A human EGFR mutation-driven mouse primary lung cancer cell line, its construction method and application
By constructing a human EGFR (L858R/T790M) mutation in C57BL/6 mice and combining in vivo passage and in vitro culture screening in nude mice, the ZST-1 cell line was obtained. This solved the problem of the lack of stable tumor formation and expression of human EGFR mutation in immune-intact mice in the existing technology, and realized stable tumor formation and in vivo and in vitro imaging monitoring in immune-intact mice, which is suitable for targeted and immunotherapy research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHONGSHAN HOSPITAL FUDAN UNIV
- Filing Date
- 2026-03-25
- Publication Date
- 2026-06-02
AI Technical Summary
Current technology lacks a mouse primary lung cancer cell line that can stably form tumors in immune intact mice, express human EGFR mutations, and possess fluorescence and bioluminescence reporter systems, for studying tumor-immune microenvironment interactions and the efficacy and mechanism of targeted combined immunotherapy.
By constructing a Rosa26-site stably expressing human EGFR (L858R/T790M) mutation in C57BL/6 background mice, and using AAV6-Scgb1a1-CRE virus to specifically activate EGFR expression in the lungs, combined with in vivo passage in nude mice and in vitro culture screening, the ZST-1 cell line was obtained. This cell line stably expresses Luciferase and tdTomato reporter genes.
It achieves stable tumor formation in immune-intact mice while possessing a fluorescence and bioluminescence reporter system, facilitating in vivo and in vitro imaging monitoring. It is suitable for in vivo and in vitro joint evaluation of human EGFR mutation-targeted drugs and for research on targeted and immunotherapy combinations.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of tumor biology and drug screening technology, specifically to a human EGFR mutation-driven mouse primary lung cancer cell line ZST-1, its construction method, and its applications. Background Technology
[0002] EGFR mutations, especially L858R and T790M mutations, are among the most important driver gene alterations in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors targeting EGFR mutations (EGFR-TKIs) have been widely used clinically and have significantly improved patient prognosis. However, almost all patients eventually develop drug resistance, limiting long-term efficacy.
[0003] In recent years, the combined use of targeted therapy and immunotherapy has been considered an important strategy for overcoming drug resistance. Studies have shown that EGFR-mutant tumors have unique immune microenvironment characteristics and have limited response to immune checkpoint inhibitor monotherapy, but may produce synergistic effects when combined with targeted therapy under certain conditions. However, the relevant mechanisms are still unclear, and there is a lack of experimental models that can simultaneously meet the following conditions: driven by human EGFR mutations, capable of accurately evaluating targeted drugs against human EGFR mutations, and able to establish tumor models in immune-intact mice for studying tumor-immune microenvironment interactions and the efficacy and mechanism of targeted combined immunotherapy.
[0004] Existing cell or animal model products of the same type and their disadvantages are as follows:
[0005] ① Human EGFR mutant cell lines (such as PC-9, HCC827, H1975): These are derived from human lung cancer and can only be used in immunodeficient mouse models. They produce severe immune rejection in immunocompetent mice (such as C57BL / 6 mice) and are difficult to form tumors in vivo. Therefore, they cannot be used for drug efficacy and immune-related studies in immunocompetent mice and are not suitable for immune microenvironment studies.
[0006] ② Mouse-derived lung cancer cell lines (such as LLC): These are usually Kras-driven or chemically induced models and do not carry human EGFR mutations; they cannot be used to directly evaluate targeted drugs against "human EGFR mutations".
[0007] ③ Transgenic mouse lung cancer model: Although it can simulate primary lung cancer, it is not convenient for in vitro research and high-throughput screening.
[0008] Therefore, there is currently a lack of a lung cancer cell line that is derived from mice, can be grown in C57BL / 6 immunized intact mice, is driven by a human EGFR (L858R / T790M) mutation, can be stably cultured in vitro, and possesses both fluorescence and bioluminescence reporter systems. Summary of the Invention
[0009] The purpose of this invention is to provide a human EGFR (L858R / T790M) mutation-driven lung cancer cell line ZST-1, derived from primary lung cancer in mice, which can be stably passaged, can stably form tumors subcutaneously in C57BL / 6 mice, and simultaneously expresses Luciferase and tdTomato reporter genes, and to provide its construction method and application.
