KRAS G12C inhibitor-resistant human pancreatic cancer cells, construction method and application thereof

Abstract

The present invention discloses KRAS G12C inhibitor-resistant human pancreatic cancer cells, their construction methods, and their applications. The KRAS G12C inhibitor-resistant human pancreatic cancer cells are MIA PaCa / KIR Homo sapiens, deposited with the China Center for Type Culture Collection under the CCTCC No. C2024358, dated October 24, 2024. MIA PaCa / KIR Homo sapiens cells exhibit significantly increased tolerance to KRAS G12C inhibitors and can be used to explore KRAS G12C resistance mechanisms in pancreatic cancer, identify KRAS G12C inhibitor resistance targets, and validate the efficacy of combination therapy.

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 highly aggressive and malignant, with a five-year overall survival rate of only approximately 12%. Over 80% of patients present with advanced disease, and surgical resection rates are only 20%-30%. For the majority of patients with unresectable pancreatic cancer, chemotherapy, targeted therapy, and immunotherapy are available treatment options. Because pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of pancreatic cancer, and over 90% of PDAC patients harbor KRAS mutations, directly targeting the KRAS protein is currently a research hotspot in pancreatic cancer treatment.

[0003] Currently, two KRAS G12C inhibitors, sotorasib and adagrasib (known in Chinese as Sotorasib and Adagrasib, respectively), have been approved by the U.S. Food and Drug Administration for the treatment of patients with advanced non-small cell lung cancer (NSCLC) carrying the KRAS G12C mutation. In August 2024, my country's National Medical Products Administration also approved the first domestically produced KRAS G12C inhibitor, fluzivac, for the clinical treatment of advanced NSCLC. Clinical studies have shown that the objective response rate of sotorasib monotherapy in pancreatic cancer patients was 21%, with no patients achieving complete remission and 63% experiencing stable or progressive disease, indicating that resistance to KRAS G12C inhibitors in pancreatic cancer develops rapidly and widely.

[0004] Therefore, there is an urgent need to explore the intrinsic regulatory mechanisms 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 yet been widely used in the clinical treatment of pancreatic cancer; (2) difficulties in recruiting, long-term tracking, and obtaining samples from resistant patients; (3) the lack of publicly shared KRAS G12C inhibitor-resistant pancreatic cancer models and their construction methods, etc., the current research on the mechanisms of KRAS G12C inhibitor resistance in pancreatic cancer has been slow. Summary of the Invention

[0005] In view of the above-mentioned 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 cells MIA PaCa / KIR Homo sapiens in screening KRAS G12C inhibitor resistance targets.

[0008] A 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] A 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 combination therapy of pancreatic cancer.

[0010] In order to achieve the above objectives, 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, which 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 line MIA PaCa / KIRHomo sapiens with high tolerance to KRAS G12C inhibitors. The human pancreatic cancer cell line MIA PaCa / KIR Homo sapiens has greatly increased tolerance to KRAS G12C inhibitors. Such human pancreatic cancer cell line 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-(prop-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 One or more of: (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 (Fozerezet).

[0015] Preferably, the human pancreatic cancer cell line MIA PaCa / KIR Homo sapiens is derived from human pancreatic cancer cell line MIA PaCa2 carrying the KRAS G12C mutation. Furthermore, the human pancreatic cancer cell line MIA PaCa2 carrying the KRAS G12C mutation does not have the BRAF V600E mutation. The BRAF V600E mutation can affect the generation and validation of KRAS-resistant cells, increasing complexity.

[0016] Furthermore, the present invention claims a method for constructing human pancreatic cancer cells MIA PaCa / KIR Homo sapiens, comprising the following steps:

[0017] Human pancreatic cancer cells MIA PaCa2 carrying KRAS G12C mutations that were cultured to the logarithmic growth phase were gradually induced using increasing concentrations of KRAS G12C inhibitors.

[0018] Each induction includes multiple stimulation cycles, with the interval between each stimulation cycle being 2-4 days. At the end of each stimulation cycle, it is determined whether the cells have returned to the logarithmic growth state. If the cells have not returned to the logarithmic growth state, the next stimulation cycle is performed. If the cells have returned to the logarithmic growth state, the next induction is performed.

[0019] Between each induction, the concentration of KRAS G12C inhibitor was increased from 10 to 100 nM.

[0020] More preferably, the number of inductions is 8-10. More 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; and during the seventh to tenth inductions, the concentration of the KRAS G12C inhibitor is increased by 95-105 nM between each induction.

[0021] 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 work such as exploring the KRASG12C resistance mechanism of pancreatic cancer, discovering KRAS G12C inhibitor resistance targets, and verifying the efficacy of combination therapy.

