Mouse EGFR (Epidermal Growth Factor Receptor) mutant lung adenocarcinoma cell line (EP Mus musculus) as well as preparation and application thereof

Abstract

The invention relates to the technical field of medicines and biology, in particular to a mouse EGFR (Epidermal Growth Factor Receptor) mutant lung adenocarcinoma cell line (EP Mus musculus) as well as preparation and application thereof. The invention provides an EP cell line ([EGFRE19delta (mutation); trp53 (deletion), EP] is derived from a lung adenocarcinoma model of a C57BL / 6 strain mouse, carries the most common EGFRE19del mutation in lung adenocarcinoma, is a first lung adenocarcinoma mouse cell line carrying EGFR mutation known in the world at present, has the in-vivo tumorigenesis ability of the C57BL / 6 strain mouse, and fills the blank of the mouse EGFR mutant cell line. The cell line provided by the invention has the background of EGFRE19del homozygous activation mutation and Trp53 homozygous deletion mutation, is sensitive to EGFR-TKI targeted therapy, can be used for in-situ tumor formation in subcutaneous and lung of an immune normal mouse C57BL / 6, and is suitable for immunotherapy and targeted therapy research of EGFR mutation lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and biotechnology, and in particular to a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) and a preparation and application thereof. Background Art

[0002] Human epidermal growth factor receptor (EGFR) is an important member of the receptor tyrosine kinase family. It can phosphorylate the tyrosine residues of target proteins, triggering a series of phosphorylation cascade reactions, thereby regulating downstream signaling pathways. At the same time, EGFR is also an important oncogene and is closely related to the occurrence and development of tumors. Targeting the EGFR signaling pathway has always been an important part of tumor treatment.

[0003] Lung adenocarcinoma is one of the most common subtypes of non-small cell lung cancer (NSCLC), and EGFR mutations are relatively common in patients with lung adenocarcinoma. EGFR mutations are closely related to the onset and development of lung adenocarcinoma, and therefore have become the focus of research and treatment. Several clinical trials have shown that EGFR tyrosine kinase inhibitors (TKIs) have superior therapeutic effects compared with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) carrying EGFR sensitive mutations. First-generation (erlotinib, gefitinib, and icotinib) or second-generation (afatinib) TKIs were found to have a median progression-free survival (mPFS) of 9 to 13 months, while the third-generation (osimertinib) EGFR TKI was 18.9 months. Therefore, EGFR TKIs are currently the first-line standard treatment for these patients. However, resistance to EGFR TKIs is inevitable, and new treatment strategies after resistance remain an unresolved issue.

[0004] Unlike EGFR TKIs, immune checkpoint inhibitors (ICIs) targeting programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) are designed to release the brakes on T cells and have shown promising antitumor effects in advanced NSCLC. However, there is considerable controversy over the antitumor effect of ICIs in patients with lung adenocarcinoma with EGFR mutations. A meta-analysis of multiple trials on PD-1 / PD-L1 inhibition in second-line treatment of NSCLC and two large retrospective analyses reported that ICIs did not improve overall survival in patients with NSCLC with EGFR mutations. In addition to their failure as monotherapy, combination therapy of ICIs and TKIs did not produce synergistic antitumor effects in patients. Some studies have found that immunosuppressive tumor microenvironment (TME), tumor mutation burden (TMB), and PD-L1 expression are possible reasons to explain the poor immune response, but these results are all correlation studies based on multi-omics analysis of patient specimens and lack mechanistic studies. It is currently unclear how EGFR affects changes in the tumor immune microenvironment of lung adenocarcinoma.

[0005] At present, the research models of EGFR-mutated lung adenocarcinoma are mainly patient-derived cell lines, organoids, and xenograft subcutaneous tumor models. These in vivo and in vitro models cannot grow in immune mice, so it is difficult to study the mechanism by which EGFR affects the immune microenvironment of lung cancer. Gene-edited mouse models (GEMM) with conditional knock-in EGFR mutants have appeared, but the time and cost of breeding and inducing tumors in GEMMs are very high; and it is difficult to further intervene and gene edit tumors in situ in mice. Therefore, there is currently no suitable model for studying the specific mechanism by which EGFR affects tumor immunity. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a mouse EGFR mutant lung adenocarcinoma cell line (EPMus musculus) and its preparation and use.

