Enzalutamide-resistant neuroendocrine prostate cancer cell line and application thereof

Abstract

The invention relates to the technical field of cell engineering, in particular to an enzalutamide drug-resistant neuroendocrine prostate cancer cell line and application thereof, the cell line is preserved, and the cell line has a drug-resistant index of 7.18 or above and meets the evaluation standard of a drug-resistant cell line. The application can be used for researching the drug resistance mechanism of the neuroendocrine prostate cancer, evaluating the activity of drugs for resisting the neuroendocrine prostate cancer, preparing a neuroendocrine prostate cancer cell model or preparing a neuroendocrine prostate cancer animal model, and screening and developing drugs for reversing the drug resistance of the neuroendocrine prostate cancer. According to the invention, a new experimental material is provided for the research on the drug resistance of the prostate cancer, and the application has important scientific research value and application prospect. The invention can be used for screening and developing the drug for treating the neuroendocrine prostate cancer and detecting the sensitivity of the drug for resisting the neuroendocrine prostate cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and specifically relates to an enzalutamide-resistant neuroendocrine prostate cancer cell line and application thereof. Background Art

[0002] Enzalutamide (MDV3100, a laboratory drug) is an oral androgen receptor antagonist. Its mechanism of action is mainly through competitive binding to the ligand binding region of the androgen receptor, thereby effectively blocking the binding of androgens such as testosterone and dihydrotestosterone to the receptor and preventing the recruitment of co-activators, thereby inhibiting gene transcription. Its chemical formula is 4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-thione-1-imidazolidinyl]-2-fluoro-N-methylbenzamide. This complex molecular structure gives it unique pharmacological properties. At present, enzalutamide is mainly used to treat advanced male castration-resistant prostate cancer that has spread or recurred and is no longer sensitive to traditional hormone therapy.

[0003] Prostate cancer is one of the most common male malignancies in the world, and its morbidity and mortality cannot be ignored. The treatment of advanced prostate cancer has always been a hot topic and difficulty in this field. Recently, the results of two important clinical trials, ARCHES and ENZAMET, showed that enzalutamide (a second-generation antiandrogen drug) combined with androgen deprivation therapy (ADT) can significantly prolong the overall survival of patients with metastatic hormone-sensitive prostate cancer (mHSPC). Similar effects have also been confirmed in the PROSPER study, which showed that enzalutamide combined with ADT can significantly prolong the metastasis-free survival of patients with metastatic castration-resistant prostate cancer (mCRPC) and reduce the risk of death by 71%. Although enzalutamide has brought hope to patients with advanced prostate cancer, about 30% of patients still develop drug resistance within a short period of time after treatment, which seriously affects the overall survival of patients. Among them, treatment-related neuroendocrine prostate cancer (t-NEPC) is a fatal resistant subtype characterized by low PSA levels, high tumor metastasis burden, and rapid resistance to androgen-targeted therapy, with a median overall survival of less than one year after diagnosis.

[0004] Since t-NEPC is resistant to endocrine therapy, the current first-line treatment is mainly platinum-based chemotherapy, but the efficacy is not ideal. Targeted therapy based on small molecule inhibitors has made some progress in clinical trials of t-NEPC. For example, alisertib, a small molecule inhibitor targeting MYCN and cell cycle kinase AURKA, showed certain efficacy in phase II clinical trials (NCT01799278). Gao Dong et al. found that the FOXA2-KIT signaling axis was significantly activated in t-NEPC, and the KIT signaling pathway may become its therapeutic target, but the current application of KIT inhibitors in prostate cancer has not yet reached the expected clinical endpoint (NCT00676650). Therefore, it is still necessary to conduct in-depth research on the potential molecular mechanisms and resistance mechanisms of t-NEPC, and improve treatment strategies, such as exploring multi-target combined therapy and solving problems such as inconsistent treatment responses, in order to improve the effectiveness and tolerability of enzalutamide treatment, thereby bringing better clinical efficacy to patients. To this end, it is particularly important to establish a stable enzalutamide-induced neuroendocrine resistance cell line for human prostate cancer.

