CN116445412A - L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof - Google Patents

L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof Download PDF

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CN116445412A
CN116445412A CN202310229872.4A CN202310229872A CN116445412A CN 116445412 A CN116445412 A CN 116445412A CN 202310229872 A CN202310229872 A CN 202310229872A CN 116445412 A CN116445412 A CN 116445412A
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李谨
冯凤兰
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First Affiliated Hospital of Guangzhou Medical University
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Abstract

The invention provides a L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof. The invention extracts the residual acquired third-generation TKI first-line drug-resistant cell strain from the postoperative tissue sample of the patient, and the cell strain carriesEGFRExon 21L 858R is mutated and carried at the same timePDGFRA10 exon mutation,KIT14 exon mutation,ATMExon 40 mutation. The L858R mutation is sensitive to the first-generation TKI, which is available, but the cell line is clinically resistant to the first-generation TKI. The cell strain can be used for researching the L858R third-generation EGFR-The TKI has higher scientific research and production application values, and can be used for researching a drug resistance mechanism of the TKI, researching a related signal path, developing a tumor drug resistance reversal drug, researching a more effective tumor treatment method and the like.

Description

L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof
Technical Field
The invention relates to the field of tumor biology and biomedical technology, in particular to a L858R mutant third-generation TKI first-line drug-resistant human non-small cell lung cancer cell strain and application thereof.
Background
Lung cancer is a common malignancy that severely jeopardizes human life and health. Lung cancer is classified into small cell lung cancer (Small lung cancer, SCLC) and Non-small cell lung cancer (Non-small lung cancer, NSCLC), wherein Non-small cell lung cancer accounts for 70-80% of lung cancer. Traditional treatments for non-small cell lung cancer include surgery, chemotherapy, and radiation therapy. With the deep research on the pathogenesis of tumor and the biological behavior thereof, people focus on the molecular targeted therapy with high specificity and light adverse reaction at present.
In the pathogenesis of the non-small cell lung cancer, the mutation of surface growth factor (Epidermal growth factor receptor, EGFR) is the most common driving gene of Asian population, and EGFR tyrosine kinase inhibitor (Epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI) is the primary treatment mode containing the mutation. Mutations in EGFR-sensitive subtypes mainly include the 19 th exon deletion (19 Del) and the 21 st exon L858R mutation, accounting for about 90% of EGFR mutations. The first EGFR-TKI used clinically at present is erlotinib, gefitinib and Ecritinib, and is mainly used for EGFR 19Del and L858R mutation. The second generation EGFR-TKI has afatinib and is suitable for the EGFR-TKI of the same generation, but is an irreversible EGFR inhibitor and mainly aims at rare mutations such as G719X, L861Q, S768I and the like of EGFR. The third generation includes compounds such as octenib (AZD 9291), CO-1686, and the like, irreversibly inhibits EGFR, and is still effective in patients suffering from T790M-resistant mutation. However, resistance to EGFR-TKI inhibitors of either the first, second or third generation may occur after a period of use.
In the face of the troublesome problem of tumor drug resistance, many researchers focus on the research of EGFR-TKI drug resistance mechanism at present, and aim at developing drugs capable of inhibiting EGFR abnormal expression. Thus, it is continually sought to fully grasp the drug resistance mechanism of the drug and find the best way to bind to EGFR kinase. However, current primary cell line models of lung cancer resistant to EGFR inhibitors are still rare. The traditional TKI is first-line one generation, and is third-generation after drug resistance, and since the third NCCN guideline in 2019 indicates that the third-generation TKI is recommended for preferential use by patients with non-small cell lung cancer EGFR mutation positive late stage, the third-generation TKI can be first-line used, so that the drug resistance condition of the first-generation TKI appears, and a first-generation drug-resistant strain model which is closer to the actual condition in the body is lacking. Accordingly, the prior art is still in need of improvement.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof, and establishes an AZD9291 drug-resistant human lung cancer cell strain NSCLC-001T which carries EGFR 21 exon L858R mutation; and simultaneously carrying PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation. The IC50 of the cell strain for the third-generation TKI is 30.29 times that of the wild cell strain carrying the same mutation H1975 for the third-generation TKI. The establishment of the first-line EGFR-TKI drug-resistant human primary lung cancer cell strain can be an important tool for researching the drug resistance mechanism, reversing the drug resistance, developing and evaluating new anti-cancer drugs and methods and the like of the L858R mutant non-small cell lung cancer.
