Epirubicin drug sensitive marker, detection kit and application of epirubicin drug sensitive marker

Abstract

The invention discloses an epirubicin drug sensitive marker, a detection reagent and related application thereof. The epirubicin drug sensitive marker is an epirubicin drug-resistant marker or / and an epirubicin sensitive marker, and the epirubicin drug-resistant marker is one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5 and NPTX1, and the epirubicin drug-sensitive marker is one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5 and NPTX1. The epirubicin star sensitive marker is one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3 and DNAAF3 (deoxyribonucleic acid-associated protein 3). The epirubicin drug sensitive marker can be used for predicting the drug effect of epirubicin. 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 epirubicin and the precise treatment of the bladder cancer.

Description

Technical Field

[0001] This invention relates to the application of molecular markers for anticancer drugs and related reagents, particularly to the application of molecular markers for the anti-bladder cancer drug epirubicin and related reagents, belonging to the field of biomedicine. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system. Although its incidence rate ranks thirteenth, due to my country's large population, new cases account for 15% of the global total. Bladder cancer is classified into non-muscle-invasive and muscle-invasive bladder cancer based on whether it invades the muscle layer. Non-muscle-invasive bladder cancer can be treated with intravesical chemotherapy, BCG instillation, and transurethral resection of the tumor. Non-muscle-invasive bladder cancer accounts for approximately 80% of all bladder cancers, with a recurrence rate of 30-40% within one year, and 15% of non-muscle-invasive bladder cancers can progress to muscle-invasive bladder cancer. Therefore, effectively controlling the recurrence and progression of non-muscle-invasive bladder cancer can effectively reduce the disease burden.

[0003] Epirubicin is one of the instillation chemotherapy drugs for treating non-muscle-invasive bladder cancer. Its core anti-cancer mechanism is that, as a cytotoxic anthracycline antibiotic, it exerts a powerful anti-tumor effect by interfering with cancer cell DNA replication and transcription, inhibiting topoisomerase II leading to DNA breaks, and generating reactive oxygen species that cause oxidative damage. Currently, epirubicin is the most commonly used instillation drug for non-muscle-invasive bladder cancer, but its clinical efficacy rate is only about 40%, making it unsuitable for most patients. Blindly using epirubicin to treat non-muscle-invasive bladder cancer may delay tumor control in ineffective patients and lead to tumor progression. Therefore, it is urgent to improve the molecular typing scheme for epirubicin drug sensitivity testing to identify the effective patient population and improve the efficacy of instillation therapy for bladder cancer.

[0004] The inventors of this invention utilize transcriptome sequencing of patient tumor tissue to refine molecular subtyping, enabling precise treatment stratification for patients. Stratification reveals which drugs a patient is potentially sensitive to and which drugs they are resistant to. This avoids the situation where all patients are treated with first-line drugs, preventing patients with primary resistance to first-line drugs from missing the optimal treatment opportunity and options.

[0005] To improve the efficacy of epirubicin chemotherapy or combined targeted therapy for bladder cancer and reduce the risk of drug resistance, it is urgent to improve the molecular subtyping scheme for epirubicin drug sensitivity, identify patients suitable for epirubicin treatment and those unsuitable for epirubicin treatment, and adopt different treatment methods for different patients. Furthermore, alternative treatment options can be provided for patients who are intolerant to epirubicin, thereby improving the efficacy of bladder cancer treatment. Summary of the Invention

[0006] The purpose of this invention is to address the technical problems encountered in the existing treatment of bladder cancer using the therapeutic drug epirubicin, such as epirubicin intolerance, low sensitivity to epirubicin, and high resistance to epirubicin, leading to poor clinical treatment effects, poor prognosis, low survival rates, and loss of optimal treatment opportunities and options for patients with primary epirubicin resistance. This invention provides an epirubicin drug sensitivity marker, detection reagent, drug, and its application. By molecularly subtyping the epirubicin drug sensitivity marker, this invention allows for different treatment approaches for different patients, enabling precise treatment for patients suitable for epirubicin treatment and alternative treatment options for patients who are resistant or insensitive to epirubicin. This provides clues for the use of epirubicin and precision treatment of bladder cancer, improves the efficacy of epirubicin chemotherapy or combined targeted therapy for bladder cancer, and reduces the risk of drug resistance.

[0007] To achieve the objectives of this invention, a first aspect of this invention provides an epirubicin susceptibility marker, wherein the epirubicin susceptibility marker is an epirubicin resistance marker or an epirubicin susceptibility marker, wherein the resistance marker is one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1; and the susceptibility marker is one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3.

[0008] High expression of the drug resistance markers SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1 is associated with epirubicin resistance. High expression of the drug sensitivity markers IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3 indicates sensitivity to epirubicin. Therefore, the expression of these genes could serve as potential drug sensitivity biomarkers to guide the treatment of primary bladder cancer.