[0010] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0011] In a first aspect, the present invention provides an EGFR mutation-driven mouse primary lung cancer cell line, named mouse lung cancer cell ZST-1, which is deposited at the China Center for Type Culture Collection on March 24, 2026, with accession number CCTCC NO: C202654.
[0012] The ZST-1 cell line of this invention stably expresses the human EGFR (L858R / T790M) mutant protein, possessing double mutations of L858R and T790M, and co-expresses the Luciferase and tdTomato reporter genes. In vitro sensitivity tests to EGFR tyrosine kinase inhibitors of this cell line are consistent with the characteristics of the L858R / T790M double mutation, which is resistant to first- and second-generation TKIs but sensitive to third-generation TKIs, indicating its dependence on the mutant EGFR signaling pathway. STR typing clearly identifies it as a mouse tumor cell line, free from human cell contamination. This cell line can stably form tumors subcutaneously in immunized C57BL / 6 mice. Furthermore, this cell line can be stably passaged after approximately 6 months of continuous in vitro culture and multiple passage selections.
[0013] Secondly, the present invention provides a method for constructing the EGFR mutation-driven mouse primary lung cancer cell line ZST-1, comprising the following steps:
[0014] (1) Obtaining transgenic mice: C57BL / 6 background transgenic mice were used, and the CAG-LSL-hEGFR(L858R / T790M)-IRES-Luc-2A-tdTomato expression construct was knocked into the Rosa26 site of the mice;
[0015] (2) Tumor induction: Eight-week-old male transgenic mice were selected and AAV6-Scgb1a1-CRE virus particles were administered intranasally after anesthesia. Eight weeks after the virus was administered intranasally, spontaneous lung tumor formation was confirmed by microCT scan.
[0016] (3) Continuous in vivo passage of tumors: lung tumor nodules were removed, cut into pieces, and inoculated into the left forelimb axilla of nude mice for subcutaneous transplantation. After the subcutaneous tumors grew, the tumor tissue was removed, cut into pieces, and inoculated into the next generation of nude mice. The passage was repeated for 8 generations to obtain tumor tissue with stable subcutaneous tumor formation ability.
[0017] (4) Cell isolation and in vitro culture: Subcutaneous tumor tissue from the 8th generation nude mice was taken, minced and digested with collagenase to prepare a single-cell suspension, which was then inoculated into a culture dish for in vitro culture. The suspension was continuously cultured and passaged for screening to obtain the cell line ZST-1, which can be passaged for a long time and grow stably.
[0018] Further, in step (1), the transgenic mouse is C57BL / 6Smoc-Gt(ROSA)26Sor purchased from Shanghai Southern Model Biotechnology Co., Ltd. tm1(CAG-LSL-EGFR(L858R-T790M)-IRES-luciferase-2A-tdTomato)Smoc Mice, catalog number NM-KI-190068.
[0019] Further, in step (2), the viral particle titer is 1×10¹¹ vg.
[0020] Furthermore, in step (4), the culture medium used for in vitro culture is DMEM medium containing 10% (volume fraction) fetal bovine serum.
[0021] Thirdly, the present invention provides the following applications of the EGFR mutation-driven mouse primary lung cancer cell line ZST-1:
[0022] ① In vitro screening and efficacy evaluation of targeted drugs for human EGFR mutations (L858R / T790M);
[0023] ② Research on EGFR-TKI resistance mechanisms;
[0024] ③ Tumor bioluminescence imaging and fluorescence tracing studies (using Luciferase and tdTomato reporter genes);
[0025] ④ In vivo tumorigenesis and drug efficacy experiments under the background of immune integrity C57BL / 6;
[0026] ⑤ Research on EGFR mutation-related signaling pathways and tumor biological mechanisms.
