Mitomycin C drug sensitive marker, detection kit and application of mitomycin C drug sensitive marker and detection kit

Abstract

The invention discloses a mitomycin C drug sensitive marker, a detection reagent and application of the mitomycin C drug sensitive marker, the mitomycin C drug sensitive marker is a mitomycin C drug-resistant marker or / and a mitomycin C sensitive marker, and the mitomycin C drug-resistant marker is one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2; and the mitomycin C sensitive marker is one or more of GABRP (gamma-aminobutyric acid binding protein), PABPC1L (poly (p-aminobutyric acid binding protein 1L) or PODXL2 (peroxidase The mitomycin C drug sensitive marker can be used for predicting the drug effect of mitomycin C. According to the present invention, the expression of the related molecules is detected by sequencing the transcriptome of the tissue of the urothelium carcinoma patient, or by using the PC R, the gene chip, the tissue chip, the NanoString technology, the immunohistochemistry and the ELISA method, such that the clue can be provided for the medication of the mitomycin C and the precise treatment of the bladder cancer.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a molecular marker of an anticancer drug and a related reagent, in particular to an application of a molecular marker of an anticancer drug mitomycin C and a related reagent, and belongs to the field of biological medicines. BACKGROUND

[0002] Bladder cancer is one of the common malignant tumors of the urinary system, and the incidence rate ranks the thirteenth, but due to the large population base in China, the number of new patients accounts for 15% of the global number of cases. Bladder cancer is divided into non-muscular and muscular invasive bladder cancer according to whether it invades the muscle layer. Non-muscular invasive bladder cancer can be treated by intravesical chemotherapy drugs, intravesical injection of bacillus Calmette-Guerin (BCG) and transurethral resection of tumor. Non-muscular invasive bladder cancer accounts for about 80% of all bladder cancers, and the one-year recurrence rate is 30-40%, and 15% of non-muscular invasive bladder cancer can progress to muscular invasive bladder cancer. Therefore, effectively controlling the recurrence and progression of non-muscular invasive bladder cancer can effectively reduce the disease burden.

[0003] Mitomycin C is one of the intravesical chemotherapy drugs for treating non-muscular invasive bladder cancer, and its core anticancer mechanism is to form a cross-linking product by combining with the DNA of cancer cells as a biological reductive alkylating agent, thereby destroying the structure and function of DNA and preventing cancer cell proliferation. At present, mitomycin C is the most commonly used intravesical drug for non-muscular invasive bladder cancer, and the clinical effective rate is only about 40%, and most of the population is not suitable for it. If mitomycin C is blindly used to treat non-muscular invasive bladder cancer, the tumor control of patients who are not suitable for it may be delayed, leading to tumor progression. Therefore, it is urgent to improve the drug sensitivity molecular typing scheme of mitomycin C, identify the effective population of the drug, and improve the efficacy of intravesical therapy for bladder cancer.

[0004] The inventors of the application improve the molecular typing by performing transcriptome sequencing on tumor tissues of patients, which can stratify the precise treatment of patients, and after stratification, it can be known which potential sensitive drugs and which drug-resistant drugs the patients have. Therefore, the use of first-line treatment drugs for all patients can be avoided, and patients with primary drug resistance to first-line drugs can lose the best treatment opportunity and treatment options.

[0005] In order to improve the efficacy of mitomycin C chemotherapy or combined targeted therapy for bladder cancer and reduce the risk of drug resistance, it is urgent to improve the drug sensitivity molecular typing scheme of mitomycin C, find patients suitable for treatment with mitomycin C and patients unsuitable for treatment with mitomycin C, and adopt different treatment methods for different patients, and also provide alternative treatment schemes for patients who are not resistant to mitomycin C, thereby improving the treatment efficacy for bladder cancer. SUMMARY

[0006] The present application aims to solve the technical problems of poor clinical treatment effect, poor prognosis, low survival rate and loss of optimal treatment opportunity and treatment options for patients with primary drug resistance to mitomycin C in the treatment of bladder cancer by using the therapeutic drug mitomycin C, by providing a mitomycin C drug sensitivity marker, a related reagent, a drug and an application thereof. The present application provides a clue for the use of mitomycin C and the precise treatment of bladder cancer by molecular typing of the mitomycin C drug sensitivity marker, different treatment methods for different patients, precise treatment for patients suitable for treatment with mitomycin C, other alternative treatment options for patients with drug resistance and insensitivity to mitomycin C, and improves the efficacy of mitomycin C chemotherapy or combined targeted drug treatment for bladder cancer and reduces the risk of drug resistance.

[0007] To achieve the object of the present application, the first aspect of the present application provides a mitomycin C drug sensitivity marker, which is a mitomycin C drug resistance marker and a mitomycin C sensitivity marker, wherein the drug resistance marker is one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2; and the sensitivity marker is one or more of GABRP, PABPC1L or PODXL2.

[0008] The high expression of the gene resistance marker PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK and DGAT2 is associated with drug resistance to mitomycin C. The high expression of the gene sensitivity marker GABRP, PABPC1L and PODXL2 is sensitive to the action of mitomycin C. Therefore, the expression of these genes can be used as potential drug sensitivity biomarkers for guiding the treatment of primary bladder cancer.

[0009] The second aspect of the present application provides the use of the above-mentioned mitomycin C drug sensitivity marker in screening patients suitable for treatment of bladder cancer with mitomycin C, and guiding the use of mitomycin C.

