AU2020103247A4

AU2020103247A4 - Third-generation EGFR-TKI drug-resistant cell strain and application thereof

Info

Publication number: AU2020103247A4
Application number: AU2020103247A
Authority: AU
Inventors: Jin Li
Original assignee: First Affiliated Hospital of Guangzhou Medical University
Current assignee: First Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-01-14
Anticipated expiration: 2028-11-05

Abstract

The invention discloses a third generation EGFR-TKI drug-resistant cell strain and application thereof. The drug-resistant cell line is a non-small cell lung cancer drug-resistant cell line to AZD9291, namely, human lung adenocarcinoma cell PC-9/AR, which has been deposited in the China Typical Culture Collection Center on June 14, 2019 with the deposit number CCTCC NO: C201983 and the deposit address: Wuhan University, Wuhan City, China. The drug resistant strain can stably grow and pass in a culture system with an action concentration of 100nmol AZD9291, and the drug-resistant multiple (RI) to AZD9291 is 11.75 times. It provides a drug-resistant cell model for studying the mechanism of drug resistance to targeted drugs in non-small cell lung cancer, finding effective therapeutic methods to overcome drug resistance in non-small cell lung cancer and guiding clinical medication, and has important functions and good application prospects.

Description

Third-generation EGFR-TKI drug-resistant cell strain and application thereof

TECHNICAL FIELD

[01] The invention belongs to the technical field of biomedicine. More particularly, it relates to a third generation EGFR-TKI drug-resistant cell line and application thereof.

BACKGROUND

[02] Lung cancer is the leading cancer mortality rate in the world. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer. The 5-year survival rate of non-small cell lung cancer is less than 15%, mainly because about 70% of patients are in advanced stage when they are first diagnosed with non-small cell lung cancer, and most of them have distant metastasis. Lung cancer is one of the first types of cancer to apply targeted therapy in clinical practice. In the past ten years, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has been an important drug for the treatment of advanced non-small cell lung cancer (NSCLC), and its target is epidermal growth factor receptor (EGFR). This kind of drug has the characteristics of definite curative effect, slight adverse reactions and convenient oral administration, which breaks through the bottleneck of traditional chemotherapy drugs.

[03] At present, the first generation of EGFR-TKI used clinically include erlotinib (trade name Trocquer), gefitinib (trade name Iressa) and exitinib (trade name Kamina). The first generation of TKI is a reversible EGFR-RTK inhibitor, mainly targeting two common mutations (exon 19 deletion mutation and exon 21 L858R mutation). The second generation of EGFR-RTK has alfatinib, which is indicative of the same generation of EGFR-TKI, but is an irreversible EGFR inhibitor, mainly targeting rare mutation sites such as G719X, L861Q and S7681 of EGFR. The third generation and subsequent EGFR-TKI include compounds such as AZD9291 and CO 1686, which irreversibly inhibit EGFR, also have inhibitory effect on wild-type EGFR, and still have high effective rate for patients with T790M drug-resistant mutation. Although the first and second generations of targeted drugs have remarkable curative effects, 2/3 of the patients will develop drug resistance after 1-2 years of drug use, and the tumor may begin to rebound. The causes of drug resistance to targeted drugs vary from patient to patient, but 50% - 60% of EGFR inhibitor resistance is related to T790M mutation. AZD9291 (ositinib) is an oral small molecule third generation EGFR TKI. It is the first lung cancer drug targeting EGFR T790M mutation. It can target EGFR gene mutation (including 18, 19, 21 mutations) and EGFR-TKI acquired drug resistance (T790M) in non-small cell lung cancer. AZD9291 (ositinib) has achieved remarkable curative effect in non-small cell lung cancer.

[04] However, after long-term use of AZD9291 (ositinib) targeted therapy, almost all patients will develop drug resistance. Therefore, it is of great significance to simulate the drug resistance process of NSCLC lung cancer AZD9291 and to deeply understand the drug resistance mechanism of NSCLC AZD9291. At present, many drug-resistant cell lines for tumors have been established at home and abroad, but there is no first-line drug-resistant cell line for non-small cell lung cancer to AZD9291.

SUMMARY

[05] The invention aims to provide a non-small cell lung cancer cell line resistant to the third generation EGFR-TKI drug ositinib (AZD9291).

[06] The object of the present invention is to provide a human lung adenocarcinoma cell line PC-9/AR resistant to the third generation EGFR-TKI drug ositinib (AZD9291).

[07] A further object of that present invention is to provide an application of the human lung adenocarcinoma cell line PC-9/AR in screen a drug for reversing tumor drug resistance.

[08] A further object of that present invention is to provide the use of the human lung adenocarcinoma cell line PC-9/AR in the preparation of an anti-tumor drug.

