Kidney cancer axitinib drug-resistant cell model as well as construction method and application thereof
By constructing a cell model of drug-resistant axitinib in renal cancer, the problem of unclear mechanism of drug resistance to renal cancer is solved, providing important tools for research and treatment, improving the efficacy of drugs, and overcoming drug resistance. Provide better treatment options for kidney cancer patients.
Patent Information
- Application Number
- CN202411749180.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-01-14
AI Technical Summary
The mechanism of drug resistance of renal cancer to axitinib is unclear, and the lack of effective targets and cellular models for research is made difficult to solve for therapeutic drug resistance.
A subcutaneous tumor-bearing model of nude mice was used to construct axitinib drug-resistant cell model of renal cancer through continuous passage method, which simulated the in vivo drug resistance environment, and was used to study drug resistance mechanisms and screen targets.
It provides a real and accurate research platform to help explore the mechanisms of axitinib resistance in renal cancer and reverse drug resistance strategies, improve the efficacy of the drug, overcome drug resistance, and provide patients with better treatment options.
Smart Images
Figure CN119307455A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a renal cancer axitinib-resistant cell model and a construction method and application thereof. Background Art
[0002] Renal cell carcinoma (RCC) originates from renal tubular epithelial cells. It is a highly malignant tumor in the urinary system and one of the most common tumors, accounting for 80% to 90% of renal malignant tumors and about 3% of all tumor cases worldwide. Renal cancer has an insidious onset, and about 30% of patients are found to have distant metastases at the time of initial diagnosis. More than one-third of renal cancer patients will still relapse after surgery. In the past, the first-line treatment strategy for advanced renal cancer was mainly targeted therapy with multi-target tyrosine kinase inhibitors. However, with the development of immune checkpoint inhibitors, the latest renal cancer guidelines at home and abroad have recommended targeted therapy combined with immunotherapy as the first choice for advanced renal cancer, especially for medium- and high-risk stratification. Although combined therapy can improve efficacy, drug resistance is still an important issue. Some patients may develop primary or acquired resistance to combined therapy. Therefore, exploring the resistance mechanism, overcoming the resistance problem, and improving drug efficacy have become key issues that need to be urgently addressed in the treatment of renal cancer.
[0003] Among the targeted drugs for combination therapy, only axitinib has been approved for marketing in my country and is used to treat advanced renal cancer. Therefore, axitinib combined with pembrolizumab has become the first-line treatment for medium- and high-risk advanced renal cancer in China. However, 60%-70% of patients with metastatic renal cancer still have inherent resistance to axitinib, and even those who are effective at the beginning of combined treatment will relapse after 16 months. At present, the biological characteristics and pathogenesis of axitinib resistance in renal cancer are still unclear. Therefore, exploring the molecular mechanism of renal cancer resistance to axitinib and finding the targets of axitinib resistance in renal cancer are of great guiding significance for clinical treatment and prognosis judgment. Summary of the invention
[0004] 1. Problems to be solved
[0005] In view of the unclear drug resistance mechanism of axitinib used for the treatment of renal cancer in the prior art, there is a lack of drug resistance targets and molecular mechanism cell models for research. This application provides a method for constructing a renal cancer axitinib-resistant cell model and its application. Using renal cancer cell lines as research objects, a nude mouse axitinib gavage continuous passage method is used to establish a renal cancer axitinib-resistant cell model, which provides a solid foundation for extensive and in-depth research on the tolerance mechanism and the exploration of more potential therapeutic targets.
[0006] 2. Technical solution
[0007] In order to achieve the above purpose, the technical solution provided is:
[0008] The method for constructing axitinib-resistant renal cancer cell model comprises the following steps:
[0009] A nude mouse subcutaneous tumor model was established using a renal cancer cell line to obtain nude mouse subcutaneous tumors;
[0010] When the subcutaneous tumor of the nude mice reaches the target volume, the nude mice are gavaged with axitinib, and the tumor tissue is separated to obtain the first generation of drug-resistant renal cancer cells;
[0011] The first generation of drug-resistant renal cancer cells were cut into tumor pieces and inoculated into the armpits of nude mice, and the above steps of oral administration, separation and inoculation were repeated to obtain the second and third generation of drug-resistant renal cancer cells respectively;
[0012] The third-generation drug-resistant renal cancer cells were cut into pieces, digested, and re-cultured in a culture dish to obtain a renal cancer axitinib-resistant cell model.
