Application of GPER1 and CD68 as prognostic markers in preparation of a kit for predicting prognosis of liver cancer

Abstract

The application discloses application of GPER1 and CD68 as a prognostic marker in preparation of a hepatocarcinoma prognosis test kit. Researches of the application show that by detecting the expression of GPER1 in macrophages of hepatocarcinoma tissues, the survival prognosis of hepatocarcinoma patients can be more accurately predicted. Specifically, a double immunofluorescence staining combined with tyramide signal amplification technology is used to detect the co-expression proportion of GPER1 and CD68 in hepatocarcinoma tissues, and it is shown that the prediction effect of the hepatocarcinoma patients on overall survival and recurrence-free survival is better than the overall GPER1 expression of tumor tissues. Compared with the prior art (only immunohistochemical staining of GPER1 is performed on tumor tissues), the method can obtain more detailed and accurate information of the GPER1 protein expression mode, and the prediction effect on the survival of hepatocarcinoma patients is better; the method is simple and easy to operate, and ordinary technicians can complete the whole operation process.

Description

Technical Field

[0001] This invention relates to the field of disease prognostic diagnostic technology, specifically to the application of GPER1 and CD68 as prognostic markers in the preparation of a liver cancer prognostic prediction kit. Background Technology

[0002] Liver cancer ranks among the top in both new cases and mortality rates among all tumor types. Currently, the most effective treatments for liver cancer are surgical resection and liver transplantation, but these are only suitable for early-stage liver cancer patients with relatively good liver function. Furthermore, due to the high recurrence rate after surgical resection, the 5-year survival rate for liver cancer patients remains low. There are significant gender differences in the occurrence and progression of liver cancer: women have a lower incidence of liver cancer, and female patients have better overall survival and a lower long-term recurrence rate. These gender differences in liver cancer are related to the effects of estrogen and its receptors. Estrogen can inhibit the occurrence, growth, and metastasis of liver cancer by acting on estrogen receptors. Therefore, assessing the expression of estrogen receptors in liver cancer tissue can predict patient survival and recurrence, helping to identify and screen patients with poor surgical outcomes and provide further personalized treatment. Human G protein-coupled estrogen receptor 1 (GPER1) is a relatively recently identified estrogen receptor, and recent studies have shown that GPER1 is closely related to tumor progression. At the same time, GPER1 expression varies in different tumors, and the signaling pathways it mediates are also different. Although existing studies have used immunohistochemical staining techniques to assess the overall GPER1 expression in liver cancer tissue and analyze its relationship with patient survival, the predictive effect on overall survival and recurrence-free survival of liver cancer patients is not very good. Summary of the Invention

[0003] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of GPER1 and CD68 as prognostic markers in the preparation of a liver cancer prognostic prediction kit.

[0004] The above-mentioned objective of this invention is achieved through the following technical solution:

[0005] Because GPER1 can be expressed in various types of tissues and cells and its role may differ in different cells, assessing GPER1 expression on specific cells is beneficial for guiding doctors to provide more personalized treatment for patients by targeting that cell. This invention demonstrates that by detecting GPER1 expression in macrophages of liver cancer tumor tissue, the survival prognosis of liver cancer patients can be predicted more accurately. Specifically, this invention uses dual immunofluorescence staining combined with tyramine signal amplification technology to detect the expression of two molecular markers, GPER1 and CD68, in surgically removed, paraffin-embedded, and sectioned tumor samples from liver cancer patients. Then, professional image analysis software is used to analyze the GPER1 expression ratio of macrophages in liver cancer nest tissue, i.e., the proportion of GPER1 and CD68 double-positive signals to CD68 single-positive signals. The median value of this ratio in the 48 liver cancer samples analyzed is used as the threshold for the GPER1 expression level of macrophages, and the samples are grouped into "high" and "low" groups based on this threshold. Finally, based on the above grouping, a survival analysis was performed on these 48 patients who underwent primary liver cancer resection, comparing the overall survival and recurrence-free survival between the two groups with high or low GPER1 expression in tissue macrophages (P<0.05 was considered statistically significant). Immunohistochemical staining for GPER1 was also performed on liver cancer tissue, and the overall GPER1 expression in the tumor tissue was assessed (H-score). The results showed that the co-expression ratio of GPER1 and CD68 in liver cancer tissue was a better predictor of overall survival and recurrence-free survival than overall GPER1 expression in the tumor tissue. In other words, detecting GPER1 expression in macrophages in situ within liver cancer tissue has a better predictive effect on patient survival compared to existing methods that assess overall GPER1 expression in liver cancer tissue. This invention employs dual immunofluorescence staining combined with tyramine signal amplification technology to detect GPER1 expression in macrophages within liver cancer tissue. By sensitively and accurately detecting GPER1 expression on macrophages, it not only obtains more refined and accurate information on GPER1 protein expression patterns but also demonstrates superior predictive power for the survival of liver cancer patients. The method is simple and easy to implement, and the entire operation can be completed by ordinary technicians.

