A method for constructing a KRAS G12C inhibitor-resistant colorectal cancer cell line

Abstract

The present application relates to the technical field of cell model, and discloses a method for constructing KRAS G12C inhibitor-resistant colorectal cancer cell line, which adopts KRAS G12C inhibitor to induce KRAS G12C mutant colorectal cancer cells in vitro through drug concentration increasing method, so that the cells are resistant to KRAS G12C inhibitor. The present application provides a more comprehensive and efficient model for scientific research and key work such as exploration of drug resistance mechanism, mining of drug resistance reversal target, new drug research and development, drug sensitivity detection and efficacy verification of combined drug therapy, and lays a foundation for further exploring the clinical treatment strategy of KRAS G12C mutant colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of cell models, and in particular to a method for constructing a KRAS G12C inhibitor-resistant colorectal cancer cell line. Background Art

[0002] Rat Sarcoma proto-oncogene RAS ) family is the earliest discovered human oncogene family, including the Kirsten rat sarcoma viral oncogene homolog ( KRAS ), Harvey rat sarcoma viral oncogene homolog ( HRAS ) and neuroblastoma RAS Viral oncogene homologs ( NRAS ) and other three major members. When the RAS protein mutates, its hydrolysis ability is inhibited and "locked" in the GTP-bound state, thereby continuously activating downstream pathways and leading to malignant cell proliferation and cancer. Clinically, approximately 20% of tumors have activating mutations in the RAS protein (the main types are KRAS G12D, KRAS G12V, KRAS G12C, and KRASG13D). In colorectal cancer, non-small cell lung cancer, and pancreatic adenocarcinoma, the proportion of RAS mutations is as high as 50%, 40%, and 90%, respectively. Therefore, directly targeting the RAS protein is the most ideal and effective therapeutic strategy for treating RAS mutation-driven malignancies.

[0003] Currently, there are two KRAS G12C inhibitors, sotorasib and adagrasib (Chinese names are Sotorasib and Adagrasib), which have been approved by the U.S. Food and Drug Administration for the clinical treatment of specific tumors carrying KRAS G12C mutations. my country also approved the first domestically produced KRAS G12C inhibitor, fluzivac, for the clinical treatment of advanced non-small cell lung cancer in August 2024. However, a large number of clinical studies have shown that there are significant differences in the efficacy of these drugs in patients with non-small cell lung cancer, colorectal cancer and pancreatic cancer. According to the results of the Codebreak100 and KRYSTAL-1 clinical trials, the objective response rates of sotorasib monotherapy in patients with non-small cell lung cancer, colorectal cancer and pancreatic cancer were 37.1%, 9.7% and 21%, respectively, and the objective response rates of adagrasib monotherapy in patients with non-small cell lung cancer and colorectal cancer were 42.9% and 19%, respectively. On the other hand, the objective response rates achieved with KRAS G12C-targeted therapy are primarily partial responses (33.9% for non-small cell lung cancer, 9.7% for colorectal cancer, and 21% for pancreatic cancer), with very few patients experiencing complete remissions (3.2% for non-small cell lung cancer, 0% for colorectal cancer, and 0% for pancreatic cancer). Over half of patients experience stable disease (43.5% for non-small cell lung cancer, 73% for colorectal cancer, and 63% for pancreatic cancer) or ongoing disease progression, indicating that resistance to KRAS G12C inhibitors develops rapidly and widely in digestive system tumors, particularly colorectal cancer. Therefore, there is an urgent need to explore the intrinsic regulatory mechanisms underlying colorectal cancer resistance to KRAS G12C inhibitors to guide the adjustment and optimization of clinical treatment regimens.

[0004] However, due to the following reasons: (1) low drug efficiency and high price, KRAS G12C inhibitors have not yet been widely used in colorectal cancer; (2) secondary sampling of drug-resistant patients is difficult; (3) there are no publicly shared KRAS G12C inhibitor-resistant colorectal cancer models and construction methods in academia, resulting in slow progress in the exploration of the KRASG12C inhibitor resistance mechanism. Therefore, the inventors proposed to construct KRAS G12C-resistant colorectal cancer cell lines to explore related resistance mechanisms and conduct drug sensitivity experiments, so as to accelerate scientific breakthroughs in the field of colorectal cancer KRAS G12C inhibitor resistance and promote the improvement of clinical treatment efficacy. Summary of the Invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a method for constructing a KRAS G12C inhibitor-resistant colorectal cancer cell line.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for constructing a KRAS G12C inhibitor-resistant colorectal cancer cell line, wherein a KRAS G12C inhibitor is used to induce colorectal cancer cells carrying the KRAS G12C mutation in vitro by increasing the drug concentration, so that the cells become resistant to the KRAS G12C inhibitor.

