KRAS G12C inhibitor drug-resistant human pancreatic cancer cells as well as construction method and application thereof

Abstract

The invention discloses KRAS G12C inhibitor drug-resistant human pancreatic cancer cells as well as a construction method and application thereof. The KRAS G12C inhibitor drug-resistant human pancreatic cancer cells are human pancreatic cancer cells MIA PaCa / KIR Homo sapiens, and are preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: C2024358, and the preservation time is October 24, 2024. The tolerance of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens to the KRAS G12C inhibitor is greatly improved, and the human pancreatic cancer cells MIA PaCa / KIR Homo sapiens can be used for exploring the drug resistance mechanism of pancreatic cancer KRAS G12C, mining the drug resistance target of the KRAS G12C inhibitor and verifying the curative effect of drug combination.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and more specifically, relates to a KRAS G12C inhibitor-resistant human pancreatic cancer cell, a construction method and an application thereof. Background Art

[0002] Pancreatic cancer is extremely invasive and highly malignant, with a 5-year overall survival rate of only about 12%. More than 80% of patients have progressed to the advanced stage when they seek medical treatment, and the surgical resection rate is only 20%-30%. For most patients with unresectable pancreatic cancer, chemotherapy, targeted therapy, and immunotherapy are available treatment options. Since pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of pancreatic cancer, more than 90% of PDAC patients have KRAS mutations. Therefore, directly targeting KRAS protein is currently a research hotspot for pancreatic cancer treatment.

[0003] Currently, there are two KRAS G12C inhibitors, sotorasib and adagrasib (Chinese names are Sotorasib and Adagrasib), approved by the US Food and Drug Administration for the treatment of patients with advanced non-small cell lung cancer (NSCLC) carrying KRAS G12C mutations. my country's National Medical Products Administration also approved the first domestically produced KRASG12C inhibitor fluzerecib for the clinical treatment of advanced NSCLC in August 2024. According to clinical studies, the objective response rate of sotorasib monotherapy in pancreatic cancer patients was 21%, no patient had a complete remission, and 63% of patients showed stable or continued progression of the disease, indicating that pancreatic cancer resistance to KRAS G12C inhibitors occurs rapidly and widely.

[0004] Therefore, it is urgent to explore the intrinsic regulatory mechanism of pancreatic cancer resistance to KRAS G12C inhibitors to guide the adjustment and optimization of clinical treatment plans. However, due to the following reasons: (1) low patient benefit and high drug prices, KRAS G12C inhibition has not been widely used in the clinical treatment of pancreatic cancer; (2) difficulties in recruiting resistant patients, long-term follow-up, and sample acquisition; (3) there are no publicly shared KRAS G12C inhibitor-resistant pancreatic cancer models and their construction methods. Therefore, the research on the resistance mechanism of pancreatic cancer to KRAS G12C inhibitors has been slow. Summary of the invention

[0005] In view of the above existing technical problems, the primary purpose of the present invention is to provide a human pancreatic cancer cell MIAPaCa / KIR Homo sapiens.

[0006] The second object of the present invention is to provide a method for constructing human pancreatic cancer cells MIA PaCa / KIR Homo sapiens.

[0007] The third object of the present invention is to provide a use of human pancreatic cancer cell MIA PaCa / KIR Homo sapiens in screening KRAS G12C inhibitor resistance targets.

[0008] The fourth object of the present invention is to provide a use of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens in screening anti-pancreatic cancer drugs.

[0009] The fifth object of the present invention is to provide a use of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens in studying the drug resistance mechanism of pancreatic cancer cells or studying the combined drug use of pancreatic cancer.

[0010] In order to achieve the above object, the present invention is implemented through the following technical solutions.