[0007] The EP cell line of the present invention ([EGFR E19del (mutation); Trp53 (deletion), EP]) is a lung adenocarcinoma model derived from C57BL / 6 mice, carrying the most common EGFR E19del Mutation, is the first lung adenocarcinoma mouse cell line known in the world to carry EGFR mutation, this cell line has the ability to form tumors in C57BL / 6 mice, filling the gap of mouse EGFR mutation cell lines. The cell line provided by the present invention is EGFR E19delThe homozygous activating mutation and Trp53 homozygous deletion mutation background are sensitive to EGFR-TKI targeted therapy, and can form tumors subcutaneously and in situ in the lungs of immune-normal C57BL / 6 mice, making it suitable for immunotherapy and targeted therapy research of EGFR mutant lung adenocarcinoma.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The first object of the present invention is to provide a mouse EGFR mutant lung adenocarcinoma cell line (EP Musmusculus), which was submitted to the China Center for Type Culture Collection on March 14, 2024, with a collection number of CCTCC NO: C2023394.

[0010] The second object of the present invention is to provide a method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus), comprising the following steps:

[0011] (S1) LSL-EGFR E19del Mice and Trp53 flox / flox Mice were bred to obtain LSL-EGFR E19del and Trp53 flox / flox Double homozygous mice: EP mice;

[0012] (S2) using AAV-Cre virus to induce treatment on the EP mice prepared in step (S1), thereby inducing tumor formation in the lungs of the EP mice;

[0013] (S3) Cultivating the tumor prepared in step (S2) to obtain a mouse EGFR mutant lung adenocarcinoma cell line (EPMus musculus).

[0014] In one embodiment of the present invention, in step (S1), the LSL-EGFR E19del The mice were obtained by inserting the human EGFR exon 19 deletion mutant gene into the ROSA26 site of C57BL / 6 mice, and inserting the LoxP-STOP-LoxP sequence into the 5' segment of the human EGFR exon 19 deletion mutant gene.

[0015] In one embodiment of the present invention, the sequence of the human EGFR exon 19 deletion mutant gene is shown as SEQ ID NO.1.

[0016] In one embodiment of the present invention, the sequence of LoxP is shown as SEQ ID NO.2.

[0017] In one embodiment of the present invention, in step (S1), the Trp53 flox / floxThe mice were obtained by inserting a LoxP gene into the 5' end of exon 5 of the Trp53 gene of C57BL / 6 mice, and inserting a LoxP gene into the 3' end of exon 7 of the Trp53 gene.

[0018] In one embodiment of the present invention, the sequence of exon 5 of the Trp53 gene is shown in SEQ ID NO.3;

[0019] The sequence of exon 7 of the Trp53 gene is shown in SEQ ID NO.4.

[0020] In one embodiment of the present invention, in step (S2), EP mice are induced by nasal drops of AAV-Cre virus.

[0021] In one embodiment of the present invention, the amount of the AAV-Cre virus is 5×10 10 vg~7×10 10 vg.

[0022] In one embodiment of the present invention, the amount of AAV-Cre virus is 6×10 10 vg.

[0023] In one embodiment of the present invention, in step (S3), the culturing includes in vitro primary culture and subculture.

[0024] In one embodiment of the present invention, during the in vitro primary culture and subculture, the tumor is cut into pieces and cultured in a 10% FBS advanced-DMEM / F-12 culture medium, and the medium is changed after tumor fragments crawl out;

[0025] Subculture was performed after 10 days of primary culture in vitro;

[0026] The culture process was placed in a 5% CO2 environment at a temperature of 37°C.

[0027] The third object of the present invention is to provide a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) for use in EGFR targeted therapy and drug resistance mechanism research.

[0028] The fourth object of the present invention is to provide a use of a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) in high-throughput in vivo and in vitro library screening.

[0029] There are many KRAS mutant lung adenocarcinoma cell lines derived from C57BL / 6 mice. These cell lines have been widely used to study the immune microenvironment of KRAS mutant lung adenocarcinoma and the mechanism of affecting ICI response. The present invention constructs an EGFR mutant lung adenocarcinoma cell line derived from C57BL / 6 mice. The cell line has a clear EGFR 19del Mutation, tissue type is lung adenocarcinoma cell line. This cell line can directly form tumors in situ in immunocompetent or immunodeficient mice, and is sensitive to EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor treatment) both in vivo and in vitro. This is a reliable and useful model for subsequent exploration of the immune microenvironment of EGFR mutant lung adenocarcinoma.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The EP cell line of the present invention currently only carries EGFR E19del Mutation and Trp53 deletion, and clear genetic background provide important research materials for the study of EGFR mutation; the EP cell line of the present invention can form EGFR-mutated lung adenocarcinoma in situ and subcutaneously in the lungs of C57BL / 6 mice with normal immunity, which solves this problem well; the EP cell line of the present invention is sensitive to EGFR-TKI treatment and is suitable for EGFR targeted therapy and drug resistance mechanism research.