[0008] In some examples, the KRAS G12C inhibitor is selected from any one of sotorasib, adagrasib, or fluazifop.

[0009] In some examples, the colorectal cancer cells carrying the KRAS G12C mutation are selected from SW837 or LIM2099.

[0010] In some examples, the colorectal cancer cells carrying the KRAS G12C mutation do not carry the BRAF V600E mutation.

[0011] In some examples, the method includes the following steps:

[0012] 1) Cultivate colorectal cancer cells carrying the KRAS G12C mutation until the logarithmic growth phase;

[0013] 2) Continuously culturing the cells in the presence of a KRAS G12C inhibitor for several cycles until the cells return to the logarithmic growth phase;

[0014] 3) Increase the concentration of KRAS G12C inhibitor and continue culturing the cells for several cycles until the cells return to the logarithmic growth phase;

[0015] 4) Repeat step 3) until the cells maintain logarithmic growth for several cycles at each concentration. This indicates that the drug-resistant strain has been successfully constructed and the cells can be frozen.

[0016] In some examples, when the colorectal cancer cells carrying the KRAS G12C mutation are SW837 cells, the terminal maintenance concentration of the KRAS G12C inhibitor is 500 nM.

[0017] In some examples, when the colorectal cancer cells carrying the KRAS G12C mutation are LIM2099 cells, the terminal maintenance concentration of the KRAS G12C inhibitor is 1000 nM.

[0018] In the second aspect, the method provided by the present invention establishes a KRAS G12C inhibitor-resistant colorectal cancer cell line, the cell line is named SW837 / KIR, and is deposited in the China Center for Type Culture Collection located in the Wuhan University Collection Center in Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: C2024360, and is classified as: Human colorectal cancer cell SW837 / KIR Homo sapiens, and the deposit date is October 24, 2024.

[0019] In the third aspect, the method provided by the present invention establishes a KRAS G12C inhibitor-resistant colorectal cancer cell line, the cell line is named LIM2099 / KIR, and is deposited in the China Center for Type Culture Collection located in the Wuhan University Collection Center in Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: C2024361, and is classified as: Human colorectal cancer cell LIM2099 / KIR Homo sapiens, and the deposit date is October 24, 2024.

[0020] In a fourth aspect, the present invention provides a method for constructing a KRAS G12C inhibitor-resistant colorectal cancer cell line, or a method for using the KRAS G12C inhibitor-resistant colorectal cancer cell line described in the second or third aspect, including:

[0021] As experimental cells for studying the resistance mechanism of KRAS G12C inhibitors;

[0022] As experimental cells for drug sensitivity testing. In particular, they are used for KRAS G12C inhibitor drug sensitivity testing and for screening single compounds or combinations that are effective against KRAS G12C inhibitor-resistant cell lines.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a method for constructing a KRAS G12C-resistant model of colorectal cancer and successfully constructs two KRAS G12C inhibitor-resistant cell lines for colorectal cancer, providing a more comprehensive and efficient model for scientific research such as exploring resistance mechanisms, mining targets for reversing resistance, developing new drugs, testing drug sensitivity, and verifying the efficacy of combined medications. It also lays the foundation for further exploring clinical treatment strategies for KRAS G12C mutant colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 IC50 value detection of KRAS G12C inhibitors in parental and resistant cells.

[0026] Figure 2The KRAS pathway activation levels were detected after parental and resistant cells were treated with sotorasib for a time gradient. DETAILED DESCRIPTION

[0027] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0028] Example 1

[0029] This example introduces the detailed information and conventional culture method of the parental KRAS G12C mutant colorectal cancer cell line, as follows:

[0030] (1) SW837 cell line: purchased from the American Type Culture Collection (ATCC) (Catalog number: CCL-235). This cell line was isolated and established from a patient with stage IV colorectal cancer. It has an epithelial-like morphology and its genomic characteristics are as follows: APC The gene has two heterozygous mutations, c.4348C>T and c.637C>T; FAM123B The gene has a c.1489C>T homozygous mutation; FBXW7 The gene has a homozygous mutation of c.1205_1206insT; KRAS The gene has a c.34G>T heterozygous mutation; TP53 The gene has a c.742C>T homozygous mutation, BRAF Gene wild type.