[0013] Preferably, the axitinib is administered intragastrically for 6 weeks at a dosage of 10 mg / kg / day.
[0014] Furthermore, the renal cancer cell line is a human renal clear cell carcinoma 786-O cell line or a human renal clear cell carcinoma 769-P cell line.
[0015] Furthermore, the inoculation amount of the renal cancer cell line is 4.5×10 6 / 100μL~5.5×10 6 / 100μL.
[0016] Furthermore, the target volume is ≥ 200 mm 3 .
[0017] Furthermore, the tumor mass was inoculated into the armpit of nude mice by gas anesthesia surgery.
[0018] Preferably, the tumor mass is 0.5 mm 3 ~1.5mm 3 .
[0019] Furthermore, the digestion was performed using ACCUMAX TM Solution.
[0020] Furthermore, in the axitinib-resistant renal cancer cell model, the growth of axitinib-resistant cells in nude mice is not inhibited by axitinib and can tolerate 15 μM axitinib concentration in vitro.
[0021] A renal cancer axitinib-resistant cell model is constructed by any of the methods described above.
[0022] Application of the axitinib-resistant cell model for renal cancer: applying the axitinib-resistant cell model for renal cancer to the study of the resistance mechanism of renal cancer cell targeted therapy.
[0023] The application of the axitinib-resistant cell model for renal cancer is to apply the axitinib-resistant cell model for renal cancer to the screening or preparation of targeted drugs for renal cancer, the screening of drug-resistant targets for targeted therapy for renal cancer, or the reversal of resistance testing for targeted therapy.
[0024] 3. Beneficial effects
[0025] Compared with the existing known technology, the technical solution provided by the present invention has the following beneficial effects:
[0026] (1) The method for constructing a renal cancer axitinib-resistant cell model of the present invention is to construct a nude mouse subcutaneous tumor model using a renal cancer cell line, and when the subcutaneous tumor of the nude mouse reaches a certain volume, the nude mouse is given axitinib by gavage. After the treatment, the tumor tissue is separated to obtain the first generation of drug-resistant renal cancer cells; the first generation of drug-resistant renal cancer cells are cut into tumor masses and inoculated in the armpits of the nude mice, and the above-mentioned gavage, separation, and inoculation steps are repeated to obtain the second and third generation of drug-resistant renal cancer cells respectively; the third generation of drug-resistant renal cancer cells are cut into pieces, digested, and re-cultured in a culture dish. Tumor-bearing nude mice are treated by gavage with axitinib, and drug pressure screening is performed using the method of continuous passage in tumor-bearing nude mice. The subcutaneous tumors of the first generation of nude mice (first generation of drug-resistant renal cancer cells) are directly cut into tumor masses and inoculated in the armpits of the second generation of nude mice, which can save a lot of operation steps and time, and the tumor cells are less stimulated by the outside world, thereby improving the success rate of tumor mass growth in mice after passage. Finally, by isolating the third-generation nude mouse subcutaneous tumor (third-generation resistant renal cancer cells) and preparing it into a tumor cell suspension for plating, a renal cancer cell line with axitinib resistance both in vivo and in vitro was obtained.
[0027] (2) Compared with the resistant cell model constructed by the in vitro concentration increment method, the renal cancer axitinib-resistant cell model of the present invention can simulate the in vivo drug-resistant environment more realistically and accurately, and provides an important research tool and platform for the study of the mechanism of renal cancer axitinib resistance and the formulation of strategies to reverse drug resistance.
[0028] (3) Application of the axitinib-resistant renal cancer cell model of the present invention: the axitinib-resistant renal cancer cell model is applied to the study of the resistance mechanism of renal cancer cell targeted therapy, and the differentially expressed downstream targets and possible resistance mechanisms are found by performing transcriptome sequencing, proteome sequencing, metabolomics measurement, signal pathway analysis, etc. on the resistant cells and control cells; the axitinib-resistant renal cancer cell model is applied to the screening or preparation of renal cancer targeted drugs or the screening of anti-renal cancer targeted therapy resistance targets. The axitinib-resistant renal cancer cell model plays a key role in the screening, preparation and research of renal cancer targeted drugs, as well as the screening of anti-renal cancer targeted therapy resistance targets, which helps to improve the efficacy of drugs, overcome drug resistance, and provide better treatment options for renal cancer patients; the axitinib-resistant renal cancer cell model is applied to the reversal of targeted therapy resistance, and the in vivo small molecule RNA interference drug is used to explore the therapeutic targets that can reverse the targeted resistance of renal cancer axitinib, and the preclinical axitinib and RNA interference drug combination treatment is carried out in mice. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 : are growth inhibition curves of control cells and axitinib-resistant cells, wherein A is the growth inhibition curve of 786-O control cells and axitinib-resistant cells under axitinib treatment, and B is the growth inhibition curve of 769-P control cells and axitinib-resistant cells under axitinib treatment.