[0009] A second aspect of the present invention provides the use of the above-mentioned epirubicin drug sensitivity markers in screening patients suitable for epirubicin treatment of bladder cancer, and guiding epirubicin medication.

[0010] Monitoring the resistance or sensitivity of the therapeutic drug epirubicin during bladder cancer treatment, i.e., using markers to determine whether a patient is resistant or sensitive to epirubicin, is used for drug selection before bladder cancer treatment and provides guidance for patients' epirubicin use.

[0011] A third aspect of the present invention provides the application of epirubicin sensitive markers in epirubicin drug research for non-therapeutic purposes.

[0012] Among them, the non-therapeutic drug use studies include: basic research on the pharmacodynamics of epirubicin and pharmacodynamic determination studies on the improvement of epirubicin.

[0013] In particular, it is used to analyze the effects of different batches of epirubicin, and organoid killing tests are performed on different batches of epirubicin to analyze the effects of different batches of epirubicin.

[0014] A fourth aspect of the present invention provides a quantitative reagent for the expression level of epirubicin susceptibility markers.

[0015] The epirubicin susceptibility markers are epirubicin resistance markers and epirubicin susceptibility markers. The epirubicin resistance markers are one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1; the epirubicin susceptibility markers are one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3.

[0016] In particular, the quantitative reagent for the expression level of the epirubicin drug sensitivity marker is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0017] In particular, the quantitative reagents include RNA extraction reagents, reverse transcription reagents, and qPCR reagents.

[0018] The fifth aspect of this invention provides a quantitative reagent for the expression level of epirubicin drug sensitivity markers in the preparation of epirubicin drug sensitivity detection reagents, wherein the drug sensitivity detection is an epirubicin resistance detection or an epirubicin drug sensitivity detection.

[0019] Specifically, the epirubicin resistance test refers to detecting the increase or decrease in the expression levels of epirubicin resistance markers SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1; an increase in expression levels indicates epirubicin resistance in the patient, while a decrease in expression levels indicates epirubicin sensitivity in the patient. The epirubicin drug sensitivity test refers to detecting the increase or decrease in the expression levels of epirubicin sensitivity markers IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3; an increase in expression levels indicates epirubicin sensitivity in the patient, while a decrease in expression levels indicates epirubicin resistance in the patient.

[0020] The sixth aspect of this invention provides a kit for detecting resistance to the therapeutic drug epirubicin during bladder cancer treatment, comprising: PCR detection primers for epirubicin drug sensitivity markers, an RNA extraction kit, and a reverse transcription kit.

[0021] In particular, the epirubicin susceptibility marker is an epirubicin resistance marker and / or an epirubicin susceptibility marker; the epirubicin resistance marker is one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1; the epirubicin susceptibility marker is one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3.

[0022] In particular, the PCR detection primers for detecting epirubicin resistance markers SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5 or NPTX1 are SEQ ID NO.1 to SEQ ID NO.14, respectively.

[0023] In particular, the preferred primers for detecting epirubicin resistance markers are:

[0024] In particular, the PCR detection primers for detecting epirubicin-sensitive markers IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3 or DNAAF3 are SEQ ID NO.15 to SEQ ID NO.36.

[0025] In particular, the preferred primers for detecting epirubicin-sensitive markers are:

[0026] The seventh aspect of this invention provides a drug sensitivity assessment kit for epirubicin, a therapeutic drug in the treatment of bladder cancer, comprising a quantitative reagent for detecting the expression level of epirubicin drug sensitivity markers, wherein the drug sensitivity assessment is a drug resistance assessment or a drug sensitivity assessment; and the epirubicin drug sensitivity markers are epirubicin resistance markers and epirubicin sensitivity markers.

[0027] In particular, the quantitative reagent for the expression level of the epirubicin drug sensitivity marker is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0028] The eighth aspect of the present invention provides a drug sensitivity analysis system for epirubicin in the treatment of bladder cancer, comprising: Target expression level detection device: used to detect the expression level of epirubicin drug sensitivity marker in a sample; the sample is bladder cancer tissue from a patient; Drug sensitivity analysis device: Determine the efficacy of epirubicin drug sensitivity markers for bladder cancer.

[0029] 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.

[0030] In particular, the target expression level detection device is a qPCR detection instrument used to detect and measure the expression level of drug-resistant or sensitive genes.

[0031] In particular, the drug sensitivity analysis device, namely the qPCR detection instrument, analyzes the increase or decrease in the expression level of drug-resistant or sensitive genes.

[0032] In particular, the result output device outputs the Ct value of the relevant drug resistance or sensitivity gene.

[0033] A ninth aspect of the present invention provides a pharmaceutical composition for treating bladder cancer, comprising epirubicin and an inhibitor or promoter of epirubicin susceptibility markers, wherein the epirubicin susceptibility markers are epirubicin resistance markers or epirubicin sensitivity markers.

