Kit for detecting malignant degree and ferroptosis sensitivity of esophageal squamous carcinoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a kit for detecting the malignant degree and ferroptosis sensitivity of esophageal squamous carcinoma. The kit comprises a PBK detection system used for detecting PBK immunohistochemical scores in esophageal squamous carcinoma tumor tissues; a vector of a mathematical model used for determining a PBK expression level high / low boundary standard is recorded, when the PBK expression level in tumor tissue of an esophageal squamous carcinoma patient is lower than a preset standard, the patient is prompted to be low in malignancy degree, sensitive to ferroptosis drug treatment and suitable for being combined with a treatment scheme of targeted ferroptosis during conventional treatment, and prognosis is good; on the contrary, when the tumor malignancy degree is not lower than the standard, the patient is prompted that the tumor malignancy degree is high, and the tumor malignancy degree is not sensitive to ferroptosis drug treatment and is not suitable for being combined with a targeted ferroptosis treatment scheme. The tumor malignancy degree and the sensitivity to ferroptosis inducer drugs are judged by detecting PBK expression, and a new thought is provided for predicting the tumor malignancy degree and treating esophageal squamous cell carcinoma through ferroptosis inducers.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is a highly prevalent digestive tract malignancy with high malignancy, strong invasiveness and easy recurrence and metastasis. There is an urgent clinical need for new detection methods to accurately evaluate the biological behavior of tumors and treatment sensitivity. Currently, the evaluation of the malignancy degree of ESCC mainly relies on histopathological grading (such as differentiation degree, Ki-67 index) and imaging staging (such as TNM staging), but the traditional methods have the following significant limitations:

[0003] 1. Staticness and lag: Tissue biopsy cannot reflect the dynamic evolution of tumors in real time and cannot predict treatment response;

[0004] 2. Lack of molecular heterogeneity: The predictive value of existing markers (such as PD-L1, TP53 mutation) for malignant phenotypes (such as metastasis, drug resistance) is limited, and it is difficult to guide individualized treatment;

[0005] 3. Blank in the evaluation of ferroptosis sensitivity: Ferroptosis, as a new type of programmed cell death, is closely related to ESCC chemotherapy resistance and immune escape, but there is a lack of a detection system in clinical practice that can quantify ferroptosis sensitivity, resulting in the inability to screen patient groups suitable for treatment with ferroptosis inducers (such as Erastin, RSL3).

[0006] In recent years, studies have suggested a molecular association between the malignancy degree of ESCC and ferroptosis sensitivity. For example, the expression level of glutathione peroxidase 4 (GPX4) can reflect both the antioxidant capacity of tumors (malignant characteristics) and the resistance to ferroptosis; the accumulation degree of lipid peroxidation metabolites (such as MDA, 4-HNE) is positively correlated with tumor invasiveness and ferroptosis sensitivity. Erastin and its derivatives, which mainly induce ferroptosis, have also shown potential in clinical trials. However, considering the strong heterogeneity of esophageal squamous cell carcinoma, not all patients can benefit from the above treatments. Therefore, accurately predicting the disease malignancy degree of esophageal squamous cell carcinoma patients and the sensitivity degree of ferroptosis drugs has practical significance for improving patient treatment and economic benefits. Summary of the Invention

[0007] To solve the above problems, one of the objectives of the present invention is to provide a kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma. The kit determines the malignancy degree of the tumor and the sensitivity to ferroptosis inducer drugs by detecting the expression of PBK in the tumors of esophageal squamous cell carcinoma patients, thereby providing new ideas for predicting the malignancy degree of tumors and using ferroptosis inducers to treat esophageal squamous cell carcinoma.

[0008] To achieve the above objective, the specific technical solution adopted by the present invention is as follows:

[0009] A kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma, comprising:

[0010] A PBK detection system for detecting the immunohistochemical score of PBK in esophageal squamous cell carcinoma tumor tissues;

[0011] A carrier recording a mathematical model for determining the high / low expression level boundary standard of PBK. When the expression level of PBK in the tumor tissues of esophageal squamous cell carcinoma patients is lower than the predetermined standard, it indicates that the patient has a lower malignancy degree, is sensitive to ferroptosis drug treatment, is suitable for combining with a ferroptosis-targeted treatment plan during conventional treatment, and has a better prognosis; conversely, when it is not lower than the standard, it indicates that the patient has a higher tumor malignancy degree, is not sensitive to ferroptosis drug treatment, is not suitable for combining with a ferroptosis-targeted treatment plan. The nucleotide sequence number of PBK is: NM_001278945.2.

