Human breast cancer Pyrotinib drug-resistant cell strain HCC1954-PR as well as construction method and application of human breast cancer Pyrotinib drug-resistant cell strain HCC1954-PR

Abstract

The invention discloses a human breast cancer Pyrotinib drug-resistant cell strain HCC1954-PR as well as a construction method and application thereof.According to the human breast cancer Pyrotinib drug-resistant cell strain HCC1954-PR, drug-resistant cells in an HCC1954 cell line are screened step by step through Pyrotinib induction culture from low concentration to high concentration, stable passage and culture can be performed in Pyrotinib with the concentration of 2 mol / L, and a cell line which is higher in growth capacity and insensitive to Pyrotinib treatment is obtained. The construction period of the whole drug-resistant cell is one year or more. After the drug-resistant cell strain is constructed, the drug-resistant cell strain is cultured for 3 weeks, and the drug resistance of the drug-resistant cell strain is detected, which shows that the drug-resistant cell strain has good drug-resistant stability. After being treated by Pyrotinib with the concentration of 1mol / L, the strain still has the capability of forming cell clone. The invention is corresponding to breast cancer patients with acquired drug resistance to Pyrotinib clinically, and provides a drug-resistant tumor cell model for researching the drug-resistant mechanism of the patients, developing tumor drug-resistant reversal drugs, researching more effective tumor treatment methods and the like.

Description

Technical Field

[0001] The present invention relates to the field of tumor biology and cell culture technology, and in particular to a human breast cancer Pyrotinib-resistant cell line HCC1954-PR, a construction method and an application thereof. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide, and breast cancer with overexpression of human epidermal growth factor receptor 2 (HER2) accounts for about 15-20% of all breast cancers, and is a more aggressive subtype of all breast cancers. Tyrosine kinase inhibitors (TKIs) targeting the HER family are used in the clinical treatment of breast cancer and have improved the prognosis of patients with HER2-positive breast cancer. However, clinical applications have found that some patients with HER2-positive breast cancer are resistant to TKI treatment, so it is of great significance to clarify its specific mechanism and effectively overcome TKI resistance.

[0003] HER family receptors are transmembrane proteins, and the intracellular segment is composed of a tyrosine kinase domain and C-terminal amino acid residues. TKIs inhibit the activity and phosphorylation of HER family tyrosine kinases by competing with ATP to bind to the ATP binding site in the receptor tyrosine kinase region, blocking the HER downstream cell signaling pathway, thereby inhibiting the proliferation of tumor cells, inducing cell cycle arrest and promoting cell apoptosis, thereby achieving the purpose of inhibiting tumors.

[0004] Pyrotinib is also a tyrosine kinase inhibitor, and there is a lack of research on the mechanism of tumor cell resistance to Pyrotinib. To clarify its resistance mechanism, it is necessary to first establish a resistant strain, and then conduct molecular mechanism research on this basis. Based on the existing method of screening resistant strains, an acquired resistant strain is constructed, so that the screening method described in this application can be generally applied to the construction of acquired resistant cell lines. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention provides a human breast cancer Pyrotinib-resistant cell line HCC1954-PR, a construction method and an application thereof.

[0006] The present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a human breast cancer Pyrotinib-resistant cell line HCC1954-PR Homosapiens, the cells are deposited in the China Center for Type Culture Collection, the address of the depository is Wuhan University, Wuhan, China, the deposit number is: CCTCC NO: C2024255, and the deposit date is August 6, 2024.

[0007] Furthermore, after the establishment of the Pyrotinib-resistant human breast cancer cell line, the cell line still maintained good drug resistance after 2 and 3 weeks of drug withdrawal and culture.

[0008] In a second aspect, the present invention provides a method for culturing a human breast cancer Pyrotinib-resistant cell line HCC1954-PR, the specific steps of which are as follows: (1) Detect the half effective inhibitory concentration (IC) of Pyrotinib on HCC1954 cell line 50 )value; (2) Using the IC obtained in step (1) 50 One third of the concentration is the initial concentration, and the method of gradually increasing the drug concentration is used to induce the growth and passage of human breast cancer cells HCC1954 in Pyrotinib from low concentration to high concentration, and finally the drug-resistant cell line HCC1954-PR is obtained; (3) One week after drug withdrawal, the IC of Pyrotinib against HCC1954-PR cell line and HCC1954 cell line was detected. 50 value; (4) After 2 and 3 weeks of drug withdrawal, the IC of Pyrotinib on HCC1954-PR cell line was detected. 50 .

