Drug-resistant cell strain of KRAS inhibitor as well as preparation and application of drug-resistant cell strain

Abstract

The invention provides a drug-resistant cell strain of a KRAS inhibitor as well as preparation and application of the drug-resistant cell strain. The preservation number of the drug-resistant cell strain is CCTCC (China Center For Type Culture Collection) NO: C2023226. A drug-resistant cell strain GP2D-R resistant to MRTX1133 is obtained by adopting a method of combining drug concentration increasing impact with drug-resistant monoclonal amplification for induction, a KRAS (G12D) inhibitor drug-resistant animal model is established, the occurrence and development microenvironment of resistance of colon cancer to a KRAS (G12D) targeted drug can be truly simulated by utilizing the animal model, and the drug-resistant cell strain GP2D-R is of great significance to clinical research of colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a KRAS inhibitor-resistant cell line and a preparation and application thereof. Background Art

[0002] RAS is one of the most common human oncogenes, with gain-of-function mutations found in 27% of cancers. Three human RAS genes: neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and Kirsten rat sarcoma viral oncogene homolog (KRAS) encode four RASGTPases (GTPases): NRAS, HRAS, KRASa, and KRASb, the latter two of which are the result of alternative splicing. KARS is an important regulatory protein for cell growth. Once activated, it can activate multiple signaling pathways and promote cell proliferation. About 30% of tumors have KRAS mutations, including 90% of pancreatic cancer, 50% of colon cancer, and 25% of lung cancer. The most common sites of gene mutations are codons 12, 13, and 61, among which mutations at codon 12 are the most common.

[0003] RAS proteins cycle between an active GTP-bound state and an inactive GDP-bound state. This process is tightly regulated by a variety of multidomain protein families: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs stimulate the dissociation of GDP and subsequent GTP binding, activating RAS proteins, while GAPs accelerate the hydrolysis of bound GTP, converting RAS to an inactive state. RAS proteins play an important role in cell proliferation, differentiation, and survival by associating with a variety of downstream pathways, the most typical of which are the MAPK and PI3K / AKT pathways.

[0004] KRAS has proven to be a difficult target to drug. The picomolar affinity of RAS proteins for binding to GTP has prevented efforts to develop GTP-competitive inhibitors. In addition, attempts to directly target KRAS are hampered by its structure: KRAS presents a smooth surface and does not have a deep hydrophobic pocket that allows tight binding. Thanks to the unremitting efforts of researchers, two targeted drugs targeting the G12C site mutation of the KRAS gene have recently been developed: Adagrasib (MRTX849) and Sotorasib (AMG510). Recent clinical trials have gradually confirmed the therapeutic effects of several drugs. However, the disease has since progressed again due to primary or secondary drug resistance. KRAS (G12D) is the most common mutation in colorectal cancer and pancreatic cancer. MRTX1133 developed by MIRATI is the first reported KRAS (G12D) inhibitor, and research on its resistance mechanism and treatment methods is still in the early stages of research.

[0005] At present, KRAS (G12C) inhibitor-resistant strains have been established both at home and abroad, but there are no reports of KRAS (G12D) inhibitor-resistant colon cancer cell lines. Therefore, an immortalized MRTX1133-resistant cell line and its resistant animal model were constructed, in order to provide a basis for studying the pathogenesis, resistance mechanism and treatment of KRAS (G12D) mutant tumors.

[0006] There are several known ways to establish drug-resistant cell lines in vitro: high-dose drug shock method, drug concentration increasing method, and a combination of drug concentration increasing and high-dose drug shock method. However, there is a problem that the drug resistance of cells is unstable. After screening, drug-resistant strains need to be cultured in a culture medium with a certain concentration of drugs to maintain the stability of drug resistance, which takes a long time. Summary of the invention

[0007] The technical problems to be solved by the present invention are that the drug resistance of drug-resistant cell lines is unstable, the screening time of drug-resistant cell lines is long, and there is a lack of KRAS (G12D) inhibitor-resistant cell lines, and a drug-resistant cell line resistant to MRTX1133 and a drug-resistant animal model thereof are provided. The drug-resistant cell line resistant to MRTX1133 obtained by the present invention has stable drug resistance and a short cell line construction time.