[0005] In existing studies, a variety of prostate cancer cell lines have been established, such as LNCaP, vCaP, C4-2, and C4-2B, to study the biological characteristics and drug response of prostate cancer. However, these cell lines often lack neuroendocrine characteristics, and treatment-induced neuroendocrine prostate cancer (t-NEPC) is a special type of prostate cancer that differs significantly from traditional adenocarcinoma in molecular characteristics and clinical behavior. t-NEPC is more resistant to enzalutamide, but there is currently a lack of resistant cell models specifically for t-NEPC. Summary of the invention

[0006] In the prior art, enzalutamide, as a second-generation hormone receptor antagonist, has been widely used in clinical practice for the treatment of prostate cancer. However, in clinical practice, some patients have developed resistance to enzalutamide, among which tumor neuroendocrine transformation (t-NEPC) caused by enzalutamide treatment is an important cause of resistance, which greatly limits the therapeutic effect of the drug. Specifically, the prior art has the following disadvantages: ① The lack of cell models of neuroendocrine prostate cancer that are resistant to enzalutamide limits the study of resistance mechanisms and the development of new treatment strategies.

[0007] ② Existing prostate cancer cell lines often do not have neuroendocrine characteristics and cannot accurately simulate the condition of patients with enzalutamide-resistant neuroendocrine prostate cancer.

[0008] ③ In the process of drug screening and new drug development, the lack of cell lines with stable drug resistance leads to low R&D efficiency and increased costs.

[0009] The present invention aims to solve the above technical problems and provide an enzalutamide-resistant neuroendocrine prostate cancer cell line, which can effectively simulate the biological characteristics of enzalutamide-resistant neuroendocrine prostate cancer.

[0010] A neuroendocrine prostate cancer cell line resistant to enzalutamide, the cell line is named Homo sapiens LNCaP-Enza, the preservation unit is Guangdong Microbial Culture Collection Center, China, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 65438, and the preservation date is November 8, 2024.

[0011] Furthermore, it was constructed by a concentration-increasing method that simulated the drug administration process.

[0012] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in studying the drug resistance mechanism of neuroendocrine prostate cancer.

[0013] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in evaluating the activity of drugs against neuroendocrine prostate cancer.

[0014] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in preparing a neuroendocrine prostate cancer cell model or a neuroendocrine prostate cancer animal model.

[0015] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in screening and developing drugs for reversing neuroendocrine prostate cancer resistance.

[0016] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in screening and developing drugs for treating neuroendocrine prostate cancer.

[0017] The present invention also provides the use of the above-mentioned enzalutamide-resistant neuroendocrine prostate cancer cell line in detecting the sensitivity of anti-neuroendocrine prostate cancer drugs.

[0018] The present invention relates to the field of cell engineering technology, and specifically provides an enzalutamide-resistant neuroendocrine prostate cancer cell line, and the cell line is preserved. The cell line has a resistance index of more than 7.18, and meets the evaluation criteria of drug-resistant cell lines. The cell line can be used to study the drug resistance mechanism of neuroendocrine prostate cancer, evaluate the anti-neuroendocrine prostate cancer activity of drugs, prepare neuroendocrine prostate cancer cell models or prepare neuroendocrine prostate cancer animal models, screen and develop neuroendocrine prostate cancer resistance reversal drugs, screen and develop drugs for treating neuroendocrine prostate cancer, and detect the sensitivity of anti-neuroendocrine prostate cancer drugs. The present invention provides new experimental materials for prostate cancer resistance research, and has important scientific research value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the IC50 determination results of LNCaP clone FGC and LNCaP-Enza cells.

[0020] Figure 2 The figure shows the PCR results of AR and neuroendocrine-related markers in LNCaP clone FGC and LNCaP-Enza cells.

[0021] Figure 3 The following are the morphological changes of LNCaP clone FGC and LNCaP-Enza.

[0022] Figure 4 Figure 2 shows the growth and proliferation curves of LNCaP clone FGC and LNCaP-Enza cells.