[0012] The above mathematical model is: The immunohistochemical staining score of PBK in the tumor tissues of esophageal squamous cell carcinoma patients is expressed as Score = percentage of positive cells × staining intensity = 3 × percentage of strongly positive cells + 2 × percentage of moderately positive cells + percentage of weakly positive cells. The final score range is between 0 and 300. The standard is: Take 150 as the critical value for Score. If <150, then PBK is lowly expressed, indicating that the patient has a lower tumor malignancy degree, is sensitive to ferroptosis drug treatment, has a better prognosis, and is suitable for combining with a ferroptosis-targeted treatment plan; if ≥150, then PBK is highly expressed, indicating that the patient has a higher tumor malignancy degree, is not sensitive to ferroptosis drug treatment, has a poor prognosis, and is not suitable for combining with a ferroptosis-targeted treatment plan.

[0013] The above carrier can be a paper instruction manual, such as an instruction manual with an APP download window, such as a QR code, or a computer program storage device or system, such as a USB flash drive, a network disk, etc.

[0014] In a preferred embodiment, the PBK detection system in the above-mentioned kit includes an immunohistochemical staining detection system and an anti-PBK antibody. The immunohistochemical staining detection system is used to detect the immunohistochemical staining score in the tumor tissue of esophageal squamous cell carcinoma patients. It can be the immunohistochemical chromogenic reagent GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit of GeneTech (Shanghai) Co., Ltd., and operate according to the kit instructions. The anti-PBK antibody can be the PBK polyclonal antibody (1:200, 16110-1-AP) of Wuhan Sanying Biotechnology Co., Ltd. The kit of the present invention can detect the immunohistochemical staining score of PBK in patient samples, judge the malignancy degree of tumors in esophageal squamous cell carcinoma patients and the sensitivity to ferroptosis drugs, and whether it is suitable for the treatment plan of inducing ferroptosis, so as to achieve the purpose of precision medicine.

[0015] In a second aspect, the present invention provides the use of a reagent for detecting the expression level of PBK in the preparation of a kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma. The reagent for detecting the expression level of PBK is the above-mentioned PBK detection system.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention first proposes to predict the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma by detecting the expression of PBK, which has important clinical significance for optimizing treatment decisions and improving patient prognosis. Judging the malignancy degree of patients and the sensitivity of ferroptosis drugs can effectively guide the individualized treatment of esophageal squamous cell carcinoma patients, improve clinical benefits, and at the same time avoid unnecessary waste of medical resources.

[0018] 2. The present invention has the advantages of simple operation, affordability, high accuracy, etc. Description of the Drawings

[0019] Figure 1 : The immunohistochemical analysis results of tissue microarrays of 34 esophageal squamous cell carcinoma patients in Example 1. In the figure, A is the immunohistochemical tissue picture; B is the comparison chart of PBK immunohistochemical scores in adjacent normal tissues and tumor tissues; C is the comparison chart of PBK immunohistochemical scores in esophageal squamous cell carcinoma patients at different stages.

[0020] Figure 2 : The experimental results of knocking out PBK in TE-1 and KYES150 cells in Example 2. In the figure, A is the western blot experimental result; B is the CCK8 experimental result; C is the Transwell experimental result; D is the colony formation experimental result.

[0021] Figure 3: Experimental results of the sensitivity of TE-1 and KYES150 cells after knocking out PBK to ferroptosis inducers IKE and RSL3 in Example 3. In the figure, A shows the experimental results of TE-1 cells; B shows the experimental results of KYES150 cells.

[0022] Figure 2 and Figure 3 Among them, ShPBK-1 and ShPBK-2 are two sets of parallel experiments. Specific implementation manners

[0023] Through comprehensive analysis of the expression data from more than 500 ESCC samples and normal esophageal tissues, and immunohistochemical analysis of tissue microarrays (including tumor tissues and adjacent normal tissue sections) from 34 esophageal cancer patients, the inventors determined that PBK is a molecule significantly overexpressed in esophageal squamous cell carcinoma and is closely related to poor patient prognosis. Patients with high PBK expression have a worse prognosis and a higher degree of tumor malignancy. Through in vitro experiments, the inventors found that PBK can regulate the Nrf2-mediated redox balance and thus regulate ferroptosis sensitivity. The research results further confirmed that knocking down PBK can, on the one hand, disrupt the redox homeostasis and enhance the ferroptosis sensitivity of cells; on the other hand, it can inhibit the JAK2 / STAT3 and PI3K / AKT pathways, thereby inhibiting tumor progression. The inventors' research shows that detecting the expression of PBK in the tumor tissues of esophageal squamous cell carcinoma patients can effectively predict the degree of tumor malignancy and ferroptosis drug sensitivity. By simultaneously evaluating the degree of malignancy and ferroptosis sensitivity of esophageal squamous cell carcinoma, it has important clinical significance for optimizing treatment decisions (such as targeted combined ferroptosis induction therapy) and improving patient prognosis.