[0008] On one hand, the present invention provides a method for constructing a drug-resistant cell line resistant to MRTX1133, wherein the drug-resistant cell line is a GP2D drug-resistant cell line. The method comprises culturing GP2D cells in a culture medium containing MRTX1133 through four steps, wherein the concentration of MRTX1133 in the culture medium in each step is 4.5-5.5 times that of the previous step.

[0009] In some embodiments, the method comprises the following steps:

[0010] Step 1) GP2D cells were cultured in a medium containing 0.01-0.03 μM MRTX1133 until single cell clones were clearly generated;

[0011] Step 2) culturing the cells obtained in step 1) using a culture medium containing 0.05-0.15 μM MRTX1133 until obvious single cell clones are generated;

[0012] Step 3) culturing the cells obtained in step 2) using a culture medium containing 0.25-0.75 μM MRTX1133 until obvious single cell clones are generated; and,

[0013] Step 4) The cells obtained in step 3) are cultured in a culture medium containing 1.25-3.75 μM MRTX1133 until single cell clones are clearly generated.

[0014] In some embodiments, step 1) culture GP2D cells in a culture medium containing 0.02 μM MRTX1133 until single cell clones are clearly generated.

[0015] In some embodiments, step 2) culture the cells obtained in step 1) using a culture medium containing 0.1 μM MRTX1133 until obvious single cell clones are generated.

[0016] In some embodiments, step 3) culture the cells obtained in step 2) using a culture medium containing 0.5 μM MRTX1133 until obvious single cell clones are generated.

[0017] In some embodiments, step 4) culture the cells obtained in step 3) in a culture medium containing 2.5 μM MRTX1133 until single cell clones are clearly generated.

[0018] In some embodiments, step 1) culturing GP2D cells in a culture medium containing 0.02 μM MRTX1133 until obvious single cell clones are generated;

[0019] Step 2) culturing the cells obtained in step 1) using a culture medium containing 0.1 μM MRTX1133 until obvious single cell clones are generated;

[0020] Step 3) culturing the cells obtained in step 2) using a culture medium containing 0.5 μM MRTX1133 until obvious single cell clones are generated; and,

[0021] Step 4) The cells obtained in step 3) are cultured in a culture medium containing 2.5 μM MRTX1133 until single cell clones are clearly generated.

[0022] In some embodiments, one or more cell culture steps in step 1) to step 4) also use a culture medium without MRTX1133 to culture cells; during the culture process, the culture medium containing MRTX1133 and the culture medium without MRTX1133 are used alternately; preferably, the number of alternations is 2.

[0023] In some embodiments, culturing cells using a medium containing MRTX1133 and then culturing the cells with a medium that does not contain MRTX1133 constitutes 1 alternation number; an alternation number of 2 means culturing cells using a medium containing MRTX1133 and then culturing the cells with a medium that does not contain MRTX1133, culturing the cells again using a medium containing MRTX1133 and then culturing the cells with a medium that does not contain MRTX1133 again.

[0024] In some embodiments, in steps 1) to 4), cells are cultured in a medium containing MRTX1133 for 18-30 hours, the culture supernatant is discarded and washed, and the medium without MRTX1133 is replaced and cultured until the cells return to a normal growth rate.

[0025] In some embodiments, washing is performed using PBS.

[0026] In some embodiments, cells that have recovered their normal growth rate are cultured in a medium containing MRTX1133 until they are adherent.

[0027] In some embodiments, the cells that have recovered their normal growth rate are digested and then seeded into a 6-well plate with culture medium at 1000 cells per well, and cultured in a CO2 incubator for 24 hours until they are attached to the plate.

[0028] In some embodiments, the adherent cells are cultured in a medium containing MRTX1133 and the medium is replaced every 3 days.

[0029] In some embodiments, the method further comprises, after step 4), culturing in a culture medium containing 2.5 μM MRTX1133 for not less than 2 weeks.

[0030] In some embodiments, in steps 1) to 4), cells in the logarithmic growth phase are used to initiate culture.

[0031] In some embodiments, in step 1) to step 4), the initial density of cell culture is 60-70%.

[0032] Another aspect of the present invention provides a drug-resistant cell strain resistant to MRTX1133, wherein the drug-resistant cell strain is obtained by the method described in one aspect of the present invention.

[0033] In some embodiments, the deposit number of the drug-resistant cell line is CCTCC NO: C2023226.

[0034] Another aspect of the present invention provides a drug-resistant animal model, which is obtained by inoculating the drug-resistant cell line provided by the present invention into an animal.

[0035] In some embodiments, the animal is a mouse.