[0023] Figure 5 The graph shows the changes in the migration ability of LNCaP clone FGC and LNCaP-Enza cells. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Example

[0026] The drug-resistant cell line provided by the present invention is a drug-resistant cell line of the anti-androgen receptor drug Enzalutamide for human prostate cancer cells. Enzalutamide (MDV3100) was purchased from APExBIO (Cat. No. A3003). Its molecular formula is: C21H16F4N4O2S, and its structural formula is as follows:

[0027] The present invention proposes a method for constructing a drug-resistant cell line, which induces human prostate cancer cells to grow and passage in an environment where the concentration of enzalutamide increases from low to high by gradually increasing the drug concentration. After a series of tests, the drug resistance and neuroendocrine phenotype of the cells are confirmed, thereby obtaining the drug-resistant cell line.

[0028] In this process, we used LNCaP clone FGC cells (Cat. No.: CL-0143) from Wuhan Pronocell as parental cells. Cell culture conditions: The complete culture medium consists of 89% RPMI-1640+10% FBS+1% penicillin / streptomycin double antibodies, and the culture environment is a cell culture incubator at 37 degrees Celsius and 5% CO2. Before building the model, we verified the correctness of the cell lineage by short tandem repeat sequence detection (STR). After successfully constructing the enzalutamide-resistant prostate neuroendocrine carcinoma (which we named LNCaP-Enza cell line), we performed STR detection again to ensure the accuracy of the cell source and that it was not contaminated by other cells.

[0029] Stepwise Increasing Dose (SID) is a commonly used method in clinical treatment, which aims to improve patients' tolerance and adaptability to drugs. The basic principle of this method is to gradually increase the drug dose to help the patient's body adapt to the drug effect and reduce adverse reactions. It is usually applicable to the use of new drugs or the adjustment of the dose of existing drugs, especially those with potential toxicity or requiring an adaptation period. In the present invention, we use the stepwise increasing dose method as a means of screening drug-resistant cell lines. By gradually increasing the drug concentration, the cells gradually adapt and screen out subpopulations with higher tolerance to the drug.

[0030] The main effects and advantages of drug concentration escalation include: (1) Simulating the treatment process: This method can simulate the actual drug treatment process, allowing cells to gradually adapt to an environment with gradually increasing drug concentrations and screen out subpopulations with higher drug tolerance.

[0031] (2) Obtaining highly resistant cell lines: By gradually increasing the drug concentration, cell populations with strong tolerance to drugs can be screened out. These resistant cell lines can be used to further study the mechanism of drug resistance, develop new treatment strategies, and evaluate the efficacy of candidate drugs.

[0032] (3) Improve research reliability: The drug concentration increment method helps reduce the frequency of cell mutations, thereby obtaining more stable and reliable drug-resistant cell lines. Compared with directly using high-dose drug screening, gradually increasing the concentration is closer to the development process of drug resistance under real treatment conditions.

[0033] (4) Studying drug resistance mechanisms: The construction of drug-resistant cell lines helps to further study drug resistance mechanisms, such as specific gene mutations, abnormal signaling pathways, etc., thereby revealing the development of drug resistance and related molecular mechanisms.

[0034] In this induction scheme, we used a method of gradually increasing the drug concentration, starting from 2uM and gradually increasing it to 12uM in increments of 2uM, to induce long-term cell resistance. However, other strategies for increasing drug concentrations may also achieve the induction of cell resistance. For example, different increments or rates can be used to induce resistance formation.

[0035] In addition, as mentioned above, in addition to the scheme proposed in the present invention, the establishment of drug-resistant models can also be achieved through the following methods: ① Short-term high-dose drug concentration shock method: Screen out cells that can survive through high concentrations of drugs, and then expand the culture to form drug-resistant cell lines. ② Gene editing technology: Directly affect the sensitivity of cells to drugs by knocking out or overexpressing specific genes, thereby constructing drug-resistant cells. However, these alternatives may have some limitations, such as insufficient stability of drug resistance or poor cell status.

[0036] In summary, the present invention uses LNCaP clone FGC prostate cancer cells as the research object, constructs an enzalutamide-resistant LNCaP-Enza cell line by an in vitro drug concentration increasing method, and confirms that it has a neuroendocrine phenotype through a series of detection methods, providing an experimental basis for studying the enzalutamide resistance mechanism and developing related targets.

[0037] The present invention is further described below by means of specific embodiments, but it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form.