[0034] In particular, the pharmaceutical composition for treating bladder cancer includes epirubicin and an inhibitor of epirubicin resistance markers. This pharmaceutical composition enhances the efficacy of epirubicin in patients with high expression of epirubicin resistance genes by inhibiting the expression of these genes.

[0035] In particular, the pharmaceutical composition for treating bladder cancer includes: epirubicin and an epirubicin sensitivity marker promoter. This pharmaceutical composition enhances the efficacy of epirubicin in patients with low expression of the sensitivity gene by promoting the expression of the epirubicin sensitivity gene.

[0036] Specifically, the epirubicin resistance gene is one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1; and the epirubicin sensitivity gene is one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3.

[0037] In addition, in order to overcome the problems of the prior art, the first objective of this invention is to provide an epirubicin drug sensitivity marker, which can be used to identify epirubicin-resistant and sensitive populations, providing a basis for precision treatment of bladder cancer.

[0038] A second objective of this invention is to propose the application of bladder cancer drug sensitivity markers in epirubicin use studies for non-therapeutic purposes.

[0039] A third objective of this invention is to provide applications of epirubicin susceptibility marker-related reagents, which include quantitative reagents for detecting its expression, inhibitors, promoters, etc. The application of these related reagents can fill the gap in the field of precision diagnosis and treatment of bladder cancer.

[0040] The above-mentioned objective of this invention is achieved by the following technical solution: The first aspect of this invention provides a set of gene resistance markers SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1, whose high expression is associated with epirubicin resistance; and gene sensitivity markers IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3, whose high expression is associated with epirubicin sensitivity, i.e., high gene expression indicates sensitivity to epirubicin. These gene expressions can serve as potential drug sensitivity biomarkers to guide the treatment of primary bladder cancer.

[0041] When the expression level of epirubicin resistance markers is significantly high, it is determined to be epirubicin resistance in bladder cancer; when the expression level of epirubicin sensitivity markers is significantly high, it is determined to be epirubicin sensitivity in bladder cancer.

[0042] This invention provides an epirubicin susceptibility marker, which can be either an epirubicin resistance marker or an epirubicin susceptibility marker. The epirubicin resistance marker is any one or more of the following groups of markers: SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1. The epirubicin susceptibility marker is IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3. The epirubicin susceptibility marker is RNA or its reverse-transcribed cDNA, or a protein.

[0043] In particular, the epirubicin susceptibility markers were screened in the following manner:

[0044] Surgical specimens from multiple patients with primary bladder cancer were collected, and samples were taken from multiple sites. A bladder cancer organoid biobank covering hundreds of sites was established using a three-dimensional culture method.

[0045] The inventors, through hematoxylin-eosin (HE) staining, found that organoids derived from patients with low-grade urothelial carcinoma also exhibited low cellular atypia, relatively uniform cells, and few mitotic figures; organoids derived from patients with high-grade urothelial carcinoma and squamous metaplasia also exhibited frequent mitotic figures, high cellular atypia, and the same histological features of squamous metaplasia, confirming that the organoids of the present invention preserve the pathological characteristics of the patients.

[0046] Immunohistochemical staining of organoids and corresponding patient surgical specimens revealed that the organoids retained the pathological characteristics of the patients, and the molecular markers of bladder cancer organoids were consistent with those of clinical bladder cancer patients.

[0047] Nearly one hundred loci were selected for exon and transcriptome sequencing of tissues and corresponding organoids to analyze their genomic characteristics. The organoid biobank effectively preserved the molecular characteristics of tissue samples and retained the mutational features of the original tissues from multiple dimensions, including somatic mutations, copy number variations, and transcriptome similarity.

[0048] The large-scale bladder cancer tissue and organoid cohort established by the inventors is consistent with previous bladder cancer studies, exhibiting high-frequency mutations characteristic of bladder cancer. Furthermore, the bladder cancer data from this invention reflect varying degrees of inter-tumor and intra-tumor heterogeneity, providing a basis for studying drug sensitivity heterogeneity in bladder cancer.

[0049] To investigate the molecular typing of epirubicin in bladder cancer, transcriptome sequencing was performed on multiple organoid loci (a total of 83). Using the Lasso model and bioinformatics analysis, the sensitive markers for epirubicin were identified as IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3; the resistance markers were identified as SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1. The training set AUC was 0.847. Subsequently, using the same method, multiple bladder cancer specimens were collected for organoid culture, drug sensitivity testing, and transcriptome sequencing for molecular typing studies, resulting in a validation set of 0.832 for epirubicin.

[0050] A second aspect of the present invention provides a quantitative reagent for the expression level of epirubicin drug sensitivity markers.

[0051] The epirubicin susceptibility testing reagent is used in the detection of bladder cancer tissue.

