EGFR-TKI drug-resistant human lung adenocarcinoma PC-9 cell strain, kit and application thereof

Abstract

The invention discloses an EGFR-TKI drug-resistant human lung adenocarcinoma PC-9 cell strain, a kit and application of the EGFR-TKI drug-resistant human lung adenocarcinoma PC-9 cell strain. The cell strain is a human lung cancer cell with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C2024220, and is named as Osi-R-PC-9-Pa. The Osi-R-PC-9-Pa and the kit thereof have relatively high application values in screening drugs for treating the drug resistance of the non-small cell lung cancer and evaluating the drugs for treating the drug resistance of the non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to an EGFR-TKI resistant cell line, a kit and applications thereof, and in particular to an EGFR-TKI resistant human lung adenocarcinoma PC-9 cell line, a kit and applications thereof. Background Art

[0002] Non-small cell lung carcinoma (NSCLC) is a relatively large type of lung cancer, among which lung adenocarcinoma is the most common subtype of non-small cell lung cancer, accounting for almost 50% of the total lung cancer, and the overall survival rate is low. At present, lung adenocarcinoma is mostly peripheral, with no obvious special symptoms in the early stage, generally with fever, hemoptysis, chest pain, shortness of breath and other respiratory symptoms, or no symptoms, so it is easy to be ignored. Some patients also have extrapulmonary manifestations in the early stage (bone and joint pain, shoulder and back pain, and even brain metastasis and other related symptoms). Late symptoms vary due to different physical conditions of patients. Common symptoms include pain, hoarseness, head and neck edema, pleural effusion, etc. At present, common diagnostic methods for lung adenocarcinoma include imaging examinations (such as X-rays, CT, etc.), PET molecular imaging, MRI, serum tumor marker detection, and pathological diagnosis. In recent years, the treatment strategy of lung adenocarcinoma has changed from the traditional staging-based method to tissue morphology and gene mutation-guided targeted therapy. Epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ROS sarcoma oncogen-receptor tyrosine kinase (ROS1), etc. are important gene targets for lung cancer. Among them, the discovery of EGFR gene mutation and the successful targeted treatment of lung adenocarcinoma using tyrosine kinase inhibitors highlight the key role of molecular biological testing in the diagnosis of lung adenocarcinoma. As the most representative type of lung adenocarcinoma gene typing, EGFR mutation has a higher mutation rate in young women and patients of East Asian ethnicity. Although the loss of exon 19 and the L858R substitution mutation of exon 21 are the most common EGFR mutations, it is still recommended to fully cover exons 18-21 during genetic testing. Experiments have shown that EGFR mutations are closely related to the new classification method of lung adenocarcinoma, and can be highly prevalent in lung adenocarcinoma subtypes such as carcinoma in situ, microinvasive carcinoma, papillary and micropapillary. In view of this, with the development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-tyrosine kinases inhibitor, EGFR-TKI) and tumor immunotherapy in the past decade, the treatment of non-small cell lung cancer, including lung adenocarcinoma, has made great progress. In general, NSCLC patients will have abnormal activation of receptor tyrosine kinases (RTKs) signals caused by EGFR abnormalities. Therefore, EGFR-TKI has become the first-line drug for the treatment of NSCLC carrying abnormal EGFR mutations. However, whether it is molecular targeted therapy or lung cancer immunotherapy, tumor resistance is inevitable. Therefore, studying the mechanism of lung cancer resistance, finding strategies to overcome it, or developing new immunotherapy methods are of great clinical significance.