The technical scheme of the invention is as follows:
a L858R mutant TKI resistant human non-small cell lung cancer cell strain is named as cell strain NSCLC-001T, and the preservation number is GDMCC No. 63192.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain, wherein the cell strain NSCLC-001T carries EGFR 21 exon L858R mutation.
The L858R mutant TKI drug-resistant human non-small cell lung cancer cell strain, wherein the cell strain with the TKI drug resistance of the third generation carries PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain is applied to preparation of three-generation TKI drug-resistant preparations.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain is applied to screening three-generation TKI drug-resistant reversal agents.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain is applied to the construction of a tumor model of the human non-small cell lung cancer in vivo and in vitro three-generation TKI drug-resistant.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain is applied to the preparation of antitumor drugs.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain is applied to the screening of anti-tumor drugs.
The beneficial effects are that: the invention provides a L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof. The invention extracts residual acquired third-generation EGFR-TKI drug-resistant cell strain from a tissue sample after operation of a patient, wherein the IC50 of a primary cell strain NSCLC-001T to the Ornitinib is 30.29 times of that of a wild cell strain carrying the same mutation H1975 to the Ornitinib, and the cell strain simultaneously carries PDGFRA 10 exon mutation, KIT 14 exon mutation and ATM 40 exon mutation. The cell strain can be used for discussing the L858R mutation three-generation TKI acquired drug resistance mechanism and researching related signal paths, analyzing the sensitivity of an anti-tumor drug, screening the anti-tumor drug or developing a tumor drug resistance reversal drug, researching a more effective tumor treatment method and the like, has higher scientific research and production application values, and is expected to generate good scientific research, economy and social benefits.
Drawings
Fig. 1 is a diagram of a treatment course of a patient according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of the primary cell strain STR detection result provided in the embodiment of the present invention.
FIG. 3 is a graph showing the overall percentage of variation in exon sequencing of the primary cell line NSCLC-001T according to the present invention.
FIG. 4 is a graph showing the results of drug sensitivity experiments on the primary cell line NSCLC-001T and the cell lines NSCLC-001T and H1975 according to the present invention.
FIG. 5 is a schematic diagram showing the cell morphology of a cell line NSCLC-001T according to an embodiment of the present invention.
Detailed Description
The invention provides a L858R mutant third-generation TKI first-line drug-resistant human non-small cell lung cancer cell strain and application thereof, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention provides a L858R mutant third-generation TKI drug-resistant human non-small cell lung cancer cell strain named cell strain NSCLC-001T which is preserved in the Guangdong province microorganism strain preservation center, address: building 5, no. 59, of Mitsui 100, guangzhou City, with a date of 2023, 2 months and 24 days, and with a preservation number of GDMCC NO. 63192.
The invention discloses an L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain NSCLC-001T, which is obtained by preparing and obtaining residual part of obtained AXatinib drug-resistant strain from a cancer tissue sample surgically excised from a 59-year-old male patient, wherein the clinical stage of the patient is IIIA-stage low-differentiation adenocarcinoma, the EGFR_21 exon L858R mutation is detected by a pre-treatment gene, and the patient is subjected to oral treatment by using a third-generation EGFR-TKI targeting drug AXatinib (AZD 9291). According to the invention, a primary cell strain is extracted from treated tissues for WES sequencing, and the cell strain NSCLC-001T carries EGFR 21 exon L858R mutation; the third generation TKI resistant cell strain carries PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation at the same time: among them, PDGFRA (mutation of the 1432T base of the 10 th exon to the C base, mutation of the 478 th amino acid from serine to proline), KIT (mutation of the 2134 th base of the 14 th exon to C, mutation of the 712 th serine to proline), ATM (mutation of the 5948 th base A of the 40 th exon to G, mutation of the 1983 rd asparagine to serine).