[0052] A third aspect of the present invention: the application of a quantitative reagent for the expression level of epirubicin drug susceptibility markers in the preparation of epirubicin drug susceptibility detection reagents, wherein the drug susceptibility detection is a drug resistance detection or a drug sensitivity detection.

[0053] The sample being tested is a bladder cancer tissue sample, and the quantitative reagent is any one of transcriptome sequencing reagents, PCR, gene chips, tissue chips, NanoString technology, immunohistochemistry, and ELISA reagents.

[0054] The present invention also provides an epirubicin drug sensitivity marker detection reagent, which is used to detect the expression level of epirubicin drug sensitivity marker.

[0055] The present invention also provides an epirubicin drug susceptibility assessment kit, comprising a quantitative reagent for detecting the expression level of the epirubicin drug susceptibility marker, wherein the drug susceptibility assessment is a drug resistance assessment or a drug sensitivity assessment.

[0056] The quantitative reagent for the expression level of the epirubicin drug sensitivity marker is a transcriptome sequencing reagent, or any one of PCR, gene chip, tissue chip, NanoString technology, immunohistochemistry or ELISA reagent.

[0057] 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 labeling 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.

[0058] 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.

[0059] This invention also provides an epirubicin susceptibility testing system for bladder cancer, comprising: Target expression level detection device: used to detect the expression level of the epirubicin drug sensitivity marker in a sample; the sample is bladder cancer tissue from a patient; Drug sensitivity analysis device: Determine the efficacy of epirubicin drug sensitivity markers for bladder cancer.

[0060] 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.

[0061] A fourth aspect of the present invention provides a pharmaceutical composition for treating bladder cancer, epirubicin, and an inhibitor or promoter of said epirubicin susceptibility marker.

[0062] The advantages and beneficial effects of this invention are: This invention screened and identified a group of genes associated with epirubicin resistance (i.e., epirubicin resistance genes), specifically SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1. This invention also screened and identified genes associated with epirubicin sensitivity (i.e., epirubicin-sensitive genes), specifically IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3.

[0063] The aforementioned drug resistance and sensitivity markers can be used to predict the efficacy of epirubicin. The epirubicin drug resistance and sensitivity markers screened in this invention can be used to predict the efficacy of epirubicin in treating bladder cancer. When the expression level of the epirubicin drug resistance marker is significantly high, it is judged that the patient has high resistance to the drug epirubicin, and the treatment effect of epirubicin is poor; conversely, it indicates no drug resistance, and the treatment effect of epirubicin is significant. When the expression level of the epirubicin sensitivity marker is significantly high, it is judged that the patient has high sensitivity to the drug epirubicin, and the treatment effect of epirubicin is significant; conversely, it indicates low sensitivity, and the treatment effect of epirubicin is poor.

[0064] By sequencing tissues from bladder cancer patients or detecting the expression of related molecules (epirarubicin susceptibility markers) using RT-qPCR or immunohistochemistry, precise guidance can be provided for bladder cancer medication and treatment. Detection of epirubicin susceptibility markers reveals that high expression of resistance genes indicates drug resistance, while high expression of sensitivity genes indicates drug sensitivity.

[0065] The epirubicin susceptibility marker of the present invention can indicate epirubicin resistance and susceptibility in individuals, providing a basis for precision treatment of bladder cancer. Moreover, the application of epirubicin susceptibility marker-related reagents can fill the gap in the field of precision diagnosis and treatment of bladder cancer. Attached Figure Description

[0066] Figure 1 Organoids were created to preserve the pathological features and tissue markers of patients with bladder cancer (BC). Figure 1A shows the construction of organoids from different sites in bladder cancer patients. The left image shows the construction of an organoid library containing both non-muscle-invasive and muscle-invasive bladder cancer. For tumors with a diameter of less than 2 cm, single-point sampling is performed, while for tumors larger than 2 cm, multiple points (superficial and invasive sites) are sampled for organoid culture. The right image shows the construction of organoids from superficial, invasive, and metastatic lymph node tumor tissues from source number 61, with bright-field observation of the organoids using an optical microscope. Figure 1 B shows the microscopic appearance and HE staining of organoids from patients with BC. Figure 1 C represents immunohistochemical staining of organoids, including the detection of culture and major subtyping molecule expression in organoid number 10: Figure 1 The left side of C shows gross observation, HE staining, and immunohistochemical staining of tumor tissue samples from the patient; the middle and right sides show bright-field microscopic observation and HE and immunohistochemical identification of organoids from different sites of the patient after successful culture.

[0067] 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.

[0068] Figure 3 This was an organoid drug sensitivity test, specifically testing 109 organoids against four bladder cancer chemotherapy drugs.