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1: Study on the expression of PBK in esophageal squamous cell carcinoma and its relationship with patient prognosis

[0026] Prepare tissue microarrays of esophageal squamous cell carcinoma patients, and analyze the expression of PBK in tumor tissues and adjacent normal tissues from 34 esophageal cancer patients by immunohistochemistry, and analyze the relationship between the PBK expression level and its malignancy degree.

[0027] The results are as Figure 1 shown. It can be seen that compared with adjacent normal tissues, the expression level of PBK is higher in tumor tissues ( Figure 1 A - B); the expression level of PBK is higher in patients with advanced esophageal squamous cell carcinoma ( Figure 1 C). Thus, it is determined that PBK is a molecule significantly overexpressed in esophageal squamous cell carcinoma and is closely related to poor patient prognosis.

[0028] Example 2: Experiment on the effects of knocking down PBK on the proliferation, migration, and invasion abilities of esophageal squamous cell carcinoma cells

[0029] After knocking out PBK in TE-1 and H1975 esophageal squamous cell carcinoma cells using shRNA, the knockdown effect of PBK was verified by western blot. The cell viability was detected using a CCK8 assay kit, and the abilities of cell invasion and migration were detected using a Transwell migration chamber. The colony formation ability after knocking out PBK was verified by a colony formation assay.

[0030] The results are as Figure 2 shown. The western blot experiment verified the decreased expression after knocking out PBK( Figure 2 A). Compared with the control, the proliferation rates of TE-1 and H197 cells with knocked-out PBK were slower( Figure 2 B), and the malignant abilities such as invasion, migration, and colony formation became weaker( Figure 2 C-D). This example confirmed that knocking down PBK would inhibit the proliferation, migration, and invasion abilities of esophageal squamous cell carcinoma cells. It can be inferred that in patients with low PBK expression levels, the malignancy of esophageal squamous cell carcinoma is lower and the prognosis is better.

[0031] Example 3: Detection of the sensitivity of esophageal squamous cell carcinoma cells to ferroptosis inducers after knocking out PBK

[0032] TE-1 and KYES150 esophageal squamous cell carcinoma cells with knocked-out PBK were used. After adding gradient concentrations of ferroptosis inducers (RSL-3 and IKE) and treating for 120 h, the cell viability was detected, and the half-maximal inhibitory concentration (IC50) of the two ferroptosis inducers for different cell lines was calculated.

[0033] The results are as Figure 3 shown. Compared with the control, the IC50 of TE-1 and KYES150 esophageal squamous cell carcinoma cells with knocked-out PBK decreased significantly. Knocking out PBK significantly increased the sensitivity of TE-1 and KYES150 cells to the two ferroptosis inducers. It can be inferred that after the increase in PBK expression level, the sensitivity of esophageal squamous cell carcinoma cells to ferroptosis drugs decreases, while patients with low PBK expression show higher sensitivity to ferroptosis drugs.

[0034] Example 4: Kit for detecting the malignancy and ferroptosis sensitivity of esophageal squamous cell carcinoma and its efficacy test

[0035] This example provides a kit for detecting the malignancy and ferroptosis sensitivity of esophageal squamous cell carcinoma. The kit includes a PBK detection system for detecting the immunohistochemical staining score in the tumor tissues of esophageal squamous cell carcinoma patients, and a carrier recording a mathematical model for determining the high / low PBK expression level boundary standard.

[0036] The PBK detection system includes an immunohistochemical staining detection system and an anti-PBK antibody. The immunohistochemical staining detection system is the GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit of GeneTech (Shanghai) Co., Ltd., and the operation is carried out according to the kit instructions. The anti-PBK antibody is the PBK polyclonal antibody (1:200, 16110-1-AP) of Wuhan Sanying Biotechnology Co., Ltd.

[0037] The scoring / mathematical model uses the H-score immunohistochemical staining scoring system to analyze the protein expression of cells by comprehensively considering the cell staining intensity and staining area. The staining intensity is divided into negative (0), weakly positive (1), moderately positive (2), and strongly positive (3). The calculation formula is Score = 3 × percentage of strongly positive cells + 2 × percentage of moderately positive cells + percentage of weakly positive cells, and the obtained score ranges from 0 to 300. This kit takes the median score as the cut-off value to judge the high / low expression of PBK, that is, 150 is taken as the critical value. If < 150, then PBK is lowly expressed, indicating that the patient's tumor malignancy degree is relatively low, is sensitive to ferroptosis drug treatment, has a good prognosis, and is suitable for the treatment plan combining targeted ferroptosis; if ≥ 150, then PBK is highly expressed, indicating that the patient's tumor malignancy degree is relatively high, is insensitive to ferroptosis drug treatment, has a poor prognosis, and is not suitable for the treatment plan combining targeted ferroptosis.

