Human lung adenocarcinoma cell line BCH-LUNK1 and application thereof

Abstract

The application belongs to the technical field of genetic engineering and cell engineering, and particularly relates to a human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion resistant to osimertinib, and further discloses a construction method and application thereof. The human lung adenocarcinoma cell line BCH-LUNK1 provided by the application is a human lung adenocarcinoma cell line with EGFR mutation and TPM3-NTRK1 gene fusion, has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.46021. The application successfully establishes a stable human lung adenocarcinoma cell line, the cell line has EGFR exon 19 mutation and TPM3-NTRK1 gene fusion at the same time, is a good model for researching an EGFR targeted therapy drug resistance mechanism, and screening and a drug resistance mechanism research of an NTRK targeted inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and cell engineering technology, specifically relating to a human lung adenocarcinoma cell line BCH-LUNK1, which is an osimertinib-resistant human lung adenocarcinoma cell line with EGFR mutation and TPM3-NTRK1 gene fusion, and further discloses its application. Background Technology

[0002] EGFR is the most common driver gene in lung adenocarcinoma, and resistance to its inhibitors has become a significant obstacle to the treatment of lung adenocarcinoma. NTRK fusions in lung adenocarcinoma, besides serving as a therapeutic target, may also be one of the mechanisms of resistance to EGFR-targeted drugs. However, the cellular materials needed for research on treatment and resistance mechanisms are currently lacking.

[0003] Lung cancer is one of the most common malignant tumors in the world today, with the highest mortality rate among all cancers, especially non-small cell lung cancer (NSCLC), which accounts for 80-85% of all lung cancers. Studies show that the most common driver gene alteration in NSCLC is the epidermal growth factor receptor (EGFR) mutation. The advent of tyrosine kinase inhibitors (TKIs) targeting this gene has revolutionized the treatment of advanced NSCLC patients. However, drug resistance caused by driver gene mutations poses a significant challenge to the treatment of NSCLC patients. To prolong patient life and improve prognosis, clarifying the drug resistance mechanism has become an urgent problem, and molecular biological research on tumor cells is a crucial step in elucidating this mechanism.

[0004] Lung cancer cell lines have made significant contributions to translational research and biomedical discoveries in lung cancer. This is because establishing a tumor cell line with a clear genetic background provides a practical model for molecular biology research, anticancer drug screening, diagnosis, and biomarker discovery. Biomedical research on tumors relies heavily on cell and animal models. Cell lines established from patient tissue or body fluid samples are a valuable cellular resource. Currently, there are approximately several hundred cell lines available for NSCLC research, such as NCI-H1299, NCI-H1975, A549, H460, and PC9. These cell lines exhibit diverse morphological characteristics and biochemical markers, representing only a portion of the biological characteristics of NSCLC. Therefore, novel cell line models derived from human primary tumors with a clear genetic background are crucial for NSCLC biological research.

[0005] Furthermore, TKI resistance remains a bottleneck in the treatment of NSCLC patients. Elucidating the mechanisms of TKI resistance is crucial for implementing alternative treatment plans for patients who no longer benefit, including dose adjustments, the development and use of next-generation TKIs, or the use of other bypass pathway inhibitors. With the rapid advancement of genomic analysis technologies, whole-exome sequencing (WES) has identified several major resistance mechanisms, such as EGFR exon 20 T790M mutations, MET duplications, and IGF1R activation. However, EGFR-TKI resistance based on NTRK1 fusions has only recently been reported. Due to a lack of relevant experimental data, research on the mechanisms by which NTRK1 fusions lead to resistance to osimertinib is currently lacking. Meanwhile, NTRK is a rare driver gene in lung cancer, and the TKI entrectinib has shown promising therapeutic effects in patients with primary NTRK fusion-related lung adenocarcinoma; however, its efficacy in patients with secondary mutations remains to be studied.

[0006] Furthermore, cellular senescence is a crucial issue that needs to be addressed to establish a stable cell line. Currently, research on cell immortalization typically uses telomerase reverse transcriptase (TERT), SV40 T antigen, and HPV E6 / E7 to immunize cells, achieving immortality. While these methods provide access to a vast amount of experimental material, they always contain artificial components, inevitably introducing some confounding factors into the research. In rare cases, most commonly with cancer cells, spontaneous immortalization occurs. Isolating these cells from tissues or body fluids, culturing and passaged them, and then testing their proliferative capacity and characteristics yields cell lines that conform to natural conditions, providing more ideal materials for experimental research.