[0069] Figure 4 For epirubicin drug sensitivity molecular typing, among which Figure 4 A represents the molecular susceptibility typing of epirubicin; Figure 4 B is the AUC of the training and validation sets of the epirubicin star.

[0070] Figure 5 This study aimed to validate the molecular typing of epirubicin in 75 bladder cancer tissue samples. Detailed Implementation

[0071] 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.

[0072] Example 1: Validation of the correlation between organoids and tissues in bladder cancer

[0073] In this embodiment, a bladder cancer organoid biobank covering 208 sites was successfully established using a 3D culture method.

[0074] 1. HE staining 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).

[0075] 2. Immunohistochemical staining Immunohistochemical staining of organoids and corresponding patient surgical specimens revealed consistent immunohistochemical expression characteristics between bladder cancer patients and their organoids. Figure 1 C) Staining results revealed that bladder cancer epithelial cell markers (CK5 / 6, CK20, GATA3, P63) and the proliferation index Ki-67 were consistently expressed in bladder cancer tissues and organoids.

[0076] This demonstrates that the organoid system established in this embodiment preserves the patient's pathological characteristics very well.

[0077] 3. Exon and transcriptome sequencing This embodiment selected 158 loci, including 75 exons and 83 transcriptome loci, for exon and transcriptome sequencing of tissues and corresponding organoids to analyze the genomic characteristics of bladder cancer tissues and corresponding organoids. The organoid biobank highly preserved the molecular characteristics of tissue samples from multiple dimensions, including somatic mutations and transcriptome similarity. The large-scale bladder cancer tissue and organoid cohort established by the team is consistent with previous bladder cancer studies. Exon sequencing results (e.g.) Figure 2 A, 2B); transcriptome sequencing results (e.g. Figure 2 C, 2D).

[0078] Organoids were treated with different concentrations of epirubicin to kill them, and the epirubicin concentration corresponding to the killing of half of the organoids was calculated, i.e., the epirubicin IC50 value. Then, based on an epirubicin IC50 value of 0.5 μmol, the groups were divided into a sensitive group (less than 0.5 μmol) and a resistant group (greater than 0.5 μmol). Machine learning models for transcriptomic characteristics were established for each group. A drug response model was built using multi-omics data, and a bootstrap strategy based on the LASSO (Least Absolute Contraction and Selection Operator) regression model was employed to identify epirubicin-sensitive and resistant genes.

[0079] The specific operating method is as follows: First, candidate genes were selected based on the association between gene expression and epirubicin response in the training set, and the correlation coefficient was gradually increased (by 0.01 each time), while limiting the number of candidate genes identified (102-103). Then, multiple sets of candidate genes were selected to construct each specific predictive model. A LASSO regression model was used to model drug sensitivity (-log2 IC50 value) against the expression profiles of a selected set of candidate genes. This model was implemented using the glmnet package (v4.1-3), and the optimal penalty parameter 'lambda' was selected through 10-fold cross-validation. Bootstrapping (n = 100) was performed during the regression modeling process; candidate genes considered important in more than 50% of cases were selected as marker genes for the best model.

[0080] By fitting selected marker genes, an optimal model with a specific correlation coefficient threshold was obtained. The AUROC of all models associated with gemcitabine was compared to find the optimal threshold for the correlation coefficient. ROC curves and AUROCs were analyzed using the ROCR software package (v1.0-11).

[0081] The organoid biobank highly preserved the molecular characteristics of tissue samples from multiple dimensions, including somatic mutations and transcriptomic similarity. Exon sequencing results showed that the large-scale bladder cancer tissue and organoid cohort established in this invention is consistent with previous bladder cancer studies, exhibiting high-frequency mutations characteristic of bladder cancer. At the transcriptomic level, organoids and corresponding patient tissues also showed a high degree of correlation.

[0082] Therefore, the results of transcriptomics, exon sequencing, immunohistochemical staining, and hematoxylin and eosin (HE) staining tests demonstrate that the organoids of this invention maintain a high degree of homology with tissues.

[0083] Example 1A Screening of epirubicin susceptibility markers

[0084] Epirubicin susceptibility markers include epirubicin sensitivity markers and epirubicin resistance markers.

[0085] 1. Drug sensitivity test

[0086] Drug sensitivity testing was performed on 109 organoids for commonly used chemotherapy drugs for bladder cancer: gemcitabine, cisplatin, mitomycin C, and epirubicin. The specific procedures are as follows: Take an appropriate amount of well-mixed resuspension containing organoids; centrifuge at 300g, 4℃ for 5min, and 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; after 24 hours of plating, add the drug (10-20μl / well), continue culturing for 6 days, observe the changes in organoid morphology and state, and test cell viability.

[0087] The IC50 values ​​of each drug were arranged, and a heatmap of the four chemotherapy drugs was plotted. Figure 3 Different colors indicate different values.