[0010] The present application provides a clue for the use of mitomycin C and the precise treatment of bladder cancer by molecular typing of the mitomycin C drug sensitivity marker, different treatment methods for different patients, precise treatment for patients suitable for treatment with mitomycin C, other alternative treatment options for patients with drug resistance and insensitivity to mitomycin C, and improves the efficacy of mitomycin C chemotherapy or combined targeted drug treatment for bladder cancer and reduces the risk of drug resistance.

[0011] The third aspect of the present application provides the use of a mitomycin C sensitivity marker in the research of non-therapeutic use of mitomycin C.

[0012] The non-treatment purpose drug research is a basic research on the pharmacodynamics of mitomycin C and a research on the pharmacodynamics determination of the improvement research of mitomycin C.

[0013] In particular, the effects of different batches of mitomycin C drugs are analyzed, the killing of different batches of mitomycin C on organoids is detected, and the effects of different batches of mitomycin C drugs are analyzed through the killing effect.

[0014] The fourth aspect of the present application provides a quantitative reagent for the expression amount of a mitomycin C drug sensitivity marker The mitomycin C drug sensitivity marker is a mitomycin C drug resistance marker, a mitomycin C sensitivity marker, the mitomycin C drug resistance marker is one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2, and the mitomycin C sensitivity marker is one or more of GABRP, PABPC1L or PODXL2.

[0015] In particular, the quantitative reagent for the expression amount of the mitomycin C drug sensitivity marker is any one of a transcriptome sequencing reagent, or a PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0016] In particular, the quantitative reagent comprises an RNA extraction reagent, a reverse transcription reagent and a qPCR reagent.

[0017] The fifth aspect of the present application provides an application of a quantitative reagent for the expression amount of a mitomycin C drug sensitivity marker in the preparation of a mitomycin C drug sensitivity detection reagent, and the drug sensitivity detection is a mitomycin C drug resistance detection or a mitomycin C drug sensitivity detection.

[0018] In particular, the mitomycin C drug resistance detection refers to detecting the expression amount of the mitomycin C drug resistance markers PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2, and an increase in the expression amount indicates that the patient is resistant to mitomycin C, and a decrease in the expression amount indicates that the patient is sensitive to mitomycin C; the mitomycin C drug sensitivity detection refers to detecting the expression amount of the mitomycin C sensitivity markers GABRP, PABPC1L or PODXL2, and an increase in the expression amount indicates that the patient is sensitive to mitomycin C, and a decrease in the expression amount indicates that the patient is resistant to mitomycin C.

[0019] The sixth aspect of the present application provides a kit for detecting the drug resistance of a therapeutic drug mitomycin C in the treatment of bladder cancer, comprising: PCR detection primers of a mitomycin C drug sensitivity marker, an RNA extraction kit and a reverse transcription kit.

[0020] In particular, the mitomycin C drug sensitivity marker is a mitomycin C drug resistance marker or / and a mitomycin C sensitivity marker; the mitomycin C drug resistance marker is one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2; and the mitomycin C sensitivity marker is one or more of GABRP, PABPC1L or PODXL2.

[0021] In particular, the PCR detection primers for detecting the mitomycin C drug resistance markers PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2 are SEQ ID NO. 1 to SEQ ID NO. 20, respectively.

[0022] In particular, the mitomycin C drug resistance marker detection primers are preferably:

[0023] In particular, the PCR detection primers for detecting the mitomycin C sensitivity markers GABRP, PABPC1L or PODXL2 are SEQ ID NO. 21 to SEQ ID NO. 26.

[0024] In particular, the mitomycin C sensitivity marker detection primers are preferably:

[0025] The seventh aspect of the present application provides a drug sensitivity evaluation kit for the therapeutic drug mitomycin C in the treatment of bladder cancer, comprising a quantitative reagent for detecting the expression amount of a mitomycin C drug sensitivity marker, wherein the drug sensitivity evaluation is drug resistance evaluation or drug sensitivity evaluation; and the mitomycin C drug sensitivity marker is a mitomycin C drug resistance marker or a mitomycin C sensitivity marker.

[0026] In particular, the quantitative reagent for detecting the expression amount of the mitomycin C drug sensitivity marker is any one of a transcriptome sequencing reagent, or a PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0027] The eighth aspect of the present application provides a drug sensitivity analysis system for the therapeutic drug mitomycin C in the treatment of bladder cancer, comprising: A target expression amount detection device for detecting the expression amount of a mitomycin C drug sensitivity marker in a sample; the sample is a bladder cancer tissue of a patient; A drug sensitivity analysis device for determining the drug effect of bladder cancer based on the expression amount of the mitomycin C drug sensitivity marker; Result output device: used for outputting the result analyzed by the drug sensitivity analysis device, which is drug resistance analysis or sensitivity analysis.

[0028] In particular, the target point expression detection device is a qPCR detection instrument, which detects and measures the expression of drug resistance or sensitivity genes.

[0029] In particular, the drug sensitivity analysis device, i.e. the qPCR detection instrument, analyzes the increase or decrease of the expression of drug resistance or sensitivity genes.

[0030] In particular, the result output device outputs the Ct value of the relevant drug resistance or sensitivity genes.

[0031] The ninth aspect of the present application provides a pharmaceutical composition for treating bladder cancer, comprising mitomycin C and an inhibitor or promoter of a mitomycin C drug sensitivity marker, wherein the mitomycin C drug sensitivity marker is a mitomycin C drug resistance marker or a mitomycin C sensitivity marker.