[0038] The actual application of this kit is as follows:

[0039] Collect the tumor tissues of 50 patients with esophageal squamous cell carcinoma, and use this kit to perform immunohistochemical scoring on some tissues. The method is as follows: The collected tumor tissues are fixed in 4% paraformaldehyde, then paraffin-embedded, and finally paraffin sections are made for immunohistochemical staining experiments. Antigen repair, blocking, primary antibody incubation (PBK polyclonal antibody, 1:200, 20385-1-AP), and secondary antibody incubation are carried out according to the instructions of the GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit. Finally, the staining is completed using DAB chromogenic agent. When necessary, hematoxylin counterstaining can be performed, and the stained section images of three fields of view are taken under the microscope. The immunohistochemical score is statistically analyzed using image j software and the IHC-Profiler plugin.

[0040] Some tissues are used to establish a xenograft model in immunodeficient mice and evaluate the clinical efficacy of solid tumors. Two weeks after the model establishment, the tumor size is measured as the baseline, and then intraperitoneal injection of the ferroptosis inducer IKE (25 mg / kg body weight / week) (injected once a week) is performed, and the tumor size is evaluated two weeks after the first injection.

[0041] According to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1), 22 models were evaluated as complete remission or partial remission, among which 2 cases had immunohistochemical staining scores of the corresponding source tumor tissues ≥ 150; 28 models were evaluated as disease progression or stable disease, among which 26 cases had immunohistochemical staining scores of the corresponding source tumor tissues ≥ 150. Therefore, it was determined that the sensitivity of this kit was 92.8% and the specificity was 90.9%.

[0042] This specific embodiment is only an interpretation of the present invention and not a limitation thereof. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A kit for detecting the malignancy and ferroptosis sensitivity of esophageal squamous cell carcinoma, characterized in that: include: PBK detection system, used to detect PBK immunohistochemical scores in esophageal squamous cell carcinoma tumor tissues; A vector recording a mathematical model for determining the high / low demarcation standard of PBK expression levels. When the PBK expression level in the tumor tissue of patients with esophageal squamous cell carcinoma is lower than the predetermined standard, it indicates that the patient has a low degree of malignancy and is sensitive to ferroptosis drug treatment, and is suitable for a combined targeted ferroptosis treatment regimen while receiving conventional treatment, and has a good prognosis. Conversely, when it is not lower than the standard, it indicates that the patient's tumor has a high degree of malignancy and is insensitive to ferroptosis drug treatment, and is not suitable for a combined targeted ferroptosis treatment regimen.

2. The kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma according to claim 1, characterized in that: The mathematical model is the PBK immunohistochemical staining score in tumor tissue of patients with esophageal squamous cell carcinoma, expressed as Score = positive cell percentage × staining intensity = 3 × strong positive cell percentage + 2 × medium positive cell percentage + weak positive cell percentage, and the resulting score range is between 0 and 300; the standard is: Score takes 150 as the critical value, if <150, PBK is lowly expressed, indicating that the patient's tumor is less malignant, sensitive to ferroptosis drug treatment, and has a better prognosis, and is suitable for a combined targeted ferroptosis treatment regimen; if ≥150, PBK is highly expressed, indicating that the patient's tumor is more malignant, insensitive to ferroptosis drug treatment, has a poor prognosis, and is not suitable for a combined targeted ferroptosis treatment regimen.

3. The kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma according to claim 1, characterized in that: The PBK detection system includes an immunohistochemical staining detection system and an anti-PBK antibody.

4. The kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma according to claim 3, characterized in that: The immunohistochemical staining detection system is an immunochromogenic reagent GTVisionTMⅢDetection System / Mo&Rb (Including DAB) secondary antibody kit.

5. The kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma according to claim 3, characterized in that: The anti-PBK antibody is a PBK polyclonal antibody (1:200, 16110-1-AP).

6. The kit for detecting the malignancy degree and ferroptosis sensitivity of esophageal squamous cell carcinoma according to claim 1, characterized in that: The carrier is a paper instruction manual, a computer program storage device or a system.

7. Use of reagents for detecting PBK expression levels in the preparation of kits for detecting the malignancy and ferroptosis sensitivity of esophageal squamous cell carcinoma.

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