[0007] Therefore, establishing new lung adenocarcinoma cell lines with relevant backgrounds will be a useful tool for exploring the molecular mechanisms of targeted therapy for lung adenocarcinoma and for screening treatment drugs after drug resistance. Summary of the Invention

[0008] Therefore, the technical problem to be solved by the present invention is to provide a human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion that is resistant to osimertinib;

[0009] The second technical problem to be solved by the present invention is to provide the application of the above-mentioned human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion resistant to osimertinib.

[0010] To solve the above-mentioned technical problems, the present invention provides a human lung adenocarcinoma cell line BCH-LUNK1, which is a human lung adenocarcinoma cell line with EGFR mutation and TPM3-NTRK1 gene fusion. It has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46021 and deposit date of July 16, 2024.

[0011] The present invention also discloses a progeny cell line obtained by passage of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion.

[0012] This invention also discloses the application of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion, or its progeny cell line, as an experimental tool in cancer research.

[0013] The present invention also discloses the application of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the daughter cell line in constructing a lung cancer drug-resistant cell model.

[0014] This invention also discloses the application of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the progeny cell line in the study of EGFR-targeted therapy resistance mechanisms.

[0015] The present invention also discloses the application of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the daughter cell line in the construction of lung cancer animal models.

[0016] The present invention also discloses the use of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the progeny cell line thereon in screening drugs for the treatment and / or prevention of lung cancer.

[0017] This invention also discloses the application of the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the progeny cell line thereon in the preparation of targeted therapy drugs for lung cancer.

[0018] Specifically, the lung cancer includes lung adenocarcinoma.

[0019] The present invention also discloses a model of EGFR-targeted therapy resistance mechanism, including the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion or the progeny cell line thereof.

[0020] This invention successfully established a stable human lung adenocarcinoma cell line that simultaneously possesses an EGFR exon 19 mutation and a TPM3-NTRK1 gene fusion. This cell line is a good model for studying EGFR-targeted therapy resistance mechanisms, as well as for screening NTRK-targeting inhibitors and studying resistance mechanisms.

[0021] This invention establishes a novel human lung adenocarcinoma cell line from the pleural effusion of a patient with advanced NSCLC who had developed resistance to osimertinib treatment and subsequently developed a TPM3-NTRK1 gene fusion. This cell line is named BCH-LCNK1. The established cell line can provide novel in vitro and in vivo experimental materials for studying the drug resistance mechanisms of NSCLC and screening for new therapeutic agents targeting NSCLC.

[0022] The cell line described in this invention was obtained from a lung adenocarcinoma patient. Initial gene testing revealed that the tumor cells carried an EGFR exon 19 mutation. After treatment with the EGFR-TKI osimertinib, the tumor progressed and developed resistance. Re-genetic testing confirmed the continued presence of the EGFR exon 19 mutation, along with a fusion of another driver gene, TPM3-NTRK1. This invention extracted tumor cells from the pleural effusion of this patient and established a novel primary lung adenocarcinoma cell line, BCH-LCNK1. This cell line carries the EGFR exon 19 mutation and secondary TPM3-NTRK1 gene fusion, providing excellent experimental material for subsequent research.

[0023] This invention utilizes the newly established BCH-LCNK1 cell line to conduct drug susceptibility tests on the third-generation EGFR-TKI osimertinib and the NTRK-TKI entrectinib. The results showed that the IC50 of osimertinib against this cell line was 404.5 nM, nearly 10 times that of two other EGFR-TKI-sensitive cell lines, indicating that the BCH-LCNK1 cell line is resistant to osimertinib. Simultaneously, the IC50 of entrectinib against this cell line was 53.49 nM, close to the IC50 reported for NTRK-TKI-sensitive cell lines in the literature, indicating that this cell line is currently sensitive to NTRK-TKIs. These experimental results suggest that this cell line is a natural cell material for studying EGFR-TKI resistance mechanisms, and also suggest that this patient may benefit from NTRK-TKI drugs, providing a basis for the selection of subsequent treatment regimens.