[0088] The drug sensitivity response of patient organoids is generally consistent with the actual clinical drug use effect in patients. Therefore, drug sensitivity testing at the in vitro organoid level can reflect the drug sensitivity level of clinical patients.

[0089] 2. Transcriptome sequencing

[0090] To investigate the molecular susceptibility typing of epirubicin, a chemotherapy drug for bladder cancer, transcriptome sequencing was performed on 56 organoid loci with susceptibility. Using the Lasso model and bioinformatics analysis, the susceptibility markers for epirubicin were identified as IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3, while the resistance markers were identified as SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1. Figure 4 A), whose training set AUC is 0.847 ( Figure 4 B).

[0091] The operation method is as follows: Total RNA was isolated using the DNeasy & RNeasy isolation kit (Qiagen) and purified using poly-T oligoligand magnetic beads. RNA libraries were sequenced using the Illumina NovaSeq 6000 platform, yielding 150 bp paired-end reads. Raw reads were quality controlled using FastQC (v0.11.9), Cutadapt (v2.5), and Trimmomatic (v0.3) tools. STAR software (v2.7.3a) was used to align reads with a reference human genome hg19 with default parameters; gene expression was quantified; differential expression analysis was performed on transcriptome data; pathway clustering analysis (e.g., Gene Ontology, KEGG, Reactome, etc.) was performed on differentially expressed genes; Gene Set Enriment Analysis (GSEA) was conducted; and comparisons were made with existing pathway datasets (MSigDB Gene Sets). After unified processing, unsupervised clustering analysis, principal component analysis, and other methods can be performed on the samples to create heatmaps. The results can then be combined with various clinical indicators, mutation information, and other data for analysis.

[0092] 3. Verification Analysis

[0093] Transcriptome sequencing was performed on 24 collected organoid sites using the same method. Using the Lasso model and bioinformatics analysis, organoid culture, drug sensitivity testing, and transcriptome sequencing were conducted on the 24 collected bladder cancer specimens. Molecular subtyping studies (i.e., transcriptome characteristics of epirubicin resistance and sensitivity) were also performed, resulting in a validation set of 0.832 for epirubicin. Figure 4 B).

[0094] The AUC of the training set was 0.847 and the validation set was 0.832 by constructing organoids and performing transcriptome sequencing. The test results show that the gene set analyzed by the model of this invention is reliable.

[0095] The independent validation set consists of other organoids.

[0096] For each drug (cisplatin, gemcitabine, epirubicin, epirubicin), the same transcriptome analysis workflow was followed to obtain the gene expression profile of the validation set. This profile was then substituted into the optimal model for drug treatment for prediction, and compared with the drug sensitivity of the validation set organoids. The prediction model was evaluated using ROC curves. The prediction model was then used to validate patient transcriptome data and patient clinical responses, showing perfect agreement with clinical treatment outcomes.

[0097] This embodiment validated the effectiveness of epirubicin molecular susceptibility typing in the tissues of 75 bladder cancer patients. In bladder cancer tissues, the AUC for epirubicin molecular susceptibility typing was 0.762 (…). Figure 5 ).

[0098] Example 2: Application of epirubicin markers in studies of gemcitabine use for non-therapeutic purposes

[0099] This embodiment is a study of epirubicin activity: 1. Bladder cancer tissue was selected, and different batches of epirubicin were reacted with selected bladder cancer organoids to determine the quantitative expression of epirubicin susceptibility markers (SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, NPTX1, IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, DNAAF3) screened in Example 1.

[0100] 2. Based on the expression level, compare the expression levels of epirubicin drug sensitivity markers in different batches, and judge the biological activity of each batch of epirubicin based on the expression level.

[0101] Example 3 Epirubicin Resistance Assessment Kit

[0102] This embodiment provides an RT-qPCR epirubicin resistance assessment kit, comprising: Quantitative reagents for detecting the expression levels of epirubicin resistance markers screened in Example 1 (different kits may contain one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, and NPTX1).

[0103] Kit 1 (i.e., SRD5A2 resistance kit) contains: upstream and downstream primers for the RNA of the resistance marker SRD5A2 (as shown in Table 1), RNA extraction kit, cDNA synthesis kit, and qPCR kit.

[0104] Kit 2 (i.e., the ATOH8 resistance kit) contains: upstream and downstream primers for the RNA of the resistance marker ATOH8 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0105] Kit 3 (i.e., the DCPS resistance marker kit) contains: upstream and downstream primers for the drug resistance marker DCPS RNA (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0106] Kit 4 (i.e., the DAPK2 resistance kit) contains: upstream and downstream primers for the DAPK2 resistance marker RNA (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0107] Kit 5 (i.e., the PON1 resistance marker kit) contains: upstream and downstream primers for the RNA of the resistance marker PON1 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0108] Kit 6 (i.e., the TLR5 resistance marker kit) contains: upstream and downstream primers for the TLR5 resistance marker RNA (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0109] Kit 7 (i.e., the NPTX1 resistance kit) contains: upstream and downstream primers for the RNA of the resistance marker NPTX1 (as shown in Table 1), an RNA extraction kit, a cDNA synthesis kit, and a qPCR kit.