[0011] The present invention claims protection for a human pancreatic cancer cell MIA PaCa / KIR Homo sapiens, wherein the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2024358, a deposit date of October 24, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0012] The present invention provides a human pancreatic cancer cell MIA PaCa / KIRHomo sapiens with high tolerance to KRAS G12C inhibitors, which has greatly increased tolerance to KRAS G12C inhibitors. Such human pancreatic cancer cell MIA PaCa / KIR Homo sapiens can accelerate scientific research breakthroughs in the field of pancreatic cancer KRAS G12C inhibitor resistance, promote the adjustment and optimization of clinical treatment plans, and benefit more patients.

[0013] Preferably, the human pancreatic cancer cells MIA PaCa / KIR Homo sapiens can tolerate a concentration of 0.5uMKRAS G12C inhibitor in in vitro culture.

[0014] Preferably, the KRAS G12C inhibitor is selected from 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-(1M)-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]-4-[(2S)-2-methyl-4-(prop-2-enoyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one (sotorasib, also known as sotorasib), {(2 S)-4-[7-(8-chloronaphthalen-1-yl)-2-{[(2S)-1-methylpyrrolidin-2-yl]-methoxy}-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl}acetonitrile (adagrasib, also known as adagrasib), (4aR,8R)-3-propenyl-11-chloro-10-(2-fluoro-6-hydroxyphenyl)-8-(2-isopropyl-4-methylpyridin-3-yl)-6-methyl-2,3,4-4a,6,8-hexahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c][1,8]naphthyridine-5,7-dione (fluzerex) or one or more thereof.

[0015] Preferably, the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens is induced by human pancreatic cancer cell MIA PaCa2 carrying KRAS G12C mutation. Further, human pancreatic cancer cell MIAPaCa2 carrying KRAS G12C mutation does not have BRAF V600E mutation. BRAF V600E mutation will affect the construction and verification of KRAS resistant cells, increasing complexity.

[0016] Furthermore, the present invention claims a method for constructing a human pancreatic cancer cell MIA PaCa / KIR Homo sapiens, comprising the following steps: The KRAS G12C inhibitor was used to gradually induce the KRAS G12C mutation in human pancreatic cancer cells MIA PaCa2 cultured to the logarithmic growth phase. Each induction includes multiple stimulation cycles, and the interval between each stimulation cycle in each induction is 2-4 days; at the end of each stimulation cycle, determine whether the cells have returned to the logarithmic growth state; if the cells have not returned to the logarithmic growth state, proceed to the next stimulation cycle; if the cells have returned to the logarithmic growth state, proceed to the next induction; Between each induction, the concentration of KRAS G12C inhibitor was increased from 10 to 100 nM.

[0017] Further preferably, the number of inductions is 8-10. Further preferably, during the first to fifth inductions, the concentration of the KRAS G12C inhibitor is increased by 15-25 nM between each induction; during the fifth to seventh inductions, the concentration of the KRAS G12C inhibitor is increased by 45-55 nM between each induction; during the seventh to tenth inductions, the concentration of the KRAS G12C inhibitor is increased by 95-105 nM between each induction.

[0018] The present invention provides a method for constructing human pancreatic cancer cell MIA PaCa / KIR Homo sapiens, and successfully constructs human pancreatic cancer cell MIA PaCa / KIR Homo sapiens based on the construction method, providing a more effective and simpler experimental model for scientific research such as exploring the drug resistance mechanism of pancreatic cancer KRASG12C, mining drug resistance targets of KRAS G12C inhibitors, and verifying the efficacy of combined medication.