[0032] In particular, the pharmaceutical composition for treating bladder cancer comprises: mitomycin C, an inhibitor of a mitomycin C drug resistance marker. The pharmaceutical composition enhances the efficacy of mitomycin C on patients with high expression of drug resistance genes by inhibiting the expression of mitomycin C drug resistance genes.

[0033] In particular, the pharmaceutical composition for treating bladder cancer comprises: mitomycin C, a promoter of a mitomycin C sensitivity marker. The pharmaceutical composition enhances the efficacy of mitomycin C on patients with low expression of sensitivity genes by promoting the expression of mitomycin C sensitivity genes.

[0034] In particular, the mitomycin C drug resistance gene is one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK or DGAT2; and the mitomycin C sensitivity gene is one or more of GABRP, PABPC1L or PODXL2.

[0035] In addition, in order to overcome the problems of the prior art, the first object of the present application is to provide a mitomycin C drug sensitivity marker, which can be used to prompt the drug resistance or sensitivity of the population to mitomycin C and provide a basis for precise treatment of bladder cancer.

[0036] The second object of the present application proposes the application of the bladder cancer drug sensitivity marker in the research of non-therapeutic purpose of mitomycin C.

[0037] The third object of the present application provides the use of a mitomycin C drug sensitivity marker related agent, the marker related agent including: a quantitative agent for detecting the expression thereof, an inhibitor, a promoter, and the like, and the use of the related agent can fill the blank in the field of precise diagnosis and treatment of bladder cancer.

[0038] The above-mentioned object of the present application is achieved by adopting the following technical solutions: The first aspect of the present application provides a group of gene drug resistance markers PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2, high expression of which is related to mitomycin C resistance; and gene sensitivity markers GABRP, PABPC1L, and PODXL2, high expression of which is related to mitomycin C sensitivity, that is, high expression of the genes is sensitive to the action of mitomycin C. The gene expression can serve as a potential drug sensitivity biomarker for guiding the treatment of primary bladder cancer.

[0039] When the expression amount of the mitomycin C resistance marker is significantly high, it is determined that the bladder cancer is mitomycin C resistant; and when the expression amount of the mitomycin C sensitivity marker is significantly high, it is determined that the bladder cancer is mitomycin C sensitive.

[0040] That is, the present application provides a mitomycin C drug sensitivity marker, which is a mitomycin C resistance marker or a mitomycin C sensitivity marker; the mitomycin C resistance marker is any one or several of a group of markers PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, and DGAT2; and the mitomycin C sensitivity marker is GABRP, PABPC1L, and PODXL2. The mitomycin C drug sensitivity marker is RNA or reverse-transcribed cDNA thereof, or a protein.

[0041] In particular, the mitomycin C drug sensitivity marker is screened in the following manner: Surgical specimens of a plurality of primary bladder cancer patients are collected, and multiple-point sampling is performed, and a bladder cancer organoid biological library covering hundreds of sites is established by a three-dimensional culture method.

[0042] The inventors find that, by Hematoxylin-Eosin (HE) staining, It is found that the organoids derived from patients with low-grade urothelial carcinoma also exhibit small cell atypia, consistent cells, and few mitotic figures; the organoids derived from patients with high-grade urothelial carcinoma with squamous epithelial metaplasia also exhibit common mitotic figures, large cell atypia, and also have the histological feature of squamous metaplasia, which confirms that the organoids of the present application retain the pathological characteristics of the patients.

[0043] By immunohistochemical staining of the organoids and the corresponding surgical specimens of patients, it is found that the organoids retain the pathological characteristics of the patients, and the bladder cancer organoids are consistent with the molecular markers of clinically bladder cancer patients.

[0044] About a hundred sites are selected for exon sequencing and transcriptome sequencing of the tissues and corresponding organoids, and the genomic characteristics of the tissues and corresponding organoids are analyzed. The molecular characteristics of the organoid library are verified from multiple dimensions such as somatic mutations, copy number variations and transcriptome similarities, and the mutation characteristics of the original tissues are retained.

[0045] The large-scale bladder cancer tissue and organoid queue established by the inventors is consistent with previous bladder cancer research, and has high-frequency mutations of bladder cancer. In addition, the bladder cancer data studied in the present application reflects inter-tumor and intra-tumor heterogeneity, which provides a basis for studying the drug sensitivity heterogeneity of bladder cancer.

[0046] In order to study the drug sensitivity molecular typing of mitomycin C for bladder cancer, the transcriptome sequencing of multiple (a total of 83) organoid sites is carried out, and through Lasso model and bioinformatics analysis, it is found that the sensitive markers of mitomycin C are GABRP, PABPC1L and PODXL2, and the drug resistance markers are PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK and DGAT2. The AUC of the training set is 0.952. Then, through the same method, multiple bladder cancer specimens are collected for organoid culture, drug sensitivity test and transcriptome sequencing, and molecular typing research is carried out, and the verification set of mitomycin C is 0.704.

[0047] The second aspect of the present application provides a quantitative reagent for the expression amount of a mitomycin C drug sensitivity marker.

[0048] The application of the quantitative reagent for the expression amount of the mitomycin C drug sensitivity marker in the detection of bladder cancer tissues.

[0049] The third aspect of the present application provides the application of the quantitative reagent for the expression amount of the mitomycin C drug sensitivity marker in the preparation of a mitomycin C drug sensitivity detection reagent, and the drug sensitivity detection is drug resistance detection or drug sensitivity detection.