[0003] The first generation of EGFR-TKIs (such as gefitinib and erlotinib) marked a major event in the history of targeted therapy for NSCLC, heralding the advent of targeted precision therapy for tumors. It has been proven to be an effective reversible inhibitor for NSCLC patients with EGFR sensitive mutations, which can effectively prolong the survival of patients. However, most patients develop drug resistance after 9-13 months of treatment. Among them, mutations in exons 19 and 20 account for 90% of the total mutations, and the T790M mutation in exon 20 is the main reason for the resistance of the first generation of EGFR-TKI. In response to this feature, the second generation of EGFR-TKIs (such as afatinib) came into being, which introduced Michael addition receptor acrylamide on the basis of aniline quinazoline. The second generation of EGFR-TKI is an irreversible inhibitor of EGFR and HER2, which overcomes the problems caused by single mutations caused by the first generation of EGFR-TKI by covalently binding to the C797 residue of the EGFR gene. However, high doses of drugs cause unacceptable side effects in patients, and inevitably produce new drug-resistant mutations. The effect on the new double mutation combinations of "L858R" combined with "T790M" and "exon19 del" combined with "T790M" is not good. Osimertinib is a third-generation EGFR-TKI. It has a good effect on patients with non-small cell lung cancer with EGFR T790M drug-resistant mutations induced by the first-generation EGFR-TKI, and shows excellent selectivity for double-mutated EGFR. However, after an average of 9 to 14 months of treatment with Osimertinib, NSCLC patients with first- and second-generation EGFR-TKI drug-resistant mutations may acquire new EGFR mutations (C797S mutations), and the possibility of this mutation is significantly increased compared with patients not treated with Osimertinib. In response to this mutation, researchers have studied and developed the fourth-generation EGFR-TKI. In response to the continuous problem of drug-resistant mutations, researchers have developed a variety of combined drug treatment approaches and achieved better tumor suppression effects by inhibiting immunosuppressive factors in the targeted tumor microenvironment, but the adverse reactions they bring seem inevitable.

[0004] The discovery of activating mutations in the epidermal growth factor receptor tyrosine kinase in patients with non-small cell lung cancer and the subsequent development of first-line treatment with small molecule tyrosine kinase inhibitors targeting EGFR have greatly changed the treatment landscape of these tumors in the clinic. Several large-scale phase 3 clinical trials have consistently demonstrated that the first-generation EGFR-TKIs (Gefitinib and Erlotinib), the second-generation EGFR-TKIs (Afatinib), and the third-generation EGFR-TKIs (Osimertinib) are superior to standard first-line platinum chemotherapy in patients with advanced non-small cell lung cancer with EGFR activating mutations. However, despite the very high objective response rate (60-70%) of patients to such treatment, acquired resistance still exists, with an average progression-free survival of 9-15 months. Studying the mechanisms of EGFR-TKI resistance that appear in the clinic is conducive to the discovery of new biomarkers and drug targets, and the development of better treatment options for patients. Summary of the invention

[0005] In order to study the drug resistance of EGFR-TKI, it is particularly important to establish a cell line resistant to EGFR-TKI. The present invention provides an EGFR-TKI resistant human lung adenocarcinoma PC-9 cell line, which is a human lung cancer cell with a deposit number of CCTCC NO: C2024220 and is named Osi-R-PC-9-Pa.

[0006] In addition, the present invention also provides the use of the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line described above in evaluating drugs for treating drug-resistant non-small cell lung cancer.

[0007] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0008] The present invention also provides the use of the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell strain described above in screening drugs for treating drug-resistant non-small cell lung cancer.

[0009] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0010] The present invention also provides a kit, comprising the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line as described above.

[0011] The present invention also provides the use of the above-mentioned kit in evaluating drugs for treating drug resistance of non-small cell lung cancer.

[0012] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0013] The present invention also provides the use of the above-mentioned kit in screening drugs for treating drug resistance of non-small cell lung cancer.

[0014] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Osi-R-PC-9-Pa cell line exhibits good in vitro resistance to the three generations of EGFR-TKIs, namely Afatinib, Erlotinib, Gefitinib and Osimertinib, and its in vitro resistance index (RI) is 10234.14, 3337.65, 865.42 and 356.18 respectively. The calculation formula of RI is: RI = Relative IC 50筛选获得细胞株 / Relative IC 50亲本 。 Relative IC 50亲本 Relative IC represents the relative half-maximal effect concentration of the parental cell line used for screening; 50筛选获得细胞株 Represents the half-maximal effect concentration of the screened cell line. For example, when calculating the in vitro resistance index of Osi-R-PC-9-Pa to Osimertinib, Relative IC 50亲本 The relative IC 50筛选获得细胞株 By taking into account the 2.7750 μmol / L obtained by experiment (see Example 2), the RI for Osimertinib was obtained to be 356.18.

[0016] Osi-R-PC-9-Pa cell line can be used as a drug-resistant tumor cell model, which is conducive to better studying the drug resistance of EGFR-TKI, studying the morphological and biological characteristics of drug-resistant lung adenocarcinoma cell lines, revealing the drug resistance mechanism of lung adenocarcinoma, screening drugs that can reverse the drug resistance of lung adenocarcinoma, and developing more effective treatment methods for lung adenocarcinoma. Osi-R-PC-9-Pa and its kit have high application value in screening drugs for the treatment of drug resistance in non-small cell lung cancer and in evaluating drugs for the treatment of drug resistance in non-small cell lung cancer.