[0027] Existing human EGFR-mutant lung cancer cell lines typically only form tumors in immunodeficient mice, failing to achieve tumor formation in immunocompetent mice or facilitate studies of the immune microenvironment. Mouse-derived lung cancer cells, on the other hand, do not carry human EGFR mutations, making it difficult to directly evaluate targeted drugs against human EGFR mutations. While existing EGFR-mutant transgenic mouse models can form primary lung cancer in vivo, they are unsuitable for in vitro studies and high-throughput screening, limiting their application in pharmacodynamic research and mechanism exploration. Therefore, there is an urgent need to establish a novel cell model with a clearly defined origin, a well-defined driving mechanism, stable tumor formation in immunocompetent mice, and suitability for combined targeted and immunotherapy studies.
[0028] This invention constructs a human EGFR mutant expression model based on a stable Rosa26 site expression system and a strong CAG promoter. CRE-dependent activation is achieved through the LSL structure, and lung-specific expression is achieved using the Scgb1a1 promoter. A combined strategy of primary lung tumor formation, continuous in vivo passage selection in nude mice, and long-term in vitro culture selection enhances the subcutaneous tumorigenicity and in vitro adaptability of primary lung cancer while preserving its biological characteristics, thereby obtaining a cell line that can be stably cultured for a long period. This strategy ensures a clear cell line origin, a well-defined driving mechanism, and applicability to both in vitro and in vivo experiments.
[0029] The ZST-1 cell line of this invention has the following advantages: its tumor growth is directly driven by human EGFR (L858R / T790M) mutation, and the driving mechanism is clear; it is derived from mouse primary lung cancer tissue and has high biological relevance; the cells are derived from the C57BL / 6 background and can form tumors in immune-intact mice, making it suitable for immune-related studies; it also has a dual reporter system of Luciferase and tdTomato, which facilitates in vivo and in vitro imaging monitoring; it can be used for in vivo and in vitro joint evaluation of human EGFR mutation-targeted drugs; the construction process is rigorous, the genetic background is clear and stable, and it can be scaled up, which is convenient for in vitro studies and in vitro high-throughput screening. Attached Figure Description
[0030] Figure 1 Example 1: Preparation process of ZST-1 cell line.
[0031] Figure 2 Example 2: Sequencing map of human EGFR gene in ZST-1 cells.
[0032] Figure 3 Example 3: Results of drug sensitivity testing of ZST-1 cells to different EGFR tyrosine kinase inhibitors.
[0033] Figure 4Figure 4: Western blot results showing the effects of different EGFR tyrosine kinase inhibitors on the expression of EGFR signaling pathway-related proteins in ZST-1 cells.
[0034] Figure 5 STR typing results of ZST-1 cells in Example 5.
[0035] Figure 6 Example 6 shows the results of Luciferase luminescence intensity detection in the ZST-1 cell line.
[0036] Figure 7 : tdTomato fluorescence imaging results of ZST-1 cell line in Example 6 (scale bar: 200 μm).
[0037] Figure 8 Photograph of solid tumors formed subcutaneously in C57BL / 6 mice using the ZST-1 cell line in Example 7.
[0038] Figure 9 HE staining results of subcutaneous tumors in ZST-1 cell line C57BL / 6 mice treated with osimertinib in Example 8. Left: Control group; Right: Osimertinib-treated group. Scale bar: 50 μm. Detailed Implementation
[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.