[0030] Figure 2 Figure 5 is a statistical graph of the half-inhibitory concentration of control cells and axitinib-resistant cells, where A is a statistical graph of the half-inhibitory concentration of 786-O control cells and axitinib-resistant cells under axitinib treatment, and B is a statistical graph of the half-inhibitory concentration of 769-P control cells and axitinib-resistant cells under axitinib treatment.
[0031] Figure 3 These are clone formation diagrams of control cells and axitinib-resistant cells, wherein A is a clone formation diagram of control cells and axitinib-resistant cells of 786-O, and B is a clone formation diagram of control cells and axitinib-resistant cells of 769-P.
[0032] Figure 4 These are statistical graphs of cell survival rates of control cells and axitinib-resistant cells in Calcein-AM experiments, where A is a statistical graph of cell survival rates of 786-O control cells and axitinib-resistant cells in Calcein-AM experiments, and B is a statistical graph of cell survival rates of 769-P control cells and axitinib-resistant cells in Calcein-AM experiments.
[0033] Figure 5 This is a growth curve of 786-O control cells and drug-resistant cells in nude mice.
[0034] Figure 6 This is the volcano plot after transcriptome sequencing of 786-O control cells and drug-resistant cells. DETAILED DESCRIPTION
[0035] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with embodiments.
[0036] Human renal clear cell carcinoma 769-P cell line (i.e., human renal cancer cell line 769-P, catalog number: SCSP-5060) and human renal clear cell carcinoma 786-O cell line (i.e., human renal cancer cell line 786-O, catalog number: SCSP-5059) were purchased from the Cell Bank of the Chinese Academy of Sciences;
[0037] Axitinib was purchased from MedChemExpress LLC, catalog number HY-10065.
[0038] Example 1
[0039] Method for constructing axitinib-resistant renal cancer cell model:
[0040] S1. Use renal cancer cell line to establish nude mouse subcutaneous tumor model;
[0041] After thawing, human renal cancer cell line 769-P and human renal cancer cell line 786-O cells were grown in RPMI-1640 medium containing 10% fetal bovine serum in an incubator at 37°C with 5% CO2. After collecting 786-O cells or 769-P cells in the logarithmic growth phase, 5×10 6 / 100μL was inoculated subcutaneously in the left anterior armpit of 4-week-old nude mice to establish a nude mouse subcutaneous renal cancer tumor model.
[0042] S2. Prepare axitinib and control group drugs
[0043] Preparation of Axitinib: Axitinib was dissolved in a solution containing 5% dimethyl sulfoxide and 95% corn oil.
[0044] Preparation of control solution: The control group used a solution of 5% dimethyl sulfoxide and 95% corn oil without axitinib.
[0045] S3. Axitinib treatment and serial passage in vivo in tumor-bearing nude mice:
[0046] First generation: When the subcutaneous tumor volume of tumor-bearing nude mice in step S1 reaches 200mm 3(Volume = long diameter × short diameter × short diameter / 2), tumor-bearing nude mice were randomly divided into 2 groups, namely, the axitinib treatment group and the control group, and the tumor-bearing nude mice were given axitinib (10 mg / kg) and an equal volume of control solution for oral gavage. The treatment lasted for 6 weeks, and the long and short diameters of the subcutaneous tumor were measured with a vernier caliper every week. After 6 weeks of medication, the nude mice were euthanized by cervical dislocation, and the tumors were separated from the subcutaneous tissue of the nude mice using ophthalmic scissors and ophthalmic forceps in a sterile environment to obtain the first-generation resistant renal cancer cells and the first-generation control renal cancer cells.