[0036] In some embodiments, the mouse is a nude mouse.

[0037] Another aspect of the present invention provides a method for constructing a drug-resistant animal model, the method comprising inoculating the drug-resistant cell line provided by the present invention into an animal.

[0038] In some embodiments, the animal is a mouse.

[0039] In some embodiments, the mouse is a nude mouse.

[0040] In some embodiments, the inoculation volume of the drug-resistant cell line is 5×10 6 cells / animal.

[0041] In some embodiments, the nude mouse is a 6-8 week old Balb / c nude mouse.

[0042] In some embodiments, the inoculation is subcutaneous inoculation into nude mice.

[0043] Another aspect of the present invention provides use of the drug-resistant cell line of the present invention in constructing a drug-resistant animal model.

[0044] In some embodiments, the drug-resistant animal model is a colon cancer drug-resistant animal model.

[0045] In some embodiments, the colon cancer drug-resistant animal model is an MRTX1133-resistant animal model.

[0046] Another aspect of the present invention provides the use of the drug-resistant cell line or the drug-resistant animal model of the present invention in preclinical research and / or development of colon cancer targeted drugs.

[0047] In some embodiments, the colon cancer targeted drug is a targeted drug for KRAS-G12D mutation.

[0048] In some embodiments, the development is to screen for drugs targeting KRAS-G12D mutations.

[0049] In some embodiments, the application is for non-diagnostic and therapeutic purposes, such as studying molecular changes in drug-resistant cell lines to discover new therapeutic targets, or studying the synergistic effects of different drug combinations on drug-resistant cell lines to develop more effective combined treatment regimens.

[0050] Another aspect of the present invention provides use of the drug-resistant cell line or the drug-resistant animal model of the present invention in studying the pathogenesis and / or drug resistance mechanism of KRAS-G12D mutant tumors.

[0051] In some embodiments, the use is for non-diagnostic therapeutic purposes.

[0052] During the construction of GP2D-R, we discovered a new and optimized way to induce drug-resistant strains. The known methods for establishing drug-resistant cell lines in vitro are mainly the following: high-dose drug shock method, increasing drug concentration method, and a combination of increasing drug concentration and high-dose drug shock method. However, there is a problem that the drug resistance of cells is unstable. After screening the drug-resistant strains, they need to be cultured in a culture medium with a certain concentration of drugs to maintain the stability of drug resistance. We adopted an induction method combining increasing drug concentration shock with drug-resistant monoclonal amplification (Example 1), and compared the commonly used method of combining increasing drug concentration with high-dose drug shock (Comparative Example 1) to screen drug-resistant cell lines. It was found that the new drug resistance induction method can quickly obtain drug-resistant cell lines with a higher resistance index and stronger resistance stability.

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available.

[0055] The positive effects of the present invention are:

[0056] The MRTX1133-resistant cell line GP2D-R of the present invention has a high resistance index, strong resistance and stable resistance; the resistant cell line obtained by the induction method of increasing drug concentration combined with drug-resistant monoclonal amplification has a short use time; the colon cancer animal model constructed by the MRTX1133-resistant cell line GP2D-R has good effect and can be used for drug screening experiments.

[0057] IC of drug-resistant cell line GP2D-R 50 Higher than 4.5μM, much higher than IC before drug resistance 50 The drug resistance index was 0.01917 μM, exceeding 300, which was also higher than the IC value of GP2D-Res cell line obtained by combining increasing drug concentration with high-dose drug shock. 50 of 2.501μM.

[0058] The induction method using increasing drug concentration combined with drug-resistant monoclonal expansion only takes 3 months, which is much shorter than the 9 months of the commonly used method combining increasing drug concentration with high-dose drug shock.

[0059] In the colon cancer animal model constructed using the drug-resistant cell line GP2D-R, the tumor size of the MRTX1133-resistant group (GP2D-R) was significantly larger than that of the MRTX1133-sensitive group (GP2D), indicating that the colon cancer animal model provided by the present invention can be used for drug screening and other experiments, and can provide a basis for studying the pathogenesis, drug resistance mechanism and treatment methods of KRAS (G12D) mutant tumors.

[0060] Biomaterial Deposit Information

[0061] The MRTX1133-resistant KRAS (G12D) mutant human colon cancer cell line GP2D-R of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on September 14, 2023. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072, the deposit number is: CCTCC NO: C2023226, the culture name is human colon cancer cell GP2D-R, and the classification name is Homo sapiens. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of MRTX1133 drug sensitivity test in GP2D cells and GP2D-R cells.

