregulated in lung metastases (see...). Figure 3b ), and is positively correlated with the secreted protein SEMA3C (see Figure 3cThis suggests that SEMA3C has potential translational value and may lead to the development of an easily detectable biomarker based on peripheral blood. Furthermore, spatial transcriptome sequencing was used to detect the tumor margin region of lung metastases, revealing a significant increase in SMYD3 and SEMA3C expression in the tumor region compared to the alveolar region (see...). Figure 3d ).

[0029] The above data preliminarily confirm that SMYD3 and SEMA3C are independent predictors of lung metastasis in colorectal cancer. In primary colorectal cancer tissue, high expression of SMYD3 is associated with poor prognosis in patients.

[0030] (ii) Validation of biomarkers In the following validation, the SMYD3 detection method was as follows: Surgically removed tumor samples were fixed in formalin, embedded in paraffin, and stored at 4°C. Paraffin sections were then dewaxed with environmentally friendly dewaxing solution and washed with distilled water. Antigen was then repaired with retrieval solution, followed by washing with PBS and blocking endogenous peroxidase with 3% hydrogen peroxide, then washing with PBS. The sections were then blocked with 3% BSA for 30 minutes and incubated with SMYD3 antibody overnight at 4°C. After washing with PBS, the sections were incubated with HRP-labeled secondary antibody at room temperature for 50 minutes. After washing with PBS, DAB staining was performed, cell nuclei were counterstained with hematoxylin, rinsed with tap water, dehydrated, and mounted.

[0031] The following verification method for detecting SEMA3C levels in serum using ELISA is as follows: (1) Collect 3-4 mL of peripheral blood from the patient, centrifuge to separate the serum, and store at -80°C; (2) Coat a 96-well plate with SEMA3C antibody and prepare a gradient working solution of standard (add 1 mL of diluent to 1 ng of standard, and then serially dilute with diluent to obtain standard solutions of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.625 pg / mL); (3) Add 100 μL of sample or standard of different concentrations to each well, and fill blank wells. Add 100uL of diluent; (4) Cover with sealing film and incubate at 37°C for 60 minutes, then discard the liquid, add 100uL of biotinylated antibody working solution to each well, cover with sealing film and incubate at 37°C for 60 minutes; (5) Wash the plate (discard the liquid, add 300uL of washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, repeat washing the plate 3 times), add 100uL of enzyme conjugate working solution / well, cover with sealing film and incubate at 37°C for 30 minutes, then wash the plate again; (6) Finally add 90uL of TMB substrate, cover with sealing film, incubate at 37°C in the dark for 15 minutes, then add 50uL of stop solution, and immediately measure the OD value of each well at a wavelength of 450nm.

[0032] The model performance was evaluated using metrics such as ROC-AUC, sensitivity, and specificity, and its performance in assessing lung metastasis risk was validated in our center's cohorts (retrospective tissue cohort n=26, retrospective plasma cohort n=82). Details are as follows: To ensure the reliability and representativeness of the validation results, the study carefully constructed two validation cohorts with different sample sources and clinical significance.

[0033] The inclusion criteria for organizing the queue are: (1) Patients diagnosed with primary colorectal cancer by pathology; (2) Pathological diagnosis confirmed lung metastasis; (3) Possess complete clinical pathology data and high-quality tumor tissue samples.

[0034] The exclusion criteria are: (1) Incomplete clinical data or follow-up information; (2) The quality of the tissue sample is substandard (e.g., severe degradation, excessive necrosis); (3) History of other malignant tumors.

[0035] Survival analysis using a Cox proportional hazards model and the Kaplan-Meier method was performed in this cohort to assess the relationship between SMYD3 and metastasis-free survival. Results showed that colorectal cancer patients with high SMYD3 expression in the primary tumor had significantly shorter metastasis-free survival, and SMYD3 expression in lung metastases was significantly higher than in the primary tumor (see...). Figure 4a and 4b SMYD3 expression was also higher in the primary lesions of patients at high risk of metastasis (lung metastasis within 2 years), and colorectal cancer patients with high SMYD3 expression had a worse prognosis (see [link to article]). Figure 4c This indicates that its tissue level can serve as a biomarker for predicting patient prognosis.

[0036] The inclusion criteria for the plasma cohort are: (1) Patients with primary colorectal cancer diagnosed by pathology, with or without lung metastasis, with lung metastasis diagnosis being an imaging diagnosis, and more than 80% of patients were also diagnosed by pathology.

[0037] (2) Possess complete clinical pathology data and high-quality tumor tissue samples.

[0038] The exclusion criteria are: (1) Incomplete clinical data or follow-up information; (2) Plasma sample quality is substandard (e.g., severe hemolysis, significantly abnormal blood biochemical indicators). This study aims to verify the feasibility of using liquid biopsy for non-invasive risk stratification, which is of great significance for future clinical applications.

[0039] The concentration of SEMA3C in the sample was calculated using a standard curve, and it was found that the serum SEMA3C level in metastatic patients was also synchronously upregulated.

[0040] Logistic regression was performed using the pROC package in R software, and ROC analysis was conducted. The area under the curve (AUC), optimal cutoff value, and sensitivity and specificity at this point were calculated. Results showed that the AUC of SEMA3C for predicting colorectal cancer lung metastases reached 0.867, significantly superior to the AUCs of traditional tumor markers (CEA, CA199) (0.532, 0.457, respectively) (see [link to documentation]). Figure 4d The combination of these two methods can more effectively identify colorectal cancer patients at high risk of lung metastasis, enabling early intervention and improving patient prognosis.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Biomarkers for detecting colorectal cancer or assessing the risk of colorectal cancer lung metastasis, the biomarkers being a combination of serum SEMA3C and tissue SMYD3.

2. The use of the reagent for detecting the biomarker described in claim 1 in the preparation of products for detecting colorectal cancer.

3. The use of the reagent for detecting the biomarker of claim 1 in the preparation of products for assessing the risk of colorectal cancer lung metastasis.

4. The application according to claim 2 or 3, characterized in that, Colorectal cancer is colon cancer.

5. The application according to claim 2 or 3, characterized in that, The reagents for detecting the biomarkers of claim 1 include reagents for IHC detection and / or reagents for ELISA detection.

6. The application according to claim 2 or 3, characterized in that, Products used to detect colorectal cancer or assess the risk of colorectal cancer metastasis to the lungs are diagnostic reagents or test kits.

7. The application according to claim 4, characterized in that, Products used to detect colorectal cancer or assess the risk of colorectal cancer metastasis to the lungs are diagnostic reagents or test kits.

8. The application according to claim 5, characterized in that, Products used to detect colorectal cancer or assess the risk of colorectal cancer metastasis to the lungs are diagnostic reagents or test kits.

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