Biomarker for evaluating prognosis of cholangiocarcinoma and use thereof

Abstract

A biomarker for evaluating prognosis of cholangiocarcinoma and a use thereof. The biomarker is a cysteine protease inhibitor SN (CST1). The prognosis of a cholangiocarcinoma patient is evaluated by measuring the concentration of CST1 in bile of the cholangiocarcinoma patient, and the result shows that the higher the concentration of CST1 in the bile is, the lower the overall survival rate of the patient is. CST1 in bile can be used as a biomarker for evaluating the prognosis of cholangiocarcinoma, and has important significance for judgment of the prognosis of a cholangiocarcinoma patient and selection of individualized treatment.

Description

A biomarker for evaluating the prognosis of cholangiocarcinoma and its application Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a biomarker for evaluating the prognosis of bile duct cancer and its application. Background Art

[0002] Cholangiocarcinoma (CCA) is a highly malignant epithelial cell tumor that can arise anywhere within the bile duct or liver. Based on primary anatomical subtype, it can be divided into intrahepatic CCA (iCCA), perihilar CCA (pCCA), and distal CCA (dCCA). Furthermore, CCA can arise from epithelial cells lining the bile duct, develop from hepatocytes, or develop from peribiliary glands, contributing significantly to its significant heterogeneity. Over the past few decades, the incidence and mortality of cholangiocarcinoma have been increasing worldwide. Furthermore, due to its insidious onset and difficulty in early diagnosis, cholangiocarcinoma is often diagnosed at an advanced stage, with extensive invasion and metastasis. Despite continuous advancements in diagnostic and treatment approaches for cholangiocarcinoma, patient prognosis has not improved significantly over the past decade, with a 5-year survival rate of only 7%-20%, and an extremely poor overall treatment outcome. Therefore, the identification of relevant biomarkers is urgently needed, which would be of great clinical significance for improving the overall efficacy of cholangiocarcinoma treatment.

[0003] Histopathologically, CCA is characterized by a cellular and stromal composition. Evidence for the origin of this matrix can be found in the regenerative microenvironment during bile duct repair. Therefore, the importance of the tumor microenvironment in cholangiocarcinoma cannot be underestimated. The CCA stroma is composed of tumor-associated endothelial cells, tumor-associated fibroblasts (CAFs), and a complex array of inflammatory cells, along with an extensive network of extracellular matrix (ECM) proteins. The TME (transmembrane membrane) (TME) undergoes continuous remodeling through the deposition of newly synthesized matricellular proteins (including fibronectin C and osteopontin) and the robust degradation of matrix metalloproteinases (MMPs) released by CAFs, tumor-associated macrophages, and CCA cells. This remodeling influences cellular interactions within the TME, enabling intercommunication between different cellular components and the exchange of paracrine factors. Furthermore, these remodeling changes enhance CCA cell invasion and metastasis. These studies demonstrate that components of the TME can interact directly or indirectly with the CCA epithelium, with CAFs and their secretory proteins playing a crucial role.