[09] The above object of that invention is realize by the following technical scheme:

[010] According to the invention, non-small cell lung cancer cells are taken as induction objects, and 100nmol of drugs are adopted to continuously treat non-small cell lung cancer cells in logarithmic growth period, and induction, domestication and culture are carried out for 6 months; The biological characteristics of non-small cell lung cancer to AZD9291 cell line were evaluated by cell morphological observation, drug sensitivity and drug resistance tests. A non-small cell lung cancer PC-9/9291 drug resistant strain carrying sensitive mutation EGFR21 exon L858R point mutation was successfully constructed and named as human lung adenocarcinoma cell line PC-9/AR. The drug-resistant cell line was deposited in the China Typical Culture Collection Center on June 14, 2019, with the collection number CCTCC NO: C201983 and the collection address: Wuhan University, Wuhan City, China.

[011] The non-small cell lung cancer drug-resistant cell strain constructed by the invention can stably grow, pass and pass in a culture system with an action concentration of 100nmol AZD9291, The drug resistance ratio (RI) to AZD9291 is 7.25 times, which provides a drug-resistant cell model for studying the mechanism of drug resistance of non-small cell lung cancer to targeted drugs, finding effective therapeutic methods to overcome drug resistance of non-small cell lung cancer and guiding clinical medication, and has an important role and good application prospect.

[012] Therefore, the application of the human lung adenocarcinoma cell line PC 9/AR in screening drugs for reversing tumor drug resistance and in preparing anti-tumor drugs should be within the scope of protection of the present invention.

[013] Preferably wherein the tumor is lung cancer. Specifically, it refers to non small cell carcinoma. More specifically lung adenocarcinoma.

[014] The invention has the following beneficial effects:

[015] A non-small cell lung canc drug resistant cell strain to AZD9291 is constructed, It can stably grow and pass in the culture system with the action concentration of 100nmol AZD9291, and the drug resistance multiple (RI) to AZD9291 is 11.75 times. It provides a drug-resistant cell model for studying the drug resistance mechanism of non-small cell lung cancer to targeted drugs, finding effective treatment methods to overcome drug resistance of non-small cell lung cancer and guiding clinical medication, and has important role and good application prospect.

BRIEF DESCRIPTION OF THE FIGURES

[016] Fig. 1 show that morphological changes of drug-resistant cell line; PC-9/AR cells: The cells grew in clusters, and some of the cells were oval or irregular spindle shaped.

[017] Fig. 2 is the detection results of drug sensitivity and drug resistance of drug resistant cell lines. IC50: PC9-pt (parent strain) 0.24 0.05 M; PC9-AR (drug resistant strain): 2.82 0. 5M RI: 11.75 (moderate drug resistant strain).

[018] Fig. 3 is a comparison before and after gene sequencing of drug-resistant cell lines. PC9-pt is before drug resistance and PC9-AR is after drug resistance.

DESCRIPTION OF THE INVENTION

[019] BRIEF DESCRIPTION OF THE DRAWINGS that present invention will be further describe below in connection with the accompanying drawing and specific embodiments of the specification, but the embodiments do not in any way limit the invention. Unless otherwise specified, the reagents, methods and apparatus employed in the present invention are conventional reagents, methods and apparatus in the art.

[020] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[021] EXAMPLE 1 induction and establishment of drug-resistant cell line

[022] Induction and Construction of Human Lung Adenocarcinoma Cell Line PC 9/AR;

[023] (1) The non-small cell lung cancer cell PC-9 was cultured in a medium containing 10% inactivated fetal bovine serum and 1% double antibody (100U/ml penicillin and 100g/ml streptomycin), and cultured in an electrothermal constant temperature incubator at 37 °C, 5% C02 and 96% humidity.

[024] (2) NSCLC cells in logarithmic growth phase were inoculated in culture flask at 5 x 10 5/mL, When cultured to 75% adherence rate, The cells were cultured in culture medium containing 100nM AZD9291 and cultured at 37 °C, 5% CO 2 and 96% humidity. The cells were cultured in culture medium containing AZD9291. The cells were cultured in culture medium containing AZD9291. The cells were cultured in culture medium containing AZD9291 at the concentration of 1 x 10 5/ml and washed once with 2ml PBS. The culture medium was cultured in culture medium containing AZD9291 at 37 °C, 5% CO 2 and 96% humidity. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. Replace the culture medium once every 2-3 days, after the cells resume growth and gradually grow into a petri dish, subculture the cells according to a ratio of 1: 3, and continuously use AZD9291 (100nM) for culture; The IC50 value was detected by MTT method every two weeks until a stable IC50 value was measured, and the drug resistant strain was obtained when the IC50 value was more than 10 times that of the primary cell. After 6 months of culture, the cell lines growing stably and passaging in the culture system with 100nmol AZD9291 as the action concentration were screened, and the results showed that the cell lines were stable and passaged in the culture system with 100nmol AZD9291 as the action concentration.

[025] The obtained cell line was named human lung adenocarcinoma cell line PC 9/AR, and was deposited in China Typical Culture Collection Center on June 14, 2019 with the deposit number CCTCC NO: C201983 and the deposit address: Wuhan University, Wuhan City, China.