[0063] Figure 2 Schematic diagram of MRTX1133 drug sensitivity test in GP2D-Res cells and GP2D-R cells.

[0064] Figure 3 Schematic diagram of MRTX1133 drug sensitivity test in GP2D-Res cells and GP2D-R cells.

[0065] Figure 4 Schematic diagram of GP2D cells and MRTX1133-resistant cells GP2D-R under a microscope.

[0066] Figure 5 Schematic diagram of the construction of the animal model for GP2D cells and the efficacy of MRTX1133.

[0067] Figure 6 Schematic diagram of the construction of the animal model for GP2D-R cells and the efficacy of MRTX1133. DETAILED DESCRIPTION

[0068] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0069] MRTX1133 was purchased from MIRATI.

[0070] Example 1 Method for constructing MRTX1133 drug-resistant cell line GP2D-R

[0071] GP2D cells (from Nanjing Kebai) were cultured in vitro (37°C, 5% CO2) in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco), and passaged twice a week.

[0072] The culture medium in the following steps refers to DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco).

[0073] Drug concentration increment shock combined with drug-resistant monoclonal expansion

[0074] First round of drug treatment: cells in the logarithmic growth phase were inoculated in a culture dish containing culture medium, and the cell density was controlled at 60-70%. MRTX1133 with an initial concentration of 0.02μM was added to the culture medium for 24 hours, the culture supernatant was discarded, PBS was washed once, and the medium without drugs was replaced. After the cell state recovered, the cells were digested and subcultured and continued to be treated with 0.02μM drugs for 24 hours, the culture supernatant was discarded, PBS was washed once, and the medium without drugs was replaced. After the cells resumed their normal growth rate, 1000 cells per well were inoculated into a 6-well plate with added culture medium after digestion, and placed in a CO2 incubator for 24 hours. After the cells attached to the wall, 0.02μM MRTX1133 was added and the treatment was continued. The medium containing 0.02μM MRTX1133 was replaced every 3 days until obvious single cell clones were generated.

[0075] Second round of drug treatment: About 2 weeks later, the cells obtained from the first round of drug treatment were digested and re-plated, and the drug concentration was increased to 0.1μM (5 times the initial concentration), and the drug treatment method of the first round was repeated: the cells grown to the logarithmic growth phase after the first round of drug treatment were inoculated in a culture dish, and the cell density was controlled at 60-70%. The culture dish contained a medium with a concentration of 0.1μM MRTX1133. The cells were treated for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cell state recovered, the cells were digested and subcultured and continued to be treated with 0.1μM drugs for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cells resumed their normal growth rate, the cells were digested and inoculated into 6-well plates with 1000 cells per well, and placed in a CO2 incubator for 24 hours. After the cells adhered, 0.1 μM MRTX1133 was added and the treatment was continued. The medium containing 0.1 μM MRTX1133 was replaced every 3 days until obvious single cell clones were generated.

[0076] The third round of drug treatment: After about 2-3 weeks, the drug concentration was increased to 0.5μM, and the second round of drug treatment was repeated: that is, the cells grown to the logarithmic growth phase after the second round of drug treatment were inoculated in a culture dish, the cell density was controlled at 60-70%, and the culture dish contained a medium with a concentration of 0.5μM MRTX1133. The cells were treated for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cell state recovered, the cells were digested and subcultured and continued to be treated with 0.5μM drugs for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cells resumed their normal growth rate, the cells were digested and inoculated into a 6-well plate with 1000 cells per well, and placed in a CO2 incubator for 24 hours. After the cells attached to the wall, 0.5μM MRTX1133 was added, and the treatment was continued. The medium containing 0.5μM MRTX1133 was replaced every 3 days until obvious single cell clones were generated.