[0038] 1. IC50 determination of enzalutamide toxicity to prostate cancer cells (1) Cell preparation: Select prostate cancer LNCaP clone FGC cells (or LNCaP-Enza cells) in the logarithmic growth phase and digest them using conventional trypsin digestion for 2-5 minutes. When the cell gap increases, cell protrusions retract, and cells become round, terminate the digestion with complete culture medium.

[0039] (2) Cell collection: Gently blow the cells to make them fall off the bottom of the culture dish, transfer the cell suspension to a 15 ml centrifuge tube, and centrifuge at 1200 rpm for 3 minutes.

[0040] (3) Cell resuspension and counting: Aspirate the supernatant, resuspend the cells in an appropriate amount of complete culture medium, and take a small amount for cell counting.

[0041] (4) Preparation of 96-well plate: Take a 96-well plate and add 200ul of PBS to the outermost circle of wells to prevent the evaporation of liquid in the plate during the experiment and affect the results.

[0042] (6) Cell inoculation: Add 200 ul of complete culture medium to the wells in column 2 as a control. Based on the cell counting results, add 5,000 cells to the wells in columns 3-11 and add an appropriate amount of culture medium to a total volume of 200 ul per well.

[0043] (7) Cell adhesion: Place the inoculated 96-well plate in an incubator overnight to allow the cells to fully adhere.

[0044] (8) Drug treatment: The next day, complete culture medium solutions of enzalutamide were prepared at different concentrations, namely 10 nM, 50 nM, 100 nM, 500 nM, 1 uM, 10 uM, 50 uM, and 100 uM.

[0045] (9) Drug addition: Take out the 96-well plate from the incubator, remove the culture medium in each well, and wash once with PBS. Add 200ul of new complete culture medium to the wells in columns 2-3, and add 200ul of drug-containing culture medium according to the concentration gradient to the wells in columns 4-11. Then, return the 96-well plate to the incubator and continue culturing for 72 hours.

[0046] (10) CCK-8 assay: After 72 hours of cell culture, a serum-free culture medium mixture containing 10% CCK-8 was prepared using RPMI-1640.

[0047] (11) Remove the culture medium and add CCK-8: Take out the 96-well plate, remove the culture medium from each well, and wash once with PBS. Add 100ul of 10% CCK-8 serum-free culture medium mixture to the wells in columns 2-11, and return the 96-well plate to the incubator for 1.5-2 hours.

[0048] (12) Result determination: Take out the 96-well plate and use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nM. The OD value of each group is imported into GraphPad Prism 9.5.1 software for calculation. The wells in the second column are used as blank controls, and the wells in the third column are used as cell control groups. The cell growth conditions under different drug concentrations are calculated, and the half-maximal inhibitory concentration (IC50) of the drug is calculated accordingly.

[0049] (13) After calculation, the resistance index of prostate cancer LNCaP clone FGC cells and enzalutamide-resistant prostate cancer LNCaP-Enza cells were 1.957uM and 14.06uM, respectively. According to the resistance index (RI) calculation formula, RI = resistant cell IC50 / parent cell IC50. It was concluded that the resistance index RI of LNCaP-Enza cells was 7.18, which met the standard of moderately resistant cells. (The cell IC50 curve is shown in Figure 2. Figure 1 shown) 2. Methods for constructing drug-resistant cell lines (1) First, a prostate cancer cell line LNCaP clone FGC was selected, which was confirmed to have a correct lineage by STR identification and was in the logarithmic growth phase. According to the method described in Embodiment 1, the cytotoxic IC50 value of the cell line to enzalutamide was determined to determine the initial dosing concentration.

[0050] (2) The above cells were inoculated into a T25 cell culture flask. When the cells were well attached and the density reached 30-40%, the cells were treated with a medium containing 2uM enzalutamide. During this period, the cell status should be closely monitored and the drug-containing medium should be replaced every 2-3 days. After about 2 weeks, when the cell density increased to 80-90%, the cells were passaged at a ratio of 1:4 and the passaged cells were inoculated into a new T25 flask and labeled as LNCaP P1.

[0051] (3) During the passaging process of LNCaP P1 cells, use complete medium without drugs for culture. After the cells are completely attached and the density reaches 30-40%, replace them with medium containing 2uM enzalutamide. Continue to replace the drug-containing medium every 2-3 days until the cell density reaches 80-90%, and then pass them into new T25 flasks according to the above method and mark them as LNCaP P2.