[0022] Furthermore, in some embodiments, a method for constructing human pancreatic cancer cell MIA PaCa / KIR Homo sapiens comprises the following steps:

[0023] (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 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.

[0024] (2) Inducing the cells that returned to the logarithmic growth state in step (1) using cell culture medium containing 25 nM KRAS G12C inhibitor. After 4 stimulation cycles, the cells returned to the logarithmic growth state.

[0025] (3) Using the operation in step (2) as a reference, the cells were induced using cell culture medium containing 50 nM, 75 nM, 100 nM, 150 nM, and 200 nM of the KRAS G12C inhibitor for 6 stimulation cycles, 4 stimulation cycles, 6 stimulation cycles, 5 stimulation cycles, and 4 stimulation cycles, respectively;

[0026] (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.

[0027] Preferably, the cell culture medium is DMEM medium. Further preferably, the DMEM medium contains 10% FBS.

[0028] 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.

[0029] 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.

[0030] 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 combination therapy of pancreatic cancer.

[0031] 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 deposit number of CCTCC NO: C2024358 and a deposit date of October 24, 2024.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a human pancreatic cancer cell MIA PaCa / KIR Homo sapiens and a construction method thereof. The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens has greatly increased tolerance to KRAS G12C inhibitors, providing a more effective and simpler experimental model for scientific research such as exploring the KRAS G12C resistance mechanism of pancreatic cancer, discovering KRAS G12C inhibitor resistance targets, and verifying the efficacy of combination therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 IC50 value detection of KRAS G12C inhibitor in MIA PaCa2 / KIR resistant cell line and MIA PaCa2 parent cell line. Figure 1 A in the figure is the IC50 value 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.

[0035] Figure 2 The 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.

[0036] Figure 3 These are fluorescence photos of the EdU cell proliferation experiment of MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells.

[0037] Figure 4 This is an apoptosis experiment of MIA PaCa2 / KIR resistant cells and MIA PaCa2 parental cells. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Example 1

[0040] (1) Detailed information and conventional culture methods of the KRAS G12C mutant pancreatic cancer cell line MIA PaCa2 are as follows:

[0041] MIA PaCa2 parental cell line: Purchased from the American Type Culture Collection (ATCC, catalog number: CRL-1420), these cells were isolated and established from tumor tissue of a 65-year-old Caucasian male patient with pancreatic cancer. These cells are poorly differentiated diploid pancreatic cancer cells with 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 wild-type BRAF.

[0042] The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, source: Thermo Fisher Scientific, Gibco, catalog number C11995500BT) supplemented with 10% fetal bovine serum (FBS) in a 37°C, 5% CO2-filled incubator. Cells were passaged when the cell density reached 80%-90%. Because the cells are semi-adherent and semi-suspended, suspension cells were harvested and digested with 0.25% trypsin (containing 0.53% ethylenediaminetetraacetic acid (EDTA)) at 37°C for 3-5 minutes. Fresh DMEM supplemented with 10% FBS was then added to terminate digestion and adherent cells were harvested. The suspension and adherent cells were centrifuged (800 rpm for 3 minutes) and resuspended in fresh DMEM supplemented with 10% FBS and plated onto new cell culture dishes. The cell ratio was 1:4-1:6, and the passage interval was 3-5 days.

[0043] (2) Method for inducing drug resistance in pancreatic cancer cells MIA PaCa2 carrying KRAS G12C mutation in vitro

[0044] The MIA PaCa2 parental 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 number HY114277) and 10% FBS. A stimulation cycle was 3 days. After three changes of culture medium containing 10 nM sotorasib, 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.

[0045] 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 new 6-well plates 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. The remaining 3 wells continued to use a 25 nM concentration to maintain stimulation as a control.

[0046] 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. The remaining 3 wells continued to use a 50 nM concentration to maintain stimulation as a control.

[0047] 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 new 6-well plates 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. The remaining 3 wells continued to use a concentration of 75 nM to maintain stimulation as a control.

[0048] 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. The remaining 3 wells continued to use a 100 nM concentration to maintain stimulation as a control.

[0049] 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. The remaining 3 wells continued to use a 150 nM concentration to maintain stimulation as a control.

[0050] After culturing for 4 cycles at a concentration of 200 nM, the cells returned to a logarithmic growth state, at which point 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 all in a logarithmic growth state. The MIA PaCa2 / KIR drug-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 deposit number of CCTCC NO: C2024358 and a deposit date of October 24, 2024.

[0051] During the subsequent culture process after the successful construction of the MIA PaCa2 / KIR drug-resistant cell line, 100 nM sotorasib needs to be added to the culture medium to maintain drug resistance.