[0009] Furthermore, the method of gradually increasing the drug concentration is used to induce the growth and passage of human breast cancer cells HCC1954 in an environment of Pyrotinib from low concentration to high concentration, thereby obtaining the drug-resistant cell line, including: ① In a 100 mm cell culture dish, human breast cancer cells HCC1954 were cultured with complete medium. When the cell density reached 70-80%, the liquid in the cell culture dish was discarded, the cell culture dish was rinsed with PBS buffer, and treated with complete medium containing an initial concentration of Pyrotinib for 23-25 ​​hours. The initial concentration was IC 50 one-third of the concentration; ② Repeat the process of rinsing with PBS buffer and treating with complete culture medium containing the initial concentration of Pyrotinib until the cell survival rate reaches 70-80% after Pyrotinib treatment. End the treatment with the initial concentration of Pyrotinib, discard the liquid in the cell culture dish, rinse the cell culture dish with PBS buffer, digest the cells with trypsin-EDTA solution and transfer them to a 100 mm cell culture dish at a ratio of 1:2.

[0010] ③ After culturing in complete medium for 23 to 25 hours, repeat the process of rinsing with PBS buffer and treating with complete medium containing the next gradient concentration of Pyrotinib, with a gradient concentration difference of 50 nmol / L, until the cell survival rate reaches 70-80% after treatment with Pyrotinib concentration of 2 µmol / L, and repeatedly culture the cell line at the final concentration of Pyrotinib to obtain a stable Pyrotinib-resistant cell line HCC1954-PR, and use the stable cell line as the resistant cell line.

[0011] In a third aspect, the present invention provides the use of a human breast cancer Pyrotinib-resistant cell line HCC1954-PR in the preparation of a product having at least one of the following functions (1) to (4): (1) Constructing tumor cell models, cell line organoid models, or preparing tumor animal models; (2) Construction of in vitro tumor resistance drugs; (3) Constructing in vitro screening and evaluation of targeted drugs or chemotherapeutic drugs; (4) Construct and develop in vitro drugs to reverse tumor resistance.

[0012] Compared with the prior art, the present invention has the following advantages: (1) HCC1954-PR of the present invention can stably grow, passage and recover in a 2µmol / L concentration of Pyrotinib. After the establishment of the drug-resistant cell line, the drug-withdrawal culture was repeated for up to 3 weeks to test its drug resistance, and the drug-resistant cell line had good drug resistance stability.

[0013] (2) The present invention provides a method for constructing a human breast cancer Pyrotinib-resistant cell line, which is feasible and easy to operate. The present invention provides a drug-resistant tumor cell model for studying tumor resistance mechanisms, analyzing chemotherapy drug sensitivity, screening and evaluating chemotherapy drugs, developing tumor resistance reversal drugs, and studying more effective tumor treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with the accompanying drawings.

[0015] Figure 1 This is the morphology of the HCC1954 cell line in the logarithmic growth phase; Figure 2 This is the morphology of the HCC1954-PR cell line in the logarithmic growth phase; Figure 3 Schematic diagram of the proliferation inhibition of HCC1954 cell line detected by CCK-8 method; Figure 4 Schematic diagram of the CCK-8 method for detecting the proliferation inhibition of HCC1954 cell line and HCC1954-PR cell line; Figure 5 Schematic diagram of the proliferation inhibition of HCC1954-PR cell line detected by CCK-8 method after 2 and 3 weeks of drug withdrawal; Figure 6 Schematic diagram of observing the cell clone formation ability after treating HCC1954 cell line and HCC1954-PR cell line without drug or with 1µmol / L concentration of Pyrotinib. DETAILED DESCRIPTION

[0016] Below in conjunction with specific embodiment, the technical scheme of the present invention is clearly and completely described. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the protection scope of the present invention. If specific conditions are not indicated in the embodiment, it is carried out according to the conditions recommended by normal conditions or manufacturers. If the manufacturer is not indicated in the reagents used or the instruments, they are all conventional products that can be obtained by commercial purchase.