[0019] Further, in some embodiments, a method for constructing human pancreatic cancer cell MIA PaCa / KIR Homo sapiens comprises the following steps: (1) After the KRAS G12C mutation-carrying human pancreatic cancer cell MIA PaCa2 cells were cultured to the logarithmic growth phase and the cells reached 40%-50% fusion, they were induced using a cell culture medium containing 10 nM KRAS G12C inhibitor. The stimulation cycle was 3 days. After 3 stimulation cycles, the cells returned to the logarithmic growth state. (2) Inducing the cells that returned to the logarithmic growth state in step (1) using a cell culture medium containing 25 nM KRAS G12C inhibitor. After 4 stimulation cycles, the cells returned to the logarithmic growth state. (3) Using the operation in step (2) as a reference, the cell culture medium containing 50 nM, 75 nM, 100 nM, 150 nM, and 200 nM of the KRAS G12C inhibitor was used to induce 6 stimulation cycles, 4 stimulation cycles, 6 stimulation cycles, 5 stimulation cycles, and 4 stimulation cycles, respectively; (4) Using the operation in step (2) as a reference, the cells that have returned to the logarithmic growth state in step (3) are induced in turn using cell culture medium containing 300 nM and 500 nM KRAS G12C inhibitor. When the cells are cultured at each concentration for at least 3 cycles and are in the logarithmic growth state, human pancreatic cancer cell MIA PaCa / KIR Homosapiens are constructed.

[0020] Preferably, the cell culture medium is DMEM culture medium. Further preferably, the DMEM culture medium contains 10% FBS.

[0021] Furthermore, the present invention seeks to protect the use of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens in screening KRAS G12C inhibitor resistance targets.

[0022] Furthermore, the present invention seeks to protect the use of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens in screening anti-pancreatic cancer drugs.

[0023] Furthermore, the present invention seeks to protect the use of human pancreatic cancer cells MIA PaCa / KIR Homo sapiens in studying the drug resistance mechanism of pancreatic cancer cells or studying the combined drug treatment of pancreatic cancer.

[0024] Furthermore, the present invention claims protection for a KRAS G12C inhibitor-resistant pancreatic cancer cell model, which is composed of human pancreatic cancer cells MIA PaCa / KIR Homosapiens with a preservation number of CCTCC NO: C2024358 and a preservation time of October 24, 2024.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a human pancreatic cancer cell MIA PaCa / KIR Homo sapiens and a construction method thereof. The tolerance of the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens to KRAS G12C inhibitors is greatly increased, thereby providing a more effective and simpler experimental model for scientific research such as exploration of the KRAS G12C resistance mechanism of pancreatic cancer, mining of KRAS G12C inhibitor resistance targets, and verification of the efficacy of combined medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 IC50 value detection of KRAS G12C inhibitor in MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells. Figure 1 A in the figure is the IC50 value test of sotolacib in MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells; Figure 1 B in the figure is the IC50 value test of adagracib in MIA PaCa2 / KIR resistant cells and MIAPaCa2 parental cells.

[0027] Figure 2The activation level of KRAS pathway was detected after MIA PaCa2 / KIR-resistant cells and MIA PaCa2 parental cells were treated with sotolacib in a time-gradient manner.

[0028] Figure 3 These are fluorescent photos of the EdU cell proliferation experiment of MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells.

[0029] Figure 4 This is an apoptosis experiment of MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Example 1 (1) Detailed information and routine culture methods of the parental cell line of KRAS G12C mutant pancreatic cancer cell MIA PaCa2 are as follows: MIA PaCa2 parental cell line: purchased from the American Type Culture Collection (ATCC, catalog number: CRL-1420). The cell was isolated and established from the tumor tissue of a 65-year-old white male pancreatic cancer patient. It is a poorly differentiated diploid pancreatic cancer cell with the following genomic characteristics: a homozygous missense mutation in the KRAS gene (p.G12C; GGT>TGT); a homozygous deletion of exons 1, 2, and 3 in the CDKN2A / p16 gene; a homozygous missense mutation in the TP53 gene (p.R248W; CGG>TGG); and a wild-type BRAF gene.