[0047] Second generation: Use pre-cooled PBS solution to wash the tumor tissue three times to remove the blood and fat tissue on the surface. Cut the non-necrotic area of the tumor into pieces of about 1mm 3 After anesthetizing new 4-week-old nude mice (hereinafter referred to as second-generation nude mice) with isoflurane, wipe the middle skin of the nude mouse chest with alcohol, cut the skin of the nude mouse with ophthalmic scissors, and then cut 1mm 3 The tumors of different sizes were implanted under the skin of the left anterior armpit of nude mice, and the wounds were sutured with sterile sutures. When the volume of the subcutaneous tumors in the second generation of nude mice reached 200 mm 3 After that, the tumor-bearing nude mice were given axitinib (10 mg / kg) and an equal volume of control solution by gavage again for 6 weeks and the volume was measured. After 6 weeks, the nude mice were euthanized by cervical dislocation, and the tumors were separated from the subcutaneous tissue of the nude mice using ophthalmic scissors and ophthalmic forceps under a sterile environment. The second-generation drug-resistant renal cancer cells and the second-generation control renal cancer cells were obtained.
[0048] Third generation: Use pre-cooled PBS solution to wash the tumor tissue three times to remove the blood and fat tissue on the surface. Cut the non-necrotic area of the tumor into pieces of about 1mm 3 After anesthetizing new 4-week-old nude mice (hereinafter referred to as third-generation nude mice) with isoflurane, wipe the middle skin of the nude mouse chest with alcohol, cut the skin of the nude mouse with ophthalmic scissors, and then cut 1mm 3 The tumors of different sizes were implanted under the skin of the left anterior armpit of nude mice, and the wounds were sutured with sterile sutures. The volume of the subcutaneous tumors in the third generation nude mice reached 200 mm 3 After that, the tumor-bearing nude mice were given axitinib (10 mg / kg) and an equal volume of control solution by gavage again for 6 weeks and the volume was measured. After 6 weeks of treatment, the subcutaneous tumor growth curve of the axitinib-treated group was close to that of the control group ( Figure 1 ), euthanized the nude mice by cervical dislocation, and the tumors were separated from the subcutaneous tissue of the nude mice using ophthalmic scissors and ophthalmic forceps under a sterile environment. The third-generation drug-resistant renal cancer cells and the third-generation control renal cancer cells were obtained.
[0049] S4. After the third generation of renal cancer cells were isolated, they were cultured again in a culture dish
[0050] Use pre-cooled PBS solution to wash the tumor tissue three times to remove the blood and fat tissue on the surface. Use sterile ophthalmic scissors to cut the non-necrotic tumor tissue into small pieces as much as possible, and place the minced tissue pieces in a container containing ACCUMAX. TM Solution (Sigma-Aldrich, Catalog No. A7089) was placed in a 15 mL centrifuge tube and placed in a 37°C shaker for digestion for 30 minutes. The centrifuge tube was then centrifuged at 300 × g for 3 minutes. The supernatant was taken and concentrated to 5 × 10 5 / well concentration was planted in a 6-well plate and grown in an incubator at 37°C with 5% CO2. Fibroblasts were removed by repeated adhesion. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 3 antibiotics (penicillin, streptomycin, amphotericin B) for the first 2 weeks. Fresh medium was replaced every 2-3 days. When the cells grew to about 80%, they were subcultured and expanded. After 2 weeks, RPMI-1640 medium containing 10% fetal bovine serum and axitinib (5μM) was used for culture. Fresh drug-containing medium was replaced every 2 to 3 days. When the cells grew to about 80%, they were subcultured and expanded. Control renal cancer cells and axitinib-resistant renal cancer cells were obtained. At this point, the construction of the axitinib-resistant cell model of renal cancer was completed.
[0051] This application directly cuts the subcutaneous tumor of the first generation mouse into pieces of about 1mm 3 The size of the tumor can be buried in the armpit of the second-generation mouse through gas anesthesia surgery, which can save a lot of operation steps and time. The tumor cells are less affected by external stimulation, thereby improving the success rate of tumor growth in mice after subculture.
[0052] The digestion system used in this application is ACCUMAX TM The solution was digested in a shaker at 37° C. The digestion system of the present application is simpler and easier to obtain, has stable ingredients, and has a better enzymatic hydrolysis effect, and can more easily obtain a cell suspension for plating.
[0053] Example 2
[0054] The growth inhibition curves of control and axitinib-resistant renal cancer cells were drawn and the half-maximal inhibitory concentration was determined.