[0024] The cell line of this invention obtained primary cell samples with characteristic mutations. The cells were not genetically modified and survived after 30-40 passages, maintaining stable proliferative activity. This established a lung adenocarcinoma cell line that can be cultured for a long time and used for clinical research. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0026] Figure 1 The results of cell proliferation characteristic detection in Example 3 are shown below; where A: shows the morphology of primary cells; B: shows that all cells exhibit an epithelial-like morphology after more than 20 passages; C: shows that the cell morphology did not change after thawing from liquid nitrogen cryopreservation; D: cell growth curve.

[0027] Figure 2 The results of the drug sensitivity tests for osimertinib and entrectinib in Example 4 are as follows: A: The drug sensitivity test results for osimertinib showed that the IC50 of the BCH-LCNK1 cell line was significantly higher than that of the other two EGFR-TKI sensitive cell lines; B: The drug sensitivity test results for entrectinib showed that the IC50 of BCH-LCNK1 was 53.49 nM.

[0028] Figure 3 The results of the tumorigenesis experiment in SCID mice in Example 5 are shown. AB: Tumor formation rate was 100% after inoculation into SCID mice; C: Tumor cells showed solid growth under low magnification (H&E); D: Tumor tissue mainly showed cribriform and micropapillary structures under high magnification; E: Intravascular tumor emboli were observed around the tumor.

[0029] Figure 4 The results show the pathological characteristics of xenograft tumors in Example 6; where A1-A2: CK7; B1-B2: TTF-1; C1-C2: Napsin A; D1-D2: pan-TRK; E1-E2: EGFR;

[0030] Figure 5 The results of gene mutation detection in Example 7 are shown; where A: gene detection shows that the main base changes in this cell line are C>T and T>C; B: it shows that there are a large number of unknown base change types in this cell line, with SBS5 being the main one in the known classification.

[0031] Figure 6 The results of gene mutation detection in Example 8 are shown; where A: shows the EGFR exon 19 mutation curve; B: shows the NTRK fusion curve.

[0032] Figure 7 The results are the quality control results of the cell line in Example 9. Detailed Implementation

[0033] Example 1 Sample Collection

[0034] In this embodiment, the clinical specimen came from a Chinese female patient diagnosed with stage IVB lung adenocarcinoma, with significant pleural effusion and multiple lymph node metastases throughout the body. The pleural effusion cytology smear was diagnosed as lung adenocarcinoma by two pathologists. An EGFR exon 19 mutation was detected in the tumor tissue.

[0035] The patient developed resistance to osimertinib. Circulating blood tumor cell gene testing revealed a co-existing EGFR exon 19 mutation and a TPM3-NTRK1 gene fusion. Primary lung adenocarcinoma cells were obtained from the patient's pleural effusion for culture. This study was approved by the Ethics Committee of Peking University Cancer Hospital, and informed consent was obtained from the patient.

[0036] Example 2 Cell Culture

[0037] In this embodiment, the pleural fluid cells from the sample in Example 1 were separated into a suspension, subjected to primary culture, and fibroblasts were removed using G418.

[0038] Collect pleural effusion samples from patients into 50ml centrifuge tubes, transfer to the laboratory within 30 minutes, centrifuge at 1000-1500 rpm for 4-5 minutes, and discard the supernatant. Add 6-10 times the volume of erythrocyte lysis buffer to remove erythrocytes, mix gently, incubate at room temperature, centrifuge at 1000-1500 rpm for 4-5 minutes, and discard the supernatant. Wash 1-2 times with 10-15ml of sterile phosphate-buffered saline (PBS) containing 2X penicillin-streptomycin (PS, Gibco, 15140122), centrifuge at 1000-1500 rpm for 4-5 minutes, and discard the supernatant. Resuspend the pellet in 1ml of DMEM / F12 medium (Gibco) containing 10% fetal bovine serum (FBS) + 1X insulin-transferrin-selenium (ITS) + 1X PS, and seed cells in 25cm... 2 Each culture flask was replenished with 7 ml of culture medium and placed in a 37°C, 5% CO2 incubator. The medium was changed the next day, and the cells were washed with PBS buffer to remove non-adherent cells. Purification was performed using a combination of 0.05% trypsin-EDTA digestion and differential adhesion. When the primary cultured cells reached sufficient growth, they were passaged every 5-7 days at a ratio of 1:3 to 1:6, and samples were taken periodically and stored in liquid nitrogen.