[0032] (2) The EP cell line gene background of the present invention is EGFR E19del and Trp53 inactivation mutations, and has a pure gene mutation background, which is suitable for high-throughput in vivo and in vitro library screening; the EP cell line of the present invention is inoculated with 1 million tumor cells and can quickly form tumors subcutaneously or in situ in the lungs of mice, which is an efficient in vivo and in vitro model for studying EGFR mutation lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the construction principle of EP mice; Figure 1 A is LSL-EGFR E19del Mice; Figure 1 B is Trp53 flox / flox Mouse.

[0034] Figure 2 For lung tumor monitoring and primary cell culture in EP mice; Figure 2 A is the CT scan result of mice 8 weeks after intranasal administration of AAV-cre virus; Figure 2 B is the process of cell line formation.

[0035] Figure 3The figure shows the results of measuring the Trp53 protein level in the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus).

[0036] Figure 4 Schematic diagram of tumor formation in different mice by mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus); Figure 4 A is a schematic diagram of the formation of tumors in the lungs of C57BL / 6 mice by a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus); Figure 4 B is a schematic diagram of the formation of tumors in the lungs of immune-deficient nude mice by a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus); Figure 4 C is the expression of TTF-1, EGFR and phosphorylated EGFR in tumor nodules of C57BL / 6 mice.

[0037] Figure 5 This is a diagram of the in vitro treatment results of osimertinib; Figure 5 A is the in vitro therapeutic sensitivity results of each cell line to osimertinib; Figure 5 B is a graph showing the protein expression of EP1 at different time periods after osimertinib treatment.

[0038] Figure 6 This is a diagram of the in vivo treatment results of osimertinib; Figure 6 A is the result graph of tumor volume changes in the osimertinib treatment group and the control group; Figure 6 B is a bar graph of tumor weight after the experiment was terminated after 18 days of treatment; Figure 6 C is the tumor size diagram after the experiment was terminated after 18 days of treatment; ****P<0.001. DETAILED DESCRIPTION

[0039] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all mice used are commercially available mice; and all detection means and methods used are conventional detection means and methods in the art.

[0041] Example 1

[0042] This example provides a method for constructing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus), which specifically includes the following steps:

[0043] (S1) LSL-EGFR E19del Mouse (specifically LSL-EGFR E19del Heterozygous mice) and Trp53 flox / floxMouse (specifically Trp53 flox / flox Homozygous mice) were bred and LSL-EGFR was obtained by screening E19del and Trp53 flox / flox Double homozygous mice: EP mice;

[0044] Among them, LSL-EGFR in this embodiment E19del The mice were constructed by Beijing Biocytogen Pharmaceuticals Co., Ltd.

[0045] Trp53 flox / flox The mice were constructed by Shanghai Model Organisms Technology Co., Ltd.;

[0046] LSL-EGFR E19del The mouse was obtained by inserting the human EGFR exon 19 deletion mutant gene (as shown in SEQ ID NO.1) into the ROSA26 site of C57BL / 6 mice, and inserting a LoxP-STOP-LoxP sequence (the sequence of LoxP-STOP-LoxP is shown in SEQ ID NO.2: 5'-ATAACTTCGTATA GCATACATTATACGAAGTTAT-3') into the 5' segment of the human EGFR exon 19 deletion mutant gene ( Figure 1 A);

[0047] Trp53 flox / flox The mice were obtained by inserting a LoxP gene into the 5' end of exon 5 (sequence as shown in SEQ ID NO.3: 5'-AGTCGCTCTGAGTTGTTATCAG-3') of the Trp53 gene of C57BL / 6 mice, and inserting a LoxP gene (sequence as shown in SEQ ID NO.5: 5'-ATAACTTCGTATA-3') into the 3' end of exon 7 (sequence as shown in SEQ ID NO.4: 5'-TGAGCATGTCTTTAAT CTACCTCGATG-3') of the Trp53 gene ( Figure 1 B).

[0048] (S2) 6×10 10 vg AAV-Cre virus was instilled into the nose of EP mice to induce EGFR E19del After 8 weeks of mutant expression and Trp53 knockout, CT scans of the mice revealed that tumors had formed in the lungs of EP mice, and tumors on the surface of the lungs of EP mice could also be seen when the lungs were dissected (e.g. Figure 2 A);

[0049] (S3) Dissect and separate the tumors formed in the mouse lungs and culture them to obtain cell lines;

[0050] In step (S3), the culture method (in vitro primary culture of tumors formed in mouse lungs to establish cell lines) is as follows:

[0051] The tumor tissue was dissected out and placed in a PBS solution containing antibiotics, and the surrounding normal tissue was removed.