[0052] Example 2

[0053] This example verifies the drug resistance of the pancreatic cancer cell line MIA PaCa2 / KIR resistant cells induced in Example 1 in multiple aspects, as follows:

[0054] (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 a rate of 5*10 3 Cells were seeded into 96-well plates at a density of 1 cell / well and cultured in a cell culture incubator at 37°C and 5% CO2. After the cells adhered, they were divided into groups of 3 wells each according to the drug concentrations of sotorasib and adagrasib (MCE, Catalog No. HY-130149) (1 nM, 10 nM, 100 nM, 1000 nM, 10 μM, and 100 μM). DMEM containing the corresponding concentrations of sotorasib and adagrasib was added to each group and cultured for an additional 72 hours. After the incubation period, CCK8 (Cell Counting Kit-8) detection reagent (GLPBIO, Catalog No. GK10001) was mixed with cell culture medium at a ratio of 1:9 (prepared immediately before use). The medium in the 96-well plate was then aspirated, and 100 μL of the prepared CCK8 mixture was added to each well. The cells were then returned to the cell culture incubator and incubated for 1-3 hours. Be careful to avoid introducing bubbles when adding the CCK8 mixture. After incubation, the absorbance of each well was detected at a wavelength of 450 nm using a microplate reader and the cell viability was calculated using the following formula: cell viability = [(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%.

[0055] Depend on Figure 1 As shown, compared with the pancreatic cancer cell line MIA PaCa2, the pancreatic cancer cell line MIA PaCa2 / KIR-resistant cell line showed a 283.76-fold decrease in sensitivity to sotorasib (IC50 value of parental cells was 51.17 nM, IC50 value of resistant cells was 14.52 uM). The pancreatic cancer cell line MIA PaCa2 / KIR-resistant cell line showed a 59.70-fold decrease in sensitivity to adagrasib (IC50 value of parental cells was 29.65 nM, IC50 value of resistant cells was 1.77 uM). These data preliminarily indicate that the pancreatic cancer cell line MIA PaCa2 / KIR-resistant cell line was successfully constructed.

[0056] (2) KRAS pathway signal detection: Pancreatic cancer cell line MIA PaCa2 / KIR-resistant strain and pancreatic cancer cell line MIA PaCa2 parent strain in the logarithmic growth phase were seeded into 6-well plates at a density of 30%-40%, and blank control group and 100nM sotorasib treatment group were set up for different treatment time. After treatment, the culture medium was discarded and 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. The cells were lysed on ice for 30 minutes and shaken frequently to fully lyse the cells. Cells were scraped off using a pre-chilled cell scraper and the cell suspension transferred to a 1.5 ml EP tube. The suspension was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was removed and subjected to BCA (bicinchoninic acid) analysis. 5× protein loading buffer (source: Dual-color Loading Buffer (DTT), Fude Biotechnology, FD006) was then added to the supernatant at a 1:4 ratio and mixed thoroughly. The sample was then heated in a metal bath at 100°C for 10 minutes to denature the proteins. Proteins were separated by SDS-PAGE using a 10% gel electrophoresis solution at 80 V for 30 minutes, then reduced to 100 V and maintained for 90 minutes. After electrophoresis, the membrane was transferred to a membrane at a constant current of 220 mA for 120 minutes. After transfer, the membrane was removed and blocked in 1× TBST (Tris-buffered saline containing 1% Tween 20) containing 8% skim milk for 1 hour at room temperature. After blocking, the membrane was washed three times with 1X TBST buffer for 10 minutes each. The desired bands were then cut based on protein size and diluted antibodies against EGFR (CST, Cat. No. 4267), p-EGFR (CST, Cat. No. 4407), ERK1 / 2 (CST, Cat. No. 4695), and p-ERK1 / 2 (CST, Cat. No. 4370) were added, respectively, and incubated overnight at 4°C on a shaker. The next day, the primary antibody was removed and the membrane was washed three times with 1X TBST buffer for 10 minutes each. A secondary antibody matching the species of the primary antibody was then used for incubation at room temperature for one hour. The secondary antibody was then removed and the membrane was washed three times with 1X TBST buffer for 10 minutes each. Chemiluminescent imaging was then performed.

[0057] Test results such as Figure 2As shown, compared with the parental MIA PaCa2 cell line, the phosphorylation level of ERK1 / 2, a downstream signaling pathway of KRAS, was increased in the MIA PaCa2 / KIR-resistant cell line, indicating significant hyperactivation of ERK1 / 2. Furthermore, under sotorasib stimulation at the same concentration and time, the phosphorylation level of ERK1 / 2 in the KRAS pathway in the MIA PaCa2 / KIR-resistant cell line did not significantly change, while the phosphorylation level of ERK1 / 2 in the parental MIA PaCa2 cell line decreased significantly after drug stimulation. These data demonstrate that the MIA PaCa2 / KIR-resistant pancreatic cancer cell line was successfully constructed.

