EGFR-TKI drug-resistant human lung adenocarcinoma HCC827 cell strain, kit and application thereof

Abstract

The invention discloses an EGFR-TKI drug-resistant human lung adenocarcinoma HCC827 cell strain, a kit and application of the EGFR-TKI drug-resistant human lung adenocarcinoma HCC827 cell strain. The cell strain is a human lung cancer cell with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C2024221, and is named as Osi-R-HCC827-Pa. The Osi-R-HCC827-Pa and the kit thereof have relatively high application values in screening of drugs for treating the drug resistance of the non-small cell lung cancer and evaluation of 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 HCC827 cell line, a kit and applications thereof. Background Art

[0002] Lung cancer is mainly divided into non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). Among them, 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. The 2015 edition of the WHO lung tumor histology reclassified lung adenocarcinoma based on the growth pattern of the tumor: ① Pre-invasive lesions, including adenocarcinoma in situ and atypical adenomatous hyperplasia; ② Early invasive lesions, named microinvasive adenocarcinoma; ③ Invasive adenocarcinoma, including lepidic, acinar, papillary, micropapillary, and solid types, and introduced invasive mucinous adenocarcinoma, colloid, fetal, and intestinal adenocarcinoma. The diagnosis of lung adenocarcinoma is a multidisciplinary process that requires comprehensive clinical, radiological, molecular biological, and pathological information to accurately determine the prognosis and find therapeutic targets. Among them, molecular biological testing is of great significance in improving the early diagnosis rate of lung adenocarcinoma and improving the prognosis of patients, and provides more possibilities for finding new diagnostic methods for lung adenocarcinoma.

[0003] Traditional treatments for lung adenocarcinoma are usually chemotherapy drugs such as platinum compounds or physical therapy, but the overall survival rate of patients has not changed much. It was not until the past decade that with the development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-tyrosine kinases inhibitor, EGFR-TKI) and tumor immunotherapy, the treatment of non-small cell cancer, including lung adenocarcinoma, has made great progress. In general, abnormalities of epidermal growth factor receptor (EGFR) in NSCLC patients will lead to abnormal activation of receptor tyrosine kinase (RTK) signals. EGFR-TKI can block the activation of intracellular tyrosine kinases and avoid the proliferation of cancer cells caused by downstream cascade reactions. At present, 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, it is of great clinical significance to study the mechanism of lung cancer resistance, find strategies to overcome it, or develop new immunotherapy methods.

[0004] The first generation of EGFR-TKI (such as gefitinib and erlotinib) is a major event in the history of targeted therapy for NSCLC. It heralds the advent of targeted precision therapy for tumors and has been proven to be an effective reversible inhibitor for NSCLC patients with EGFR sensitive mutations, which can effectively prolong the survival of patients. Unfortunately, however, most patients develop drug resistance after 9-13 months of treatment. EGFR gene mutations are closely related to the development of this resistance. Among these gene mutations, mutations in exons 19 and 20 of EGFR account for 90% of the total mutations, and the T790M mutation in exon 20 is the main cause of resistance to the first generation of EGFR-TKI. In response to this feature, the second generation of EGFR-TKI (such as afatinib) was introduced, which introduced the Michael addition acceptor 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 patients to have unacceptable toxic side effects, and inevitably produce new resistance 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 of EGFR-TKI, which has a good effect on non-small cell lung cancer patients with EGFR T790M resistance mutations induced by the first generation of EGFR-TKI, and shows excellent selectivity for double mutation EGFR. However, after an average of 9 to 14 months of treatment with Osimertinib, NSCLC patients with resistance mutations to the first and second generation EGFR-TKI 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 developed the fourth-generation EGFR-TKI. In response to the continuous drug-resistant mutation problem, researchers have developed a variety of combined drug therapies and achieved better tumor suppression effects by inhibiting immunosuppressive factors in the targeted tumor microenvironment, but the adverse reactions seem to be inevitable.

[0005] Several large-scale phase 3 clinical trials have consistently demonstrated that the first-generation EGFR-TKI (Gefitinib and Erlotinib), the second-generation EGFR-TKI (Afatinib), and the third-generation EGFR-TKI (Osimertinib) are more effective than 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 such treatments in patients, acquired resistance still exists, with an average progression-free survival of 9-15 months. Studying the EGFR-TKI resistance mechanism that occurs clinically is conducive to the discovery of new biomarkers and drug targets, and the development of better treatment options. Summary of the invention

[0006] 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 HCC827 cell line, which is a human lung cancer cell with a deposit number of CCTCC NO: C2024221 and is named Osi-R-HCC827-Pa.

[0007] In addition, the present invention also provides the use of the EGFR-TKI-resistant human lung adenocarcinoma HCC827 cell line described above in evaluating drugs for treating drug-resistant non-small cell lung cancer.

[0008] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0009] The present invention also provides the use of the EGFR-TKI-resistant human lung adenocarcinoma HCC827 cell line described above in screening drugs for treating drug-resistant non-small cell lung cancer.

[0010] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0011] The present invention also provides a kit, comprising the EGFR-TKI-resistant human lung adenocarcinoma HCC827 cell line as described above.

[0012] 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.

[0013] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0014] 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.