[0017] Example 1 The steps for establishing the drug-resistant cell line of the present invention are as follows: (1) Human breast cancer HCC1954 cell line, after recovery (cell recovery in this step is similar to cell culture, etc., which is a basic and commonly used operation method), use RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (100×) and culture in a 5% vol CO2, 37°C incubator as usual; (2) Cytotoxicity experiments were performed using the Cell Counting Kit 8 (CCK-8) produced by Wuhan Elior Biotech Co., Ltd.

[0018] Collect logarithmic phase cells of human breast cancer HCC1954 cell line and adjust the cell suspension to a concentration of 1×10 4 / ml, add 100μL of cell suspension to each well of a 96-well plate, and plate the cells to be tested to a density of 1000 cells / well. Culture overnight in a 37°C, 5% volCO2 incubator until the cell monolayer adheres to the wall, then use Pyrotinib at concentrations of 0.01nmol / L to 10µmol / L, with 5 replicate wells for each concentration. Measure the absorbance 5 days after drug treatment. Add 10μL of CCK-8 solution to each well. After continuing to incubate in the cell culture incubator for 2h, use an enzyme reader (Tecan, Switzerland) to measure the absorbance at 450nm and calculate the IC of the drug 50 .

[0019] (3) Human breast cancer cells HCC1954 were cultured in complete culture medium in a 100 mm cell culture dish. When the cell density reached 70-80%, the liquid in the cell culture dish was discarded, the cell culture dish was rinsed with PBS buffer, and treated with complete culture medium containing an initial concentration of Pyrotinib for 23-25 ​​hours. The initial concentration was IC 50 one-third of the concentration; (4) Repeat the process of rinsing with PBS buffer and treating with complete culture medium containing the initial concentration of Pyrotinib until the cell survival rate reaches 70-80% after Pyrotinib treatment. End the treatment with the initial concentration of Pyrotinib, discard the liquid in the cell culture dish, rinse the cell culture dish with PBS buffer, digest the cells with trypsin-EDTA solution and transfer them to a 100 mm cell culture dish at a ratio of 1:2.

[0020] (5) After culturing in complete medium for 23 to 25 hours, the cells were repeatedly washed with PBS buffer and treated with complete medium containing the next gradient concentration of Pyrotinib, with a gradient concentration difference of 50 nmol / L, until the cell survival rate reached 70-80% after treatment with a Pyrotinib concentration of 2 µmol / L, and the cell line was repeatedly cultured and passaged at the final concentration of Pyrotinib to obtain a stable Pyrotinib-resistant cell line HCC1954-PR, and the stable cell line was used as the resistant cell line. The resistant cell construction cycle is 1 year or more.

[0021] The CCK-8 method was used to detect cell proliferation and the stability of the human breast cancer Pyrotinib-resistant cell line HCC1954-PR. It was found that the human breast cancer Pyrotinib-resistant cell line had good drug resistance. After the construction of the resistant cell line, the drug resistance was retested after 2 and 3 weeks of culture with withdrawal of the drug, showing good stability of drug resistance. After being treated with 1µmol / L concentration of Pyrotinib, it still had the ability to form cell clones. It can provide new research methods for constructing a human breast cancer Pyrotinib-resistant model, studying the resistance mechanism of human breast cancer to Pyrotinib, screening and evaluating a variety of targeted drugs or chemotherapeutic drugs, and studying more effective tumor treatment methods.

[0022] Specifically, the established HCC1954-PR cell line was subjected to morphological observation and biological characteristics identification: 1. Morphological observation Take one cell culture dish each of HCC1954 and HCC1954-PR cell lines in the logarithmic growth phase, change the medium, and observe the cell morphology under an inverted microscope and take photos. Figure 1 and Figure 2As shown, the parental cell HCC1954 has a consistent morphology and a larger cell volume; the drug-resistant cell HCC1954-PR has a consistent morphology and a smaller cell volume.