[0039] After 40 generations of passage, it was considered a continuous cell line. The characteristics of the cell line were then tracked to study its biological characteristics.

[0040] The cell line, designated BCH-LUNK1, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46021, deposited on July 16, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] Example 3: Detection of Cell Proliferation Characteristics

[0042] Take 100 μl of suspension containing 5 × 10 4 / ml or 1×10 5 / ml of exponentially growing cells were seeded in eight parallel wells on each of the 96-well plates and cultured in growth medium.

[0043] Starting from the second day, cell proliferation was monitored every two days according to the instructions of the Cell Counting Kit-8 (CCK-8, Dojindo, CK04), for a total of 5 times, to obtain cell growth curves. Doubling time was calculated using online algorithm software provided at http: / / www.doubling-time.com / compute.php. The calculation formula is as follows:

[0044]

[0045] As attached Figure 1 The results show that, under an inverted microscope, some cells in the primary culture exhibited an epithelial-like morphology, while others showed atypical morphology. Figure 1 After more than 20 passages, the cells (A) firmly adhered to the culture flask, growing in an island-like pattern, and all cells exhibited an epithelial cell-like morphology. Figure 1 (B) It can be seen that the entire cell line grows relatively slowly in vitro, with a cell doubling time of 5.6-7.4 days ( ). Figure 1 (D). After recovery from liquid nitrogen, cryopreserved cells can proliferate in culture, with no significant changes in growth and morphology. Figure 1 (C)

[0046] As can be seen, the cell line established in this invention does not show any signs of aging and is therefore considered immortal. Furthermore, in this embodiment, no signs of fungal or bacterial contamination were observed, and the culture method indicates that mycoplasma is absent from the cell line.

[0047] Example 4: Drug Sensitivity Test for Osimertinib and Entrectinib

[0048] In this embodiment, osimertinib was purchased from Selleck Chemicals (USA), and entrectinib was purchased from Roche Pharmaceuticals (USA).

[0049] All compounds were dissolved in dimethyl sulfoxide for in vitro experiments, and the inhibitory effect of the drugs on cell growth was determined using the CCK8 assay.

[0050] Cells were seeded at a density of 2000-6000 cells / well in 96-well plates and continuously exposed to each drug for 96 hours. Data analysis was performed using nonlinear regression in GraphPad Prism 7 (GraphPad Software, USA). In this example, two primary cultured EGFR-TKI sensitive cell lines (221102-LXL and 220929-WQM) were selected as control cell lines for the osimertinib susceptibility test.

[0051] As attached Figure 2 The results showed that BCH-LCNK1 cells were resistant to osimertinib, with an IC50 of 404.5 nM, significantly higher than the other two EGFR-TKI-sensitive cell lines. Figure 2 (A); while entrectinib's IC50 is approximately 53.49 nM ( Figure 2 The IC50 value of the NTRK-TKI sensitive cell line (B) is close to that reported in the literature.

[0052] Example 5: Tumor formation experiment in SCID mice

[0053] In this embodiment, the tumorigenicity of the cell line was assessed by its ability to form tumors at the subcutaneous injection site in SCID mice. Cells in the exponential growth phase were collected, suspended in PBS, and 6 × 10⁶ cells were prepared. 7 Cell suspension at 1 cell / ml. 0.1 ml of cell suspension was subcutaneously injected into the right axilla of five 6-week-old female SCID mice (Beijing Hanfukang Biotechnology Co., Ltd.). The cells were examined and measured weekly during tumor formation. Mice were sacrificed when the tumor diameter approached 1.5 cm. This tumorigenesis experiment was repeated using 5-week-old male SCID mice.

