Method for circularly screening tumor drug-resistant cell line in vivo by using mouse model and application of tumor drug-resistant cell line

Abstract

The invention provides a method for circularly screening tumor drug-resistant cell lines in vivo by using a mouse model and application thereof, and the method comprises the following steps: injecting human glioma cells U87MG into the brain of a BALB / c nude mouse in situ, and applying temozolomide (TMZ) drug pressure to screen tumor cell lines with different drug-resistant levels. The TMZ drug-resistant tumor cells are obtained step by step through multiple times of circulating in-vivo screening, and the cells continuously keep drug resistance in vitro. Then, through function verification, drug sensitivity analysis and transcriptome sequencing, the reliability of the screened drug-resistant cells on gene and function levels is confirmed, and differential expression non-coding RNA related to drug resistance, including newly found non-coding RNA, is found. According to the invention, the research on tumor drug resistance can be accelerated, a drug resistance related molecular mechanism is disclosed, and a new thought and a new tool are provided for new drug research and development and personalized treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a method for establishing a tumor resistance screening model, and specifically relates to a method for establishing an evolutionary tumor resistance screening model in mice and an application thereof. Background Art

[0002] The development of drug resistance is a major challenge in cancer treatment, which can lead to decreased treatment efficacy, tumor recurrence, and poor patient prognosis. Especially for highly invasive malignant tumors such as gliomas, chemotherapy drugs are the main treatment method outside of surgery. Once drug resistance occurs, it will lead to recurrence and death of patients. Therefore, researchers have constructed a tumor resistance model in the laboratory to study the mechanism of resistance, screen new drugs, and guide clinical treatment.

[0003] At present, commonly used tumor resistance models are mainly based on in vitro cell line models, in vivo animal models, and three-dimensional organoid models. These models have played an important role in tumor resistance research, but there are still significant technical limitations. In vitro cell line models usually screen out drug-resistant cells by long-term exposure to drugs. They are easy to operate and have fast data output, but their biggest disadvantage is that it is difficult to simulate the complex tumor microenvironment in the body and cannot fully reflect the interaction between the immune system, matrix and tumor, thus affecting the physiological relevance of the research results. With the development of technology, three-dimensional organoid models have gradually emerged. Although this model can better simulate the cell-to-cell interactions and microenvironment characteristics in tumor tissues, organoid models have problems such as complex culture conditions, poor reproducibility, and difficulty in fully reproducing the dynamic environment in the body. Long-term culture of organoids may cause changes in cell heterogeneity and may also affect the reliability of research results.

[0004] In contrast, in vivo animal models can better simulate the human microenvironment and have a high potential for clinical translation in tumor resistance research. However, the current techniques and methods for establishing animal resistance models require a lot of time and resources, especially for human tumor tissue-derived transplants (PDX) models, which require a long tumor transplantation and growth cycle, resulting in high experimental costs and low success rates. Due to the complex development process of tumor resistance and large individual differences, the acquisition of resistant populations is not always stable or consistent. Tumor resistance often involves complex gene networks and signaling pathways. Although existing models provide important tools for studying tumor resistance, they are difficult to systematically reveal the multiple mechanisms of resistance formation. Studies have shown that drug resistance of tumor cells is not only related to changes in protein-coding genes, but non-coding RNA (such as long non-coding RNA, microRNA) also plays a key role in the regulation of resistance. Existing technologies lack systematic research on the role of non-coding RNA in tumor resistance and its regulatory network, especially the lack of screening models based on in vivo evolution to reveal its function in resistance.

[0005] Therefore, there is an urgent need for more comprehensive and reliable tumor resistance technology solutions that combine the advantages of in vitro and in vivo environments, simulate the evolution of tumor cells under drug pressure, and further explore the gene regulatory network related to drug resistance. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a method for screening tumor-resistant cell lines in mice, which simulates the drug selection process in vivo, can capture the evolutionary dynamics of tumor cells under stressful environments, and significantly accelerate the screening speed of drug-resistant cell populations.

[0007] Another object of the present invention is to provide an application of a tumor resistant cell line in screening anti-drug resistance drugs.

[0008] In order to achieve the above object, the present invention provides a method for screening tumor resistant cell lines by circulating in vivo in a mouse model, comprising the following steps:

[0009] (1) Tumor cells were injected into mice and then fed for 7 days;

[0010] (2) Treating mice with chemotherapy drugs to induce tumor resistance pressure, then killing the mice, isolating the tumors in the mice, chopping them up, and further digesting them with trypsin to obtain the first generation of drug-resistant tumor cells;

[0011] (3) First-generation drug-resistant tumor cells were injected into mice and then fed for 7 days;

[0012] (4) Continue to give the mice chemotherapy drugs to cause tumor resistance pressure, then kill the mice, isolate the tumors in the mice, cut them into pieces, and further digest them with trypsin to obtain the second generation of drug-resistant tumor cells;

[0013] (5) The second-generation drug-resistant tumor cells were injected into mice and then fed for 7 days;

[0014] (6) The mice were continued to be treated with chemotherapy drugs to induce tumor resistance pressure, and then the mice were killed, the tumors in the mice were isolated, cut into pieces, and further digested with trypsin to obtain the second generation of drug-resistant tumor cells.

[0015] (7) The first to third generations of drug-resistant tumor cells were cultured in a culture medium containing 10% FBS, 1% penicillin-streptomycin and 10 μM chemotherapeutic drugs to maintain their drug resistance under drug pressure.

[0016] Furthermore, the tumor cells in step (1) are human glioma cells.

[0017] Furthermore, the tumor cells in step (1) stably express luciferase or fluorescent protein, such as GFP, RFP, etc.

[0018] Furthermore, the mouse in step (1) is a nude mouse.

[0019] Furthermore, the chemotherapy drug is temozolomide.

[0020] Furthermore, the treatment in step (2), step (4) and step (6) is to administer the chemotherapy drug at 50 mg / kg / day, 5 days a week, and rest for 2 days, for 3 to 4 weeks.