[0023] 2. Determination of the proliferation inhibition curve of human breast cancer HCC1954 cell line The cytotoxicity experiment used the CCK-8 kit of Wuhan Eliorite Biotechnology Co., Ltd. The logarithmic phase cells of human breast cancer HCC1954 cell line were collected and the cell suspension concentration was adjusted to 1×10 4 / ml, add 100μL of cell suspension to each well of a 96-well plate, and plate the cells to be tested so that the density is 1000 cells / well. Culture overnight in a 37°C, 5% vol CO2 incubator until the cell monolayer adheres to the wall, then use Pyrotinib at concentrations of 0.01nmol / L to 10µmol / L, and set up 5 replicate wells for each concentration. Measure the absorbance 5 days after drug treatment. Add 10μL of CCK-8 solution to each well. After continuing to incubate in the cell culture incubator for 2h, use an enzyme reader (Tecan, Switzerland) to measure the absorbance at 450nm. Repeat each group of experiments 3 times or more, and draw a cell growth curve. Figure 3 shown.

[0024] 3. Determination of the drug resistance of human breast cancer pyrotinib-resistant cell lines The cytotoxicity experiment used the CCK-8 kit of Wuhan Eliorite Biotechnology Co., Ltd. The logarithmic phase cells of human breast cancer HCC1954 cell line and human breast cancer Pyrotinib-resistant cell line HCC1954-PR were collected and the cell suspension concentration was adjusted to 1-3×10 4 / ml, add 100μL cell suspension to each well of a 96-well plate, and plate the cells to be tested to a density of 1000~3000 cells / well. Culture overnight in a 37℃, 5% vol CO2 incubator until the cell monolayer adheres to the wall, and then use Pyrotinib at concentrations of 0.01nmol / L~10µmol / L, with 5 replicates for each concentration. Measure the absorbance value 5 days after drug treatment. Add 10μL CCK-8 solution to each well. After continuing to incubate in the cell culture incubator for 2h, use an enzyme reader (Tecan, Switzerland) to measure the absorbance at 450nm. Repeat each group of experiments 3 times or more, and draw a cell growth curve. The results showed that the proliferation of human breast cancer HCC1954 cell line was inhibited after treatment with Pyrotinib, while the inhibitory effect of Pyrotinib on the proliferation of HCC1954-PR was weak, indicating that the human breast cancer Pyrotinib-resistant cell line HCC1954-PR has obvious characteristics of a resistant tumor cell line. Figure 4 shown.

[0025] 4. Determination of the drug resistance stability of the human breast cancer pyrotinib-resistant cell line HCC1954-PR The cytotoxicity experiment used the CCK-8 kit of Wuhan Eliorite Biotechnology Co., Ltd. The logarithmic phase cells of the human breast cancer Pyrotinib-resistant cell line HCC1954-PR were collected 2 weeks and 3 weeks after withdrawal of the drug, and the cell suspension concentration was adjusted to 1-3×10 4 / ml, add 100μL of cell suspension to each well of a 96-well plate, and plate the cells to be tested so that the density is 1000~3000 / well. Culture overnight in a 37℃, 5% vol CO2 incubator until the cell monolayer adheres to the wall, and then use Pyrotinib at concentrations of 0.01nmol / L~10µmol / L, with 5 replicate wells for each concentration. Measure the absorbance value 5 days after drug treatment. Add 10μL of CCK-8 solution to each well. After continuing to incubate in the cell culture incubator for 2h, use an enzyme reader (Tecan, Switzerland) to measure the absorbance at 450nm. Repeat each group of experiments 3 times or more, and draw a cell growth curve. The results showed that after the construction of the HCC1954-PR cell line, the drug was withdrawn and cultured for 2 and 3 weeks, and the cells had good drug resistance. Figure 5 shown.

[0026] 5. Determination of the ability of the drug-resistant cell line HCC1954-PR to form cell clones after drug treatment The logarithmic phase cells of human breast cancer HCC1954 cell line and the logarithmic phase cells of human breast cancer Pyrotinib-resistant cell line HCC1954-PR cell line after withdrawal of drug for 1 week were collected respectively, and the concentration of cell suspension was adjusted to 1×10 4 / ml, add 100μL of cell suspension to each well of a 6-well plate, and plate the cells to be tested so that the density is 1000 cells / well. Culture overnight in a 37°C, 5% vol CO2 incubator until the cell monolayer adheres to the wall, and treat with drug-free culture medium or culture medium containing 1µmol / L Pyrotinib for 5 days, then replace the drug-free culture medium and continue culturing for 1 week. Then fix with 4% paraformaldehyde at room temperature for 30 minutes, and then stain with crystal violet at room temperature for 30 minutes and take a picture. The results showed that the HCC1954-PR cell line can still form cell clones after treatment with 1µmol / L concentration of Pyrotinib. Figure 6 shown.