The primary tumor cell strain NSCLS-001T with the first-line mutation of L858R is sensitive to the first-line TKI of patients carrying L858R, the first-line TKI is used for one generation, the third-line TKI is used after drug resistance, and since the third NCCN guideline in 2019 indicates that the third-line TKI is a preferential recommendation of patients with the positive late EGFR mutation of non-small cell lung cancer, the third-line TKI can be used for one generation, so that the drug resistance condition of the first-line TKI of the third-line TKI appears, and a primary drug resistant strain model which is more close to the actual condition in vivo is lacked. Patients were given an oral regimen of "pemetrexed disodium + carboplatin + bevacizumab + octreotide 2 times (24 days apart) followed by a surgical procedure with oral octreotide for 89 days, with a specific course of treatment as shown in fig. 1. H1975 is a commercial cell line carrying the exon 21L 858R mutation, sensitive to the first-generation TKI, available for the third generation. The invention extracts the residual part of the obtained third-generation TKI drug-resistant cell strain from the postoperative tissue sample of the patient (the IC50 of the primary cell strain NSCLC-001T to AZD9291 is 30.29 times of the IC50 of the wild cell strain carrying the same mutation H1975 to the AXitinib). The third generation TKI resistant cell strain carries PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation.
The method for establishing the L858R mutant tertiary TKI drug-resistant primary tumor cell NSCLC-001T comprises the following steps:
first extraction of primary cells
1. Preparation of support cells
1.1 preparing a complete culture medium, wherein the formula is DMEM+10% FBS+double antibody;
1.2, taking out 3T3-J2 mouse fibroblasts from liquid nitrogen, and immediately putting the fibroblasts into a water bath pot at 37 ℃ for thawing as soon as possible;
1.3 adding 3T3-J2 cells into 5ml of complete medium at 1000rpm/min, centrifuging at room temperature for 5min;
1.4 removing the supernatant, and adding 1ml of complete culture medium, and gently beating uniformly;
1.5 adding into T25 flask for culturing
1.6, after culturing the cells until the fitting degree reaches 80-90%, adding 3ml containing mitomycin C (2-4 ug/ml) for 2 hours to make the cells become non-proliferation cells which can be used as supporting cells;
1.7 removal of the drug-containing Medium, addition of DMEM complete Medium for cultivation, and further primary cell culture as support cells.
2. Tissue digestion into single tumor cells for culture
1.1 Collection of tumor tissue 0.5X0.5X0.5 cm after surgery in patients with non-small cell lung cancer 3 Tumor tissue;
1.2, treating tumor tissue with absolute ethanol for no more than 3 seconds;
1.3, the tumor tissue is clamped into physiological saline with double antibodies for washing;
1.4 1.0ml of the prepared collagen digestive enzyme was added to a 2.0ml centrifuge tube;
1.5 adding the washed tissue into collagenase, shearing the tissue by scissors, and placing the tissue in a water bath kettle at 37 ℃ for digestion for 25 minutes;
1.6 adding 10% PBS to the digested tissue to terminate digestion;
1.7 the digested tissue was screened through a 70 mesh screen and the cell screen was washed with PBS;
1.8 Centrifuging at 1000rpm/min at room temperature for 5min, removing supernatant, adding 5ml erythrocyte lysate, and lysing for 5-10min;
1.9 adding 10ml PBS to stop erythrocyte lysis, centrifuging at 1000rpm/min at room temperature for 5min, and removing the supernatant;
1.10 adding the tumor complete culture medium to gently blow the cells;
1.11 cells were aspirated into T25 flasks with support cells at 37℃with 5% CO 2 Culturing in an incubator;