[0032] The culture conditions of the cells are: Dulbecco's Modified Eagle's Medium (DMEM, source: Thermo Fisher Scientific, Gibco, catalog number C11995500BT) containing 10% fetal bovine serum (FBS), and placed in a 37°C 5% CO2 ventilation incubator. When the cell density grows to 80%-90%, the cells are passaged. Since the cells have the characteristics of semi-attached and semi-suspended, the suspended cells in the culture medium are first collected during passage, and then 0.25% trypsin (containing 0.53% ethylenediaminetetraacetic acid (EDTA)) is added to digest for 3-5 minutes at 37°C. Fresh DMEM medium containing 10% FBS is then added to terminate the digestion and collect the adherent cells. After the suspension cells and adherent cells are centrifuged (centrifuged at 800 rpm for 3 minutes), they are resuspended with fresh DMEM medium containing 10% FBS and inoculated into a new cell culture dish. The passage ratio is 1:4-1:6, and the passage cycle is 3-5 days.

[0033] (2) Method for inducing drug resistance in vitro in pancreatic cancer cells MIA PaCa2 carrying KRAS G12C mutation The MIA PaCa2 parent cell line carrying the KRAS G12C mutation cultured to the logarithmic growth phase in step (1) was inoculated into a 6-well plate. After the cells were fused to 40%-50%, 3 wells were added with DMEM culture medium containing 10 nM sotorasib (MCE, Catalog No. HY114277) and 10% FBS. A stimulation cycle was 3 days. After the culture medium containing 10 nM sotorasib was replaced three times, that is, after 3 stimulation cycles (if the cell density exceeded 80% during the stimulation period, normal passage was allowed, the same below), it was observed that the cells returned to the logarithmic growth state. At this time, the cells were inoculated into a new 6-well plate and the remaining cells were frozen for seed preservation. 3 wells in the 6-well plate were stimulated and cultured with 25 nM sotorasib, and the remaining 3 wells continued to be stimulated with 10 nM sotorasib as a control.

[0034] After culturing for 4 cycles at a concentration of 25 nM, the cells returned to a logarithmic growth state. At this time, the cells were seeded into a new 6-well plate and the remaining cells were frozen for seed preservation. In addition, the cells in the 10 nM stimulation group were discarded, and the sotorasib stimulation concentration in 3 wells of the 6-well plate was increased to 50 nM, and the remaining 3 wells continued to use 25 nM concentration to maintain stimulation as a control.

[0035] After culturing for 6 cycles at a concentration of 50 nM, the cells returned to a logarithmic growth state. At this time, the cells were seeded into a new 6-well plate and the remaining cells were frozen for seed preservation. In addition, the cells in the 25 nM stimulation group were discarded, and the sotorasib stimulation concentration in 3 wells of the 6-well plate was increased to 75 nM, and the remaining 3 wells continued to use 50 nM concentration to maintain stimulation as a control.

[0036] After culturing for 4 cycles at a concentration of 75 nM, the cells returned to a logarithmic growth state. At this time, the cells were seeded into a new 6-well plate and the remaining cells were frozen for seed preservation. In addition, the cells in the 50 nM stimulation group were discarded, and the sotorasib stimulation concentration in 3 wells of the 6-well plate was increased to 100 nM, and the remaining 3 wells continued to use 75 nM concentration to maintain stimulation as a control.

[0037] After culturing for 6 cycles at a concentration of 100 nM, the cells returned to a logarithmic growth state. At this time, the cells were seeded into a new 6-well plate and the remaining cells were frozen for seed preservation. In addition, the cells in the 75 nM stimulation group were discarded, and the sotorasib stimulation concentration in 3 wells of the 6-well plate was increased to 150 nM, and the remaining 3 wells continued to use 100 nM concentration to maintain stimulation as a control.

[0038] After culturing for 5 cycles at a concentration of 150 nM, the cells returned to a logarithmic growth state. At this time, the cells were seeded into a new 6-well plate and the remaining cells were frozen for seed preservation. In addition, the cells in the 100 nM stimulation group were discarded, and the sotorasib stimulation concentration in 3 wells of the 6-well plate was increased to 200 nM, and the remaining 3 wells continued to use 150 nM concentration to maintain stimulation as a control.