[0017] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the cell growth morphology pictures of PC-9-Parental cell line and Osi-R-PC-9-Pa cell line.

[0019] Figure 2This is the cell proliferation inhibition curve of Afatinib, Erlotinib, Gefitinib, and Osimertinib in PC-9-Parental cell line.

[0020] Figure 3 This is the cell proliferation inhibition curve of Afatinib, Erlotinib, Gefitinib and Osimertinib in Osi-R-PC-9-Pa cell line.

[0021] Figure 4 These are the growth curves of PC-9-Parental cell line and Osi-R-PC-9-Pa cell line. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0023] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0024] The human lung adenocarcinoma cell line PC-9 (RCB4455) used in the examples of the present invention was purchased from the RIKEN BRC cell bank, and Afatinib, Erlotinib, Gefitinib, and Osimertinib were purchased from Selleck Chemicals, Anage Chemicals, Sann, and Haoyuan Pharmaceuticals, respectively.

[0025] Example 1: Construction of EGFR-TKI-resistant human lung adenocarcinoma cell line

[0026] The screening method is as follows:

[0027] (1) Routine thawing of cryopreserved human lung adenocarcinoma cell line PC-9 (referred to herein as "PC-9-Parental" because it is the parent cell line) was routinely cultured and passaged in a 37°C, 5% CO2 incubator in 1640 medium containing 10% fetal bovine serum to expand the cells.

[0028] (2) Take cells in the logarithmic growth phase, count them and prepare a cell suspension with a concentration of 1×10 4Cells were plated in 6-well plates at a density of 2 mL per well. After centrifugation on the second day, the cell culture medium containing a final concentration of 20 nmol / L Osimertinib was replaced. Every 4 days, the cells were passaged once to 6-well plates and the cell culture medium containing the same concentration of Osimertinib was added.

[0029] (3) Repeat this process for 5 weeks, which is one drug screening process. Thereafter, three drug screening processes were performed, with the dose increased to 200% every 5 weeks, and the screening was continued for 20 weeks. The Osimertinib concentrations were 20 nmol / L, 40 nmol / L, 80 nmol / L, and 160 nmol / L, respectively. Finally, a cell line resistant to Osimertinib was obtained, named human lung cancer cell Osi-R-PC-9-Pa Homo sapiens, with a deposit number of CCTCC NO: C2024220, a deposit date of July 31, 2024, a deposit institution of China Center for Type Culture Collection (CCTCC), and a deposit address of Wuhan University, Wuhan, China.

[0030] from Figure 1 It can be seen that the drug-resistant strain Osi-R-PC-9-Pa is morphologically similar to the parental cell line PC-9-Parental.

[0031] Example 2: Determination of the Growth Inhibition Rate of Afatinib, Erlotinib, Gefitinib and Osimertinib on Osi-R-PC-9-Pa Resistant Strain

[0032] (1) The cell line Osi-R-PC-9-Pa obtained in Example 1 and its parental cell line PC-9-Parental were respectively subjected to in vitro expansion culture, and cells in the logarithmic growth phase were used for plating, and the cells were stained with trypan blue and the living cells were counted, and the cell concentration was adjusted to 1500 cells per well. 90 μL / well of the cell suspension was added to the 96-well culture plate as shown in Table 1, but a cell culture medium without cells was added to the blank control; the 96-well culture plate was cultured overnight in an incubator at 37° C., 5% CO2, and 100% relative humidity.

[0033] (2) 10 μL of drug working solution of different concentrations was added to the above-mentioned cell culture plate to make the final concentration as shown in Table 1. There were 2 replicate wells in each group. 10 μL of DMSO-cell culture medium mixture was added to the solvent control and blank control. The final DMSO concentration was 0.25% by volume.

[0034] Table 1 Schematic diagram of cell plating and drug treatment in in vitro cell activity experiment

[0035]

[0036] Note: Drug concentrations were measured in μmol / L; Osi-R-PC-9-Pa and PC-9-Parental were loaded into 96-well culture plates as shown in Table 1.

[0037] (3) The 96-well culture plate was returned to the incubator for 72 h. Then, 50 μL (equal to half the volume of the cell culture medium in each well) of the Cell Titer-Glo working solution of the Promega CellTiter-Glo Luminescent Cell Activity Assay Kit (Promega-G7573) was added to each well, and the cell plate was wrapped with aluminum foil to avoid light.