[0040] Example 1: Construction process of ZST-1 cell line
[0041] This embodiment provides a method for constructing the ZST-1 cell line, the process of which is as follows: Figure 1 As shown, the specific steps are as follows:
[0042] (1) Obtaining transgenic mice: The ZST-1 cell line described in this invention is derived from genetically engineered C57BL / 6 background transgenic mice, which are C57BL / 6Smoc-Gt(ROSA)26Sor mice purchased from Shanghai Southern Model Biotechnology Co., Ltd. tm1(CAG-LSL-EGFR(L858R-T790M)-IRES-luciferase-2A-tdTomato)SmocMouse (Catalog No.: NM-KI-190068). This mouse had a CAG-LSL-hEGFR(L858R / T790M)-IRES-Luc-2A-tdTomato expression construct knocked into the Rosa26 site. The CAG promoter is a strong promoter, and the LSL (LoxP-STOP-LoxP) structure is used to achieve CRE-dependent activation. Under the action of CRE recombinase, the STOP sequence is cleaved, thereby initiating the expression of the human EGFR (L858R / T790M) mutant gene. Simultaneously, Luciferase and the tdTomato reporter gene are co-expressed through the IRES and 2A peptide structures. This design ensures the synchronous expression of the mutant EGFR and the fluorescent / bioluminescent reporter system, and allows for dynamic monitoring using imaging technology.
[0043] (2) Tumor induction: Eight-week-old male transgenic mice were anesthetized with isoflurane and then instilled 1×10¹¹ vg AAV6-Scgb1a1-CRE viral particles (Shanghai Jikai Gene, catalog number: GOSV5002568) via nasal drip. The Scgb1a1 promoter in this viral vector drives CRE expression, thereby specifically activating human EGFR (L858R / T790M) expression in lung epithelial cells. Eight weeks after the viral instillation, microCT scans were used to detect the formation of lung tumors in the mice, confirming the formation of spontaneous lung tumors.
[0044] (3) Continuous in vivo passage of tumors: After tumor formation, mice were sacrificed, lung tumor nodules were dissected and removed, and then minced and inoculated into the left forelimb axilla of BALB / c Nude mice (Shanghai Jicui Pharmaceutical Co., Ltd., catalog number: D000521) for subcutaneous transplantation. After the subcutaneous tumor grew to a suitable size, the tumor tissue was removed again, mechanically minced, and inoculated into the next generation of BALB / c Nude mice. Through eight consecutive generations of subcutaneous passage in BALB / c Nude mice, tumor tissue with stable subcutaneous tumorigenesis ability was obtained, laying the foundation for the subsequent establishment of in vitro cell lines. Among them, nude mice are immunodeficient mice lacking T cells, and play a transitional role in the cell line construction process to assist the growth of primary cells. Without the cells obtained in this step, it is difficult for them to grow subcutaneously in C57BL / 6 mice.
[0045] (4) Cell isolation and in vitro culture: Subcutaneous tumor tissue from 8th generation nude mice was mechanically minced and digested with collagenase to prepare a single-cell suspension, which was then inoculated into culture dishes for in vitro culture. The culture medium was DMEM basal medium + 10% fetal bovine serum. The culture conditions were 37℃ and 5% CO2. The cells were continuously cultured and passaged multiple times in vitro for about 6 months (passing was generally 3-7 days, and 30-40 passages were performed) to stabilize their genetic characteristics and growth characteristics, and finally a cell line that could be passaged for a long time and grow stably was obtained, named ZST-1, where ZS stands for Zhongshan Hospital and T stands for Thoracic. The cell line ZST-1 was deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on March 24, 2026, with accession number CCTCC NO: C202654.
[0046] Example 2: Sangon sequencing detection of human EGFR mutations in ZST-1 cells
[0047] Genomic DNA was extracted from ZST-1 cells using a blood-cell-tissue genomic DNA extraction kit (centrifuge column type) (Beijing Tiangen Biotech Co., Ltd., catalog number: DP304). Human EGFR fragments were amplified using Taq PCR Mix premix (2X, containing blue dye) (Shanghai Sangon Biotech Co., Ltd., catalog number: #B639295) and the following primers and reaction conditions.