[0004] CAFs are a heterogeneous population of spindle-shaped cells of mesenchymal origin that comprise a significant portion of the TME cell pool and dynamically interact with tumor cells, particularly those in aggressive tumors. CAFs primarily support tumor progression and phenotypic transitions through signaling via paracrine soluble factors. This intercellular communication promotes cellular plasticity and metastasis, maintenance of tumor cell stemness, reprogramming, and extracellular matrix remodeling. Numerous studies have demonstrated that CAF abundance in cholangiocarcinoma is significantly positively correlated with CCA tumor growth and poor survival. CAFs can be derived from fibrocytes of various origins through transdifferentiation and activation. This activation process leads to metabolic reprogramming, altering CCA proliferation, motility, and secretion of extracellular matrix components. The signaling molecules secreted by CAFs have been shown to promote CCA progression by enhancing proliferation, survival, chemotaxis, and angiogenesis. Furthermore, CAFs have been shown to promote CCA progression through short-range or direct cell-to-cell morphogenetic signaling, such as through the NOTCH3 and Hedgehog pathways. In cholangiocarcinoma, CAFs can also regulate innate immunity by secreting immunomodulatory factors that support M2 macrophages and reduce NK cell activation. They can also secrete proteins that promote the activation of regulatory T cells and helper T cells and inactivate dendritic cells and cytotoxic T cells. In vitro, researchers have also found that CAFs can secrete VEGF-A and VEGF-C, which recruit and assemble lymphatic endothelial cells in vascular structures susceptible to CCA cell intravasation. More importantly, studies in a CCA rat model have demonstrated that selectively targeting and promoting apoptosis of CAFs in the tumor microenvironment can significantly improve host survival. These studies demonstrate that elucidating key pathways in the CAF-secreted protein-cholangiocarcinoma interaction network can provide new insights and approaches for prognostic assessment and therapeutic intervention in cholangiocarcinoma. Studies have shown that CST1 (Cystatin SN) can be secreted by CAFs into the tumor microenvironment to exert its biological effects. CST1 is a secreted peptide encoded by the CST1 gene. Its cystatin domain, consisting of five amino acid residues, binds to the active site of cysteine ​​proteases, thereby inhibiting their hydrolytic activity. It is widely distributed in human body fluids and tissues and plays a key regulatory role in the development and progression of various human tumors, as well as drug resistance. Currently, there are no reports of using CST1 as a biomarker for assessing the prognosis of cholangiocarcinoma.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a biomarker for evaluating the prognosis of bile duct cancer and its application to solve the problems existing in the above-mentioned prior art. The present invention has found that CST1 in bile can be used as a biomarker for evaluating the prognosis of bile duct cancer. The present invention provides a new marker for evaluating the prognosis of bile duct cancer, which is of great significance for the judgment of the prognosis of bile duct cancer patients and the selection of individualized treatment.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a biomarker for evaluating the prognosis of cholangiocarcinoma, wherein the biomarker is a cysteine ​​protease inhibitor SN.

[0009] Furthermore, an increased concentration of the biomarker in the bile of a cholangiocarcinoma patient indicates a poor prognosis for the cholangiocarcinoma patient.

[0010] The present invention also provides use of a reagent for detecting the concentration of cysteine ​​protease inhibitor SN in bile in the preparation of a product for evaluating the prognosis of bile duct cancer.

[0011] Furthermore, the product includes a reagent or a kit.

[0012] The present invention also provides a product for evaluating the prognosis of bile duct cancer, comprising a reagent for detecting the concentration of cysteine ​​protease inhibitor SN in bile.

[0013] Furthermore, the product includes a reagent or a kit.

[0014] Furthermore, the product detects the concentration of the cysteine ​​protease inhibitor SN in bile by enzyme-linked immunosorbent assay.

[0015] The present invention discloses the following technical effects:

[0016] The present invention assesses the prognosis of bile duct cancer patients by measuring the concentration of CST1 in their bile. The results show that higher bile CST1 concentrations are associated with lower overall survival rates. Therefore, the present invention has discovered that bile CST1 can be used as a biomarker for assessing the prognosis of bile duct cancer. This invention provides a new biomarker for assessing the prognosis of bile duct cancer, which is of great significance for determining the prognosis of bile duct cancer patients and selecting individualized treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 shows the results of single-cell sequencing dimensionality reduction and cluster analysis of tumor tissue (CCA) and adjacent adjacent tissue (Non-CCA) samples from patients with cholangiocarcinoma. The red color represents CST1, and the box represents the CAF cell population.