[0052] (4) Repeat the above process until the cells reach the LNCaP P3 stage. At this point, in the medium containing 2uM enzalutamide, the cell growth rate is significantly accelerated, and it only takes about 5 days to grow from 30% density to 80%. At this point, the cells are passaged into a new T25 flask and labeled as LNCaP P4.

[0053] (5) During the passaging process from LNCaP P3 to LNCaP P4, maintain an enzalutamide concentration of 2uM. After the cells are completely attached and the density reaches 30-40%, replace the medium with 4uM enzalutamide. Continue to replace the drug-containing medium every 2-3 days until the cell density reaches 80-90%, then continue to pass the cells according to the above passaging plan, and use the medium containing 4uM enzalutamide to maintain the culture after the cells are attached.

[0054] (6) When the cell growth rate increases again, the drug concentration is increased by 2uM for maintenance culture. This process is repeated, and the low dose (2uM) drug concentration is maintained throughout the subculture process. After the cells attach, the drug is administered according to the current tolerance concentration.

[0055] (7) As the cells were passaged to LNCaP P7, P10, P13 and P16, the drug concentration was gradually increased to 6uM, 8uM, 10uM and 12uM. Since the drug concentration had reached more than 5 times the initial concentration, the IC50 determination was performed at this time. The results are as follows: Figure 1 Based on these data, we labeled this cell line LNCaP-Enza.

[0056] (8) After being frozen for 3 months and then revived, LNCaP-Enza cells still maintained good resistance to 12uM enzalutamide and the cells grew well.

[0057] (9) LNCaP-Enza cells were cultured and the expression changes of neuroendocrine-related molecules were detected using real-time fluorescence quantitative PCR technology to determine their neuroendocrine properties (qPCR results are shown in Figure 2 STR identification confirmed that the cell line was of the correct origin and not contaminated by other cells, and it was finally identified as the enzalutamide-resistant neuroendocrine prostate cancer cell line LNCaP-Enza.

[0058] 3. Changes in morphological characteristics of parental cell lines and drug-resistant cell lines (1) First, the untreated prostate cancer cell line LNCaP clone FGC and the drug-resistant cell line LNCaP-Enza induced by enzalutamide were inoculated into T25 cell culture flasks respectively. In order to maintain the drug resistance characteristics of the drug-resistant cell line LNCaP-Enza, it was cultured in a culture medium containing 10uM enzalutamide. The parent cell line LNCaP cloneFGC was cultured in a standard complete culture medium. During the culture process, the growth status of the two cell lines needs to be continuously monitored.

[0059] (2) Use an inverted phase contrast microscope to observe and record the morphological changes of the two cell lines at different densities. Pay special attention to the morphological maintenance of the drug-resistant cell lines in drug-containing culture medium and the growth performance of the parental cell lines in conventional culture medium.

[0060] (3) If Figure 3As shown in the figure, LNCaP clone FGC cells present a typical long oval shape with multiple antenna-like protrusions. In contrast, the morphology of LNCaP-Enza cells has changed significantly, with a reduced cell size, a short spindle shape, some cells with irregular morphology, a reduced number of antennae, and a more obvious island growth pattern.

[0061] 4. Proliferation curve determination of parental cell lines and drug-resistant cell lines (1) Select the drug-untreated prostate cancer cell line LNCaP clone FGC and the drug-resistant cell line LNCaP-Enza induced by enzalutamide from the logarithmic growth phase. Use trypsin for routine digestion and centrifugation, then resuspend the cells in complete medium and count the cells. The cells were seeded into a 24-well plate at a density of 5×10^4 cells per well. Set up the following groups: LNCaP clone FGC group, LNCaP clone FGC+5uM enzalutamide group, LNCaP-Enza group, LNCaP-Enza+5uM enzalutamide group, and set up 3 replicate wells in each group to ensure that the final culture medium volume in each well is 2 ml.

[0062] (2) On the day after inoculation, remove the 24-well plate, remove the culture medium, and wash once with PBS. Next, add 450ul of serum-free culture medium and 50ul of CCK-8 solution to each well, and then place the plate in an incubator and incubate for 1.5 to 2 hours.