[0050] The sample to be detected is a bladder cancer tissue sample, and the quantitative reagent is any one of transcriptome sequencing reagent, PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry, ELISA reagent.

[0051] The present invention also provides a mitomycin C drug sensitivity marker detection reagent, which is used to detect the expression level of mitomycin C drug sensitivity marker.

[0052] The present invention also provides a mitomycin C drug susceptibility assessment kit, comprising a quantitative reagent for detecting the expression level of the mitomycin C drug susceptibility marker, wherein the drug susceptibility assessment is a drug resistance assessment or a drug sensitivity assessment.

[0053] The quantitative reagent for the expression level of the mitomycin C drug sensitivity marker is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0054] The basic principle of real-time quantitative PCR (qPCR) is to utilize the property of DNA polymerase to synthesize new DNA strands during the PCR process, combined with a... Fluorescent label The probe or dye is used to measure the progress of the PCR reaction by monitoring the increase in fluorescence signal in real time. Since there is a linear relationship between the Ct value of the template and the initial copy number of the template during the exponential phase of PCR amplification, it serves as the basis for quantification.

[0055] The relative expression levels of relevant drug-resistant or drug-sensitive genes can be calculated by comparing them with the CT values ​​of adjacent normal tissue. A relative gene expression greater than twice that of adjacent normal tissue is defined as indicating increased gene expression, while a relative expression less than 0.5 times that of adjacent normal tissue is defined as indicating decreased gene expression.

[0056] This invention also provides a mitomycin C susceptibility testing system for bladder cancer, comprising: Target expression level detection device: used to detect the expression level of the mitomycin C drug sensitivity marker in a sample; the sample is bladder cancer tissue from a patient; Drug sensitivity analysis device: Determine the efficacy of drugs for bladder cancer based on the expression level of mitomycin C drug sensitivity markers;

[0057] Result output device: used to output the results obtained by the drug sensitivity analysis device, wherein the drug sensitivity analysis is a drug resistance analysis or a sensitivity analysis.

[0058] A fourth aspect of the present invention provides a pharmaceutical composition for treating bladder cancer, comprising mitomycin C and an inhibitor or promoter of said mitomycin C susceptibility marker.

[0059] The advantages and beneficial effects of this invention are: This invention screened and identified a group of genes associated with mitomycin C resistance (i.e., mitomycin C resistance genes), specifically PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, or DGAT2. The application also screens and determines genes associated with mitomycin C sensitivity (i.e., mitomycin C sensitivity genes), specifically GABRP, PABPC1L, or PO DXL2.

[0060] The above-mentioned drug resistance and sensitivity markers can be used to predict the efficacy of mitomycin C. The mitomycin C drug resistance markers and sensitivity markers screened by the application can be used to predict the efficacy of mitomycin C in treating bladder cancer. When the expression level of the mitomycin C drug resistance marker is significantly high, it is determined that the patient has high drug resistance to mitomycin C, and the treatment effect of mitomycin C is poor. Conversely, the patient has no drug resistance, and the treatment effect of mitomycin C is significant. When the expression level of the mitomycin C sensitivity marker is significantly high, it is determined that the patient has high sensitivity to mitomycin C, and the treatment effect of mitomycin C is significant. Conversely, the sensitivity is low, and the treatment effect of mitomycin C is poor.

[0061] By sequencing the tissues of bladder cancer patients, or by detecting the expression of related molecules (mitomycin C drug sensitivity markers) by RT-qPCR or immunohistochemical methods, accurate guidance can be provided for drug use and treatment of bladder cancer. The detection of mitomycin C drug sensitivity markers, high expression of drug resistance genes, and high expression of sensitivity genes can provide a basis for precise treatment of bladder cancer.

[0062] The mitomycin C drug sensitivity markers of the application can provide guidance for mitomycin C drug resistance and sensitivity populations, and provide a basis for precise treatment of bladder cancer. Moreover, the application of mitomycin C drug sensitivity marker-related reagents can fill the gap in the field of precise diagnosis and treatment of bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 The organoids of bladder cancer (BC) patients retain the pathological characteristics and tissue markers of the patients. Among them, Figure 1 A is to establish BC patient organoids at different sites. The left side shows that a library of non-muscular invasive and muscular invasive bladder cancer organoids is constructed. For tumors less than 2 cm in diameter, single-point sampling is performed. For tumors greater than 2 cm in diameter, multi-point (superficial site and invasive site) sampling is performed, and organoid culture is performed. The right side shows that the superficial site, invasive site, and metastatic lymph node tumor tissue of source No. 61 are used to construct organoids, and bright field observation of the organoids is performed using an optical microscope; Figure 1 B is the state of the BC patient organoids under a microscope and HE staining; Figure 1 C is the immunohistochemical staining of the organoids, in which the organoids from source No. 10 are cultured and the expression of the main typing molecules is detected; Figure 1 The left side of C is the general observation, HE, and immunohistochemical staining of the tumor tissue of the patient; the middle and right sides are the bright field observation and HE and immunohistochemical identification of the successfully cultured organoids from different sites of the patient.

[0064] Figure 2 To preserve the exon and transcriptome characteristics of patients for organoids, among which, Figure 2 A shows that exon sequencing revealed that the characteristics of mutations in bladder cancer tissue were preserved in organoids. 2B shows the concordance rate of oncogene mutations between organoids and tissues, with a median of 85.71% (the median concordance rate of gene mutations between organoids and cancer tissues is 85.71%). Figure 2 C represents transcriptome sequencing of cultured organoids and their corresponding cancer tissues; Heatmap shows the correlation between tumor tissues and paired organoids. Figure 2 D represents the correlation coefficient between the cultured organoids and the corresponding cancer tissue transcriptomes.