[0058] Example 3

[0059] This example verifies the differences between pancreatic cancer cell line MIA PaCa2 / KIR drug-resistant cells and MIA PaCa2 parental cell line cells in multiple aspects, as follows.

[0060] (1) EdU cell proliferation assay

[0061] Logarithmically growing pancreatic cancer cells (MIA PaCa / KIR-resistant) and the parental pancreatic cancer cell line (MIAPaCa2) 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, they were treated with sotorasib for 24 hours at concentrations of 0µM and 0.1µM, respectively. Using the BeyoClick™ EdU-555 Cell Proliferation Assay Kit (Beyotime, Cat. No. C0075S), a 20µM EdU working solution (2X) was prepared and pre-warmed to 37°C. An equal volume of solution was added to the 24-well plate and incubated for another 2 hours for EdU labeling. After EdU labeling, the culture medium was removed, and 1ml of 4% paraformaldehyde fixative (Beyotime, Cat. No. P0099) was added to each well for 15 minutes at room temperature. After fixation, the fixative was removed, and the wells were washed three times with 1ml of PBS buffer for 3-5 minutes each. Remove PBS and add 1 ml of Immunostaining Intense Permeabilization Buffer (Biyuntian, Cat. No. P0097) to each well. Incubate at room temperature for 10-15 minutes. Remove the permeabilization buffer and wash the cells one to two times with 1 ml of PBS per well, each time for 3-5 minutes. Prepare a click reaction solution containing 860 µl of Click Reaction Buffer, 40 µl of CuSO₄, 2 µl of Azide 555, and 100 µl of Click Additive Solution per ml of click reaction solution. Remove PBS and add 1000 µl of click reaction solution to each well. Gently shake the plate to ensure even coverage of the sample with the reaction mixture. Incubate at room temperature for 30 minutes in the dark. At the end of the incubation, remove the reaction solution and wash the cells three times with PBS, each for 3-5 minutes. Dilute Hoechst 33342 at a 1:1000 ratio with PBS. After removing the PBS, add 500 µl 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.

[0062] Test results such as Figure 3 As shown, blue represents cell nuclei, and red represents cells in the proliferating phase. Sotorasib treatment significantly reduced the number of proliferating cells in the parental MIA PaCa2 strain, significantly inhibiting its proliferation. However, treatment with the same concentration of sotorasib did not significantly inhibit the proliferation of the MIA PaCa2 / KIR-resistant strain. Therefore, the MIA PaCa2 / KIR-resistant strain exhibited significant resistance to the antiproliferative effects of sotorasib.

[0063] (2) Annexin V-AF647 / PI (propidium iodide) apoptosis assay

[0064] Logarithmically growing pancreatic cancer cells (MIA PaCa / KIR-resistant) and the parental pancreatic cancer cell line (MIAPaCa2) were seeded into 12-well plates at a density of 40%-50% and cultured overnight at 37°C in a cell culture incubator with 5% CO2. After the cells adhered, they were treated with sotorasib for 24 hours at concentrations of 0µM, 0.1µM, and 1µM. Cell apoptosis was assessed using the Annexin V-AF647 / PI Apoptosis Kit (GOONIE, Cat. No. 100-102). After digestion, the cells were collected into a centrifuge tube, the supernatant removed by centrifugation, and washed once with 1ml of 4°C pre-chilled PBS. The supernatant was carefully aspirated. Bingding Buffer was diluted 1:4 with deionized water and the cells were resuspended in 1X Bingding Buffer to adjust the cell concentration to 1-5 x 106 / ml. For each sample, transfer 100µl of cell suspension to a 5ml flow cytometry tube. Add 5µl of Annexin V-AF647 and 5µl of PI solution, mix gently, and incubate at room temperature in the dark for 5 minutes. Add 400µl of Binding Buffer and immediately begin flow cytometry analysis.

[0065] 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).

[0066] 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%.

[0067] In contrast, in MIA PaCa / KIR-resistant cells treated with 0µM sotorasib, the proportion of early apoptotic cells was 0.97% and the proportion of late apoptotic cells was 1.35%; in 0.1µM sotorasib, the proportion of early apoptotic cells was 1.09% and the proportion of late apoptotic cells was 1.99%; in 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 are significantly resistant to the pro-apoptotic effects of sotorasib.

[0068] The foregoing examples are merely illustrative, serving to illustrate some of the features of the method of the present invention. The appended claims are intended to claim the widest possible scope that can be envisioned, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the present invention. Some numerical ranges used in the claims also include subranges therein, and variations in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A human pancreatic cancer cell line MIA PaCa / KIR Homo sapiens, characterized in that: The human pancreatic cancer cell MIA PaCa / KIR Homo sapiens was 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.

Images