[0027] In summary, the HCC1954-PR of the present invention can stably grow, passage, and recover in a 2µmol / L concentration of Pyrotinib. After the establishment of the resistant cell line, the drug was withdrawn and cultured for up to 3 weeks to retest its drug resistance. The resistant cell line has good drug resistance stability and still has the ability to form cell clones after being treated with 1µmol / L concentration of Pyrotinib. The present invention also provides a method for constructing a human breast cancer Pyrotinib-resistant cell line, and the construction method is feasible and easy to operate. The present invention provides a resistant tumor cell model for studying tumor resistance mechanisms, analyzing chemotherapy drug sensitivity, screening and evaluating chemotherapy drugs, developing tumor resistance reversal drugs, and studying more effective tumor treatment methods.

[0028] Example 2 A use of a human breast cancer Pyrotinib-resistant cell line HCC1954-PR, comprising one or more of the following: (1) Construction of tumor cell models, cell line organoid models, tumor animal models, etc.; wherein, tumor cell models include progeny cells established from this cell line or cells established from progeny cells established from this cell line by transfection of fluorescently labeled genes. Animal tumor models include animal models of human breast cancer established by subcutaneous tumor loading or tail vein injection.

[0029] (2) Studying the mechanism of tumor resistance in vitro; the method for studying the mechanism of tumor resistance can be: using the progeny cells and the constructed model established by the human breast cancer Pyrotinib-resistant cell line HCC1954-PR, after treatment with or without Pyrotinib, extracting DNA, RNA, non-coding RNA, and protein for sequencing, and then analyzing to obtain differential signaling pathways or molecules, and using drugs that can specifically target the differential signaling pathways or molecules to treat the progeny cells and the constructed model established by the human breast cancer Pyrotinib-resistant cell line HCC1954-PR to determine its resistance mechanism.

[0030] (3) In vitro screening and evaluation of multiple targeted drugs or chemotherapeutic drugs; the method for screening tumor targeted drugs or chemotherapeutic drugs can be: adding different targeted drugs or chemotherapeutic drugs to the culture medium of the human breast cancer Pyrotinib-resistant cell line HCC1954-PR, observing the drug cytotoxicity, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, and the IC of the screened effective drugs is calculated. 50 , select IC 50 The lowest drug was further applied to animal models and compared with the survival period, tumor size, metastasis, etc. of animals in the non-drug group to screen for potential drugs for treating Pyrotinib-resistant human breast cancer cell lines.

[0031] (4) Developing tumor resistance reversal drugs in vitro. The method for developing tumor resistance reversal drugs can be: adding resistance reversal drugs to the culture medium of the human breast cancer Pyrotinib-resistant cell line HCC1954-PR, observing the drug cytotoxicity, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, and the IC of the screened effective drugs is calculated. 50 , select IC 50 The lowest drug was further applied to animal models and compared with the survival period, tumor size, metastasis, etc. of animals in the non-drug group to screen for potential resistance-reversing drugs.

Claims

1. A human breast cancer Pyrotinib-resistant cell line HCC1954-PR, characterized in that: The cells are deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: C2024255 and the deposit date is August 6, 2024.

2. The human breast cancer Pyrotinib-resistant cell line HCC1954-PR according to claim 1, characterized in that: After the establishment of the human breast cancer Pyrotinib-resistant cell line, the cell line still maintained good drug resistance after 2 and 3 weeks of drug withdrawal and still had the ability to form cell clones after treatment with 1µmol / L Pyrotinib.