[0065] Figure 3 This was an organoid drug sensitivity test, specifically testing 109 organoids against four bladder cancer chemotherapy drugs.

[0066] Figure 4 For mitomycin C drug sensitivity molecular typing, among which Figure 4 A represents the drug sensitivity molecular typing of mitomycin C; Figure 4 B is the AUC of mitomycin C on the training and validation sets.

[0067] Figure 5 This study validated the mitomycin C molecular typing in 75 bladder cancer tissue samples. Detailed Implementation

[0068] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0069] Example 1: Validation of the correlation between organoids and tissues in bladder cancer

[0070] In this embodiment, a bladder cancer organoid biobank covering 208 sites was successfully established using a 3D culture method.

[0071] 1. HE staining

[0072] Taking surgical specimens from patients with bladder cancer (BC) as an example ( Figure 1 A), HE staining was performed, confirming that the organoids preserved the patient's pathological features. Figure 1 B).

[0073] 2. Immunohistochemical staining

[0074] By immunohistochemical staining of the organoids and corresponding patient surgical specimens, it was found that bladder cancer patients and their organoids maintained consistent immunohistochemical expression characteristics Figure 1 C), and the staining results found that bladder cancer epithelial cell markers (CK5 / 6, CK20, GATA3, P63), proliferation index Ki-67, were consistently expressed in bladder cancer tissues and organoids.

[0075] It is shown that the organoid system established in this embodiment well retains the pathological characteristics of the patient.

[0076] 3. Exons, transcriptome sequencing

[0077] In this embodiment, 158 sites were selected, of which 75 were exons and 83 were transcriptome sites, for exome and transcriptome sequencing of tissues and corresponding organoids to analyze the genomic characteristics of bladder cancer tissues and corresponding organoids. From the somatic mutations and transcriptome similarities, it was verified that the organoid library highly retains the molecular characteristics of the tissue sample. The large-scale bladder cancer tissue and organoid queue established by the team is consistent with previous bladder cancer research. Exome sequencing results (such as Figure 2 A, 2B); transcriptome sequencing results (such as Figure 2 C, 2D).

[0078] The organoids were subjected to different concentrations of mitomycin C drug killing treatment, and the concentration of mitomycin C corresponding to the killing of half of the organoids, i.e. the mitomycin C IC50 value, was calculated; then according to the mitomycin C IC50 value 0.5 micromole, it was divided into: sensitive group (less than 0.5 micromole) and drug-resistant group (greater than 0.5 micromole), and the machine learning model of the transcriptome characteristics of different groups was established. Use multiple omics data to establish drug response model, use LASSO (Least Absolute Shrinkage and Selection Operator) regression model based on bootstrap strategy to find mitomycin C sensitive genes and drug-resistant genes.

[0079] The specific operation method is as follows:

[0080] First, based on the correlation between gene expression and mitomycin C response in the training set, candidate genes are selected, the correlation coefficient is gradually increased (increased by 0.01 each time), and the number of identified candidate genes is limited (102-103);

[0081] Then, multiple sets of candidate genes were selected to build each specific predictive model, using LASSO regression model to model drug sensitivity (-log2 IC50 value) with the expression profile of a selected set of candidate genes, which was implemented using the glmnet package (v4.1-3), and the optimal penalty parameter 'lambda' was selected by 10 fold cross validation. The regression modeling process was bootstrapped (n = 100), and the candidate genes that were considered important genes in more than 50% of the cases were selected as the signature genes of the best model.

[0082] The best model with a specific correlation coefficient threshold was obtained by fitting the selected signature genes. AUROC of all models related to gemcitabine drug was compared to find the best threshold of correlation coefficient. ROC curve and AUROC were analyzed by ROCR package (v1.0-11).

[0083] The organoid biobank was verified to highly retain the molecular characteristics of the tissue samples from multiple dimensions such as somatic mutations and transcriptome similarity. Exon sequencing results show that the large-scale bladder cancer tissue and organoid queue established by the present application is consistent with previous bladder cancer research, and has high-frequency mutations of bladder cancer. At the transcriptome level, the organoids and the corresponding patient tissues also have high correlation.

[0084] Therefore, the results of transcriptome, exon sequencing, immunohistochemical staining, hematoxylin-eosin staining (HE staining) tests show that the organoids of the present application maintain high consistency with the tissues.

[0085] Example 1A Screening of mitomycin C drug sensitivity markers

[0086] The mitomycin C drug sensitivity marker is a mitomycin C sensitive marker or a mitomycin C drug resistance marker.

[0087] 1. Drug sensitivity test

[0088] Drug sensitivity tests of 109 organoids for commonly used chemotherapeutic drugs for bladder cancer: gemcitabine, cisplatin, mitomycin C, epirubicin were performed, and the specific operation method is as follows: Take an appropriate amount of resuspended liquid containing organoids and beat evenly; 300g, 4℃ centrifugation for 5min, carefully remove the supernatant; add an appropriate amount of culture medium containing 5% Matrigel to resuspend the organoids; add 40μl of organoid suspension to each well of a 384-well plate, and add 50μl of sterile pure water to the peripheral wells and wells without suspension; add drugs (10-20μl / well) after plating for 24 hours, continue to culture for 6 days, observe the morphological and state changes of the organoids, and test the cell survival rate.