1.12 The cell culture flask is not moved within 48 hours, and the support cells are replaced once in 4 days;
1.13 About 8-16 days, it was observed whether primary tumor cell clones were formed.
(II) Primary cell culture
2.1 sucking out the culture medium in a flask containing primary tumor cells, and washing the cells with 3ml PBS;
2.2 removing PBS, adding 3ml EDTA cell digestive juice, placing in a 5% CO2 incubator at 37 ℃ for digestion for 5min;
2.3 After 5min, the digestion was stopped by pipetting 10% FBS in PBS, the support cells were removed, and the support cells were completely removed by washing with PBS;
2.4 sucking 1ml of pancreatin, adding the pancreatin into a culture flask, placing the culture flask at 37 ℃ and digesting the pancreatin in a 5% CO2 incubator for 5min, and observing the digestion condition of cells during the digestion period;
2.5 adding 3ml containing 10% FBS PBS to terminate the pancreatin digestion reaction;
2.6 sucking cells, placing the cells into a 15ml centrifuge tube, centrifuging at 1000rpm/min at room temperature for 5min, and removing the supernatant;
2.7 cells were passaged 1:2 or 1:3 in T75 flasks, or frozen as required.
(III) cell line STR detection
STR loci consist of short tandem repeats of 3 to 7 base pairs in length, which are widely found in the human genome, can be used as highly polymorphic markers, and can be detected by PCR (polymerase chain reaction). Alleles at STR loci can be distinguished by differences in copy number of the repeat sequences within the amplified region, which can be identified by fluorescent detection after separation by capillary electrophoresis. And then comparing the STR parting result with a professional cell STR database by a certain calculation method to calculate the names of the cell lines to which the sample belongs or the cell lines possibly cross-contaminated.
Sample processing and inspection methods:
extracting whole genome DNA from cultured cell suspension, cell sediment and the like by using a genome extraction kit, and detecting the DNA concentration by using a micro ultraviolet visible spectrophotometer QC. Multiplex amplification using IGE-STR20A, capillary electrophoresis using ABI3730XL genetic Analyzer, fragment separation and employingThe ID software performs genotyping. Genotyping results for the 8 core STR sites and 1 unique site that were required to be examined according to (ASN-0002-2011).
The embodiment of the invention also provides application of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain NSCLC-001T in preparation of a third-generation TKI drug-resistant preparation.
The embodiment of the invention also provides application of the L858R mutant third-generation TKI drug-resistant human non-small cell lung cancer cell strain NSCLC-001T in screening of third-generation TKI drug-resistant reversal agents.
The embodiment of the invention also provides application of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain NSCLC-001T in constructing a tumor model of the human non-small cell lung cancer in-vivo and in-vitro TKI-resistant human non-small cell lung cancer.
The embodiment of the invention also provides application of the L858R mutant third-generation TKI drug-resistant human non-small cell lung cancer cell strain NSCLC-001T in preparing antitumor drugs.
The embodiment of the invention also provides application of the L858R mutant third-generation TKI drug-resistant human non-small cell lung cancer cell strain NSCLC-001T in anti-tumor drug screening.
The invention obtains a residual part acquired drug-resistant tumor cell strain obtained after the use of the Ornitinib for the human non-small cell lung cancer, can be used for exploring and researching the mechanism of the Ornitinib acquired drug resistance, researching the morphological and biological characteristics of the Ornitinib drug-resistant human non-small cell lung cancer cells, researching the tumor drug-resistant mechanism, analyzing the sensitivity of the anti-tumor drug, screening and evaluating the anti-tumor drug, developing the tumor drug-resistant reversing drug, researching more effective tumor treatment method and the like, can be used for discussing the non-small cell lung cancer drug-resistant mechanism and researching related signal paths, has higher scientific research and production application values, and is expected to generate good scientific research, economy and social benefit.