[0063] (3) After incubation, transfer 100ul of the reaction solution from each well of the 24-well plate to a 96-well plate, with 3 replicates per group. After transfer, temporarily store the 96-well plate away from light.

[0064] (4) Wash the cells in the 24-well plate with PBS, then add the corresponding culture medium according to the grouping, 2 ml per well, and return the cells to the incubator for continued culture.

[0065] (4) Use an ELISA reader to measure the absorbance of each group in the 96-well plate at a wavelength of 450 nm.

[0066] (5) From the second day to the fifth day, measure the absorbance every day according to the scheme described in step (2), and use the absorbance data of 5 consecutive days to draw a cell proliferation curve.

[0067] (6) Figure 4As shown, the cells in the LNCaP clone FGC group showed a slow growth trend; in the LNCaP cloneFGC+5uM enzalutamide group, cell growth was significantly inhibited after the third day; while the cell growth rates of the LNCaP-Enza group and the LNCaP-Enza+5uM enzalutamide group were significantly faster than those of the LNCaP clone FGC group, and 5uM enzalutamide treatment did not inhibit the growth of LNCaP-Enza cells.

[0068] 5. Differences in Transwell migration ability between parental cell lines and drug-resistant cell lines (1) As described above, untreated prostate cancer cell line LNCaPclone FGC and enzalutamide-induced drug-resistant cell line LNCaP-Enza were selected from cells in the logarithmic growth phase. Routine digestion and centrifugation were performed using trypsin, and then the cells were resuspended in serum-free medium and counted to adjust the cell density to 25×10^4 cells / ml.

[0069] (2) The following experimental groups were set up: LNCaP clone FGC group, LNCaP clone FGC + 5μM enzalutamide group, LNCaP-Enza group, LNCaP-Enza + 5μM enzalutamide group, with three replicate wells in each group. First, add the corresponding complete culture medium to the outer chamber of the Transwell chamber. Then, add 200μl of cell suspension to the inner chamber of each chamber, and place the chamber in a cell culture incubator for 48 hours.

[0070] (3) After the culture is completed, remove the Transwell chamber, carefully remove the liquid inside and outside the chamber, and wash it twice with PBS. Then, place the chamber in 4% paraformaldehyde for 20 minutes and wash it twice with PBS. Next, place the chamber in crystal violet stain for 30 minutes. After staining, wash it several times with PBS until the washing solution is clear and no stain remains. Finally, use a cotton swab to gently wipe the cells on the surface of the chamber, wash it once with PBS, and dry the chamber upside down at room temperature.

[0071] (4) Use a 10× objective lens to observe the migration of cells in each group, randomly select five fields of view, and count the number of migrating cells in each field of view.

[0072] (5) Experimental results are as follows Figure 5As shown in the figure, the number of cells migrating in the LNCaP clone FGC group was significantly lower than that in the LNCaP-Enza group. Under the action of 5μM enzalutamide, the cell migration of the LNCaP clone FGC group was significantly inhibited, while this concentration had no significant inhibitory effect on the cell migration of the LNCaP-Enza group. These data indicate that the migration ability of LNCaP-Enza cells is significantly enhanced compared with its parental cells.

[0073] In some embodiments, some uses of enzalutamide-induced neuroendocrine prostate cancer cells LNCaP-Enza are also defined, including one or more of the following: (1) Used to study the drug resistance mechanism of tumors in vitro, especially to analyze the drug resistance characteristics of treatment-induced neuroendocrine prostate cancer (t-NEPC).

[0074] (2) Used to evaluate the sensitivity of chemotherapeutic drugs in vitro, thereby providing a reference for clinical drug use.

[0075] (3) Used to prepare tumor cell models, cell line organoid models, and tumor animal models. The tumor cell model can be a progeny cell established on the basis of the LNCaP-Enza cell line, or a cell established by transfecting a gene with a fluorescent marker. Animal tumor models can be established by subcutaneous inoculation or tail vein injection to simulate the growth and metastasis of neuroendocrine prostate cancer.