[0048] Forward primer: 5'-TGCGTTCGGCACGGTGTATAAG-3';
[0049] Backward primer: 5'-GGCGACTATCTGCGTCTATCATCC-3';
[0050] PCR reaction system: 10 μL of Sangon 2X Taq PCR Mix premix, 0.5 μL of forward primer, 0.5 μL of backward primer, 9 μL of DNA template, and ddH2O to bring the total volume to 20 μL;
[0051] PCR reaction program: 94 ℃ for 4 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 1 min, for a total of 35 cycles; 72 ℃ for 5 min; 4 ℃.
[0052] The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for Sangon sequencing. The sequencing results are as follows: Figure 2 As shown, the results, when compared with the human EGFR gene sequence, indicate that mutations in the human EGFR gene L858R and T790M exist in ZST-1 cells.
[0053] Example 3: Detection of the inhibitory effects of different EGFR-TKIs on ZST-1 cell proliferation
[0054] To verify the drug sensitivity of ZST-1 cells to different EGFR tyrosine kinase inhibitors (EGFR-TKIs), cell viability assays were used to evaluate the in vitro efficacy of various TKIs.
[0055] ZST-1 cells were seeded in 96-well plates at a density of 2000 cells per well and allowed to adhere overnight before drug treatment. Different concentrations of gefitinib, erlotinib, afatinib, osimertinib, almonertinib, furmonertinib, and befotertinib were added, with final concentrations set at 0.1, 1, 10, 100, and 1000 nM. Each drug was used in duplicate.
[0056] After 5 days of drug treatment, cell viability was assessed using the AlamarBlue cell viability assay kit. 10 μL of the kit was added to each well after medium replacement, and the cells were incubated for 2 hours. Fluorescence values were then measured using a microplate reader to evaluate cell viability.
[0057] The results are as follows Figure 3 As shown, ZST-1 cells exhibited high sensitivity to third-generation EGFR-TKIs (osimertinib, ametinib, vormetinib, befotinib) (IC50 1-10 nM), while the inhibitory effects of first- and second-generation EGFR-TKIs (gefitinib, erlotinib, afatinib) were relatively weak (IC50 > 200 nM). This is consistent with the molecular characteristics of the L858R / T790M double mutation, which is resistant to first- and second-generation TKIs but sensitive to third-generation TKIs. This further verifies the dependence of this cell line on the human EGFR (L858R / T790M) signaling pathway.
[0058] Example 4: Molecular verification of EGFR signaling pathway inhibition
[0059] To further verify the inhibitory effect of EGFR-TKI on the EGFR signaling pathway in ZST-1 cells at the molecular level, Western blot was used to detect changes in EGFR phosphorylation levels.
[0060] ZST-1 cells were seeded in culture dishes. In the first group of experiments, cells were treated with control, osimertinib 2, 20, and 200 nM for 48 hours, respectively. In the second group of experiments, cells were treated with control, 20 nM osimertinib, 20 nM vormetinib, 20 nM ametinib, and 20 nM befotinib for 48 hours, respectively. The control group was treated with an equal volume of solvent.
[0061] After drug treatment, cells were collected, lysed and total protein was extracted. After separation by SDS-PAGE electrophoresis, the cells were transferred to a membrane. Specific antibodies were used to detect the expression levels of EGFR phosphorylation sites p-EGFR (Y1068, Y1092, Y1197), total EGFR, and human EGFR (L858R mutant) proteins.
[0062] The results are as follows Figure 4 As shown, human EGFR (L858R mutant) protein is expressed in the cells. With increasing osimertinib concentration, the phosphorylation levels of all sites on p-EGFR significantly decreased. Different third-generation EGFR-TKIs (osimertinib, vormetinib, ametinib, and befotinib) can all significantly inhibit EGFR phosphorylation levels at a concentration of 20 nM, suggesting that this cell line is significantly dependent on the EGFR signaling pathway and has a good response to third-generation EGFR-TKIs.
[0063] The above results further demonstrate that the ZST-1 cell line is a human EGFR (L858R / T790M) mutation-driven cell line, which can be used for the efficacy evaluation and mechanism study of EGFR-targeted drugs.