[0055] 3000 786-O and 769-P control cells and axitinib-resistant cells were inoculated in a 96-well plate, with three replicates per group, and cultured in a 37°C incubator containing 5% CO2. 1, 10, 100, 1000, 10000 and 100000nM axitinib were used for 48 hours. 100μL CCK-8 working solution was added to each well for 2 hours, and the OD450 value was read in an ELISA reader. The growth inhibition curve was drawn according to the OD450 value, and the half inhibition concentration IC50 was calculated.
[0056] The analysis results showed that compared with the control cells, the growth inhibition of axitinib-treated cells was significantly reduced, and the half inhibitory concentration IC50 was significantly increased (e.g. Figure 1 , Figure 2 shown).
[0057] Example 3
[0058] Figure 1. Control and axitinib-resistant renal cancer cell plate cloning experiment.
[0059] 786-O and 769-P control cells and axitinib-resistant cells in the logarithmic growth phase were seeded at 500 / well in a 12-well plate and cultured in a 37°C incubator with 5% CO2. After 14 days of treatment with 5μM axitinib, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. The cells were then washed again with PBS and stained with 0.1% crystal violet for 15 minutes. After removing the staining solution, the cells were washed three times with laboratory running water to remove excess dye and then dried at room temperature. The number of clones with >50 cells was counted under a microscope.
[0060] The analysis results showed that compared with the control cells, the number of clone colonies under axitinib treatment increased significantly (e.g. Figure 3 shown).
[0061] Example 4
[0062] Calcein-AM experimental analysis of control and axitinib-resistant renal cancer cells.
[0063] 3000 786-O and 769-P control cells and axitinib-resistant cells were inoculated in a 96-well plate and cultured in a 37°C incubator containing 5% CO2. 5μM axitinib was used for 48 hours, and three replicate wells were set for each group. The culture medium was removed, the cells were washed once with PBS, and 100μl of staining solution (Calcein-AM final concentration was 4μmol / l, PI final concentration was 6μmol / l) was added to each well. Mix well and incubate at 37°C for 15min. The ratio of yellow-green live cells and red dead cells was observed under a fluorescence microscope.
[0064] The analysis results showed that compared with the control cells, the number of cell deaths in the axitinib-treated cells was significantly reduced (e.g. Figure 4 As shown, Figure 4 The control mixture in the sample is the control solution).
[0065] Example 5
[0066] In vivo growth curve analysis of control and axitinib-resistant renal cancer cells.
[0067] 786-O and 769-P control cells and axitinib-resistant cells in the logarithmic growth phase were collected and cultured at 5×10 6 / 100μL was inoculated into the subcutaneous tissue of the left anterior armpit of 4-week-old nude mice. 3 Afterwards, the nude mice were randomly divided into 2 groups, 5 in each group, and the tumor-bearing nude mice were treated with axitinib (10 mg / kg) by gavage for 6 weeks and the volume was measured. After 6 weeks, the nude mice were euthanized by cervical dislocation, and the tumors were surgically separated from the subcutaneous tissue of the nude mice, and the tumor volume was measured.
[0068] The results showed that compared with the control group, the growth of transplanted tumors formed by resistant cells was not inhibited by axitinib, further demonstrating the resistance of resistant cells to axitinib in vivo (e.g. Figure 5 shown).
[0069] The control cells in the figure are control renal cancer cells, and the resistant cells are axitinib-resistant renal cancer cells.
[0070] The drug-resistant cells and control cells obtained by re-culturing the third-generation renal cancer cells in the axitinib-treated group and the control group were subjected to in vivo and in vitro drug sensitivity experiments. It was found that the renal cancer cells in the axitinib-treated group showed obvious drug resistance under conventional doses of axitinib, and could tolerate 15 μM drug concentration in vitro (such as Figure 2 ), and growth in nude mice was not inhibited by axitinib (as shown in Figure 5 As shown), it indicates that the renal cancer cells in the third-generation axitinib-treated group have axitinib resistance, and the drug-resistant model was successfully constructed.
[0071] This application detects the biological characteristics of the resistant cells constructed by the axitinib resistance model through growth inhibition curve and IC50 determination, clone formation experiment, Calcein-AM experiment and nude mouse tumor bearing experiment, so as to determine that the axitinib resistance model is successfully constructed.
[0072] Example 6
[0073] Application of the axitinib resistance model: Transcriptome sequencing was performed on control and axitinib-resistant renal cancer cells to identify downstream resistance targets.