[0021] The present invention also provides a use of the above-mentioned tumor drug-resistant cell line in screening anti-drug resistance drugs, comprising the following steps:

[0022] (1) administering an anticancer candidate drug to the tumor-resistant cell line;

[0023] (2) Detect the sensitivity of the tumor-resistant cell line to candidate anticancer drugs.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a method for screening tumor-resistant cell lines in mice, which simulates the drug selection process in vivo, can capture the evolutionary dynamics of tumor cells under stressful environments, and significantly accelerate the screening speed of drug-resistant cell populations; at the same time, the screened resistant cells are stable and reliable, and can simulate the evolutionary process of tumors in vivo, providing a new tool for in-depth understanding of drug resistance mechanisms, screening new drugs and personalized treatment, which can then help find drug resistance target sites and can screen new drugs against drug resistance in a targeted manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A flow chart of a method for screening tumor-resistant cell lines in mice provided by the present invention.

[0027] Figure 2A To use small animal in vivo imaging methods to show the growth of transplanted tumors in the mouse brain over time.

[0028] Figure 2B The statistical method of two-way analysis of variance was used to Figure 2A Statistics of photon flux in images Tumor growth curves at different screening rounds.

[0029] Figure 2C This is the survival curve of tumor-bearing mice after TMZ screening with different screening times.

[0030] Figure 3A The second-generation drug-resistant glioma cells (T2) obtained in the second round of screening were treated with different TMZ concentrations for 72 hours. The CCK8 method was used to detect the cell viability and calculate the median lethality (IC50) value of TMZ.

[0031] Figure 3B The third-generation drug-resistant glioma cells (T3) obtained in the third round of screening were treated with different TMZ concentrations for 72 hours. The CCK8 method was used to detect the cell viability and calculate the half lethality (IC50) value of TMZ.

[0032] Figure 3C These are light microscopic photographs of cell clones after crystal violet staining of colonies of P0 and T2 cells treated with TMZ (10 μM).

[0033] Figure 3D This is the statistical result of cell clone counting in different treatment groups after P0 and T2 cells were treated with TMZ (10 μM).

[0034] Figure 3E These are light microscopic photographs of cell clones after crystal violet staining of colonies of P0 and T3 cells treated with TMZ (10 μM).

[0035] Figure 3F This is the statistical result of cell clone counting in different treatment groups after P0 and T3 cells were treated with TMZ (10 μM).

[0036] Figure 4A Correlation analysis of transcriptome library sequencing data for bioinformatics analysis of P0, T2, and T3.

[0037] Figure 4B Heat map of differentially expressed genes for transcriptome sequencing analysis.

[0038] Figure 5A A bioinformatics analysis was performed to characterize the overall coding capacity of long noncoding RNA genes that were differentially expressed in glioma drug-resistant cells.

[0039] Figure 5B Gene distribution statistics of differentially expressed long noncoding RNA genes in glioma drug-resistant cells were analyzed by bioinformatics.

[0040] Figure 5C The gene expression statistics of differentially expressed long non-coding RNA genes in glioma resistant cells were analyzed by bioinformatics.

[0041] Figure 5D This is a visualization result of the TUG1 gene, which is known to have no significant changes in expression in glioma resistant cells.

[0042] Figure 5E The figure shows the visualization result of UCA1 gene, which is known to have increased expression level in glioma resistant cells.

[0043] Fig. 5F This is a visualization screenshot of the new long non-coding gene ATRIG newly discovered and assembled through transcriptome sequencing analysis of glioma-resistant cells obtained through the screening model in the present invention. DETAILED DESCRIPTION

[0044] The following describes a number of preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied by many different forms of embodiments, not limited to the establishment of related drug resistance models for other tumors other than glioma and chemotherapeutic drugs other than temozolomide. These embodiments are exemplary descriptions, and the scope of protection of the present invention is not limited to the embodiments mentioned in the text.

[0045] Gliomas are the most common and most aggressive type of primary brain tumors, especially glioblastoma (GBM), which has an extremely poor prognosis. Temozolomide is the most commonly used chemotherapy drug in the current standard treatment of glioblastoma, usually used in combination with radiotherapy. However, TMZ resistance is common in clinical practice and is an important cause of treatment failure and tumor recurrence. However, the mechanism of TMZ resistance is complex, and the main research involves the enhancement of DNA repair mechanisms; gene mutations related to DNA repair and changes in the tumor microenvironment, and the regulatory mechanism involving non-coding RNA is also unclear.

[0046] The U87MG cell line is the most commonly used human glioblastoma cell line and is widely used in the study of gliomas, including drug screening, gene function analysis, and molecular mechanism exploration. The specific embodiments of the present invention mainly take the commercialized U87MG cell line as an example, and can also be replaced by other human glioma cell lines, other tumor cell lines, or primary isolated tumor cells from other sources (patients). At the same time, the experimental methods and transcriptome library sequencing used in the present invention are relatively mature technologies and methods for laboratory application.

[0047] The present invention proposes a method for screening tumor-resistant cell lines by circulating in vivo in a mouse model, which specifically comprises the following steps:

[0048] 1. Method for establishing an in vivo circulation screening model

[0049] The mice used were immunodeficient BALB / c nude mice as the experimental model, and U87 glioma cells (P0) that stably expressed luciferase were implanted into the mouse brain by intracranial injection. The chemotherapy drug TMZ (temozolomide) or DMSO control group was applied for five days a week and two days of drug withdrawal for 3-4 weeks. During this period, the weight and tumor growth of the mice were monitored, and bioluminescence imaging was performed using the IVIS imaging system to confirm tumor growth and treatment effects.

[0050] After the treatment cycle, tumor cells are isolated from mice, extracted and cultured to complete the first screening cycle (T1). These cells are injected back into nude mice again, and drug pressure is continued for the second and third rounds of screening (T2 and T3). Through multiple SELEX screening cycles, cell populations with different levels of drug resistance are gradually obtained. These cells can be cultured in vitro and continue to maintain drug resistance in a culture medium containing TMZ.