[0089] The IC50 values of each drug obtained were arranged, and a heat map of the above 4 chemotherapeutic drugs was drawn, as shown in FIG. 6. Figure 3 Different colors represent different values.

[0090] The drug sensitivity of patient organoids is generally consistent with the actual drug effect of patients in the clinic, and therefore, the drug sensitivity test at the level of in vitro organoids can reflect the drug sensitivity level of patients in the clinic.

[0091] 2. Transcriptome sequencing

[0092] In order to study the drug sensitivity molecular typing of the chemotherapeutic drug mitomycin C for bladder cancer, 56 organoid sites with drug sensitivity effects were selected for transcriptome sequencing. Through Lasso model and bioinformatics analysis, the sensitive markers of mitomycin C were GABRP, PABPC1L and PODXL2, and the drug resistance markers were PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK and DGAT2 (A), and the AUC of the training set was 0.95 (B). Figure 4 Figure 4

[0093] The operation method is as follows: Total RNA was isolated using DNeasy & RNeasy isolation kits (Qiagen) and purified using poly-T oligo magnetic beads. RNA libraries were sequenced by the Illumina NovaSeq 6000 platform and produced 150 bp paired-end reads. Raw reads were subjected to quality control by FastQC (v0.11.9), Cutadapt (v2.5) and Trimmomatic (v0.3) tools. STAR software (v2.7.3a) was used to align the reads to the reference human genome hg19 with default parameters; quantitative gene expression, differential expression analysis of transcriptome data, pathway clustering analysis (such as Gene Ontology, KEGG, Reactome, etc.) of differentially expressed genes, Gene Set EnrimentAnalysis (GSEA), comparison with existing pathway data sets (MSigDB Gene Sets). After uniform processing, unsupervised clustering (Unsupervised clustering) analysis, principal component analysis, etc. can be performed on the samples, and a heat map can be made, and the results can be analyzed in combination with various clinical indicators, mutation information, etc.

[0094] 3. Verification analysis

[0095] ​​The same method was used to perform transcriptome sequencing on 24 organoid sites collected therein. Through Lasso model and bioinformatics analysis, the 24 collected bladder cancer specimens were subjected to organoid culture, drug sensitivity test and transcriptome sequencing, and molecular typing study (i.e. transcriptome characteristics of mitomycin C resistance and sensitivity), and the verification set of mitomycin C was 0.70 Figure 4 B).

[0096] The training set AUC was 0.95 and the verification set was 0.70 by constructing organoids for transcriptome sequencing. The test results showed that the gene set analyzed by the model was reliable.

[0097] The independent verification set was composed of another organoid.

[0098] For each drug (cisplatin, gemcitabine, mitomycin C, epirubicin), the same transcriptome analysis process was used to obtain the gene expression profile of the verification set, which was substituted into the best model for drug treatment to predict and compare with the drug sensitivity of the verification set organoid; the prediction model was evaluated by ROC curve. The prediction model was used for verification of patient transcriptome data and patient clinical response, which perfectly matched the treatment effect of the patient in the clinic.

[0099] The effectiveness of the drug sensitivity molecular typing of mitomycin C was verified in the tissues of 75 bladder cancer patients in this embodiment. In the bladder cancer tissue, the drug sensitivity molecular typing AUC of mitomycin C was 0.79 Figure 5 ).

[0100] Example 2 Application of Mitomycin C Marker in Gemcitabine Drug Research for Non-therapeutic Purpose

[0101] In this embodiment, the study of the activity of mitomycin C is as follows: 1. Select bladder cancer tissues, and allow different batches of mitomycin C to act on the selected bladder cancer organoids, and determine the quantitative expression of the drug sensitivity markers of mitomycin C ((PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2, GABRP, PABPC1L, PODXL2)) screened in Example 1. 2. According to the expression amount, compare the expression amounts of the drug sensitivity markers of different batches of mitomycin C, and determine the biological activity of each batch of mitomycin C according to the expression amount.

[0102] Example 3 Mitomycin C Resistance Evaluation Kit

[0103] The RT-qPCR mitomycin C resistance evaluation kit provided in this embodiment comprises: A quantitative reagent for detecting the expression amount of the mitomycin C resistance marker screened in Example 1 (one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2 in different kits).

[0104] Kit 1 (i.e. marker PRODH resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker PRODH (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0105] Kit 2 (i.e. marker ANKRD35 resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker ANKRD35 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0106] Kit 3 (i.e. marker ADAMTSL5 resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker ADAMTSL5 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0107] Kit 4 (i.e. marker PTPRQ resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker PTPRQ (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0108] Kit 5 (i.e. marker NOXA1 resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker NOXA1 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0109] Kit 6 (i.e. marker PRAC1 resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker PRAC1 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0110] Kit 7 (i.e. marker CRYM resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker CRYM (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0111] Kit 8 (i.e. marker NFIB resistance kit) comprises: upstream and downstream primers of RNA of the resistance marker NFIB (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0112] Kit 9 (i.e. marker SYK resistance kit) comprises: the upstream and downstream primers of the RNA of the resistance marker SYK (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit and a qPCR kit.

[0113] Kit 10 (i.e. marker DGAT2 resistance kit) comprises: the upstream and downstream primers of the RNA of the resistance marker DGAT2 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit and a qPCR kit.