[0052] Place the tumor tissue in a 1.5mL EP tube and cut into pieces to obtain 1-2 cubic millimeters of tumor fragments. Resuspend the tumor fragments with 1mL 10% FBS advanced-DMEM / F-12 medium and place them in a 6cm culture dish. Then add 4mL 10% FBS advanced-DMEM / F-12 medium and culture them at 37℃ 5% CO2 for 5 days. After observing that cells have crawled out of the tumor fragments, change the medium; and perform subculture after 10 days.

[0053] During the first ten generations of culture of this cell line, the cell morphology was observed. Of course, when the fibroblasts began to increase, 0.25% trypsin was used for short-term digestion for 30-90s and then the culture dish was washed twice with PBS to reduce the enrichment of fibroblasts. After 10 generations, the tumor cells were basically very pure and could be stably passaged. At this time, monoclonal selection was performed to obtain cell lines (such as Figure 2 As shown in B, the subsequent experiments were carried out with the screened EP1 and EP2);

[0054] Using the first-generation sequencing Sanger Sequencing technology, the constructed cell line was detected to carry EGFR E19del Mutation. At the same time, Western blot detected that the Trp53 protein level in the primary cell line was missing (such as Figure 3 As shown, LLC cell line and KP cell line were used as controls); this shows that this example successfully constructed EGFR E19del Mutation / Trp53-deficient cell lines.

[0055] The cell line prepared in this example was submitted to the China Center for Type Culture Collection on March 14, 2024, with the collection number CCTCC NO: C2023394, and was named mouse EGFR mutant lung adenocarcinoma cell line (EP Musmusculus).

[0056] This example tested LSL-EGFR E19del Heterozygous mice, EGFR E19del Heterozygous mice and Trp53 flox / flox Homozygous mice (LSL-EGFR E19del Mice and Trp53 flox / floxThe mice obtained by breeding and screening mice) were found to form lung tumors but could not be further cultured into in vitro cell lines; only LSL-EGFR E19del and Trp53 flox / flox The tumors formed in the double homozygous mice can be used to establish and culture EP cell lines.

[0057] Performance Analysis:

[0058] 1) Mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) can form adenocarcinoma in the lungs of immunocompetent and immunodeficient mice

[0059] The mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) is derived from the C57BL / 6 mouse strain and is therefore theoretically capable of forming tumors in the immune-competent murine microenvironment.

[0060] One million mouse EGFR mutant lung adenocarcinoma cell lines (EP Mus musculus) constructed in Example 1 were implanted into immune-competent C57BL / 6 mice and immune-deficient Nude mice by tail vein injection. After 10 weeks, CT scanning of the mice revealed that visible tumors had formed in the lungs of both C57BL / 6 mice and Nude mice. The lung tissues of the mice were further dissected out, revealing that scattered tumor nodules ( Figure 4 A and 4B);

[0061] The tumor nodules from dissected C57BL / 6 mice were fixed with formalin and then subjected to IHC analysis. It was found that the tumor nodules highly expressed TTF-1, a marker of lung adenocarcinoma, as well as EGFR and phosphorylated EGFR protein (pho-EGFR), which verified the activation of EGFR signaling and the pathological characteristics of lung adenocarcinoma (such as Figure 4 C); confirmed that the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) can form tumors not only in immunodeficient mice, but also in immune-competent mice.

[0062] 2) EGFR-TKI osimertinib can significantly inhibit the growth and downstream signaling pathways of mouse EGFR mutant lung adenocarcinoma cell line (EP Musmusculus)

[0063] hEGFR exon 19 deletion mutation is a sensitive mutation for EGFR-TKI therapy in EP cells, which is applicable to lung adenocarcinoma. In order to test the drug sensitivity of mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) to EGFR-TKI osimertinib, human PC9 cell line known to carry EGFR mutation was used as positive control, and human A549 cell line and mouse LLC cell line known not to carry EGFR mutation were used as negative controls.

[0064] The specific method is: by determining the half lethal concentration IC 50 In this way, PC9 cell lines with EGFR mutations and sensitive to EGFR-TKI, mouse EGFR mutant lung adenocarcinoma cell lines (EP Mus musculus), and A549 cell lines and LLC cell lines without EGFR mutations and insensitive were plated into cell well plates, and culture medium containing EGFR-TKI osimertinib was added according to a concentration gradient of 0nM, 5nM, 10nM, 20nM, 50nM, 100nM, 200nM, 500nM, 1000nM, 5000nM, and 10000nM. After culturing for 48h, CCK8 solution was added to each well to a final concentration of 10% (volume ratio); the cells were then incubated for 1h, and the absorbance was measured at a wavelength of 450nm using a microplate reader (BioTek) to further detect the IC50 of each cell line.