[0039] After culturing for 4 cycles at a concentration of 200 nM, the cells returned to a logarithmic growth state, at which time the cell resistance tended to be stable. The subsequent drug sotorasib stimulation concentration gradients were 300 nM and 500 nM, respectively. The passaging and freezing requirements were the same as above. The cells were cultured for 3 cycles at each concentration and were in a logarithmic growth state. The MIA PaCa2 / KIR resistant cell line was constructed, which is a human pancreatic cancer cell line MIA PaCa / KIR Homo sapiens, deposited in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: C2024358, and a preservation time of October 24, 2024.

[0040] During the subsequent culture process after the successful construction of the MIA PaCa2 / KIR resistant cell line, 100 nM sotorasib needs to be added to the culture medium to maintain drug resistance.

[0041] Example 2 This example verifies the drug resistance of the pancreatic cancer cell MIA PaCa2 / KIR drug-resistant strain induced by Example 1 in many aspects, as follows: (1) Cell drug sensitivity test: Pancreatic cancer cell MIA PaCa2 / KIR resistant strain cells and pancreatic cancer cell MIA PaCa2 parent strain cells in the logarithmic growth phase were cultured at 5*10 3The cells were inoculated into 96-well plates at a density of 100 cells / well and cultured in a cell culture incubator at 37°C and 5% CO2. After the cells adhered to the wall, the groups were set according to the drug concentrations of sotorasib and adagrasib (MCE, Catalog No. HY-130149) (1nM, 10nM, 100nM, 1000nM, 10uM and 100uM), with 3 wells in each group. DMEM medium containing the corresponding concentrations of sotorasib and adagrasib was added to each group and cultured for 72 hours. After the culture was completed, CCK8 (Cell Counting Kit-8) detection reagent (GLPBIO, Catalog No. GK10001) and cell culture medium were mixed at a ratio of 1:9 (prepared and used immediately), and then the culture medium in the 96-well plate was aspirated, and 100uL of the above-configured CCK8 mixture was added to each well and placed back in the cell culture incubator for incubation for 1-3 hours. Note that bubbles should be avoided when adding the CCK8 mixture. After incubation, the absorbance of each well was detected at a wavelength of 450 nm using an enzyme reader and the cell viability was calculated. The conversion formula was: cell survival rate = [(absorbance value of the experimental well - absorbance value of the blank well) / (absorbance value of the control well - absorbance value of the blank well)] × 100%.

[0042] Depend on Figure 1 It can be seen that compared with the pancreatic cancer cell MIA PaCa2 parental strain cells, the sensitivity of the pancreatic cancer cell MIA PaCa2 / KIR resistant strain cells to sotorasib is reduced by 283.76 times (the half-inhibitory concentration IC50 value of the parental cells is 51.17nM, and the IC50 value of the resistant cells is 14.52uM); the sensitivity of the pancreatic cancer cell MIA PaCa2 / KIR resistant strain cells to adagrasib is reduced by 59.70 times (the IC50 value of the parental cells is 29.65nM, and the IC50 value of the resistant cells is 1.77uM). The above data preliminarily indicate that the pancreatic cancer cell MIA PaCa2 / KIR resistant strain was successfully constructed.