[0064] Example 5: Identification of ZST-1 cell line origin and detection of cross-contamination
[0065] To verify the species origin of the ZST-1 cell line and rule out the possibility of human cell contamination or cross-contamination, the cell line was identified by short tandem repeat (STR) typing.
[0066] A suitable amount of ZST-1 cell samples were collected, and genomic DNA was extracted using the Axygen Genomic DNA Extraction Kit. The extracted DNA was used as a template for multiplex PCR amplification using a STR amplification fluorescence detection kit. The PCR amplification products were analyzed using an ABI 3730xl genetic analyzer (Applied Biosystems) to detect STR loci and the sex-identifying gene Amelogenin.
[0067] The test results show that ( Figure 5 This test found no multiple alleles, no cross-contamination, and no human-caused contamination in the ZST-1 cell line.
[0068] The above results further demonstrate that the ZST-1 cell line has a clear origin, is a human EGFR mutation-driven cell line derived from mice, and can be safely used for subsequent in vivo and in vitro experimental studies.
[0069] Example 6: Detection of Luciferase and tdTomato in ZST-1 cell line
[0070] Luciferase in the ZST-1 cell line was detected using a firefly luciferase reporter gene assay kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: RG005). 50,000 cells were seeded in 24-well plates, and after 24 hours, 200 μL of reporter gene cell lysis buffer was added to fully lyse the cells. 100 μL of the lysis supernatant was collected, and 100 μL of firefly luciferase assay kit was added. After gentle mixing, the luminescence intensity was measured using a FlexStation6 microplate reader (Molecular Devices). PC-9 cells without Luciferase were used as a negative control. Results are as follows: Figure 6 As shown, ZST-1 cells exhibit strong Luciferase activity.
[0071] tdTomato fluorescence in ZST-1 cells was detected using the Operetta CLS high-content imaging system (PerkinElmer, USA). The excitation wavelength was 554 nm and the emission wavelength was 581 nm. The results are as follows: Figure 7 As shown, ZST-1 cells emit strong orange fluorescence.
[0072] The above results demonstrate the successful expression of the Luciferase and tdTomato reporter genes, which can be used in multiple fields such as in vivo imaging monitoring, cell tracing, gene function verification, drug screening, and tumor metastasis research.
[0073] Example 7: Tumor formation in C57BL / 6 mice using the ZST-1 cell line
[0074] A mixture of 10 million ZST-1 cell pellets and 200 μL of Ceturegel® Matrix High Concentration, LDEV-Free (Shanghai Yisheng Biotechnology, catalog number 40187ES) was injected into the right upper limb axilla of four-week-old male C57BL / 6 mice (Shanghai Jicui Pharmaceutical, catalog number: N000013). Tumors were dissected four weeks later. Figure 8 As shown, this demonstrates that the ZST-1 cell line can successfully form tumors in immunocompetent C57BL / 6 mice.
[0075] Example 8: Osimertinib treatment for tumorigenesis in C57BL / 6 mice of ZST-1 cell line
[0076] 10 million ZST-1 cell pellets were mixed with 200 μL of Ceturegel® Matrix High Concentration, LDEV-Free (Shanghai Yisheng Biotechnology, catalog number 40187ES) and inoculated into the right upper limb axilla of four-week-old male C57BL / 6 mice. Two weeks later, mice were treated with either a control or 5 mg / kg osimertinib via gavage. The control was administered 2% DMSO + 98% saline. Two weeks after that, the tumors were dissected and stained with hematoxylin and eosin (HE). The staining results are shown below. Figure 9 As shown, the control group formed typical adenocarcinoma structures, while the osimertinib treatment group showed significant necrosis and fibrosis, indicating that the subcutaneous tumors of C57BL / 6 mice formed by the ZST-1 cell line were sensitive to osimertinib.