[0114] Table 1 RNA primers of the resistance marker of mitomycin C

[0115] wherein, The cDNA synthesis kit (i.e. reverse transcription kit) comprises dNTPmix: containing dATP, dCTP, dGTP, dTTP each 2mM; Taq DNA polymerase.

[0116] The qPCR reagent comprises: Taq hot start enzyme, reaction buffer, SYBR Green fluorescent dye, dNTPs.

[0117] The qPCR reagent further comprises PCR enhancer, PCR stabilizer and Universal Passive Reference Dye.

[0118] The existing known qPCR kits are all suitable for the present application, and are used according to the operation instructions of the kits, for example: Fullengen Bio Co., Ltd: PerfectStart® Universal Green qPCR SuperMix; Universal Dye Method Fluorescent Quantitative PCR Premix (Item No: AQ631); Tian Gen Biological Technology Co., Ltd: SuperReal Fluorescent Quantitative Premix Reagent Enhanced Edition (SYBR Green) (Item No: FP205).

[0119] The existing known RNA extraction kits are all suitable for the present application, for example, TransZol Up High Performance RNA Extraction Kit (Item No: ET111-01-V2) of Fullengen Bio Co., Ltd, and Magnetic Bead Method High Efficiency Tissue / Cell Total RNA Extraction Kit (Item No: DP771) of Tian Gen Biological Technology Co., Ltd; All existing known cDNA synthesis kits are applicable to this invention, such as the FastKing cDNA First Strand Synthesis Kit II (Genomic De-generated) (Catalog No.: KR126) from Tiangen Biotech Co., Ltd.; the TransScript One-Step GDNA Removal and First Strand cDNA Synthesis Kit from TransGen Biotech Co., Ltd. (Catalog No.: AT311); or the SuperScript Preamplification System for First Strand cDNA Synthesis Kit from GIBICOL Co., Ltd., etc.

[0120] All existing known qPCR kits are applicable to this invention. Follow the instructions on the kits. For example: TransGen Biotech Ltd.: PerfectStart® Universal Green qPCR SuperMix; Universal Dye-Based Quantitative PCR Premix (Catalog No.: AQ631); Tiangen Biotech Ltd.: SuperReal Quantitative PCR Premix Enhanced Version (SYBR Green) (Catalog No.: FP205).

[0121] Using the kit of this invention in combination with commonly used RNA extraction reagents and reverse transcription reagents, the expression levels of mitomycin C resistance markers (PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2) in samples can be specifically detected. This helps to determine the resistance of bladder cancer patients to mitomycin C in advance. If the test results show an increase in the expression level of the resistance markers, it indicates that the patient is resistant to mitomycin C; if the expression level is decreased, it indicates that the patient is sensitive to mitomycin C.

[0122] Using the mitomycin C resistance detection kit for bladder cancer of the present invention, mitomycin C resistance can be detected immediately after obtaining bladder cancer tissue through surgery or pathological biopsy in bladder cancer patients. This allows for the evaluation of drug efficacy before treatment with mitomycin C, providing patients with personalized and precise treatment plans and ensuring the best possible treatment. Conversely, without using the resistance assessment kit of the present invention to evaluate drug efficacy, patients resistant to mitomycin C may be treated with mitomycin C, delaying tumor treatment and leading to tumor progression.

[0123] Example 4 Mitomycin C Sensitivity Assessment Kit

[0124] This embodiment provides an RT-qPCR mitomycin C susceptibility assessment kit, comprising: A quantitative reagent for detecting the expression amount of the mitomycin C sensitive marker (GABRP, PABPC1L, PODXL2) screened in Example 1.

[0125] The kit 1 (i.e. the sensitive marker GABRP kit) comprises: the upstream and downstream primers of the RNA of the sensitive marker GABRP (as shown in Table 2); an RNA extraction kit; a reverse transcription kit (cDNA synthesis kit) and a qPCR kit.

[0126] The kit 2 (i.e. the sensitive marker PABPC1L kit) comprises: the upstream and downstream primers of the RNA of the sensitive marker PABPC1L (as shown in Table 2); an RNA extraction kit; a reverse transcription kit and a qPCR kit.

[0127] The kit 3 (i.e. the sensitive marker PODXL2 kit) comprises: the upstream and downstream primers of the RNA of the sensitive marker PODXL2 (as shown in Table 2); an RNA extraction kit; a reverse transcription kit and a qPCR kit.

[0128] Table 2 RNA primers of the mitomycin C sensitive marker

[0129] By using the kit of the present application in combination with the commonly used RNA extraction reagent and reverse transcription reagent, the expression amount of the mitomycin C sensitive marker (GABRP, PABPC1L, PODXL2) in the sample can be specifically detected, which helps to judge the sensitivity of the bladder cancer patient to mitomycin C in advance. If the detection result shows that the expression amount of the sensitive marker increases, it indicates that the patient is sensitive to mitomycin C; if the expression amount decreases, it indicates that the patient is resistant to mitomycin C.

[0130] By using the bladder cancer treatment drug mitomycin C drug sensitivity detection kit of the present application, the drug sensitivity of mitomycin C can be detected after the bladder cancer tissue is obtained by surgery or pathological puncture of the bladder cancer patient, and the drug efficacy of the patient is evaluated before the treatment with mitomycin C, so as to provide the patient with personalized and precise treatment plan and to provide the patient with the best treatment mode.