[0043] (2) KRAS pathway signal detection: Pancreatic cancer cell MIA PaCa2 / KIR-resistant strain cells and pancreatic cancer cell MIA PaCa2 parent strain cells in the logarithmic growth phase were seeded into 6-well plates at a density of 30%-40%, and a blank control group and a 100 nM sotorasib treatment group were set up for different treatment time. After the treatment, the culture medium was discarded, and the cells were washed twice with pre-cooled phosphate buffered saline (PBS). Then, protein lysis buffer containing 1× protease inhibitor (source: Cocktail (EDTA-Free, 100×in DMSO), MCE, catalog number HY-K0010) and phosphatase inhibitor (source: Cocktail II (100×in ddH2O), MCE, catalog number HY-K0022) was added, and the cells were lysed on ice for 30 min, and shaken frequently to fully lyse the cells. Scrape the cells with a pre-cooled cell scraper and transfer the cell suspension to a 1.5 ml EP tube, then centrifuge at 12000 rpm for 10 minutes at 4°C, transfer the supernatant and perform BCA (Bicinchoninic Acid), then add 5× protein loading buffer (source: dual-color loading buffer (DTT), Fude Biotechnology, FD006) to the protein supernatant at a ratio of 1:4 and mix well, then place the sample in a metal bath and heat at 100°C for 10 minutes to denature the protein. Perform polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein. The electrophoresis conditions are 10% electrophoresis gel, first run at a constant voltage of 80V for 30 minutes, then adjust the voltage to 100V and maintain it for 90 minutes. After the electrophoresis, transfer the membrane, and the conditions are a constant current of 220mA for 120 minutes. After the transfer, take out the membrane and place it in 1X TBST (Tris-buffered saline containing 1% Tween 20) containing 8% skim milk, and block it at room temperature for 1 hour. After blocking, wash the membrane three times with 1X TBST buffer for 10 minutes each time, then cut the required bands according to the size of the protein molecule, add the diluted EGFR antibody (antibody CST, cat. No. 4267), p-EGFR antibody (CST, cat. No. 4407), ERK1 / 2 antibody (CST, cat. No. 4695) and p-ERK1 / 2 antibody (CST, cat. No. 4370), respectively, and place it in a shaker at 4°C overnight. The next day, recover the primary antibody and wash the membrane three times with 1X TBST buffer for 10 minutes each time. Incubate with a secondary antibody that matches the species of the primary antibody at room temperature for 1 hour, then recover the secondary antibody and wash the membrane three times with 1X TBST buffer for 10 minutes each time, and then perform chemiluminescence development.

[0044] Test results such as Figure 2As shown: Compared with MIA PaCa2 parental cells, the phosphorylation level of KRAS downstream signal ERK1 / 2 in MIA PaCa2 / KIR resistant cells increased, indicating that ERK1 / 2 showed significant hyperactivation; in addition, under the stimulation of sotorasib at the same concentration and time, the phosphorylation level of ERK1 / 2 in KRAS in MIA PaCa2 / KIR resistant cells did not change significantly, while the phosphorylation level of ERK1 / 2 in MIA PaCa2 parental cells decreased significantly after drug stimulation. The above data show that the pancreatic cancer cell MIA PaCa2 / KIR resistant strain was successfully constructed.

[0045] Example 3 This example verifies the differences between pancreatic cancer cell line MIA PaCa2 / KIR drug-resistant cells and MIA PaCa2 parental cell line cells in many aspects, as follows.

[0046] (1) EdU cell proliferation assay Pancreatic cancer cell MIA PaCa / KIR drug-resistant strain cells and pancreatic cancer cell MIAPaCa2 parent strain cells in logarithmic growth phase were seeded into 24-well plates at a density of 40%-50% and cultured overnight in a cell culture incubator at 37°C and 5% CO2. After the cells adhered to the wall, sotorasib was added for 24 hours, and the sotorasib concentration was set to 0µM and 0.1µM. Using BeyoClickTMEdU-555 Cell Proliferation Detection Kit (Biyuntian, Cat. No. C0075S), 20µM EdU working solution (2X) was prepared and preheated to 37 degrees. An equal volume was added to the 24-well plate and cultured for 2 hours for EdU labeling. After EdU labeling, the culture medium was removed, and 1ml of 4% paraformaldehyde fixative (Biyuntian, Cat. No. P0099) was added to each well, and fixed at room temperature for 15 minutes. After fixation, the fixative was removed, and 1ml of PBS buffer was added to each well for washing 3 times, 3-5 minutes each time. Remove PBS, add 1ml of immunostaining strong permeabilization solution (Biyuntian, catalog number P0097) to each well, and incubate at room temperature for 10-15 minutes. Remove the permeabilization solution and wash the cells 1-2 times with 1ml PBS per well, each time for 3-5 minutes. Prepare the Click reaction solution according to the ratio of 860µl Click Reaction Buffer, 40ul CuSO4, 2µl Azide 555, and 100µl Click Additive Solution per ml of Click reaction solution. Remove PBS, add 1000µl of Click reaction solution to each well, gently shake the culture plate to ensure that the reaction mixture evenly covers the sample, and incubate at room temperature in the dark for 30 minutes. At the end of incubation, remove the reaction solution and wash the cells 3 times with PBS, each time for 3-5 minutes. Dilute Hoechst 33342 with PBS at a ratio of 1:1000. After removing PBS, add 500µl of the diluted Hoechst solution to each well and incubate at room temperature in the dark for 10 minutes. The Hoechst solution was removed and the cells were washed three times with PBS for 3-5 minutes each time, followed by observation and photography using a fluorescence microscope.