[0051] 2. Functions of cells with different drug resistance levels obtained and verification of drug tolerance

[0052] The present invention evaluates the selected cells (T1, T2, T3) with different drug resistance levels through functional verification and drug sensitivity analysis. These cells are exposed to different concentrations of TMZ and other anticancer drugs, and their response to the drugs is analyzed to confirm the performance of drug-resistant cells in vitro. Functional verification methods such as cell proliferation experiments and apoptosis analysis are used to ensure that the selected cells have obvious drug resistance characteristics.

[0053] 3. Reliability verification of gene expression

[0054] In order to ensure that the selected drug-resistant cells have a reliable gene expression profile, the present invention performs transcriptome sequencing analysis on cells with different drug resistance levels. By comparing the gene expression changes of drug-resistant cells of different levels, the key genes, signaling pathways, and gene regulatory networks related to drug resistance are determined. Particular attention is paid to mechanisms related to drug resistance such as DNA repair and drug efflux channels to ensure the correlation between the selected drug-resistant cells and drug resistance at the molecular level.

[0055] 4. Discover new differentially expressed noncoding RNAs

[0056] The present invention further uses transcriptome data to screen out differentially expressed non-coding RNAs related to drug resistance, including lncRNA, miRNA, etc. By analyzing the changes in non-coding RNA expression in cells with different drug resistance levels, new drug resistance regulatory molecules are discovered, and through functional verification, the role of these non-coding RNAs in the formation of drug resistance is determined. This provides a theoretical basis for exploring new therapeutic targets and developing new anti-resistance drugs.

[0057] Glioma is the most common and most aggressive malignant tumor of the central nervous system in adults, with a high recurrence rate and extremely poor prognosis. Temozolomide (TMZ) is a standard chemotherapy drug for glioma, but most patients will gradually develop resistance after TMZ treatment, leading to tumor recurrence and treatment failure. To date, researchers have used a variety of methods to study the resistance mechanism of glioma cells, including in vitro cell culture and mouse xenograft models. However, existing research methods are difficult to accurately simulate the evolution of tumor cells under the action of chemotherapeutic drugs in vivo, especially the formation process of different resistance levels and their molecular mechanisms. Therefore, the development of a screening model that can simulate the evolution of tumor cell resistance in vivo is of great significance for understanding the resistance mechanism of glioma and exploring new therapeutic targets.

[0058] Material:

[0059] 1. Human glioma cell line U87MG (Cat. No.: HTB-14) and human 293T cells (Cat. No.: CRL-3216) were purchased from the American Type Culture Collection (ATCC).

[0060] 2. High-glucose DMEM medium (Cat. No.: 11966025), Opti-MEM medium for transfection (Cat. No.: 31985070), penicillin-streptomycin (10,000U / ml) (Cat. No.: 15140122), 0.25% trypsin (Cat. No.: 25200072) and Lipofectamine TM2000 transfection reagent (Cat. No.: 11668027) was purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0061] 3. CCK8 detection kit (Cat. No.: 11203ES08) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. Temozolomide (Cat. No.: 85622-93-1), hygromycin B (Cat. No.: 31282-04-9) and crystal violet (Cat. No.: C3886) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0062] 4. Packaging vector plasmids pMD.2G (#12259) and psPAX2 (#12260) were purchased from Addgene, Inc. TRIZOL REAGENT (Cat. No. 15596026) was purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0063] 5. VivoGlo substrate for in vivo imaging detection TM Luciferin (Cat. No.: P1041) was purchased from Promega (Beijing) Biotechnology Co., Ltd.

[0064] 6. Experimental mice: Balb / c nude mice, male, aged 4-6 weeks, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. In a sterile animal room, under standard conditions of constant temperature of 25-27°C and constant humidity of 45-50%, placed in a laminar flow clean rack, 3-5 nude mice were kept in each cage. The operation of animal experiments strictly complied with the Guidelines for the Management and Use of Laboratory Animals of the Institute of Biophysics, Chinese Academy of Sciences, and was approved by the Animal Ethics Committee and the Animal Use Committee.

[0065] instrument:

[0066] 1. Surgical instruments were purchased from Shanghai Mingyuan Industrial Co., Ltd. Suture needles and threads were purchased from Shanghai Pudong Jinhuan Medical Supplies Co., Ltd.

[0067] 2. The small animal in vivo imaging system (IVIS Spectrum) was purchased from PerkinElmer, USA.

[0068] 3. The mouse lightweight brain stereotaxic instrument (Cat. No. 68805) was purchased from Shenzhen Ruiwode Life Science Co., Ltd.

[0069] Example 1: Method for screening tumor-resistant cell lines using in vivo circulation in mouse models

[0070] Since the human glioma cell line is selected to construct a mouse model, the human glioma cells need to be injected into the mouse skull, so the human glioma cells need to be labeled so that the tumor growth can be displayed. In this embodiment, luciferase is selected to label the human glioma cells. If the tumor cells are only injected subcutaneously, it is easier to observe the tumor formation of the tumor cells, and then the tumor cells do not need to be labeled.

[0071] 1. Construction of a human glioma cell line stably expressing luciferase.

[0072] 1) Construction of pLenti-CMV-luciferase expression plasmid:

[0073] Using the luciferase reporter vector pGL3.0-basic (product number: 3666650) purchased from Promega as a template, the luciferase fragment was cloned into the lentivirus (pLenti) system (Addgene, #658-5) by PCR amplification to construct a lentivirus plasmid expressing luciferase.

[0074] plenti-CMV-luciferase-EGFP, and the next step of the experiment was carried out after the sequence was confirmed to be completely correct by Sanger sequencing (Beijing BGI).

[0075] PCR primer sequences:

[0076] Luci-F (Seq ID No. 1): 5'

[0077] -CATGCTAGCGCCACCATGGAAGATGCCAAAAAC-3'.