[0077] Based on the results of the above embodiments, the ZST-1 cell line of the present invention can be used in vitro and in vivo in the following ways: In in vitro experiments, ZST-1 cells can be seeded in 96-well plates, and the drug to be tested can be added. The drug effect can be evaluated by cell viability detection, Luciferase or tdTomato signal detection. In in vivo experiments, ZST-1 cells can be seeded subcutaneously in C57BL / 6 mice. After tumor formation, the mice can be treated with the drug, and tumor growth and treatment response can be monitored by measuring tumor size using an IVIS imaging system or vernier calipers.
[0078] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.
Claims
1. A human EGFR mutation-driven mouse primary lung cancer cell line, characterized in that, The cell line, named mouse lung cancer cell ZST-1, is deposited at the China Center for Type Culture Collection on March 24, 2026, with accession number CCTCC NO: C202654.
2. The human EGFR mutation-driven mouse primary lung cancer cell line according to claim 1, characterized in that, This cell line stably expresses the human EGFR (L858R / T790M) mutant protein and co-expresses the Luciferase and tdTomato reporter genes.
3. The human EGFR mutation-driven mouse primary lung cancer cell line according to claim 1, characterized in that, This cell line is resistant to first- and second-generation EGFR tyrosine kinase inhibitors but sensitive to third-generation EGFR tyrosine kinase inhibitors, indicating that it is dependent on the mutant EGFR signaling pathway.
4. The human EGFR mutation-driven mouse primary lung cancer cell line according to claim 1, characterized in that, This cell line can stably form tumors subcutaneously in immune-intact C57BL / 6 mice.
5. The method for constructing a human EGFR mutation-driven mouse primary lung cancer cell line according to claim 1, characterized in that, Includes the following steps: (1) Obtaining transgenic mice: C57BL / 6 background transgenic mice were used, and the CAG-LSL-hEGFR(L858R / T790M)-IRES-Luc-2A-tdTomato expression construct was knocked into the Rosa26 site of the mice; (2) Tumor induction: Eight-week-old male transgenic mice were selected and AAV6-Scgb1a1-CRE virus particles were administered intranasally after anesthesia. Eight weeks after the virus was administered intranasally, spontaneous lung tumor formation was confirmed by microCT scan. (3) Continuous in vivo passage of tumors: lung tumor nodules were removed, cut into pieces, and inoculated into the left forelimb axilla of nude mice for subcutaneous transplantation. After the subcutaneous tumors grew, the tumor tissue was removed, cut into pieces, and inoculated into the next generation of nude mice. The passage was repeated for 8 generations to obtain tumor tissue with stable subcutaneous tumor formation ability. (4) Cell isolation and in vitro culture: Subcutaneous tumor tissue from the 8th generation nude mice was taken, minced and digested with collagenase to prepare a single-cell suspension, which was then inoculated into a culture dish for in vitro culture. The suspension was continuously cultured and passaged for screening to obtain the cell line ZST-1, which can be passaged for a long time and grow stably.
6. The construction method according to claim 5, characterized in that, In step (1), the transgenic mouse is C57BL / 6Smoc-Gt(ROSA)26Sor, purchased from Shanghai Southern Model Biotechnology Co., Ltd. tm1(CAG -LSL-EGFR(L858R-T790M)-IRES-luciferase-2A-tdTomato)Smoc Mice, catalog number NM-KI-190068.
7. The construction method according to claim 5, characterized in that, In step (2), the viral particle titer is 1×10¹¹ vg.
8. The construction method according to claim 5, characterized in that, In step (4), the culture medium used for in vitro culture is DMEM medium containing 10% fetal bovine serum.
9. Any one or more of the following applications of the human EGFR mutation-driven mouse primary lung cancer cell line of claim 1: ① Application in in vitro screening and efficacy evaluation of human EGFR mutation-targeting drugs, wherein the human EGFR mutation is L858R and / or T790M mutation; ② Application in the study of EGFR-TKI resistance mechanisms; ③ Application in tumor bioluminescence imaging and fluorescence tracing research; ④ Application in in vivo tumorigenesis and pharmacodynamic experiments in an immune-intact C57BL / 6 background; ⑤ Application in the study of EGFR mutation-related signaling pathways and tumor biological mechanisms.