[0031] The cell culture conditions are: DMEM cell culture medium + 10% fetal bovine serum and placed in a 37°C, 5% CO2 ventilation incubator. When the cell density reaches 80%-90%, the culture medium is removed and the cells are digested with 0.25% trypsin (containing 0.53% EDTA) at 37°C for 8-10 minutes. Fresh complete culture medium is then added to terminate the digestion and the cells are collected, centrifuged (800 rpm for 3 minutes), resuspended and inoculated into a new cell culture dish. The subculture ratio is 1:2-1:5 and the subculture cycle is 5-7 days.

[0032] (2) LIM2099 cell line: purchased from the European Collection of Authenticated Cell Cultures (ECACC) (Catalog number: 12062002). This cell line was obtained from a liver metastasis of colorectal cancer and is a moderately differentiated sclerosing adenocarcinoma. Its genomic characteristics are as follows: KRAS The gene has a homozygous mutation of c.34G>T; BRAF Gene wild type.

[0033] The cell culture conditions are as follows: RPMI-1640 cell culture medium + 10% fetal bovine serum and placed in a 37°C, 5% CO2 ventilation incubator. When the cell density reaches 80%-90%, the culture medium is removed and the cells are digested with 0.25% trypsin (containing 0.05% EDTA) at 37°C for 3-5 minutes. Fresh complete culture medium is then added to terminate the digestion and the cells are collected, centrifuged (800 rpm for 3 minutes), resuspended and inoculated into a new cell culture dish. The subculture ratio is 1:3-1:5 and the subculture cycle is 3-5 days.

[0034] Example 2

[0035] This example describes methods for inducing drug resistance in SW837 and LIM2099 cells in vitro, respectively, as follows:

[0036] (1) Induction of SW837 drug-resistant cells (SW837 / KIR):

[0037] S1) SW837 parental cells in the logarithmic growth phase were seeded into 6-well plates. After adherence, cell culture medium containing sotorasib (MCE, Catalog No. HY114277) was added to 3 wells of the plate. The initial induction concentration of sotorasib was 10 nM. After 3 days of continuous induction, a small number of cells (approximately 10%) were observed to die.

[0038] S2) Replace the culture medium with fresh medium and continue stimulating with 10 nM sotorasib. After two more changes of fresh medium containing 10 nM sotorasib (each stimulation cycle lasting 3 days), the cells should return to the logarithmic growth phase, with cell mortality remaining less than 5% even under sotorasib stimulation. At this point, the cells should be passaged 1:4 into new 6-well plates (the remaining cells should be frozen for seed storage). In three wells, the sotorasib stimulation concentration should be increased to 30 nM, while the remaining three wells should continue to be stimulated at 10 nM (to prevent forced termination of the experiment due to cell contamination, similarly below).

[0039] S3) After three cycles of stimulation at 30 nM (passaged 1:4 when cell density exceeds 80%), cells returned to the logarithmic growth phase and were again passaged 1:4 into new 6-well plates (remaining cells in the 30 nM stimulation group were frozen, and cells in the 10 nM stimulation group were discarded). In three wells, the sotorasib stimulation concentration was increased to 70 nM, and the remaining three wells continued to use 30 nM to maintain stimulation.

[0040] S4) After culturing at 70 nM for four cycles, the cells returned to the logarithmic growth phase. At this point, the cells were passaged again at a 1:4 ratio into fresh 6-well plates (the remaining cells in the 70 nM stimulation group were frozen, and the cells in the 30 nM stimulation group were discarded). The sotorasib stimulation concentration was increased to 100 nM in three wells, while the stimulation was maintained at 70 nM in the remaining three wells. Notably, increasing the sotorasib stimulation concentration from 70 nM to 100 nM resulted in significant growth inhibition of SW837 cells (approximately 30% inhibition), but this inhibition was significantly less than the inhibition at 100 nM in the original parental cells (>50%). Therefore, culturing at 100 nM for 6-8 cycles was required for the cells to return to the logarithmic growth phase. At this point, cells were passaged at a ratio of 1:4 into new 6-well plates (the remaining cells in the 100 nM stimulation group were frozen, and the cells in the 70 nM stimulation group were discarded). In 3 wells, the sotorasib stimulation concentration was increased to 150 nM, and the other 3 wells continued to use 100 nM concentration to maintain stimulation.