[0015] In some embodiments, the non-small cell lung cancer drug resistance is lung adenocarcinoma drug resistance.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: Osi-R-HCC827-Pa cell line showed good in vitro resistance to the three generations of EGFR-TKIs, namely Afatinib, Erlotinib, Gefitinib and Osimertinib, and their in vitro resistance indexes (RI) were 11018.73, 33223.19, 5867.67 and 1610.02 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 RelativeIC represents the half-maximal effect concentration of the screened cell line. For example, when calculating the in vitro resistance index of Osi-R-HCC827-Pa to Osimertinib, RelativeIC 50亲本 The relative IC 50 EBC-1-筛选 The 3.278 μmol / L obtained by experiment (see Example 2) was taken into account to obtain an RI of 1610.02 for Osimertinib.

[0017] Osi-R-HCC827-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 lung adenocarcinoma treatment methods. Osi-R-HCC827-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.

[0018] 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

[0019] Figure 1 It is the cell growth morphology diagram of HCC827-Parental cell line and Osi-R-HCC827-Pa cell line.

[0020] Figure 2This is the cell proliferation inhibition curve of Afatinib, Erlotinib, Gefitinib, and Osimertinib in HCC827-Parental cell line.

[0021] Figure 3 This is the cell proliferation inhibition curve of Afatinib, Erlotinib, Gefitinib and Osimertinib in Osi-R-HCC827-Pa cell line.

[0022] Figure 4 These are the growth curves of HCC827-Parental cell line and Osi-R-HCC827-Pa cell line. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] The human lung adenocarcinoma cell line HCC827 (ATCC-CRL-2868) used in the embodiments of the present invention was purchased from the ATCC cell bank, and Afatinib, Erlotinib, Gefitinib, and Osimertinib were purchased from Selleck Chemicals, Anage Chemical, Sane, and Haoyuan Pharmaceutical Company, respectively.

[0026] Example 1: Construction of EGFR-TKI-resistant human lung adenocarcinoma cell line

[0027] The screening method is as follows:

[0028] (1) The frozen human lung adenocarcinoma cell line HCC827 (referred to herein as "HCC827-Parental" because it is the parental cell line) was routinely thawed and cultured and passaged in a 37°C, 5% CO2 incubator using 1640 medium containing 10% fetal bovine serum (i.e., the cell culture medium used in Examples 1-3) to expand the cells.

[0029] (2) Take cells in the logarithmic growth phase, count them and prepare a cell suspension with a concentration of 1×10 4 Cells 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.

[0030] (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-HCC827-Pa Homo sapiens, with a deposit number of CCTCC NO: C2024221, 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.

[0031] from Figure 1 It can be seen that the resistant strain Osi-R-HCC827-Pa is morphologically similar to the parental cell line HCC827-Parental.

[0032] Example 2: Determination of the Growth Inhibition Rate of Afatinib, Erlotinib, Gefitinib and Osimertinib on Osi-R-HCC827-Pa Resistant Strain

[0033] (1) The cell line Osi-R-HCC827-Pa obtained in Example 1 and its parent cell line HCC827-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.

[0034] (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.

[0035] Table 1 Schematic diagram of cell plating and drug treatment in in vitro cell activity experiment

[0036]

[0037] Note: Drug concentration unit is μmol / L; Osi-R-HCC827-Pa and HCC827-Parental were loaded into 96-well culture plates as shown in Table 1.

[0038] (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.

[0039] (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.

[0040] (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 .

[0041] Table 2 Growth inhibition rate of Gefitinib, Erlotinib, Afatinib and Osimertinib on HCC827 drug-resistant tumor cells

[0042]

[0043] It was determined that the cell line Osi-R-HCC827-Pa screened in vitro using Osimertinib showed in vitro resistance to Afatinib, Erlotinib, Gefitinib, and Osimertinib, and its resistance index (RI) was 11018.73, 33223.19, 5867.67, and 1610.02, respectively, all greater than 5. Therefore, the screened EGFR-TKI-resistant human lung adenocarcinoma HCC827 cell line Osi-R-HCC827-Pa was resistant to Afatinib, Erlotinib, Gefitinib, and Osimertinib in vitro, and the resistance performance was stable.

[0044] Example 3: Growth curve of human lung adenocarcinoma HCC827 cell line resistant to EGFR-TKI (1) The cell line Osi-R-HCC827-Pa, which has been continuously screened for resistance to Osimertinib in Example 1, was cultured in vitro, and cells in the logarithmic growth phase were used for plating. The cells were stained with trypan blue and the living 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-HCC827-Pa was added to each well, but HCC827-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]

[0048] (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;

[0049] (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.

[0050] (3) The growth curve of EGFR-TKI-resistant human lung adenocarcinoma HCC827 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.

[0051] The growth curve drawn ( Figure 4 ) The results showed that under the same culture conditions, the growth rate of Osi-R-HCC827-Pa was similar to that of the parent HCC827-Parental.

[0052] 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 HCC827 cell line, characterized in that: The cell line is a human lung cancer cell with a deposit number of CCTCCNO: C2024221 and is named Osi-R-HCC827-Pa.

2. Use of the EGFR-TKI-resistant human lung adenocarcinoma HCC827 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 HCC827 cell line according to claim 1 in screening drugs for treating drug resistance in 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: Comprising the EGFR-TKI-resistant human lung adenocarcinoma HCC827 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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