[0077] Fourth round of drug treatment: After about 2-3 weeks, the drug concentration was increased to 2.5μM, and the drug treatment method of the third round was repeated, that is, the cells grown to the logarithmic growth phase after the third round of drug treatment were inoculated in a culture dish, the cell density was controlled at 60-70%, and the culture dish contained a medium with a concentration of 2.5μM MRTX1133. The cells were treated for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cell state recovered, the cells were digested and subcultured and continued to be treated with 2.5μM drugs for 24 hours, the culture supernatant was discarded, the cells were washed once with PBS, and the medium without drugs was replaced. After the cells resumed their normal growth rate, the cells were digested and inoculated into a 6-well plate with 1000 cells per well, and placed in a CO2 incubator for 24 hours. After the cells attached to the wall, 2.5μM MRTX1133 was added, and the treatment was continued. The medium containing 2.5μM MRTX1133 was replaced every 3 days until obvious single cell clones were generated. The cells were continuously cultured in a medium containing 2.5 μM MRTX1133, and after the cells recovered, they were subcultured for 2 weeks under 2.5 μM MRTX1133 pressure to obtain the MRTX1133-resistant cell line GP2D-R. The drug induction lasted about 3 months.

[0078] Comparative Example 1 Construction method of GP2D-Res of MRTX1133 drug-resistant cell line

[0079] GP2D cells were cultured in vitro in culture medium (37°C, 5% CO2) and passaged twice a week.

[0080] Combination of increasing drug concentration with high-dose drug shock

[0081] The drug concentration was increased and induced by shock, and the drug concentration gradient was 0.02μM, 0.04μM, 0.08μM, 0.16μM, 0.32μM, 0.64μM, 1.28μM, and 2.56μM. Cells in the logarithmic growth phase were inoculated in a culture dish containing culture medium, and the cell density was controlled at 60-70%. MRTX1133 with an initial concentration of 0.02μM was added to start the first shock, and the cells were acted for 24 hours. The culture supernatant was discarded, PBS was washed once, and the medium without drugs was replaced. After the cell state recovered, the digestion and passage continued for the second shock. The cells were acted with medium containing 0.02μM drugs for 24 hours, the culture supernatant was discarded, PBS was washed once, and the medium without drugs was replaced. After the cells proliferated to the normal morphology, the above drug shock was repeated, and each concentration was shocked 8 times. After the cells were stably grown in the medium with 0.02μM drug, the drug concentration was increased in the order of 0.04μM, 0.08μM, 0.16μM, 0.32μM, 0.64μM, 1.28μM, and 2.56μM. After increasing the drug concentration, the cells were cultured in a shock mode of 0.02μM. The drug induction lasted for about 9 months until the cells were able to grow stably under the pressure of 2.5μM MRTX1133 drug, and the MRTX1133-resistant cell line GP2D-Res was obtained.

[0082] Example 2 Drug sensitivity detection of GP2D cells before and after drug resistance induction, morphological observation, biological characteristics identification and gene mutation detection of drug-resistant cells GP2D-R

[0083] 1) Drug sensitivity analysis and cell proliferation assay of MRTX1133-resistant cell lines GP2D-R and GP2D

[0084] The method for drug sensitivity testing is as follows:

[0085] By CellTiter-Glo ® The Luminescent Cell Viability Assay Kit quantitatively measures intracellular ATP to detect the number of live cells in culture.

[0086] The first step is to culture cells. GP2D-R and GP2D cells were inoculated into 384-well plates containing culture medium at a density of 1000 cells per well, with a volume of 45 μL per well. The plates were placed in an incubator at 37°C and 5% carbon dioxide for overnight culture.

[0087] The second step is to treat cells with MRTX1133. The starting concentration of MRTX1133 is 20 μM, and it is diluted 3 times. A total of 10 concentration gradients are set, namely 20.000, 6.667, 2.222, 0.741, 0.247, 0.082, 0.027, 0.009, 0.003, and 0.001 μM; 5 μL of DMSO (D2650, Sigma) diluted in culture medium (the final concentration of DMSO is controlled at 0.5%) or the compound to be tested is added to each well, and 3 replicates are set for each concentration. The cells are placed in a 37°C 5% carbon dioxide incubator for 4 days.

[0088] Step 3: CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega, G7570) was used to detect cell viability in the control and treatment groups. 50 μL CellTiter-Glo was added to each well, mixed, and incubated at room temperature for 10 min. The full-wavelength luminescence signal was read using a multifunctional microplate reader (Infinite200 Pro, Tecan). The cell viability percentage (%) was calculated using the following formula:

[0089] Cell viability percentage (%) = signal value of compound-treated group / signal value of DMSO-treated group*100.

[0090] The MRTX1133 drug-resistant cell lines GP2D-R and GP2D cells obtained in Example 1 were subjected to drug sensitivity tests according to the above method. The results are as follows: Figure 1 shown.