[0041] S5) After five cycles of stimulation at 150 nM, cells return to the logarithmic growth phase, at which point drug resistance stabilizes. Subsequent stimulations are performed at concentrations of 200 nM, 300 nM, 400 nM, and 500 nM, respectively. Passaging and cryopreservation follow the same procedures as above. A drug-resistant strain is considered successfully constructed if four cycles at each concentration are maintained while maintaining logarithmic growth.

[0042] During the subsequent culture process after the successful construction of the drug-resistant strain, 500nM sotorasib needs to be added to the drug-resistant cell culture medium to maintain drug resistance.

[0043] (2) Induction of LIM2099-resistant cells (LIM2099 / KIR):

[0044] S1) LIM2099 parental cells in the logarithmic growth phase were plated in 6-well plates. After attachment, 3 wells were inoculated with cell culture medium containing sotorasib at an initial induction concentration of 50 nM. After 2 cycles of stimulation, cells returned to the logarithmic growth phase (an additional 1:3 passage was performed when the cell density was >80%, the same below). At this point, the cells were passaged 1:3 into new 6-well plates (the remaining cells were cryopreserved for seed stock). The sotorasib stimulation concentration was increased to 100 nM in 3 wells, and the remaining 3 wells were maintained at 50 nM.

[0045] S2) After 3 cycles, cells returned to the logarithmic growth phase and were passaged again at a 1:3 ratio into new 6-well plates (remaining cells from the 100 nM stimulation group were frozen, and cells from the 50 nM stimulation group were discarded). In 3 wells, the sotorasib stimulation concentration was increased to 250 nM, and the remaining 3 wells continued to use 100 nM to maintain stimulation.

[0046] S3) After 4 cycles of culture, cells were passaged again at a 1:3 ratio into new 6-well plates (remaining cells from the 250 nM stimulation group were frozen, and cells from the 100 nM stimulation group were discarded). In 3 wells, the sotorasib stimulation concentration was increased to 500 nM, and the remaining 3 wells were maintained at 250 nM.

[0047] S4) Stimulation at a 500 nM concentration gradient requires eight cycles for cells to stably return to the logarithmic growth phase. Subsequent stimulations are performed at concentrations of 750 nM, 1000 nM, 1500 nM, and 2000 nM, with the same passaging and cryopreservation requirements as above. A successful drug-resistant strain is considered established if logarithmic growth is maintained for four cycles at each concentration.

[0048] During the subsequent culture process after the successful construction of the drug-resistant strain, 1000nM sotorasib needs to be added to the drug-resistant cell culture medium to maintain drug resistance.

[0049] Example 3

[0050] This example verifies the drug resistance of SW837 / KIR and LIM2099 / KIR resistant cells in multiple aspects, as follows:

[0051] (1) Cell drug sensitivity test:

[0052] S1) The resistant strains SW837 / KIR and LIM2099 / KIR and their paired parental strains in the logarithmic growth phase were cultured at a rate of 5×10 3 The cells were seeded into 96-well plates at a density of 100 cells / well and placed in a cell incubator for further culture.

[0053] S2) After cells adhered (24 hours after seeding), the medium was replaced with fresh medium and cells were stimulated with 1 nM, 10 nM, 100 nM, 1000 nM, 10 μM, and 100 μM of sotorasib and adagrasib (MCE, Catalog No. HY-130149), respectively, for 72 hours.

[0054] S3) After treatment, aspirate the cell culture medium from the 96-well plate and prepare a 1:9 mixture of CCK8 (CellCounting Kit-8) detection reagent (Dongren, Cat. No. CK04) and cell culture medium (prepared immediately). Add 100 μL of this mixture to each well and return the plate to the incubator for 1-3 hours. After incubation, measure the absorbance of each well using a microplate reader (450 nm wavelength) and convert it to cell viability using the following formula: Cell viability = [(experimental well - blank well) / (control well - blank well)] × 100%.