[0065] The results showed that both 48-hour and 72-hour drug sensitivity tests confirmed that the monoclonal (EP1 and EP2) of the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) were sensitive to the treatment of EGFR-TKI osimertinib, and their half-lethal concentration (IC50) was comparable to that of the positive control PC9 and significantly lower than that of the negative controls A549 and LLC ( Figure 5 A).

[0066] EP1 was treated with osimertinib at a drug concentration of 100 nM, and the effects of phosphorylated EGFR (pEGFR) and downstream ERK (pERK) in the EP1 cell line at different time points were collected; it was found that the phosphorylated EGFR level in the EP1 cell line was significantly inhibited 2 hours after treatment and lasted until 48 hours during the 48 hours of drug treatment, and the phosphorylated ERK level was observed to be significantly inhibited 2 hours after the initial treatment, and then slowly recovered within 48 hours of treatment ( Figure 5 B), indicating that the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) is sensitive to osimertinib and exhibits a corresponding significant response in the EGFR downstream signaling pathway.

[0067] 3) EGFR-TKI osimertinib can inhibit the growth of EP-derived tumors in vivo

[0068] The mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) prepared in this example was subcutaneously injected into the dorsal part of nude mice (BALB / c-Nude Strain NO.D000521 6-week-old nude mice). When the subcutaneous tumor grew to a volume of nearly 300 mm 3 At 4 pm, osimertinib treatment was started (5 mg / kg, 100 μL, intragastrically once every two days); mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) was subcutaneously injected into the dorsal part of nude mice (6-week-old nude mice from BALB / c-Nude Strain NO.D000521). When the subcutaneous tumor grew to a volume of nearly 300 mm 3 The mice treated with placebo (normal saline, 100 μL, intragastric administration once every two days) served as the control group. The results showed that osimertinib could significantly inhibit tumor growth (e.g. Figure 6 The weight and volume of the tumor in the osimertinib-treated group were significantly smaller than those in the control group (as shown in Figure 2A). Figure 6 B and Figure 6 C).

[0069] The sequence (5'-3') of SEQ ID NO.1 is as follows:

[0070]

[0071] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus), characterized in that: It was sent to the China Center for Type Culture Collection on March 14, 2024, and the preservation number is CCTCC NO: C2023394.

2. A method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) as claimed in claim 1, characterized in that: The following steps are involved: (S1) LSL-EGFR E19del Mice and Trp53 flox / flox Mice were bred to obtain LSL-EGFR E19del and Trp53 flox / flox Double homozygous mice: EP mice; (S2) using AAV-Cre virus to induce treatment on the EP mice prepared in step (S1), thereby inducing tumor formation in the lungs of the EP mice; (S3) culturing the tumor prepared in step (S2) to obtain a mouse EGFR mutant lung adenocarcinoma cell line (EP Musmusculus).

3. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 2, characterized in that: In step (S1), the LSL-EGFR E19del The mice were obtained by inserting the human EGFR exon 19 deletion mutant gene into the ROSA26 site of C57BL / 6 mice, and inserting the LoxP-STOP-LoxP sequence into the 5' segment of the human EGFR exon 19 deletion mutant gene.

4. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 2, characterized in that: In step (S1), the Trp53 flox / flox The mice were obtained by inserting a LoxP gene into the 5' end of exon 5 of the Trp53 gene of C57BL / 6 mice, and inserting a LoxP gene into the 3' end of exon 7 of the Trp53 gene.

5. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 2, characterized in that: In step (S2), EP mice are induced by nasal drops of AAV-Cre virus.

6. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 5, characterized in that: The amount of AAV-Cre virus used was 5×10 10 vg~7×10 10 vg.

7. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 6, characterized in that: The amount of AAV-Cre virus used was 6×10 10 vg.

8. The method for preparing a mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) according to claim 2, characterized in that: In step (S3), the culture includes in vitro primary culture and subculture.

9. Use of the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) as claimed in claim 1 in EGFR targeted therapy and research on the mechanism of drug resistance.

10. Use of the mouse EGFR mutant lung adenocarcinoma cell line (EP Mus musculus) as claimed in claim 1 in high-throughput in vivo and in vitro library screening.

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