The L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof are further explained by the following specific examples:
example 1 screening of the third-generation TKI-resistant primary tumor cell lines mutated with L858R
The obtained Ornitinib resistant strain is prepared from a cancer tissue sample surgically excised from a 59-year-old male patient, the clinical stage of the patient is a period IIIA hypodifferentiation adenocarcinoma, the gene detection result of the puncture tissue sample before treatment is EGFR_21 exon L858R mutation, the patient is treated for 2 times (24 days apart) by a scheme of 'pemetrexed disodium + carboplatin + bevacizumab + Ornitinib oral administration', and then the operation is carried out by taking Ornitinib for 89 days all the time, and the specific treatment course is shown in figure 1. Finally obtaining the residual part of the Ornitinib drug-resistant tumor cell strain which is named as cell strain NSCLC-001T. Wherein, WES sequencing finds that the cell strain NSCLC-001T carries EGFR No. 21 exon L858R mutation; simultaneously carrying PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation: among them, PDGFRA (mutation of the 1432T base of the 10 th exon to the C base, mutation of the 478 th amino acid from serine to proline), KIT (mutation of the 2134 th base of the 14 th exon to C, mutation of the 712 th serine to proline), ATM (mutation of the 5948 th base A of the 40 th exon to G, mutation of the 1983 rd asparagine to serine).
First extraction of primary cells
1. Preparation of support cells
1.1 preparing a complete culture medium, wherein the formula is DMEM+10% FBS+double antibody;
1.2, taking out the 3T3-J2 fibroblast cells from liquid nitrogen, and immediately putting the 3T3-J2 fibroblast cells into a water bath pot at 37 ℃ for thawing as soon as possible;
1.3 adding 3T3-J2 cells into 5ml of complete medium at 1000rpm/min, centrifuging at room temperature for 5min;
1.4 removing the supernatant, and adding 1ml of complete culture medium, and gently beating uniformly;
1.5 adding into T25 flask for culturing
1.6, after culturing the cells until the fitting degree reaches 80-90%, adding 3ml containing mitomycin C (2-4 ug/ml) for 2 hours to make the cells become non-proliferation cells which can be used as supporting cells;
1.7 removal of the drug-containing Medium, addition of DMEM complete Medium for cultivation, and further primary cell culture as support cells.
2. Tissue digestion into single tumor cells for culture
1.1 Collection of tumor tissue 0.5X0.5X0.5 cm after surgery in patients with non-small cell lung cancer 3 Tumor tissue;
1.2, treating tumor tissue with absolute ethanol for no more than 3 seconds;
1.3, the tumor tissue is clamped into physiological saline with double antibodies for washing;
1.4 1.0ml of the prepared collagen digestive enzyme was added to a 2.0ml centrifuge tube;
1.5 adding the washed tissue into collagenase, shearing the tissue by scissors, and placing the tissue in a water bath kettle at 37 ℃ for digestion for 25 minutes;
1.6 adding 10% PBS to the digested tissue to terminate digestion;
1.7 the digested tissue was screened through a 70 mesh screen and the cell screen was washed with PBS;
1.8 Centrifuging at 1000rpm/min at room temperature for 5min, removing supernatant, adding 5ml erythrocyte lysate, and lysing for 5-10min;
1.9 adding 10ml PBS to stop erythrocyte lysis, centrifuging at 1000rpm/min at room temperature for 5min, and removing the supernatant;
1.10 adding the tumor complete culture medium to gently blow the cells;
1.11 cells were aspirated into T25 flasks with support cells at 37℃with 5% CO 2 Culturing in an incubator;