[0055] Depend on Figure 1 It can be seen that compared with the parental cell line, the sensitivity of SW837 / KIR-resistant cells to sotorasib was reduced by 143.5 times (the half maximal inhibitory concentration (IC50) of parental cells was 356.45nM, and the IC50 value of resistant cells was 51.17uM), and the sensitivity to adagrasib was reduced by 24 times (the IC50 value of parental cells was 70.79nM, and the IC50 value of resistant cells was 1.75uM). The sensitivity of LIM2099 / KIR-resistant cells to sotorasib was reduced by 194.9 times (the IC50 value of parental cells was 97.72nM, and the IC50 value of resistant cells was 19.05uM), and the sensitivity to adagrasib was reduced by 3.8 times (the IC50 value of parental cells was 459.20nM, and the IC50 value of resistant cells was 1.73uM). The above data preliminarily indicate that the SW837 / KIR and LIM2099 / KIR resistant strains were successfully constructed.

[0056] (2) KRAS pathway signal detection:

[0057] S1) SW837 / KIR, LIM2099 / KIR-resistant strains and their paired parental strains in logarithmic growth phase were seeded into 6-well plates at a density of 30%-40%. A blank control group and a sotorasib-treated group (SW837 parental strain and resistant strains were treated with 1000 nM sotorasib, and LIM2099 parental strain and resistant strains were treated with 1000 nM sotorasib) were set up for 24 and 48 hour time gradient treatments.

[0058] S2) After the treatment period, cells from each group were collected into 1.5 ml EP tubes, washed twice with phosphate-buffered saline (PBS), and then added with protein lysis buffer containing 1× protease inhibitors and phosphatase inhibitors. Lysed on ice for 30 minutes, the tubes were centrifuged at 12,000 rpm and 4°C for 10 minutes. The supernatant was collected and quantified by BCA (bicinchoninic acid) analysis.

[0059] S3) Add 5× protein loading buffer according to the sample volume, mix thoroughly, and incubate at 100°C for 10 minutes. Perform protein separation by polyacrylamide gel electrophoresis (10% gel electrophoresis, constant voltage 80 V, then increase to 100 V after 30 minutes and maintain for 90 minutes).

[0060] S4) After electrophoresis, transfer the membrane (the transfer condition is a constant current of 220 mA for 120 minutes);

[0061] S5) After transfer, place the membrane in 8% skim milk solution and block at room temperature for 1 hour.

[0062] S6) After blocking, wash the membrane three times with 1× TBST buffer (Tris-buffered saline with Tween 20). Cut the desired bands and add antibodies against EGFR (CST, Catalog No. 4267), p-EGFR (to detect EGFR phosphorylation and thus reflect activation, CST, Catalog No. 4407), ERK1 / 2 (CST, Catalog No. 4695), and p-ERK1 / 2 (to detect ERK1 / 2 phosphorylation and thus reflect activation, CST, Catalog No. 4370), respectively. Incubate at 4°C in a shaker overnight.

[0063] S7) The next day, remove the primary antibody and wash the membrane three times with 1× TBST buffer for 10 minutes each. Incubate with the secondary antibody at room temperature for 1 hour. Then, wash the membrane three times with TBST buffer for 10 minutes each. After washing, perform chemical development.

[0064] The test results are as follows Figure 2 As shown in the results, compared with parental cells, the KRAS downstream signaling ERK1 / 2 in drug-resistant cells showed significant hyperactivation. Furthermore, under the same concentration and duration of sotorasib stimulation, the phosphorylation level of ERK1 / 2 in drug-resistant cells did not change significantly, while the phosphorylation level of ERK1 / 2 in parental cells decreased significantly after drug stimulation. These results further confirmed that the drug resistance of the drug-resistant cells constructed by the above method is reliable.

[0065] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A KRAS G12C inhibitor-resistant colorectal cancer cell line, characterized in that: The cell line was named SW837 / KIR and deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024360.

2. A KRAS G12C inhibitor-resistant colorectal cancer cell line, characterized in that: The cell line was named LIM2099 / KIR and deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024361.

3. The use of the KRAS G12C inhibitor-resistant colorectal cancer cell line according to claim 1 or 2, comprising: As experimental cells for studying the resistance mechanism of KRAS G12C inhibitors; As experimental cells for drug sensitivity testing.

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