[0078] Luci-R (Seq ID No. 2): 5'-AACCTCGAGTTACACGGCGATCTTGCC-3'.

[0079] 2) Lentivirus packaging

[0080] 293T cell plating: The constructed lentiviral expression plasmid pLenti-CMV-luciferase-EGFP and auxiliary packaging vector plasmid pMD.2G, psPAX2 were extracted with high purity and endotoxin-free plasmid mini-extraction kit (Cat. No.: DP118, Tiangen Biochemical Technology (Beijing) Co., Ltd.) and stained with Lipofectamine TM293T cells were transfected with 2000 transfection reagent. After 8 hours of transfection, the medium was replaced with DMEM medium containing 10% fetal bovine serum. The cell supernatant rich in lentiviral particles was collected once after 24 and 48 hours, combined and filtered with a 0.45 μm filter membrane, and centrifuged at 4 degrees at 20,000 revolutions per minute for 2 hours to concentrate the virus.

[0081] 3) Lentivirus infection of human glioma cell line U87MG

[0082] U87 cells were collected at 5×10 5 The cells were inoculated at a density of 10 cells / well in a six-well plate and cultured in a 37°C, 5% CO2 incubator. After 18 hours, an appropriate amount of virus was added according to the measured virus titer, and the culture medium was replaced with serum-containing DMEM cell culture medium after 24 hours. After 2 to 3 days of virus infection, hygromycin B was added at a final concentration of 50 μg / ml for resistance screening. After 3 days of screening, a portion was taken for luciferase quantitative determination. The specific method was as follows: 1×10 cells were plated in a 24-well plate, and 1×10 cells were plated. 5 cells / well, with three replicate wells in each group. On the second day, after the cells adhered to the wall, the substrate luciferin was added. The fluorescence value was measured by 20 / 20 luminescence detector (Promega). When the fluorescence value reached 4 million or more, the human glioma cell line U87-luc stably expressing luciferase was obtained. This cell line can be expanded as P0 generation cells for the next animal experiment.

[0083] 2. Establishment of drug-resistant cell lines of human glioma cells by in vivo circulation screening in mouse models

[0084] The main process of using mouse models to screen tumor-resistant cell lines in vivo is as follows: Figure 1 As shown, the main steps are as follows:

[0085] (1) Establishment of a mouse brain orthotopic glioma transplantation model

[0086] 1) Mice were selected from male BALB / c nude mice aged 4 to 6 weeks (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) and were raised under standard pathogen-free conditions with a 12-h light-dark cycle, a temperature of 20-22°C, and a humidity range of 40-50%. All animal experiments were performed under sodium pentobarbital anesthesia, and all measures were based on minimizing animal suffering and were approved by the Animal Experiment Ethics Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences.

[0087] 2) Orthotopic transplantation of tumor cells into the brain:

[0088] After the mouse was anesthetized with isoflurane inhalation anesthesia machine, the animal was fixed to the brain positioning device to keep the head immobile. The skin outside the skull was cut longitudinally with a scalpel to find the bregma point and the needle was inserted at the right anterior temporal side. The needle of a 5 ml syringe was used to rotate and pierce the hole to destroy the skull layer. 5×10 5 U87-luc cells (P0) that stably express luciferase were resuspended in 10 μl of saline. The cells were aspirated with a 10 μl microsyringe. The injection needle was inserted about 2 mm from the right anterior temporal side of the nude mouse. The injection needle was fixed and the cells were injected at a speed of 1 μl per minute. After the injection was completed, the needle was slowly withdrawn and the skin was sutured. The animal was returned to the animal room after it woke up.

[0089] On day 7 after orthotopic cell transplantation, in vivo imaging of mouse brain xenograft tumors was performed using IVIS Spectrum (PerkinElmer, USA) to confirm successful tumor implantation.

[0090] 3) Seven days after successful orthotopic transplantation of the tumor in the mouse brain, oral DMSO (50 mg / kg / day) or TMZ (50 mg / kg / day) treatment was started, with DMSO as a negative control. The treatment regimen was 5 days of treatment per week, 2 days of drug withdrawal, and continued for 3 to 4 weeks. Six mice were selected from each treatment group, and a total of three repeated treatment cycles were performed. The treatment cycle of drug administration-drug withdrawal cycles was used to simulate the treatment cycle of tumor chemotherapy drugs, making the cell resistance process closer to the actual clinical situation.

[0091] 4) Bioluminescence imaging was performed on days 7, 14, 21, and 28 of treatment to monitor intracranial tumor growth. At the same time, the weight of mice was recorded every other day, and the data were normalized to the luminescence value detected for each animal at the beginning of treatment.

[0092] 5) Separation of tumors and tumor cells: After completing a treatment cycle, mice were killed and the intact xenografted gliomas were peeled off for cell extraction. First, the tumor was mechanically separated and cut into 1 cubic millimeter tissue fragments, then washed with cold PBS containing penicillin (500U / ml), digested with 0.05% trypsin and DNase (1mg / ml) at 37°C for 15 minutes, and intermittently shaken to obtain a cell suspension. The cells were further filtered through a 100μm filter membrane (Life Science, 352360) and centrifuged at 300g per minute for 5 minutes at 4°C. The resulting cell pellet was resuspended and inoculated into a 6-well plate, completing the first round of in vivo screening under TMZ drug pressure. The first generation of drug-resistant tumor cells obtained was named T1-U87-luc, referred to as T1.

[0093] (2) In vivo circulation screening

[0094] 1) The T1 drug-resistant cells obtained above were transplanted back into the brains of another batch of mice according to the above steps, and the mice were treated with TMZ or DMSO at the same dose and treatment cycle and then killed; the tumor and tumor cells were separated to obtain the second-generation drug-resistant tumor cells, named T2-U87-luc glioma cells, referred to as T2.