[026] EXAMPLE 2 Morphological observation of drug-resistant cell strain

[027] 1. Test cell line materials:

[028] The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely human lung adenocarcinoma cell line PC-9/AR, and the parent non small cell lung cancer PC-9/pt.

[029] 2. Test method:

[030] The two kinds of cells were inoculated into 6-well plates respectively, cultured to logarithmic growth period, and the cell morphology was observed under inverted phase contrast microscope.

[031] 3. The test results are shown in Figure 1.

[032] Microscopically, PC-9/pt and PC-9/AR cells grew in epithelium-like monolayer arrangement and adherence. PC-9/pt cells had clear boundaries, mostly round and clustered, while PC-9/AR cells were slightly larger in volume, with slightly blurred boundaries, and mostly oval or irregular spindle-shaped cells grew in clusters.

[033] Compared with parent cells, lung cancer drug-resistant cell lines are enlarged in volume, easy to form clusters and irregular in shape. This indicates that the drug-resistant cell line of tube cancer has changed in morphology.

[034] EXAMPLE 3 detection of drug sensitivity and drug resistance of drug resistant cell strain

[035] 1. Test cell line materials:

[036] The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely human lung adenocarcinoma cell line PC-9/AR, and the parent non small cell lung cancer PC-9/pt.

[037] 2. Test method:

[038] The logarithmic non-small cell lung cancer resistant cell line PC-9/AR and the parent non-small cell lung cancer cell PC-9/pt were prepared into cell suspension with a concentration of 30000 cells/ml respectively, and inoculated into a 96-well cell culture plate with 2001 cell suspension (5000 cells) added to each well. The 96-well cell culture plate was placed in an incubator with 37 °C, 5% C02 and 96% humidity. After the cells were cultured for 24 hours and adhered to the wall, the old culture medium was carefully sucked out. 2001 of different concentrations of drug-containing culture medium solution was added to each well (the concentration gradient of PC-9 cell AZD9291 was 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5uM/ml). Six multiple pores were set up at each concentration. The negative control group was cultured with DMSO-containing culture medium. After 48 hours of continuous culture in the incubator, the old culture medium was carefully sucked out. 200ul of MTT solution (MTT: PBS = 1: 9) was added to each well, cultured at 37 °C for 4 hours, 150L of dimethyl sulfoxide (DMSO) was added, and mixed evenly in a shaking table for 10 minutes. The OD value of each well was measured by enzyme labeling instrument at = 490nm, and the inhibition rate and drug resistance index were calculated.

[039] Inhibition rate (%) = (OD value of control group-OD value of test group)/OD value of control group x 100%

[040] Drug resistance index (RI) = IC50 of drug-resistant cells/IC50 of parent cells.

[041] 3. The test results are shown in Fig. 2.

[042] The drug resistance multiple (RI) of the non-small cell lung cancer cell PC 9/AR cell strain constructed by the invention to AZD9291 is 11.75 times.

[043] EXAMPLE 4 detection of gene mutation in drug-resistant cell strain

[044] 1. Test cell line materials:

[045] The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely human lung adenocarcinoma cell line PC-9/AR, and the parent non small cell lung cancer PC-9/pt.

[046] 2. Test method:

[047] Illumina cell genome high-throughput sequencing and new generation sequencing (NGS) technology analyze the whole genome, which can provide base views of all genome changes, including single nucleotide site variation (SNV), insertion and deletion, copy number change and structural variation.

[048] 3. The test results are shown in Fig. 3.

[049] PC-9/pt cells: interchromosome translocation (CTX) 11%, intra chromosome translocation (ITX) 6%, inversion (INV) 0%; Delete (DEL) 67%; Insertion (INS) 16%.

[050] PC-9/AR cells: Interchromosomal translocation (CTX) 11%, Intra chromosome translocation (ITX) 8%, Inversion (INV) 0%; Delete (DEL) 66%; Insertion (INS) 15%.

[051] The sequencing results show that the genetic background of PC-9/AR cell has completely changed compared with PC-9/pt cell, and it is a new cell line.

[052] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[053] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A human lung adenocarcinoma cell PC-9/AR resistant to the third generation EGFR TKI drug ositinib (AZD9291), characterized in that the human lung adenocarcinoma cell PC 9/AR was deposited in the China Typical Culture Collection Center on June 14, 2019, with the deposit number CCTCC NO: C201983 and the deposit address: Wuhan University, Wuhan City, China.

2. That use of the human lung adenocarcinoma cell line PC-9/AR of claim 1 for screen a drug for reversing tumor drug resistance.

3. That use of the human lung adenocarcinoma cell line PC-9/AR accord to claim 1 in the preparation of an anti-tumor drug.

4. That application accord to claim 2 or 3, wherein the tumor is lung cancer.

5. That use of claim 4, wherein the lung canc is a non-small cell carcinoma.

6. That method accord to claim 5, wherein the lung cancer is a lung adenocarcinoma.