[0006] Therefore, the present invention first provides the application of GPER1 and CD68 as prognostic markers in the preparation of a liver cancer prognostic prediction kit, wherein the kit predicts the survival of liver cancer patients by detecting the expression ratio of GPER1 in macrophages of liver cancer tissue.

[0007] This invention also provides the application of reagents for detecting GPER1 expression levels and CD68 expression levels in the preparation of a hepatocellular carcinoma (HCC) prognostic prediction kit. The clinical prognosis of HCC patients is predicted by the proportion of CD68 and GPER1 co-expressing cells to CD68-only expressing cells. The median proportion of CD68 and GPER1 co-expressing cells to CD68-only expressing cells in the tumor tissues of different patients who underwent primary HCC resection was used as the threshold for the GPER1 expression proportion in macrophages. The proportion of CD68 and GPER1 co-expressing cells to CD68-only expressing cells in the tumor tissues of the test patients was then detected. If this proportion is higher than the threshold, a better prognostic survival is predicted; if it is lower than the threshold, a poorer prognostic survival is predicted.

[0008] Furthermore, the reagent used to detect GPER1 expression levels is an antibody against GPER1.

[0009] Furthermore, the reagent used to detect CD68 expression levels is an antibody against CD68.

[0010] Furthermore, the reagent also includes horseradish peroxidase-labeled secondary antibodies that can bind to either an antibody against CD68 or an antibody against GPER1. The expression of GPER1 in macrophages within liver cancer tissue can be detected by dual immunofluorescence.

[0011] Furthermore, the reagent also includes a tyramine signal amplification reagent. The expression of GPER1 in macrophages within liver cancer tissue can be detected by combining dual immunofluorescence staining with tyramine signal amplification technology.

[0012] Preferably, the tyramine signal amplification reagent is PPD520 and PPD570.

[0013] Furthermore, it also includes reagents required for immunofluorescence detection.

[0014] The present invention also provides a kit having two first antibodies for immunoassay multilabeling detection, the protein molecules to be detected being CD68 (cell differentiation antigen 68) and GPER1 (G protein-coupled estrogen receptor 1); the kit also has a second antibody labeled with horseradish peroxidase; PPD520 and PPD570 (tyramine signal amplification reagent).

[0015] This invention also provides a method for analyzing results based on the above-mentioned detection kit. By counting the number of CD68-positive cells and the number of CD68 and GPER1 double-positive cells in liver cancer tumor tissue, the method aims to assess the GPER1 expression ratio of macrophages in the tissue.

[0016] The detection principle of the kit is as follows:

[0017] First, the expression of two molecular markers, GPER1 and CD68, in surgically removed, paraffin-embedded, and sectioned tumor samples from liver cancer patients was detected using tyramine signal amplification technology based on immunohistofluorescence.

[0018] Then, professional image analysis software was used to analyze the GPER1 expression ratio of macrophages in liver cancer nest tissue, that is, the proportion of GPER1 and CD68 double positive signals to CD68 single positive signals. The median value of the proportion of the 48 liver cancer samples analyzed was used as the threshold for the GPER1 expression level of macrophages, and the samples were grouped into "high" and "low" groups based on this threshold.

[0019] Finally, based on the above groupings, a survival analysis was performed on patients who underwent resection of primary liver cancer.

[0020] This invention predicts and assesses the survival of liver cancer patients by evaluating the proportion of GPER1 expression on macrophages in tumor tissue, rather than simply assessing the overall GPER1 expression in the tissue. GPER1 can be expressed in various cells within tumor tissue (including parenchymal cells and various types of stromal cells), and its functional effects may differ. Existing techniques only assess the overall GPER1 expression in tissue through immunohistochemical staining, which loses information about the GPER1 expression levels of specific cell populations, masks the differences in GPER1 expression patterns in tumor tissues from different patients, and weakens or even negates the ability of GPER1 expression to assess and predict the survival of liver cancer patients due to differences in GPER1 function within different cells. Assessing the co-expression ratio of GPER1 and CD68 can more effectively predict patient survival.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Compared with existing technologies (which only perform immunohistochemical staining of GPER1 on tumor tissue), this invention, by detecting the expression level of GPER1 in macrophages of liver cancer tissue, can not only obtain more refined and accurate information on the GPER1 protein expression pattern, but also has a better predictive effect on the survival prognosis of liver cancer patients. By assessing the GPER1 expression of macrophages in the tumor tissue of liver cancer patients, researchers can more accurately predict the survival and recurrence of patients, which is beneficial for identifying and screening patients with poor surgical efficacy and providing them with further personalized treatment.