3. A method for culturing a human breast cancer Pyrotinib-resistant cell line HCC1954-PR, characterized in that: The specific steps are as follows: (1) Detection of IC of Pyrotinib against HCC1954 cell line 50 concentration; (2) Using the IC obtained in step (1) 50 One third of the concentration is the initial concentration, and the method of gradually increasing the drug concentration is used to induce the growth and passage of human breast cancer cells HCC1954 in Pyrotinib from low concentration to high concentration, thereby obtaining the drug-resistant cell line HCC1954-PR; (3) One week after drug withdrawal, the IC of Pyrotinib against HCC1954-PR cell line and HCC1954 cell line was detected. 50 ; (4) After 2 and 3 weeks of drug withdrawal, the IC of Pyrotinib on HCC1954-PR cell line was detected. 50 ; (5) After treatment with 1 µmol / L pyrotinib, the ability of HCC1954-PR cell lines and HCC1954 cell lines to form cell clones was detected.

4. The construction method according to claim 3, characterized in that: The drug-resistant cell line is obtained by inducing human breast cancer cells HCC1954 to grow and passage in an environment of Pyrotinib from low concentration to high concentration by gradually increasing the drug concentration, including: (1) Human breast cancer cells HCC1954 were cultured in complete culture medium in a 100 mm cell culture dish. When the cell density reached 70-80%, the liquid in the cell culture dish was discarded, the cell culture dish was rinsed with PBS buffer, and the dish was treated with complete culture medium containing the initial concentration of Pyrotinib for 23-25 ​​hours; (2) Repeat the process of rinsing with PBS buffer and treating with complete medium containing the initial concentration of Pyrotinib until the cell survival rate reaches 70% to 80% after Pyrotinib treatment. End the treatment with the initial concentration of Pyrotinib, discard the liquid in the cell culture dish, rinse the cell culture dish with PBS buffer, digest the cells with trypsin-EDTA solution and transfer them to a 100 mm cell culture dish at a ratio of 1:2; (3) After culturing with complete medium for 23 to 25 hours, the cells were rinsed with PBS buffer and treated with complete medium containing the next gradient concentration of Pyrotinib, with a gradient concentration difference of 50 nmol / L, until the cell survival rate reached 70% to 80% after treatment with Pyrotinib at a concentration of 2 µmol / L. The cell line was repeatedly cultured at the final concentration of Pyrotinib to obtain a stable Pyrotinib-resistant cell line HCC1954-PR, and the stable cell line was used as the resistant cell line.

5. The construction method according to claim 3, characterized in that: The specific operation of step (3) is as follows: After withdrawing Pyrotinib from the HCC1954-PR cell line for 1 week, the CCK-8 kit is used to test the IC of Pyrotinib against the HCC1954-PR cell line and the HCC1954 cell line. 50 concentration.

6. The construction method according to claim 3, characterized in that: The specific operation of step (4) is as follows: After withdrawing Pyrotinib from the HCC1954-PR cell line for 2 weeks and 3 weeks, the CCK-8 kit is used to test the IC of Pyrotinib on the HCC1954-PR cell line. 50 concentration.

7. The construction method according to claim 3, characterized in that: The specific operation of step (5) is as follows: collect logarithmic phase cells of human breast cancer HCC1954 cell line and logarithmic phase cells of human breast cancer Pyrotinib-resistant cell line HCC1954-PR after withdrawal of drug for 1 week, and adjust the cell suspension concentration to 1×10 4 / ml, add 100μL cell suspension to each well of a 6-well plate, and plate the cells to be tested to a density of 1000 cells / well; culture in a 37℃, 5% vol CO2 incubator overnight until the cell monolayer adheres to the wall, and treat with drug-free medium or medium containing 1µmol / L Pyrotinib for 5 days, then replace with drug-free medium and continue culturing for 1 week, then fix with 4% paraformaldehyde at room temperature for 30 minutes, and then stain with crystal violet at room temperature for 30 minutes and take pictures.

8. Use of the human breast cancer Pyrotinib-resistant cell line HCC1954-PR according to claim 1 in the preparation of a product having at least one of the following functions (1) to (4): (1) Constructing tumor cell models, cell line organoid models, or preparing tumor animal models; (2) Construction of in vitro tumor resistance drugs; (3) Constructing in vitro screening and evaluation of targeted drugs or chemotherapeutic drugs; (4) Construct and develop in vitro drugs to reverse tumor resistance.

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