[0095] 2) The T2 drug-resistant cells obtained above were transplanted back into the brains of another batch of mice according to the above steps, and the mice were treated with TMZ or DMSO at the same dose and treatment cycle and then killed; the tumor and tumor cells were separated to obtain the third-generation drug-resistant tumor cells, named T3-U87-luc glioma cells, referred to as T3.

[0096] 3) T2 and T3 cells were cultured in DMEM medium containing 10% FBS, 1% penicillin-streptomycin and 10 μM TMZ for subsequent experiments.

[0097] like Figure 2A The following are photos of the growth of three mouse brain transplanted tumors over time, taken using a small animal in vivo imaging system. Figure 2B The statistical method of two-way ANOVA is shown. Figure 2A Statistics of photon flux in the image Tumor growth curves at different screening rounds, where **P<0.01. Figure 2C The survival curves of tumor-bearing mice after TMZ screening with different screening times. FIG. 2A to FIG. 2C It can be seen that compared with the P0-U87 cell transplantation group (DMSO group in T1), the tumor cells transplanted into the mouse brain for the first time are sensitive to TMZ drugs. By using IVIS live imaging to monitor tumor growth every week, it can be seen that the tumor has shrunk on the 14th day; compared with the oral DMSO group, the tumor growth in the TMZ treatment group was significantly inhibited 14 to 21 days after transplantation, which indicates that the first round of screening killed most tumor cells due to the effect of the drug, and most of the cells (T1) obtained by the final screening may be insensitive to TMZ and survive in vivo. T2 and T3 cells obtained in the second and third rounds of in vivo cyclic transplantation screening using T1 cells have shown significant tolerance to TMZ through their in vivo growth. As the administration time prolonged, the tumors transplanted in the third round (T3) grew faster than those in the DMSO-treated group. At the same time, a survival curve analysis was performed on tumor-bearing mice with different treatments and cyclic screening times. Each group had 6 nude mice injected with cells from different screening rounds. The administration method was the same as that for separating cells. The results also showed that the survival time of mice in the T3 group after TMZ treatment was less than 28 days, even shorter than that of the group not treated with DMSO.

[0098] The above results show that the three-round cyclic screening of the present invention can promote the drug resistance of glioma cells in vivo. By constructing a mouse tumor model through the present invention, and then obtaining a method of simulating the in vivo environment-induced drug-resistant tumor cells through a cyclic drug administration screening method, the tumors or cells obtained in vivo and in vitro can be stably used in related research.

[0099] Example 2: TMZ tolerance and function of glioma cells with different drug resistance levels

[0100] IC50 (Inhibitory Concentration 50%) is a measure of the inhibitory effect of a drug or other chemical substance on biological activity. Specifically, the IC50 value refers to the drug concentration required to reduce a certain biological function or activity of the target cell or organism by 50% under experimental conditions. The IC50 value is often used in drug screening and evaluation of drug efficacy. A high IC50 value means that a higher drug concentration is required to achieve a 50% inhibitory effect, which usually indicates that the drug is less effective.

[0101] In this example, the IC50 values ​​of temozolomide were measured on the T2 and T3 cells screened in Example 1, and the drug resistance generation and degree of drug resistance of the drug-resistant cells obtained by establishing the above-mentioned in vivo evolutionary tumor resistance screening model were analyzed to determine the reliability of the in vivo resistance screening model.

[0102] 1.CCK8 detection of cell viability and IC50 value calculation:

[0103] (1) Cell culture:

[0104] U87MG and TMZ-resistant cells T2 and T3 obtained by in vivo screening were seeded into 96-well plates at 2000 cells per well and cultured for 24 hours to reach the logarithmic growth phase. U87MG was used as a negative control and marked as P0.

[0105] (2) TMZ treatment:

[0106] Prepare a series of drug solutions with different concentrations (100 μM, 200 μM, 300 μM, 400 μM, 500 μM), and add these different concentrations of TMZ-containing culture medium to different wells of the cell culture plate. Set up 3 replicate wells for each concentration to ensure the reliability of the data. Continue to culture the cells for 48 hours to ensure that the drug can fully exert its effect.

[0107] (3) CCK8 staining:

[0108] CCK8 reagent (Cell Counting Kit-8) was added to each well. Since WST-8 in CCK8 reagent would be reduced to orange-yellow formazan product by dehydrogenase in cells, the optical density (OD) of each well at 450nm wavelength could be measured. The OD value was proportional to the activity of cells.

[0109] (4) Calculate the IC50 values ​​of different cells' responses to TMZ based on the OD values:

[0110] According to the OD values ​​at different drug concentrations, a curve of TMZ concentration and cell viability (ratio of OD value to control group) is drawn. The drug concentration that reduces cell viability by 50%, i.e., IC50 value, is calculated by data fitting (e.g., using nonlinear regression analysis).

[0111] The results are as follows FIG. 3A to FIG. 3B As shown, Figure 3A and Figure 3B After 72 hours of exposure to different TMZ concentrations, T2 and T3, the cell viability was detected by CCK8 method, and the half lethality (IC50) value of TMZ was calculated. Figure 3A and Figure 3B It can be seen that compared with U87 cells before transplantation (P0-U87), cell viability assays showed that the half-maximal inhibitory concentration (IC50) of T2-U87 and T3-U87 cells to temozolomide (TMZ) increased by 2.7 and 4.8 times, respectively, indicating that in vivo circulation screening of resistant cells can promote the in vivo evolution of glioma cells under TMZ pressure and make the cells TMZ-resistant.

[0112] 2. The crystal violet-stained cell colony formation assay (Clonogenic Assay) is an experimental method used to evaluate cell proliferation ability and survival rate. The specific steps are as follows:

[0113] (1) Cell inoculation and culture:

[0114] After culturing T2 and T3 cells for 24 hours until the logarithmic growth phase, the cells were counted by trypan blue staining. 5000 cells were seeded per well in a 6-well plate to ensure uniform cell seeding.