[0110] Table 1. RNA primers for rubibrine resistance markers

[0111] in, The cDNA synthesis kit (i.e., reverse transcription kit) includes dNTPmix: containing 2 mM each of dATP, dCTP, dGTP, and dTTP; and Taq DNA polymerase.

[0112] The qPCR reagents include: Taq hot-start enzyme, reaction buffer, SYBR Green fluorescent dye, and dNTPs.

[0113] qPCR reagents also include PCR enhancers, PCR stabilizers, and Universal Passive Reference Dye.

[0114] 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).

[0115] All existing known RNA extraction kits are applicable to this invention, such as the TransZol Up high-performance RNA extraction kit (catalog number: ET111-01-V2) from TransGen Biotech and the magnetic bead method high-efficiency tissue / cell total RNA extraction kit (catalog number: DP771) from Tiangen Biotech. 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.

[0116] 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).

[0117] Using the kit of this invention in combination with commonly used RNA extraction reagents and reverse transcription reagents, the expression levels of epirubicin resistance markers (SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, NPTX1) in samples can be specifically detected. This helps to determine the resistance of bladder cancer patients to epirubicin 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 epirubicin; if the expression level is decreased, it indicates that the patient is sensitive to epirubicin.

[0118] Using the bladder cancer epirubicin resistance detection kit of this invention, epirubicin drug 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 epirubicin treatment, providing patients with personalized and precise treatment plans and ensuring the best possible treatment. Without using the resistance assessment kit of this invention to evaluate drug efficacy, patients resistant to epirubicin may be treated with epirubicin, delaying tumor treatment and leading to tumor progression.

[0119] Example 4 Epirubicin Sensitivity Assessment Kit

[0120] This embodiment provides an RT-qPCR epirubicin susceptibility assessment kit, comprising: Quantitative reagents for detecting the expression levels of epirubicin-sensitive markers (IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, DNAAF3) screened in Example 1.

[0121] Kit 1 (i.e., the IGFBP3 sensitive marker kit) contains: upstream and downstream primers for the RNA of the sensitive marker IGFBP3 (as shown in Table 2); RNA extraction kit; reverse transcription kit (cDNA synthesis kit); and qPCR kit.

[0122] Kit 2 (i.e., the CCZ1 sensitive marker kit) contains: upstream and downstream primers for the CCZ1 sensitive marker RNA (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0123] Kit 3 (i.e., the TRIAP1 sensitive marker kit) contains: upstream and downstream primers for the RNA of the sensitive marker TRIAP1 (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0124] Kit 4 (i.e., the LSR Sensitive Marker Kit) contains: upstream and downstream primers for the RNA of the LSR sensitive marker (as shown in Table 2); RNA extraction kit; reverse transcription kit (cDNA synthesis kit); and qPCR kit.

[0125] Kit 5 (i.e., the CCNE1 sensitive marker kit) contains: upstream and downstream primers for the CCNE1 sensitive marker RNA (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0126] Kit 6 (i.e., the PCSK1N sensitive marker kit) contains: upstream and downstream primers for the PCSK1N sensitive marker RNA (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0127] Kit 7 (i.e., the NUDT5 sensitive marker kit) contains: upstream and downstream primers for the RNA of the NUDT5 sensitive marker (as shown in Table 2); RNA extraction kit; reverse transcription kit (cDNA synthesis kit); and qPCR kit.

[0128] Kit 8 (i.e., the CDKL2 sensitive marker kit) contains: upstream and downstream primers for the CDKL2 sensitive marker RNA (as shown in Table 2); RNA extraction kit; reverse transcription kit and qPCR kit.

[0129] Kit 9 (i.e., the LRRC73 sensitive marker kit) contains: upstream and downstream primers for the RNA of the sensitive marker LRRC73 (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0130] Kit 10 (i.e., the RAC3 sensitive marker kit) contains: upstream and downstream primers for the RNA of the RAC3 sensitive marker (as shown in Table 2); RNA extraction kit; reverse transcription kit (cDNA synthesis kit); and qPCR kit.

[0131] Kit 11 (i.e., the DNAAF3 sensitive marker kit) contains: upstream and downstream primers for the DNAAF3 sensitive marker RNA (as shown in Table 2); RNA extraction kit; reverse transcription kit; and qPCR kit.