[0131] Example 5 Mitomycin C resistance analysis system

[0132] The present embodiment provides an analysis system for evaluating the therapeutic effect of using mitomycin C to treat bladder cancer, comprising: 1) a mitomycin C resistance evaluation kit, comprising one or more of the kits 1-10 described in Example 3; 2) a mitomycin C sensitivity evaluation kit, comprising one or more of the kits 1-3 described in Example 4; 3) RT-qPCR target expression detection device: for detecting the expression of mitomycin C drug sensitive markers in the sample, wherein the mitomycin C drug sensitive markers include mitomycin C drug resistant markers and mitomycin C sensitive markers, the mitomycin C drug resistant markers are one or more of (PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2); the mitomycin C sensitive markers are one or more of (GABRP, PABPC1L, PODXL2); The RT-qPCR target expression detection device is a device commonly used in the art, i.e. a qPCR detector, a real-time fluorescent quantitative PCR instrument, such as AriaMx Real-Time PCR System (Agilent Technologies); The sample is a bladder cancer tissue of a patient; The reagent used by the target expression detection device is an RT-qPCR quantitative reagent, including upstream and downstream primers of mitomycin C drug resistant and mitomycin C sensitive marker genes; and reverse transcription reagent, qPCR reagent (such as PerfectStart® Green qPCR SuperMix); It also includes a dye method fluorescent quantitative PCR premix (item number: AQ601-01-V2) 4) Drug resistance analysis device: based on the expression of mitomycin C drug resistant markers (PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2), determine the drug effect of bladder cancer; based on the expression of mitomycin C sensitive markers (GABRP, PABPC1L, PODXL2), determine the drug effect of bladder cancer.

[0133] 5) Result output device: for outputting the results analyzed by the drug resistance analysis device.

[0134] The drug efficacy can be evaluated before the patient uses mitomycin C, and a personalized and precise treatment plan is provided for the patient.

[0135] Example 6 Drug composition for treating bladder cancer

[0136] A drug composition for treating bladder cancer, comprising: Mitomycin C, an inhibitor of mitomycin C drug resistant markers (i.e. any one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, DGAT2).

[0137] The pharmaceutical composition can be an injection preparation for a bladder cancer patient. The medication can improve the treatment effect of a patient who is resistant to mitomycin C during the treatment process.

[0138] Example 6A Pharmaceutical composition for treating bladder cancer

[0139] A pharmaceutical composition for treating bladder cancer, comprising: Mitomycin C, a promoter of a marker sensitive to mitomycin C (i.e. any one or more of GABRP, PABPC1L, and PODXL2).

[0140] The pharmaceutical composition can be an injection preparation for a bladder cancer patient. The medication can improve the treatment effect of a patient who is resistant to mitomycin C during the treatment process.

[0141] The above examples of the present application are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

Claims

1. A mitomycin C susceptibility marker, characterized in that, The mitomycin C susceptibility markers are mitomycin C resistance markers and mitomycin C susceptibility markers. The resistance markers are one or more of PRODH, ANKRD35, ADAMTSL5, PTPRQ, NOXA1, PRAC1, CRYM, NFIB, SYK, or DGAT2. The susceptibility markers are one or more of GABRP, PABPC1L, or PODXL2.

2. The use of the mitomycin C susceptibility marker as described in claim 1 in screening patients suitable for treatment of bladder cancer with mitomycin C, and guiding the use of mitomycin C.

3. The application of the mitomycin C susceptibility marker as described in claim 1 or 2 in studies of mitomycin C use for non-therapeutic purposes.

4. The quantitative reagent for the expression level of mitomycin C susceptibility marker as described in claim 1.

5. The application of the quantitative reagent for the expression level of mitomycin C drug susceptibility marker as described in claim 4 in the preparation of mitomycin C drug susceptibility detection reagent, wherein the drug susceptibility detection is a mitomycin C drug resistance detection or a mitomycin C drug sensitivity detection.

6. A kit for detecting resistance to the therapeutic drug mitomycin C during bladder cancer treatment, characterized in that, include: PCR detection primers for mitomycin C drug sensitivity markers, RNA extraction kit, and reverse transcription kit.

7. A drug sensitivity assessment kit for mitomycin C, a therapeutic drug used in the treatment of bladder cancer, characterized in that, The reagent includes a quantitative reagent for detecting the expression level of mitomycin C drug susceptibility markers, wherein the drug susceptibility assessment is a drug resistance assessment or a drug sensitivity assessment; and the mitomycin C drug susceptibility marker is a mitomycin C resistance marker or a mitomycin C sensitivity marker.

8. The drug susceptibility assessment kit as described in claim 7, characterized in that, The quantitative reagent for the expression level of the mitomycin C drug sensitivity marker is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

9. A mitomycin C susceptibility testing system for bladder cancer, characterized in that, include: Target expression level detection device: used to detect the expression level of mitomycin C susceptibility marker in samples; The sample was bladder cancer tissue from a patient. Drug sensitivity analysis device: Determine the efficacy of drugs for bladder cancer based on the expression level of mitomycin C drug sensitivity markers; Result output device: used to output the results obtained by the drug sensitivity analysis device, wherein the drug sensitivity analysis is a drug resistance analysis or a sensitivity analysis.

10. A pharmaceutical composition for treating bladder cancer, characterized in that, Including mitomycin C and inhibitors or promoters of mitomycin C susceptibility markers, wherein the mitomycin C susceptibility markers are mitomycin C resistance markers or mitomycin C sensitivity markers.

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