[0076] (4) Used for in vitro screening and evaluation of chemotherapeutic drugs for neuroendocrine prostate cancer. The screening method includes adding different chemotherapeutic drugs to the culture medium of LNCaP-Enza cell line and observing the toxicity of the drugs to cells, thereby screening out preliminary effective candidate drugs. The most effective drug is further determined by calculating the half inhibitory concentration (IC50) and verifying its efficacy in animal models.

[0077] (5) Used for the development of tumor resistance reversal drugs in vitro. By adding resistance reversal drugs and enzalutamide to the culture medium of LNCaP-Enza cell line, observing the toxicity of drugs to cells, and screening out preliminary effective candidate drugs. Calculate the IC50 of effective drugs, and further evaluate their effect of reversing resistance in animal models.

[0078] The beneficial effect obtained so far is that the LNCaP-Enza cell line can stably grow, passage and recover at a concentration of 12uM enzalutamide, showing good resistance to enzalutamide. The cells recovered after 3 months of withdrawal of the drug and freezing in liquid nitrogen still maintain the original drug resistance, showing good drug resistance stability. The present invention provides a stable drug-resistant tumor cell model for studying the drug resistance mechanism of neuroendocrine prostate cancer, evaluating the sensitivity of chemotherapy drugs, screening and evaluating chemotherapy drugs, and developing drug resistance reversal drugs.

[0079] The present invention successfully cultivated a prostate cancer cell line with stable resistance to enzalutamide, and its resistance index can reach 7.18, showing good drug resistance and persistence. The cell line has neuroendocrine characteristics and provides a stable and effective cell model for studying enzalutamide resistance, especially the mechanism of neuroendocrine transformation. This achievement significantly shortens the research cycle and greatly reduces the investment of research resources. Compared with its parental cells, the resistant cell line shows better passaging and recovery ability, and can be passaged multiple times to obtain a large number of stable daughter cells, providing rich cell resources for experiments. The resistant cell line has a wide range of applications and can be used for research in the following fields: in vitro study of tumor resistance mechanisms; in vitro analysis of the sensitivity of chemotherapeutic drugs and cross-resistance between drugs; preparation of tumor cell models, cell line organoid models and tumor animal models; in vitro screening and evaluation of new chemotherapeutic drugs; in vitro development of tumor resistance reversal drugs.

[0080] The cell lines cultivated by the present invention have the following key advantages: High efficiency: The enzalutamide-resistant prostate cancer cell lines cultured by the present invention have stable drug resistance, avoiding the drawbacks of unstable drug resistance induced by traditional high-dose drug concentration surges or gene editing methods, and providing an efficient and reliable experimental model for research. Economical: By optimizing the culture process, the present invention significantly reduces the cost and time of using the model, providing an economical and efficient option for scientific research. Safety: The adopted technology and conditions help to reduce the risk of contamination that may occur in long-term cultured cells, ensuring the accuracy and reliability of experimental data. Accuracy: Through precise screening and verification steps, the present invention ensures the neuroendocrine transformation of the cell line, providing a highly accurate model for research. Application prospects: This cell line is not only suitable for basic research, but also provides a powerful tool for drug screening, efficacy evaluation and preclinical research, which helps to promote the transformation of scientific research results into clinical applications. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An enzalutamide-resistant neuroendocrine prostate cancer cell line, characterized in that: The cell line was named LNCaP-Enza and deposited in Guangdong Microbiological Culture Collection Center, China with the deposit number GDMCC No: 65438 and the deposit date is November 8, 2024.

2. The enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1, characterized in that: It was constructed by a concentration-increasing method that simulated the drug administration process.

3. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in studying the drug resistance mechanism of neuroendocrine prostate cancer.

4. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in evaluating the activity of a drug against neuroendocrine prostate cancer.

5. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in preparing a neuroendocrine prostate cancer cell model or a neuroendocrine prostate cancer animal model.

6. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in screening and developing drugs for reversing drug resistance of neuroendocrine prostate cancer.

7. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in screening and developing drugs for treating neuroendocrine prostate cancer.

8. Use of the enzalutamide-resistant neuroendocrine prostate cancer cell line according to claim 1 in detecting the sensitivity of anti-neuroendocrine prostate cancer drugs.

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