[0047] Test results such as Figure 3 As shown, blue is the cell nucleus and red is the cell in the proliferation phase. The number of proliferating cells of the MIA PaCa2 parental strain cells was significantly reduced under sotorasib treatment, and the proliferation was significantly inhibited. However, under the same concentration of sotorasib treatment, the proliferation of the MIA PaCa2 / KIR resistant strain cells was not significantly inhibited. Therefore, the MIA PaCa2 / KIR resistant strain cells have obvious resistance to the inhibitory effect of sotorasib on proliferation.

[0048] (2) Annexin V-AF647 / PI (propidium iodide) apoptosis experiment Pancreatic cancer cell MIA PaCa / KIR drug-resistant strain cells and pancreatic cancer cell MIAPaCa2 parent strain cells in logarithmic growth phase were seeded into 12-well plates at a density of 40%-50% and cultured overnight in a cell culture incubator at 37°C and 5% CO2. After the cells adhered to the wall, sotorasib was added for 24 hours, and the concentration of sotorasib was set to 0µM, 0.1µM, and 1µM. The level of cell apoptosis was detected using the Annexin V-AF647 / PI apoptosis kit (GOONIE, Cat. No. 100-102). After digestion, the cells were collected in a centrifuge tube, centrifuged to remove the supernatant, and then 1ml of 4°C pre-cooled PBS was added to wash once, and the supernatant was carefully removed. Dilute Bingding Buffer with deionized water at a ratio of 1:4, resuspend the cells with 1X Bingding Buffer, and adjust the cell concentration to 1-5X 106 / ml. Take 100µl of cell suspension from each sample into a 5ml flow cytometry tube, add 5µl Annexin V-AF647 and 5µl PI solution, mix gently and incubate at room temperature for 5 minutes in the dark. Add 400µl BingdingBuffer and perform flow cytometry detection immediately.

[0049] The experimental results are as follows Figure 4 As shown, the lower left quadrant represents live cells (Annexin V negative, PI negative), the upper left quadrant represents necrotic cells (Annexin V negative, PI positive), the lower right quadrant represents early apoptotic cells (Annexin V positive, PI negative), and the upper right quadrant represents late apoptotic cells (Annexin V positive, PI positive).

[0050] It can be seen that when the MIA PaCa2 parental strain cells were treated with 0µM sotorasib, the proportion of early apoptotic cells was 1.44%, and the proportion of late apoptotic cells was 1.16%; when treated with 0.1µM sotorasib, the proportion of early apoptotic cells was 9.77%, and the proportion of late apoptotic cells was 4.11%; when treated with 1µM sotorasib, the proportion of early apoptotic cells was 11.7%, and the proportion of late apoptotic cells was 4%.

[0051] In comparison, when MIA PaCa / KIR resistant cells were treated with 0µM sotorasib, the proportion of early apoptotic cells was 0.97%, and the proportion of late apoptotic cells was 1.35%; when treated with 0.1µM sotorasib, the proportion of early apoptotic cells was 1.09%, and the proportion of late apoptotic cells was 1.99%; when treated with 1µM sotorasib, the proportion of early apoptotic cells was 1.02%, and the proportion of late apoptotic cells was 1.9%. Therefore, MIA PaCa / KIR resistant cells have obvious resistance to the pro-apoptotic effect of sotorasib.