[0091] Figure 1 It was shown that the GP2D cell line had a resistance-pre-IC 50 The IC after drug resistance is 0.01917μM. 50 It is 5.974μM, and the resistance index is 311.63.

[0092] The MRTX1133 drug-resistant cell lines GP2D-R and GP2D cell lines obtained in Example 1 were frozen at -196°C (normal pressure, liquid nitrogen). After 5 months of freezing, the drug sensitivity test was performed together with the MRTX1133 drug-resistant cell line GP2D-Res obtained in Comparative Example 1 according to the above method. The results are as follows: Figure 2 shown.

[0093] Figure 2 It was shown that the GP2D-Res cell line IC 50The GP2D-R cell line obtained by increasing the drug concentration and inducing drug resistance by expanding the drug-resistant monoclonal clones had an IC of 2.501 μM after being frozen for 5 months. 50 It is 4.555μM.

[0094] The MRTX1133-resistant cell lines GP2D-Res and GP2D cell lines obtained in Comparative Example 1 were cultured for one month using a medium without MRTX1133, and the results of drug sensitivity tests were as follows: Figure 3 shown.

[0095] Figure 3 It showed that after 1 month of drug-free culture, the IC 50 The IC value of GP2D-R cell line is 0.906μM. 50 It is 3.516μM.

[0096] Compared with the combination of increasing drug concentration and high-dose drug shock, the combination of increasing drug concentration and expanding drug-resistant monoclonal clones greatly reduces the time required for screening drug-resistant cell lines, shortening the time from 9 months to 3 months. On the other hand, the cell lines obtained in Example 1 are more resistant than the cell lines obtained in Comparative Example 1. The drug-resistant cells screened are expanded from monoclonal cells with strong drug resistance. After one month of drug-free culture, the drug resistance of the cell lines obtained in Example 1 is better maintained. At the same time, the drug-resistant cell lines obtained in Example 1 were frozen for 5 months and then revived. IC 50 Still maintained at 4.555μM.

[0097] 2) Morphological observation of drug-resistant cells GP2D-R

[0098] GP2D cells were cultured in vitro in a culture medium (37°C, 5% CO2) and passaged twice a week. GP2D-R cells obtained in Example 1 were cultured in vitro in a culture medium containing 2.5 μM MRTX1133 (37°C, 5% CO2). GP2D cells grew adherently and were spindle-shaped when observed under an inverted microscope. After MRTX1133 drug-induced culture, the growth rate of GP2D-R cells slowed down. Under an inverted microscope, the morphology of GP2D-R cells became shorter and rounder (see Figure 4 ).

[0099] 3) Identification of biological characteristics

[0100] The GP2D-R cells obtained in Example 1 were subjected to cell STR identification. The results are shown in Table 1. The induced GP2D-R cells completely matched the STR typing data of the GP2D cells in the cell database.

[0101] Table 1

[0102]

[0103] Example 3 Colon cancer animal model constructed with MRTX1133-resistant cell line GP2D-R

[0104] (1) GP2D and GP2D-R cell lines obtained in Example 1 were cultured in vitro (37°C, 5% CO2) and subcultured twice a week.

[0105] (2) The GP2D and GP2D-R cells were cultured at 5×10 6 The inoculation amount of cells / mouse was transplanted subcutaneously into 6-8 week old Balb / c nude mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) to obtain transplanted mice;

[0106] (3) Mice were treated with 5 mg / kg of MRTX1133 by intraperitoneal injection twice a day for 28 days, and the tumor growth curve was recorded.

[0107] (4) Tumor size was measured using a vernier caliper (No. 105688, Sylvac Dantsin) on Mondays, Wednesdays, and Fridays. Tumor volume was recorded in mm. 3 The calculation formula of tumor volume is: V=0.5×a×b 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the relative tumor proliferation rate T / C (%) value. T / C (%) is calculated using the formula: T / C (%) = (Ti-T0) / (Vi-V0) × 100, where Ti is the average tumor volume of the treatment group on a certain day, T0 is the average tumor volume of the treatment group on the first day of treatment (before medication), Vi is the average tumor volume of the control group on the day Ti was measured, and V0 is the average tumor volume of the control group on the first day of treatment.

[0108] (5) Use the formula: Tumor growth inhibition rate TGI (%) = [100-T / C ​​(%)] to calculate the TGI (%) of each group.