[0132] Table 2. RNA primers for rubistar-sensitive markers

[0133] Using the kit of this invention in combination with commonly used RNA extraction reagents and reverse transcription reagents, the expression levels of epirubicin-sensitive markers (IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, DNAAF3) in samples can be specifically detected, which helps to determine the sensitivity of bladder cancer patients to epirubicin in advance. If the test results show an increased expression level of the sensitive markers, it indicates that the patient is sensitive to epirubicin; if the expression level is decreased, it indicates that the patient is resistant to epirubicin.

[0134] Using the epirubicin drug sensitivity test kit for bladder cancer treatment of the present invention, epirubicin drug sensitivity testing can be performed immediately after obtaining bladder cancer tissue through surgery or pathological puncture in bladder cancer patients. Before using epirubicin for treatment, the efficacy of the drug can be evaluated, providing patients with personalized and precise treatment plans and ensuring the best treatment for them.

[0135] Example 5 Epirubicin Resistance Analysis System

[0136] This embodiment provides an analytical system for evaluating the efficacy of epirubicin in the treatment of bladder cancer, including: 1) Epirubicin resistance assessment kit, including one or more of kits 1-7 described in Example 3; 2) Epirubicin sensitivity assessment kit, including one or more of kits 1-11 described in Example 4; 3) RT-qPCR target expression level detection device: used to detect the expression level of epirubicin drug susceptibility markers in samples. The epirubicin drug susceptibility markers include epirubicin resistance markers and epirubicin sensitive markers. Epirubicin resistance markers are one or more of (SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, NPTX1); epirubicin sensitive markers are one or more of (IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, DNAAF3). The RT-qPCR target expression level detection device is a commonly used device in this field, namely a qPCR detector, a real-time fluorescence quantitative PCR instrument, such as the AriaMx Real-Time PCR System (Agilent Technologies); The sample was bladder cancer tissue from a patient. The target expression level detection device uses RT-qPCR quantitative reagents, including upstream and downstream primers for epirubicin resistance and epirubicin sensitivity marker genes; as well as reverse transcription reagents and qPCR reagents (such as full-gold reagent and PerfectStart® Green qPCR SuperMix). Also includes dye-based quantitative PCR premix (catalog number: AQ601-01-V2). 4) Drug resistance analysis device: The drug efficacy for bladder cancer is determined based on the expression levels of epirubicin resistance markers (SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, NPTX1); the drug efficacy for bladder cancer is also determined based on the expression levels of epirubicin sensitivity markers (IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, DNAAF3).

[0137] 5) Result output device: Used to output the results obtained from the drug resistance analysis device.

[0138] The efficacy of the drug can be assessed before the patient uses epirubicin, providing the patient with a personalized and precise treatment plan.

[0139] Example 6: Pharmaceutical composition for treating bladder cancer

[0140] A pharmaceutical composition for treating bladder cancer, comprising: Inhibitors of epirubicin and epirubicin resistance markers (i.e., any one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, NPTX1).

[0141] The pharmaceutical composition can be an injectable formulation for bladder cancer patients. Oral administration during treatment can improve the treatment outcome in patients resistant to epirubicin.

[0142] Example 6A: Pharmaceutical composition for treating bladder cancer

[0143] A pharmaceutical composition for treating bladder cancer, comprising: Promoters of epirubicin and epirubicin-sensitive markers (i.e., any one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, and DNAAF3).

[0144] The pharmaceutical composition can be an injectable formulation for bladder cancer patients. Oral administration during treatment can improve the therapeutic effect in patients receiving epirubicin.

[0145] The above embodiments of the present invention 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 thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. An epirubicin susceptibility marker, characterized in that, The epirubicin susceptibility markers are epirubicin resistance markers and epirubicin susceptibility markers. The resistance markers are one or more of SRD5A2, ATOH8, DCPS, DAPK2, PON1, TLR5, or NPTX1. The susceptibility markers are one or more of IGFBP3, CCZ1, TRIAP1, LSR, CCNE1, PCSK1N, NUDT5, CDKL2, LRRC73, RAC3, or DNAAF3.

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

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

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

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

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

7. A drug sensitivity assessment kit for epirubicin, 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 epirubicin drug susceptibility markers, wherein the drug susceptibility assessment is a drug resistance assessment or a drug sensitivity assessment; and the epirubicin drug susceptibility markers are epirubicin resistance markers and epirubicin sensitivity markers.

8. The drug susceptibility assessment kit as described in claim 7, characterized in that, The quantitative reagent for the expression level of the epirubicin 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 system for analyzing the susceptibility of epirubicin to bladder cancer, characterized in that, include: Target expression level detection device: used to detect the expression level of epirubicin drug sensitivity markers in samples; The sample was bladder cancer tissue from a patient. Drug sensitivity analysis device: Determine the efficacy of epirubicin drug sensitivity markers for bladder cancer. 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 epirubicin and inhibitors or promoters of epirubicin susceptibility markers, wherein the epirubicin susceptibility markers are epirubicin resistance markers or epirubicin sensitivity markers.

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