[0019] Figure 2 shows the difference in CST1 expression in bile from patients with cholangiocarcinoma (CCA) and non-cancerous patients (Non-CCA);

[0020] Figure 3 shows the survival analysis results of patients in the CST1 high expression group (High-CST1) and the CST1 low expression group (Low-CST1);

[0021] Figure 4 shows the receiver operating characteristic curve for CST1 in predicting the prognosis of patients with cholangiocarcinoma. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1

[0028] (1) By comparing and analyzing single-cell transcriptome sequencing data of tumor tissue samples and non-cancerous tissue samples from patients with cholangiocarcinoma, it was found that the level of CST1 in cholangiocarcinoma was relatively high. By tracing its cell source, it was found that it mainly originated from CAF cells. The results are shown in Figure 1.

[0029] (2) Bile samples from 30 patients with cholangiocarcinoma and 30 patients with cholelithiasis were collected clinically. The CST1 concentration was detected using the Human Cystatin SN ELISA Kit (SIGMA, RAB1036) and the differences were analyzed. The results are shown in Figure 2. The results showed that the CST1 concentration in the bile of patients with cholangiocarcinoma was significantly higher than that in the bile of patients without cholangiocarcinoma.

[0030] The specific steps for detecting CST1 concentration are as follows:

[0031] Remove the required plates from the sealed bag that has been equilibrated to room temperature, add standard and specimen diluent to the wells, seal the reaction wells with sealing tape, and incubate at 36°C for 90 minutes; prepare the biotinylated antibody working solution 20 minutes in advance, wash the plate 5 times, add biotinylated antibody diluent to the blank wells, and add biotinylated antibody working solution (100 μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 36°C for 60 minutes; prepare the enzyme conjugate working solution 20 minutes in advance, place it at room temperature in the dark, wash the plate 5 times, add the enzyme conjugate diluent to the blank wells, add the enzyme conjugate working solution (100 μL / well) to the remaining wells, seal the reaction wells with new sealing tape, incubate at 36°C in the dark for 30 minutes, and wash the plate 5 times; add 100 μL / well of chromogenic substrate (TMB), incubate at 36°C in the dark for 15 minutes; add 100 μL / well of stop solution, mix well and immediately detect the OD value with a microplate reader, and calculate the average net OD of each standard (average net OD = average assay OD - average NSB OD).

[0032] (3) The CST1 concentration in the bile of patients with cholangiocarcinoma was divided into a high-CST1 expression group (High-CST1) and a low-CST1 expression group (Low-CST1) based on the median value. That is, when the CST1 concentration was ≥5 ng / mL, it was defined as the high-CST1 expression group; when the CST1 concentration was <5 ng / mL, it was defined as the low-CST1 expression group. The relationship between the CST1 level in bile and the overall survival (OS) of patients was analyzed (all patients underwent surgical treatment). The results are shown in Figure 3. The results showed that the higher the CST1 level in bile, the shorter the patient's overall survival; the overall survival of patients in the low-CST1 expression group was significantly longer than that of patients in the high-CST1 expression group.

[0033] (4) The predictive value of CST1 for the prognosis of patients with cholangiocarcinoma:

[0034] The predictive value of CST1 for the prognosis of patients with cholangiocarcinoma is demonstrated by the prognostic receiver operating characteristic curve (AUC), as shown in Figure 4 , where AUC = 0.903. These results demonstrate that CST1 has a high predictive value for the prognosis of cholangiocarcinoma.

[0035] Specifically, the method for evaluating the prognosis of bile duct cancer using the above method is:

[0036] S1: Extract bile from patients with cholangiocarcinoma and perform sample pre-experimental treatment (dilute with phosphate buffer at a certain ratio);

[0037] S2: Detect the concentration of CST1 in bile;

[0038] S3: Evaluate the prognosis of patients with cholangiocarcinoma based on the concentration of CST1 in bile.

[0039] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the concentration of a biomarker for evaluating the prognosis of cholangiocarcinoma in bile in the preparation of a product for evaluating the prognosis of cholangiocarcinoma, characterized in that: The biomarker is cysteine ​​protease inhibitor SN.

2. The use according to claim 1, characterized in that: The product is a test kit.

Images