[0109] The results are as follows Figure 5 and Figure 6 As shown, after 28 days of drug treatment, the tumor growth of mice in the MRTX1133 sensitive group (GP2D) was significantly inhibited, with a TGI of 102.64%. The tumors of mice in the MRTX1133 resistant group (GP2D-R) continued to grow, with a TGI of 57.99%, and the tumor size was significantly larger than that in the drug sensitive group.

Claims

1. A method for constructing a drug-resistant cell resistant to MRTX1133, wherein the drug-resistant cell line is a GP2D drug-resistant cell line, characterized in that: The method comprises culturing GP2D cells in a culture medium containing MRTX1133 through four steps, wherein the concentration of MRTX1133 in the culture medium in each step is 4.5-5.5 times that in the previous step.

2. The method according to claim 1, characterized in that The method comprises the following steps: Step 1) GP2D cells were cultured in a medium containing 0.01-0.03 μM MRTX1133 until single cell clones were clearly generated; Step 2) culturing the cells obtained in step 1) using a culture medium containing 0.05-0.15 μM MRTX1133 until obvious single cell clones are generated; Step 3) culturing the cells obtained in step 2) using a culture medium containing 0.25-0.75 μM MRTX1133 until obvious single cell clones are generated; and, Step 4) culturing the cells obtained in step 3) using a culture medium containing 1.25-3.75 μM MRTX1133 until obvious single cell clones are generated; Preferably, step 1) culturing GP2D cells in a culture medium containing 0.02 μM MRTX1133 until obvious single cell clones are generated; and / or, step 2) culturing the cells obtained in step 1) using a culture medium containing 0.1 μM MRTX1133 until obvious single cell clones are generated; and / or, step 3) culturing the cells obtained in step 2) using a culture medium containing 0.5 μM MRTX1133 until obvious single cell clones are generated; And / or, step 4) culturing the cells obtained in step 3) using a culture medium containing 2.5 μM MRTX1133 until obvious single cell clones are generated.

3. The method according to claim 2, characterized in that In one or more of the cell culture steps in step 1) to step 4), cells are cultured in a medium without MRTX1133, and during the culture process, the medium containing MRTX1133 and the medium without MRTX1133 are used alternately; preferably, the number of alternations is 2; Preferably, in steps 1) to 4), the cells are cultured in a medium containing MRTX1133 for 18-30 hours, the culture supernatant is discarded and washed, and the medium without MRTX1133 is replaced and cultured until the cells recover to a normal growth rate; More preferably, the cells that have recovered their normal growth rate are cultured in a medium containing MRTX1133 until they are in an adherent state; More preferably, the adherent cells are cultured in a medium containing MRTX1133 and the medium is replaced every 3 days.

4. The method according to claim 2 or 3, characterized in that The method further comprises, after step 4), culturing for no less than 2 weeks using a culture medium containing 2.5 μM MRTX1133; and / or, in step 1) to step 4), cells in the logarithmic growth phase are taken to start culture; And / or, in step 1) to step 4), the initial density of cell culture is 60-70%.

5. A drug-resistant cell line resistant to MRTX1133, characterized in that: The drug-resistant cell line is obtained by the method according to any one of claims 1 to 4; Preferably, the deposit number of the drug-resistant cell line is CCTCC NO: C2023226.

6. A drug-resistant animal model, characterized in that: The drug-resistant animal model is obtained by inoculating the drug-resistant cell line as claimed in claim 5 into an animal, such as a mouse.

7. A method for constructing a drug-resistant animal model, characterized in that: The construction method comprises inoculating the drug-resistant cell line according to claim 1 or 5 into an animal; the animal is, for example, a mouse, preferably a nude mouse; Preferably, the inoculation amount of the drug-resistant cell line is 5×10 6 cells / animal; And / or, the nude mice are 6-8 week old Balb / c nude mice.

8. Use of the drug-resistant cell line according to claim 5 in constructing a drug-resistant animal model.

9. Use of the drug-resistant cell line according to claim 5 or the drug-resistant animal model according to claim 6 in preclinical research and / or development of targeted drugs for colon cancer; Preferably, the colon cancer targeted drug is a targeted drug for KRAS-G12D mutation; And / or, the development is to screen targeted drugs for KRAS-G12D mutations.

10. Use of the drug-resistant cell line according to claim 5 or the drug-resistant animal model according to claim 6 in studying the pathogenesis and / or drug resistance mechanism of KRAS-G12D mutant tumors.

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