[0052] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A human pancreatic cancer cell MIA PaCa / KIR Homo sapiens, characterized in that: The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024358 and a deposit date of October 24, 2024.

2. The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to claim 1, characterized in that: The human pancreatic cancer cells MIA PaCa / KIR Homo sapiens tolerate 0.5uM KRAS G12C inhibitor concentration in vitro culture.

3. The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to claim 2, characterized in that: The KRAS G12C inhibitor is selected from 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-(1M)-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]-4-[(2S)-2-methyl-4-(prop-2-enoyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one, {(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-{[(2 S)-1-methylpyrrolidin-2-yl]-methoxy}-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl}acetonitrile, (4aR,8R)-3-propenyl-11-chloro-10-(2-fluoro-6-hydroxyphenyl)-8-(2-isopropyl-4-methylpyridin-3-yl)-6-methyl-2,3,4-4a,6,8-hexahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c][1,8]naphthyridine-5,7-dione or more.

4. The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to claim 1, characterized in that: The human pancreatic cancer cells MIA PaCa / KIR Homo sapiens are obtained by inducing human pancreatic cancer cells MIAPaCa2 carrying KRAS G12C mutation.

5. The method for constructing the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to any one of claims 1 to 4, characterized in that: The following steps are involved: The KRAS G12C inhibitor was used to gradually induce the KRAS G12C mutation in human pancreatic cancer cells MIA PaCa2 cultured to the logarithmic growth phase. Each induction includes multiple stimulation cycles, and the interval between each stimulation cycle in each induction is 2-4 days; at the end of each stimulation cycle, determine whether the cells have returned to the logarithmic growth state; if the cells have not returned to the logarithmic growth state, proceed to the next stimulation cycle; if the cells have returned to the logarithmic growth state, proceed to the next induction; Between each induction, the concentration of KRAS G12C inhibitor was increased from 10 to 100 nM.

6. The construction method according to claim 5, characterized in that: The following steps are involved: (1) After the KRAS G12C mutation-carrying human pancreatic cancer cell MIA PaCa2 cells were cultured to the logarithmic growth phase and the cells reached 40%-50% fusion, they were induced using a cell culture medium containing 10 nM KRAS G12C inhibitor. The stimulation cycle was 3 days. After 3 stimulation cycles, the cells returned to the logarithmic growth state. (2) Inducing the cells that returned to the logarithmic growth state in step (1) using a cell culture medium containing 25 nM KRAS G12C inhibitor. After 4 stimulation cycles, the cells returned to the logarithmic growth state. (3) Using the operation in step (2) as a reference, the cell culture medium containing 50 nM, 75 nM, 100 nM, 150 nM, and 200 nM KRASG12C inhibitor was used to induce 6 stimulation cycles, 4 stimulation cycles, 6 stimulation cycles, 5 stimulation cycles, and 4 stimulation cycles, respectively; (4) Using the operation in step (2) as a reference, the cells that have returned to the logarithmic growth state in step (3) are induced in turn using cell culture medium containing 300 nM and 500 nM KRAS G12C inhibitor. When the cells are cultured at each concentration for at least 3 cycles and are in the logarithmic growth state, human pancreatic cancer cell MIA PaCa / KIR Homosapiens are constructed.

7. The construction method according to claim 6, characterized in that: The cell culture medium is DMEM culture medium.

8. Use of the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to any one of claims 1 to 4 in screening KRASG12C inhibitor resistance targets.

9. Use of the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to any one of claims 1 to 4 in screening anti-pancreatic cancer drugs.

10. Use of the human pancreatic cancer cell MIA PaCa / KIR Homo sapiens according to any one of claims 1 to 4 in studying the drug resistance mechanism of pancreatic cancer cells or studying the combined drug treatment of pancreatic cancer.

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