1.12 The cell culture flask is not moved within 24 hours, and the support cells are replaced once within 3-5 days;
1.13 It was observed about 7-14 days whether primary tumor cell clones were formed.
(II) Primary cell culture
2.1 sucking out the culture medium in a flask containing primary tumor cells, and washing the cells with 3ml PBS;
2.2 removing PBS, adding 3ml EDTA cell digestive juice, placing in a 5% CO2 incubator at 37 ℃ for digestion for 5min;
2.3 After 5min, the digestion was stopped by pipetting 10% FBS in PBS, the support cells were removed, and the support cells were completely removed by washing with PBS;
2.4 sucking 1ml of pancreatin, adding the pancreatin into a culture flask, placing the culture flask at 37 ℃ and digesting the pancreatin in a 5% CO2 incubator for 5min, and observing the digestion condition of cells during the digestion period;
2.5 adding 3ml containing 10% FBS PBS to terminate the pancreatin digestion reaction;
2.6 sucking cells, placing the cells into a 15ml centrifuge tube, centrifuging at 1000rpm/min at room temperature for 5min, and removing the supernatant;
2.7 cells were passaged 1:2 or 1:3 in T75 flasks, or frozen as required.
(III) cell line STR detection
Extracting whole genome DNA from cultured cell suspension, cell sediment and the like by using a genome extraction kit, and detecting the DNA concentration by using a micro ultraviolet visible spectrophotometer QC. Using IGE-SMultiplex amplification of TR20A by multiple fluorescent systems, capillary electrophoresis and fragment separation using ABI3730XL genetic Analyzer and employingThe ID software performs genotyping. Genotyping results for the 8 core STR sites and 1 unique site that were required to be examined according to (ASN-0002-2011).
The genotyping results for the 8 core STR loci and 1 trait locus that the present embodiment detects 21 loci, requiring the necessary detection, are expressed as "locus/allele length: amelogenin/X/Y, vWA/15/18, D7S820/9.2/10, CSF1PO/12/12, D16S539/9/11, TH01/6/7, D13S317/8/12, TPOX/8/11, D5S818/10/11. The purified primary lung cancer cells were subjected to STR detection, and the results are shown in fig. 2, which confirm that they were single human-derived cells and free of cross-contamination.
EXAMPLE 2 detection of Whole exons of cell lines
DNA extraction was performed on cell suspensions, cell pellets, etc. cultured with the cell strain obtained in example 1 using a Qiagen DNeasy Blood & Tissue Kit. Sequencing libraries were constructed following disruption of the DNA using a Covaris M220Focused-ultrasonicator (Covaris). Each sample was captured using the agilent SureSelect Human All Exon V Kit and the results are shown in fig. 3: wherein indel is an indel, an exon (exonic) mutation accounts for 8.02% of the indels (number 586), an intron (intronic) mutation accounts for 65.45% of the indels, and other mutations account for 26.53% of the indels; SNPs are single nucleotide polymorphisms (refer to DNA sequence polymorphisms at the genomic level caused by single nucleotide variation), in which the exon (exonic) mutation accounts for 34.82% (number 24051), the intron (intronic) mutation accounts for 33.46%, synonymous single nucleotide mutation (synomous_snv) accounts for 19.53%, and other mutations account for 12.17%.
Example 3 cell line growth Curve assay
1. The cell line NSCLC-001T was cultured according to the method provided in example 1, and the morphology thereof is shown in FIG. 4, and it is understood that the tumor cells were in a three-dimensional morphology and the cell state was good.
2. The cultured cell lines (primary tumor cells) were digested into single cell suspensions with pancreatin containing 0.25% edta and counted;
3. 1000 cells per well were plated into 96-well plates, 6 wells per group; after adding 10. Mu.L of CCK8 reagent every 24 hours and incubating at 37℃for 1 hour, the OD value of each well was measured at λ=490 nm using an enzyme-labeled instrument, and the measurement was continued for 5 days to obtain a growth curve of the cell line, as shown in FIG. 5A.