[0115] (2) Cell treatment and colony formation

[0116] After the cells adhered, 10 μM TMZ or DMSO was added for treatment. After 48 hours, the culture medium was replaced with fresh drug-free culture medium. The cells were cultured for another 10 days. Cell proliferation was observed and colonies visible to the naked eye were formed (one colony usually consisted of more than 50 cells).

[0117] (3) Fixed cells

[0118] Prepare methanol or ice-cold methanol:ethanol (1:1) solution as fixative. Pour off the culture medium, gently wash the cells twice with PBS, and then add the fixative. Fix the cells for 15 minutes at room temperature, aspirate the fixative, and keep the culture plate dry at room temperature.

[0119] (4) Crystal violet staining

[0120] Prepare 0.5% crystal violet staining solution (0.5g crystal violet dissolved in 100ml 20% methanol aqueous solution). Add 1ml crystal violet staining solution to the fixed cells and stain for 15 minutes at room temperature. Pour out the staining solution and rinse the culture plate with tap water until the background becomes clear and transparent with no excess dye residue.

[0121] (5) Drying and observation and photography

[0122] After drying the stained culture plate at room temperature, observe and photograph it under a microscope or with the naked eye, and count the number of cell colonies formed in each well or dish.

[0123] (6) Data analysis

[0124] Count the number of colonies in each well or culture dish. Calculate the colony formation rate according to the colony formation rate calculation formula: colony formation rate (%) = number of colonies in the experimental group / number of cells inoculated × 100. Compare the colony formation rates of the treatment group and the control group to evaluate the effect of TMZ on cell proliferation ability.

[0125] The results are as follows FIG. 3C to FIG. 3F As shown, Figure 3C These are photos of cell clones under a light microscope after crystal violet staining of cell colonies after TMZ (10 μM) P0 and T2; Figure 3D This is the statistical result of cell clone counting in different treatment groups after P0 and T2 cells were treated with TMZ (10 μM); Figure 3E These are photos of cell clones under a light microscope after crystal violet staining of cell colonies after TMZ (10 μM) P0 and T3; Figure 3F The statistical results of cell clone counting in different treatment groups after TMZ (10 μM) treatment of P0 and T3 cells. FIG. 3C to FIG. 3F It can be seen that under the action of TMZ, the proliferation of U87 cells of the P0 generation was significantly inhibited, while the cell proliferation ability of the resistant cells T2 and T3 was not affected by the drug under the action of the drug, and the proliferation rate was even accelerated. From the above results, it can be seen that the method of the present invention for in vivo drug-resistant cell screening through 2 to 3 cycles can successfully separate drug-resistant tumor cells and obtain tumor cells with different drug resistance levels. These tumor cells with different drug resistance levels are obviously resistant to TMZ under drug pressure, and the cell proliferation ability also shows tolerance to the drug.

[0126] Example 3: Application of the method for establishing a glioma drug resistance model by in vivo evolution screening

[0127] In order to confirm the reliability of the obtained drug-resistant cells at the gene expression level and reveal the key molecular mechanisms related to drug resistance, this example obtained RNA extracted from glioma cell lines with different drug resistance levels through in vivo evolution screening, and constructed a transcriptome library to confirm the changes in gene expression in drug-resistant cells obtained by using the mouse in vivo circulation screening method. Further combined with high-throughput transcriptome analysis, the present invention also discovered differentially expressed non-coding RNAs in drug-resistant cells and sensitive tumor cells, especially newly assembled differentially expressed non-coding RNAs. These newly discovered non-coding RNAs are potential therapeutic targets and can also open up new avenues for tumor resistance research and personalized treatment strategies.

[0128] 1. Construction of transcriptome library

[0129] (1) RNA extraction

[0130] P0 (U87) cells before transplantation and T2 and T3 cells obtained after three rounds of in vivo evolution screening were collected, and 5×10 5 Add 750μl Trizol reagent to each cell pellet, mix well and place at room temperature for 5 minutes, add 200μl chloroform, mix vigorously for 15 seconds, and place at room temperature for 5 minutes. Centrifuge at 13000rpm per minute at 4℃ for 15 minutes, transfer 500μl of supernatant to a 1.5ml centrifuge tube, add 500μl isopropanol, mix well, and place the centrifuge tube in a -20℃ refrigerator to precipitate RNA overnight. The next day, take out the precipitated sample, centrifuge at 4℃ and 13000rpm for 20 minutes, discard the supernatant, add 500μl 75% ethanol to wash the precipitate, repeat this step once, aspirate the ethanol, dry the precipitate at room temperature, add 20μl DEPC water to dissolve the RNA precipitate, take 1μl sample and use NanoDrop to measure RNA concentration and quality.

[0131] (2) Construction of transcriptome library:

[0132] 1) Removal of ribosomal RNA

[0133] Take out 2 μg of total cell RNA from the RNA sample obtained above, add 10 μl rRNA probe mix (2 μg / μl), 4 μl 5× Hybridization buffer, mix well, put the PCR tube into the PCR instrument, set the reaction program: 95°C for 2 minutes, -0.1°C / s to 22°C, 22°C for 5 minutes, and put the sample on ice immediately after the reaction.

[0134] Add 3μl 10×RNase H buffer, 5μl RNase H (10U) and 2μl DEPC water to the sample after the reaction, mix well, put the sample into the PCR instrument, and set the program: 37℃30 minutes. Put the sample on ice immediately after the reaction. Purify the sample with RNAclean beads, and finally elute the RNA in 10μl denuclearized water and transfer it to a 0.2ml PCR tube. Add 4μl 10×TURBO buffer, 5μl TURBO DNase and 1μl DEPC water to the sample, mix well, put the sample into the PCR instrument, and set the program: 37℃30 minutes. Put the sample on ice immediately after the reaction. Purify the sample again with RNA clean beads, and finally elute the RNA in 10μl denuclearized water and transfer it to a 0.2ml PCR tube.