[0023] (2) The expression of GPER1 in macrophages in liver cancer tissue can be objectively, accurately and efficiently detected by using dual immunofluorescence staining combined with tyramine signal amplification technology. The method is simple and easy to implement, and ordinary technicians can complete the entire operation process. Attached Figure Description

[0024] Figure 1 To detect GPER1 expression in macrophages of hepatocellular carcinoma tissue using dual immunofluorescence staining. Red fluorescence indicates CD68 molecules, and green fluorescence indicates GPER1 molecules. The combined image shows the co-localization of CD68 and GPER1.

[0025] Figure 2 To analyze the relationship between the expression ratio of GPER1 in macrophages in tumor tissues of liver cancer patients and overall survival and recurrence-free survival using survival curves.

[0026] Figure 3 The results of GPER1 immunohistochemical staining in liver cancer tissue.

[0027] Figure 4 Comparison of the predictive power of two indicators, namely the proportion of macrophages expressing GPER1 and the GPER1H-Score, on overall survival or recurrence-free survival in patients with liver cancer. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1

[0031] A combination antibody assay kit for assessing GPER1 expression in macrophages from tumor tissue samples of patients with primary liver cancer who have undergone hepatocellular carcinoma resection includes two primary antibodies (detecting the protein molecules CD68 and GPER1, respectively), a horseradish peroxidase-labeled secondary antibody, and PPD520 and PPD570 (tyramine signal amplification reagent). The assay was performed on 48 patients who underwent primary liver cancer resection. The specific usage steps include the following three parts:

[0032] (I) Specimen Testing

[0033] 1. Select paraffin sections of liver cancer tissue with a thickness of 4 μm containing the tumor area, ensuring that there is no large area of ​​necrosis;

[0034] 2. Bake the slices at 60℃ for 2 hours;

[0035] 3. Dewax the tissue sections twice with xylene at room temperature, 10 minutes each time;

[0036] 4. Wash away the xylene on the tissue in 100% ethanol, then pass it through 100% ethanol, 95% ethanol, 80% ethanol, and 70% ethanol in sequence, for 3 minutes each time;

[0037] 5. Rinse in double-distilled water for 3 minutes;

[0038] 6. Immerse the tissue sections in 0.3% (v / v) H2O2 and react at room temperature for 15 minutes. This step is to block endogenous peroxidase activity.

[0039] 7. Rinse four times in double-distilled water, five minutes each time;

[0040] 8. Antigen retrieval: Tris / EDTA retrieval solution (pH 9.0), autoclaved heat retrieval for 10 minutes;

[0041] 9. Allow to cool naturally to room temperature for 30 minutes;

[0042] 10. Wash 4 times with TBS buffer containing 0.1% (v / v) Tween-20, 3 minutes each time;

[0043] 11. Block with PBS solution containing 5% (w / v) BSA and 0.1% (v / v) Triton X100 at room temperature for 30 min;

[0044] 12. Add the first antibody GPER1 (purchased from Invitrogen, catalog number PA5-109319) diluted 1:100 (volume ratio) to the tissue section and incubate overnight at 4°C;

[0045] 13. Wash four times with TBS buffer containing 0.1% Tween-20, five minutes each time;

[0046] 14. Add horseradish peroxidase-labeled secondary antibody (purchased from Dako, catalog number K5007) and incubate at 37 degrees Celsius for 30 minutes;

[0047] 15. Wash four times with TBS buffer containing 0.1% Tween-20, five minutes each time;

[0048] 16. Add tyramine signal amplification reagent PPD520 (purchased from PANOVUE, catalog number 0144100050) diluted 1:400 (volume ratio) and incubate at room temperature in the dark for 13 minutes;

[0049] 17. Wash four times with TBS buffer containing 0.1% Tween-20, five minutes each time;

[0050] 18. Place the slide in antigen retrieval solution (10mM citrate buffer (pH 6.0)) and microwave on high for 5 minutes + on low for 15 minutes;

[0051] 19. Allow to cool naturally to room temperature for 30 minutes;