[0074] Inoculate 3*10 5 786-O control cells and axitinib-resistant cells were cultured in an incubator at 37°C with 5% CO2 and treated with 5 μM axitinib for 48 hours. Three replicate culture dishes were set up for each group. The culture medium was removed, the cells were washed once with PBS, and the cells were scraped and collected into EP tubes and quickly frozen with liquid nitrogen. The samples were sequenced for RNA transcriptome to find downstream drug resistance targets.
[0075] The sequencing results showed that there were significantly more highly expressed and less expressed genes in the axitinib-resistant group compared with the control group in the volcano plot, from which drug-resistant targets could be found for research (such as Figure 6 shown).
[0076] The axitinib-resistant cell model of renal cancer in the present application can be used to study the resistance mechanism of renal cancer cells to targeted therapy. By performing transcriptome sequencing, proteome sequencing, metabolomics measurement, signal pathway analysis, etc. on resistant cells and control cells, differentially expressed downstream targets and possible resistance mechanisms can be discovered; it can be used to screen or prepare targeted drugs for renal cancer, and screen for resistance targets against targeted therapy for renal cancer. The axitinib-resistant cell model of renal cancer plays a key role in the screening, preparation and research of targeted drugs for renal cancer, as well as in the screening of resistance targets against targeted therapy for renal cancer, which helps to improve the efficacy of drugs, overcome resistance, and provide better treatment options for renal cancer patients; the axitinib-resistant cell model of renal cancer can be used to reverse resistance to targeted therapy. In vivo small molecule RNA interference drugs are used to explore therapeutic targets that can reverse the targeted resistance of renal cancer axitinib, and preclinical axitinib and RNA interference drugs are used in combination for treatment in mice.
[0077] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person skilled in the art, several modifications, improvements and substitutions can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.
Claims
1. A method for constructing axitinib-resistant renal cancer cell model, characterized in that: The following steps are involved: A nude mouse subcutaneous tumor model was established using a renal cancer cell line to obtain nude mouse subcutaneous tumors; When the subcutaneous tumor of the nude mice reaches the target volume, the nude mice are intragastrically administered with axitinib, and the tumor tissues are separated to obtain the first generation of drug-resistant renal cancer cells; Cutting the first-generation drug-resistant renal cancer cells into tumor masses and inoculating them into the armpits of nude mice, repeating the steps of oral gavage, separation and inoculation to obtain second-generation and third-generation drug-resistant renal cancer cells respectively; The third-generation drug-resistant renal cancer cells are cut into pieces, digested and then cultured to obtain a renal cancer axitinib-resistant cell model.
2. The method for constructing axitinib-resistant renal cancer cell model according to claim 1, characterized in that: The renal cancer cell line is a human renal clear cell carcinoma 786-O cell line or a human renal clear cell carcinoma 769-P cell line.
3. The method for constructing axitinib-resistant renal cancer cell model according to claim 2, characterized in that: The inoculation amount of the renal cancer cell line was 4.5×10 6 / 100μL~5.5×10 6 / 100μL.
4. The method for constructing axitinib-resistant renal cancer cell model according to claim 1, characterized in that: The target volume is ≥ 200 mm 3 .
5. The method for constructing axitinib-resistant renal cancer cell model according to claim 1, characterized in that: The tumor mass was inoculated into the axilla of nude mice by gas anesthesia surgery.
6. The method for constructing axitinib-resistant renal cancer cell model according to claim 1, characterized in that: The digestion was performed using ACCUMAX TM Solution.
7. The method for constructing the axitinib-resistant renal cancer cell model according to any one of claims 1 to 6, characterized in that: The axitinib-resistant renal cancer cell model has axitinib-resistant cells whose growth in nude mice is not inhibited by axitinib and can tolerate 15 μM axitinib concentration in vitro.
8. A renal cancer axitinib-resistant cell model constructed by the method described in any one of claims 1 to 7.
9. Application of axitinib-resistant renal cancer cell model, characterized by: The renal cancer axitinib-resistant cell model described in claim 8 is applied to the study of the mechanism of drug resistance in targeted therapy of renal cancer cells.
10. Application of axitinib-resistant renal cancer cell model, characterized by: The axitinib-resistant renal cancer cell model of claim 8 is used in screening or preparing renal cancer targeted drugs, screening anti-renal cancer targeted therapy resistance targets or reversing targeted therapy resistance testing.