[0135] 2) RNA fragmentation and single-strand synthesis

[0136] Transfer the sample to a PCR tube, add 4μl 5×First strand buffer, mix well, put into a PCR instrument, react at 94℃ for 10 minutes, and immediately put on ice for first-strand cDNA synthesis. Add 0.5μl N6 primer (0.1μg / μl) to the RNA, mix well, incubate at 65℃ for 5 minutes in the PCR instrument, and quickly put on ice for at least 2 minutes. Prepare the following reaction mixture in a PCR tube: 1μl dNTP mix (10mM), 0.5μl 100mM DTT, 0.5μl RNase Inhibitor (40U / μl), 0.5μl Superscript II (200U / μl), mix well, put the PCR tube into the PCR instrument, and set the program: 25℃ for 10 minutes, 42℃ for 40 minutes, and 70℃ for 15 minutes. Put the sample on ice after the reaction is completed.

[0137] 3) Synthesis of cDNA Second Strand

[0138] Transfer the sample obtained in the previous step to a new 1.5 ml centrifuge tube, add 10 μl 5× Second strand buffer, 0.8 μl dNTP (dUTP) (25 mM), 0.2 μl RNase H (5 U / μl), 2.5 μl DNA pol I (10 U / μl), put the centrifuge tube into a thermomixer, and set the reaction program: 16°C for 2 hours, 300 rpm, 15 s / 2 min.

[0139] After the reaction is completed, AMPure XP magnetic beads (abbreviated as XP magnetic beads, Beckman) are mixed and balanced at room temperature for 30 minutes in advance. Then add 90μl (1.8×) XP magnetic beads to the reaction solution obtained in step 25 and mix gently. Let stand at room temperature for 5 minutes, transfer to a magnetic stand and let stand for 5 minutes, remove the supernatant, and rinse the magnetic beads twice with 200μl fresh 80% ethanol solution. Place the magnetic beads on the magnetic stand to dry for 2 minutes, add 43μl TE buffer to resuspend the magnetic beads, and pipette 50 times. Let stand at room temperature for 5 minutes, then place it on a magnetic stand and let stand for 5 minutes, aspirate 42μl supernatant, and add it to a 1.5ml centrifuge tube.

[0140] 4) DNA end repair, adding "A"

[0141] After the cDNA double-strand synthesis and purification, add 5μl 10×PNK buffer, 0.4μl dNTPs (25mM), 1.2μl T4 DNA polymerase (3U / μl), 0.2μl Klenowfragment (5U / μl), 1.2μl T4 PNK, mix well, put the centrifuge tube into the thermomixer, and react for 30 minutes at 20℃. After the reaction, add 90μl AMPurebeads for purification. The specific steps are the same as step 26. Finally, elute with 20.5μl TE buffer, aspirate 19.7μl supernatant, and add it to a new 1.5ml centrifuge tube.

[0142] Add 2.3 μl 10× blue buffer, 0.5 μl dATP (5 mM) and 0.5 μl Klenow exo-(3ˋto 5ˋexo minus) to the sample, mix well, place the centrifuge tube in a thermomixer, and react at 37°C for 30 minutes.

[0143] 5) Connector connection

[0144] After completing step 4), add 1.4μl 2×Rapid buffer, 0.1μl 10mMATP, 1μl adapter (2μM) and 1μl T4 quick DNA ligase (1U / μl) to the obtained sample, mix well, put the centrifuge tube into the thermomixer, and react for 15 minutes at 20℃. After the reaction, add 47.7μl AMPure beads for purification. The specific steps are the same as step 26. Finally, elute with 26μl TE buffer, aspirate 25μl supernatant, and add it to a new 1.5ml centrifuge tube. Add 45μl AMPure beads for secondary purification, and finally elute with 16.5μl TE buffer, aspirate 15.7μl supernatant, and add it to the PCR tube.

[0145] 6) Library construction

[0146] Step 5) The supernatant obtained was used as a template for PCR reaction in a PCR tube to obtain a PCR reaction solution (25 μl). The PCR reaction system was 25 μl: 15.7 μl of supernatant; 2.5 μl of 10×pfx buffer (Invitrogen); 1 μl of 10 μM upstream and downstream primers; 1 μl of 50 mM MgSO4 solution; 0.4 μl of 25 mM dNTP; 0.4 μl of pfx enzyme (Invitrogen); 3 μl of USER enzyme (NEB). The reaction procedure was: 37°C for 15 minutes; 94°C for 2 minutes; 94°C denaturation for 15 seconds, 62°C annealing for 30 seconds, 72°C extension for 30 seconds, 12 cycles; 72°C for 10 minutes. The PCR product was purified by adding 25 μl AMPure beads and dissolved in 25 μl TE solution. 1 μl of sample was quantified by Qubit 3.0, and the qualified samples were used for sequencing.

[0147] 2. Sequencing

[0148] The constructed RNA-seq library was sequenced using PE150 paired-end sequencing using an Illumina Xten sequencer.