[0052] 20. Wash 4 times with TBS buffer containing 0.1% Tween-20, 3 minutes each time;

[0053] 21. Block with PBS solution containing 5% BSA and 0.1% Triton-100 at room temperature for 30 minutes;

[0054] 22. Add the second primary antibody CD68 (purchased from Zhongshan Jinqiao, catalog number ZM-0060) diluted 1:200 (volume ratio) to the tissue section and incubate overnight at 4°C;

[0055] 23. Wash four times with TBS buffer containing 0.1% Tween-20, five minutes each time;

[0056] 24. Add horseradish peroxidase-labeled secondary antibody (purchased from Dako, catalog number K5007) and incubate at 37 degrees Celsius for 30 minutes;

[0057] 25. Wash 4 times with TBS buffer containing 0.1% Tween-20, 5 minutes each time;

[0058] 26. Add tyramine signal amplification reagent PPD570 (purchased from PANOVUE, catalog number 0144100050) diluted 1:400 (volume ratio) and incubate at room temperature in the dark for 13 minutes;

[0059] 27. Wash 4 times with TBS buffer containing 0.1% Tween-20, 5 minutes each time;

[0060] 28. Add the nuclear dye DAPI and stain at room temperature for 3 minutes;

[0061] 29. Wash twice with TBS buffer containing 0.1% Tween-20, 5 minutes each time;

[0062] 30. Seal the sheet with an anti-quenching agent.

[0063] (II) Image Acquisition

[0064] 1. Five images of the cancer nest region were obtained from each sample under a high-power field (200x) fluorescence microscope;

[0065] 2. Analytical software was used to identify single-positive signal images of CD68 (red fluorescence) and GPER1 (green fluorescence), as well as double-positive signal images of both (e.g., ...). Figure 1 );

[0066] 3. Analyze the number of CD68 single positive signals and the number of GPER1 and CD68 double positive signals, and calculate the proportion of GPER1 and CD68 double positive signals to CD68 single positive signals (i.e. the proportion of macrophages expressing GPER1).

[0067] 4. The average proportion of macrophages expressing GPER1 in the five photos was taken as the final result for this sample.

[0068] (III) Results Analysis

[0069] 1. The above analysis results of 48 samples were arranged in order of size, and the samples were divided into two groups with high and low expression of GPER1 in macrophages using the median value as the threshold.

[0070] 2. Survival analysis showed significant differences in overall survival and relapse-free survival between different patient groups (macrophages: P<0.01, e.g., ...). Figure 2 ).

[0071] 3. To compare with existing technologies, this invention also performed GPER1 immunohistochemical staining on liver cancer tissue (…). Figure 3 The proportion of GPER1 expressed in macrophages and the overall tumor tissue were evaluated, and the GPER1 histochemical score (GPER1H-Score) was obtained. By plotting ROC curves (receiver operating characteristic curves), this invention compared the predictive ability of the proportion of macrophages expressing GPER1 and the GPER1H-Score for overall survival or recurrence-free survival in liver cancer patients. The larger the area under the curve (AUC), the better the predictive effect of the indicator. Through comparison, this invention found that, for both overall survival and recurrence-free survival, the proportion of macrophages expressing GPER1 was superior to the overall tumor tissue GPER1 score in predicting patient survival. Figure 4 ).

Claims

1. Use of a reagent for detecting a marker in the manufacture of a kit for predicting the prognosis of a patient who has undergone resection of a liver cancer, characterized in that, The reagents are for detecting GPER1 expression level and CD68 expression level; by analyzing the number of CD68 single positive signals and the number of GPER1 and CD68 double positive signals in liver cancer nest tissue, the proportion of GPER1 and CD68 double positive signals to CD68 single positive signals is calculated, that is, the proportion of macrophages expressing GPER1, and survival prognosis is determined.

2. Use according to claim 1, characterized in that, The reagent used to detect GPER1 expression levels is a GPER1 antibody.

3. Use according to claim 1, characterized in that, The reagent used to detect CD68 expression levels is a CD68 antibody.

4. Use according to claim 2 or 3, characterized in that, The reagent also includes horseradish peroxidase-labeled second antibodies that can bind to either CD68 antibody or GPER1 antibody.

5. Use according to claim 4, characterized in that, The reagents also include tyramine signal amplification reagents.

6. Use according to claim 5, characterized in that, The tyramine signal amplification reagents are PPD520 and PPD570.

7. Use according to claim 6, characterized in that, It also includes reagents required for immunofluorescence detection.

Images