Example 4 cell line drug sensitivity detection
The H1975 cell strain and NSCLC-001T cell strain grown in logarithmic phase are respectively configured into 2000 cells per well; after cell attachment, the medium was aspirated, 100. Mu.l of complete medium containing 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1. Mu.M, 500nM, 250nM, 50nM, 25nM, 12.5nM, 0nM AZD9291 was added, and 3 wells were added at the same concentration. 48 hours after dosing, 10 μl of CCK8 was added per well and absorbance was measured with an microplate reader after 2 hours. Wherein, inhibition (%) = (control OD value-test OD value)/control OD value x 100%; drug Resistance Index (RI) =primary cell IC 50/control cell IC50. As shown in FIG. 5B, the IC50 of the primary cell strain NSCLC-001T to AZD9291 is 30.29 times, more than 10 times, that of the wild cell strain carrying the same L858R mutation H1975 to AZD9291, so that the cell strain NSCLC-001T is a first-line third-generation TKI acquired drug-resistant cell strain clinically.
In summary, the invention provides a L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof. The L858R mutant third-generation TKI drug-resistant non-small cell lung cancer primary cell strain NSCLS-001T is sensitive to the first-generation TKI of a patient carrying L858R, and is used for the first generation, three generations after drug resistance, and since the third NCCN guideline in 2019 indicates that the third-generation TKI can be used as a preferential recommendation of a patient with the non-small cell lung cancer EGFR mutant positive late stage, the third-generation TKI can be used for the first generation, so that the first-generation TKI drug resistance condition appears, and a primary drug-resistant strain model which is closer to the in-vivo actual condition is lacked. The patient of the present invention was given an "oral pemetrexed disodium + carboplatin + bevacizumab + octreotide" regimen 2 times (24 days apart) followed by an oral administration of octreotide for up to 89 days for surgery. The invention extracts the acquired third-generation EGFR-TKI drug-resistant cell strain from the postoperative tissue sample of the patient. The cell strain carries EGFR No. 21 exon mutation L858R, PDGFRA No. 10 exon mutation, KIT No. 14 exon mutation and ATM No. 40 exon mutation. The IC50 of the cell line NSCLC-001T to the Ornitinib is 30.29 times that of the wild cell line carrying the same mutation H1975. The cell strain can be used for researching an L858R third-generation TKI drug resistance mechanism, analyzing the sensitivity of an anti-tumor drug, screening the anti-tumor drug, developing a tumor drug resistance reversal drug, researching a more effective tumor treatment method and the like; the method can be used for discussing pathogenesis of the L858R mutant third-generation EGFR-TKI non-small cell lung cancer and researching related signal paths, has higher scientific research and production application value, and is expected to generate good scientific research, economic and social benefits.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims (8)

1. A L858R mutant TKI resistant human non-small cell lung cancer cell strain is named as cell strain NSCLC-001T, and the preservation number is GDMCC No. 63192.
2. The L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1, wherein the cell strain NSCLC-001T carries an EGFR exon L858R mutation.
3. The L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1, wherein the third-generation TKI-resistant cell strain carries both PDGFRA exon 10 mutation, KIT 14 exon mutation, ATM 40 exon mutation.
4. The use of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1 in the preparation of a third-generation TKI-resistant formulation.
5. The use of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1 in screening for third-generation TKI resistance reversal agents.
6. The use of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1 in constructing a tumor model of the in vivo and in vitro third-generation TKI-resistance of human non-small cell lung cancer.
7. The use of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1 for preparing an antitumor drug.
8. The use of the L858R mutant third-generation TKI-resistant human non-small cell lung cancer cell strain of claim 1 in the screening of antitumor agents.
CN202310229872.4A 2023-03-10 2023-03-10 L858R mutant three-generation TKI drug-resistant human non-small cell lung cancer cell strain and application thereof Pending CN116445412A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116355851A (en) * 2023-03-13 2023-06-30 广州医科大学附属第一医院（广州呼吸中心） Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116355851A (en) * 2023-03-13 2023-06-30 广州医科大学附属第一医院（广州呼吸中心） Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof
CN116355851B (en) * 2023-03-13 2023-09-08 广州医科大学附属第一医院（广州呼吸中心） Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof

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