[0038] (4) The 96-well culture plate was shaken on an orbital shaker for 2 min to induce cell lysis. The 96-well culture plate was placed at room temperature for 10 min to stabilize the luminescent signal, and the luminescent signal was detected on a 2104 EnVision plate reader.

[0039] (5) Calculate the inhibition rate (IR) of the test drug: IR (%) = (1 – (RLU 药物 –RLU 空白对照 ) / (RLU 溶媒对照 –RLU 空白对照 ))*100%. RLU represents luminescence value. The inhibition rate of different drug concentrations was calculated in Excel, and then the inhibition curve was plotted and related parameters were calculated using GraphPad Prism software, including the minimum inhibition rate, maximum inhibition rate and relative IC 50 The results are shown in Table 2. Figure 2 and Figure 3 .

[0040] Table 2 Growth inhibition rate of Gefitinib, Erlotinib, Afatinib and Osimertinib on PC-9 resistant tumor cells

[0041]

[0042] It was determined that the cell line Osi-R-PC-9-Pa screened in vitro using Osimertinib showed in vitro resistance to Afatinib, Erlotinib, Gefitinib, and Osimertinib, and its resistance index (RI) was 10234.14, 3337.65, 865.42, and 356.18, respectively, all greater than 5. Therefore, the screened EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line Osi-R-PC-9-Pa was resistant to Afatinib, Erlotinib, Gefitinib, and Osimertinib in vitro, and the resistance performance was stable.

[0043] Example 3: Growth curve of EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line

[0044] (1) The cell line Osi-R-PC-9-Pa, which has been continuously screened for resistance to Osimertinib in Example 1, was cultured in vitro, cells in the logarithmic growth phase were used for plating, cells were stained with trypan blue and viable cells were counted, and the cell concentration was adjusted to 1000 cells per well. In the experimental wells of the 96-well culture plate, 100 μL of the cell suspension of Osi-R-PC-9-Pa was added to each well, but PC-9-Parental was added to the control wells. 7 plates were laid as shown in Table 3 (to facilitate sampling and detection in 7 time periods), and the culture plates were cultured in an incubator at 37° C., 5% CO2, and 100% relative humidity, and the cell viability was measured at 6h, 1d, 2d, 3d, 4d, 5d, and 6d, respectively.

[0045] Table 3 Schematic diagram of cell plating for growth curve experiment

[0046]

[0047] (1) Take out the 96-well culture plate, add 50 μL (equal to half the volume of the cell culture medium in each well) of the Cell Titer-Glo working solution of the Promega Cell Titer-Glo Luminescent Cell Activity Assay Kit (Promega-G7573) to each well, and wrap the cell plate with aluminum foil to avoid light;

[0048] (2) The 96-well culture plate was shaken on an orbital shaker for 2 min to induce cell lysis. The culture plate was placed at room temperature for 10 min to stabilize the luminescent signal, and the luminescent signal was detected on a 2104 EnVision plate reader.

[0049] (3) The growth curve of EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line was plotted with culture time as the horizontal axis and luminescence value (RLU) as the vertical axis. The results are as follows: Figure 4 shown.

[0050] The growth curve drawn ( Figure 4 ) The results showed that under the same culture conditions, there were certain differences in cell proliferation. Osi-R-PC-9-Pa grew slower than its parent PC-9-Parental.

[0051] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line, characterized in that: The cell line is a human lung cancer cell with a deposit number of CCTCC NO: C2024220 and is named Osi-R-PC-9-Pa.

2. Use of the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line according to claim 1 in evaluating drugs for treating drug resistance in non-small cell lung cancer.

3. The use according to claim 2, characterized in that: The drug resistance of non-small cell lung cancer is lung adenocarcinoma drug resistance.

4. Use of the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line according to claim 1 in screening drugs for treating drug resistance of non-small cell lung cancer.

5. The use according to claim 4, characterized in that: The drug resistance of non-small cell lung cancer is lung adenocarcinoma drug resistance.

6. A kit, characterized in that: The invention comprises the EGFR-TKI-resistant human lung adenocarcinoma PC-9 cell line according to claim 1.

7. Use of the kit according to claim 6 in evaluating drugs for treating drug resistance in non-small cell lung cancer.

8. The use according to claim 7, characterized in that: The drug resistance of non-small cell lung cancer is lung adenocarcinoma drug resistance.

9. Use of the kit according to claim 6 in screening drugs for treating drug resistance of non-small cell lung cancer.

10. The use according to claim 9, characterized in that: The drug resistance of non-small cell lung cancer is lung adenocarcinoma drug resistance.

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