[0149] 3. Transcriptome Library Data Analysis

[0150] (1) Library quality analysis

[0151] (2) Assembly and identification of long noncoding RNA

[0152] The RNA-seq data were analyzed in depth, and multiple steps were used to assemble and identify new non-coding RNAs. First, HISAT2 was used for efficient alignment to normalize the sequencing reads with the reference genome. StringTie was then used to assemble the transcripts, and through its transcriptome assembly function, potential new transcripts were identified and annotated. To further identify new non-coding RNAs, Cuffcompare was used to perform comparative analysis with known gene annotation databases to filter out known coding genes and annotated non-coding RNAs. CPC2 (Coding Potential Computational Tool) was used to evaluate the protein coding potential of these transcripts and screen out transcripts with low coding potential. Finally, after this series of analyses, 289 new non-coding RNAs were successfully identified, such as Figure 4A and Figure 4B As shown, Figure 4A (a), (b), and (c) are the correlation analysis of transcriptome library sequencing data from bioinformatics analysis of P0, T2, and T3, respectively. Figure 4B Heat map of differentially expressed genes for transcriptome sequencing analysis. Figure 4A and Figure 4B It can be seen that the above RNAs show low protein coding potential and are not recorded in the existing gene annotation database. Some lncRNAs whose expression levels have not changed, such as Figure 5A The figure shows the characteristic diagram of the overall coding capacity of the long non-coding RNA genes that are differentially expressed in glioma resistant cells through bioinformatics analysis, where the red line represents protein coding genes, the green line represents non-coding genes (annotated in the GENECODE database), and the blue line represents the non-coding genes newly discovered by transcriptome sequencing of the present invention. The results show that the coding capacity of non-coding genes is significantly lower than that of coding genes. Figure 5B The figure shows the distribution statistics of long non-coding RNA genes with differential expression in glioma resistant cells through bioinformatics analysis, where the red line represents protein-coding genes, the green line represents non-coding genes (annotated in the GENECODE database), and the blue line represents the non-coding genes newly discovered by transcriptome sequencing of the present invention, where (a) shows the distribution of genes in the promoter region, and (b) shows the distribution of genes in the gene body. Figure 5C Shown is a statistical graph of gene expression of differentially expressed long non-coding RNA genes in glioma resistant cells analyzed by bioinformatics, wherein the red line represents protein-coding genes, the green line represents non-coding genes (annotated in the GENECODE database), and the blue line represents the non-coding genes newly discovered by transcriptome sequencing of the present invention.

[0153] like Figure 5D This is a visualization result diagram of the TUG1 gene, which is known to have no significant changes in expression in glioma resistant cells. Figure 5DIt can be seen that TUG1 in the constructed transcriptome library was also found to have the same expression trend as previously reported after bioinformatics analysis, indicating the reliability of the library construction and data and analysis results used in Example 3 of the present invention.

[0154] It is worth noting that among the lncRNAs whose expression levels gradually increased with the increase of drug resistance level, 85 were newly discovered, while 204 were already annotated, such as Figure 5E Compared with mRNA, these newly discovered lncRNAs showed lower protein coding probability and lower conservation in coding and promoter regions, and the expression level of UCA1 gradually increased with the increase of drug resistance level. In addition, some genes with the same expression level and trend as UCA1 were also found in the newly assembled lncRNAs. Among them, a newly discovered lncRNA was named lncATRIG ( L ong N on c odingRNAs A associated with T MZ- R esistance i n G lioma), a long noncoding RNA associated with TMZ resistance in glioma. Fig. 5F In the visualization data screenshot of , it can be clearly seen that the expression level of lncATRIG gradually increased during the cyclic evolution process. This expression pattern is similar to Figure 5D This is in stark contrast to the potential tumor suppressor TUG1 shown in Figure 5E The expression of the positive control UCA1 in each cycle remains consistent. From the above results, it can be seen that the establishment of the in vivo evolution screening cell line provided by the present invention can identify the molecular mechanism related to TMZ resistance.

[0155] From the above examples, it can be seen that the present invention successfully constructs a model that can effectively screen tumor resistant cells through the in vivo evolution screening method. This model is of great significance in accelerating drug resistance research, exploring new drug resistance mechanisms and discovering potential therapeutic targets. The present invention discovers and verifies the regulatory role of non-coding RNA related to drug resistance through transcriptome analysis, providing new ideas for future anti-drug resistance treatment.

[0156] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention and these improvements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for screening tumor-resistant cell lines by in vivo circulation in a mouse model, characterized in that: The steps include: (1) Tumor cells were injected into mice and then fed for 7 days; (2) Treating mice with chemotherapy drugs to induce tumor resistance pressure, then killing the mice, isolating the tumors in the mice, chopping them up, and further digesting them with trypsin to obtain the first generation of drug-resistant tumor cells; (3) First-generation drug-resistant tumor cells were injected into mice and then fed for 7 days; (4) Continue to give the mice chemotherapy drugs to cause tumor resistance pressure, then kill the mice, isolate the tumors in the mice, cut them into pieces, and further digest them with trypsin to obtain the second generation of drug-resistant tumor cells; (5) The second-generation drug-resistant tumor cells were injected into mice and then fed for 7 days; (6) The mice were continued to be treated with chemotherapy drugs to induce tumor resistance pressure, and then the mice were killed, the tumors in the mice were isolated, cut into pieces, and further digested with trypsin to obtain the second generation of drug-resistant tumor cells. (7) The first to third generations of drug-resistant tumor cells were cultured in a culture medium containing 10% FBS, 1% penicillin-streptomycin, and 10 μM chemotherapeutic drugs to maintain their drug resistance under drug pressure.

2. The method according to claim 1, characterized in that The tumor cells in step (1) are patient-derived tumor cells or commercialized tumor cells.

3. The method according to claim 1, characterized in that The mice in step (1) are nude mice.

4. The method according to claim 1, characterized in that: The tumor cells in step (1) stably express luciferase or fluorescent protein tags.

5. The method according to claim 4, characterized in that The method for stably expressing luciferase or fluorescent protein in tumor cells comprises: a) constructing a lentiviral vector expressing luciferase or fluorescent protein; b) packaging the lentiviral vector; and c) infecting tumor cells with the packaged lentivirus to obtain tumor cells stably expressing luciferase or fluorescent protein.

6. The method according to claim 1, characterized in that The chemotherapy drug is temozolomide.

7. The method according to claim 1, characterized in that The chemotherapy drug treatment in step (2), step (4) and step (6) is to administer the chemotherapy drug at 50 mg / kg / day, 5 days a week, and stop for 2 days, for 3 to 4 weeks.

8. Use of the tumor drug-resistant cell line according to any one of claims 1 to 7 in screening anti-drug resistance drugs, characterized in that: The steps include: (1) Extract RNA from tumor-resistant cell lines to construct transcriptome libraries; (2) Sequencing the transcriptome library; (3) Analyze library quality and identify novel long noncoding RNAs.

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