[0054] As attached Figure 3 The results showed that two months after cell inoculation in female SCID mice, a tumor mass was palpable subcutaneously at the inoculation site. One month later, the tumor diameter was nearly 1.5 cm, and the tumor formation rate was 100%. Figure 3 (A, B). The same results were replicated in male SCID mice. H&E staining showed that the tumor was poorly differentiated adenocarcinoma, with solid, cribriform, and micropapillary growth; some cells showed mucus secretion. Figure 3 (C, D) Intravascular tumor emboli were observed around the tumor. Figure 3 (E).

[0055] Example 6: Pathological features of xenograft tumors

[0056] In this embodiment, the transplanted tumor tissue was fixed with 4% formaldehyde, embedded in paraffin, and stained with H&E. Tissue sections were stained using characteristic markers of lung adenocarcinoma: CK7, TTF-1, Napsin A; neuroendocrine markers: Syn, CgA, CD56; and abnormally expressed proteins: EGFR, pan-TRK (EPR17341, Roche), ALK, ROS1, HER2, and c-MET, according to the instructions of the Dako (Link 48, USA) and Leica (BOND-III, Germany) automated immunohistochemistry analyzers.

[0057] like Figure 4 The immunohistochemical results showed that the tumor cells expressed lung adenocarcinoma markers CK7, TTF-1, and Napsin A (which were respectively...). Figure 4 In the A1-A2, B1-B2, and C1-C2 groups, pan-TRK immunohistochemistry showed diffuse cytoplasmic 3+ positivity. Figure 4 D1-D2), high expression of EGFR ( Figure 4 (E1-E2). It did not express the neuroendocrine markers Syn, CgA, and CD56; treatment-related markers such as ALK, ROS1, and c-MET were all negative; HER2 was 1+ (not shown).

[0058] Example 7 Gene Mutation Detection

[0059] In this embodiment, NGS is used to detect gene alterations in cell lines.

[0060] Genomic DNA samples from cell lines were screened for 618 specific gene mutations using NGS sequencing on the Illumina sequencing platform (AccBio, Beijing, China). Of these 618 genes, 232 were associated with molecularly targeted therapies, such as EGFR, KRAS, NRAS, ALK, ROS1, and MET; 439 were associated with signaling pathways and immunotherapy; 53 were associated with chemotherapy; and 123 cancer-related genes were also detected.

[0061] like Figure 5 As shown in the results, a total of 291 gene variants were detected in this embodiment. The single nucleotide variants were mainly SBS5, and the main driver gene change was the EGFR exon 19 mutation (exon19:c.2237_2255>T:p.E746_S752>V). At the same time, the TPM3-NTRK1 fusion (TPM3_exon 8:NTRK1_exon 12) was also detected.

[0062] Example 8 Gene Mutation Detection

[0063] In this embodiment, EGFR exon 19 mutation and TPM3-NTRK1 gene fusion in mouse xenograft tumor tissue were detected by RT-PCR.

[0064] The altered status of EGFR and NTRK genes in mouse tumor tissues was detected using a human lung cancer 11 mutation gene detection kit (Aid, China). The procedure was performed according to the instructions of the commercial kit.

[0065] like Figure 6 The results showed that the RT-PCR results indicated that the xenografted tumor tissue still contained EGFR exon 19 mutations and TPM3-NTRK1 gene fusions.

[0066] Example 9: Cell Line Quality Control

[0067] Quality control is crucial for cell culture. During the establishment of the BCH-LCNK1 cell line, samples were repeatedly collected for cell species identification, STR analysis, and mycoplasma detection to ensure that there was no contamination from other cells or foreign microorganisms.

[0068] Mycoplasma testing

[0069] Referring to the Chinese Pharmacopoeia, we used arginine broth culture and semi-fluid culture methods to detect mycoplasma in the supernatant of P20 cell culture within 2 hours. Simultaneously, all samples were tested using PCR.

[0070] In this embodiment, PCR testing showed that there were no mycoplasma in the cell line.

[0071] STR genotype analysis

[0072] Eighteen STR loci (D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, vWA, D8S1179, D16S539, TPOX, TH01, D19S433, D18S51, FGA, D13S317, Penta E, D6S1043, and D12S391) and amelogenin sex markers were amplified and analyzed by capillary electrophoresis. STR data were analyzed using the online STR databases of the German Center for Microbiology and Cell Culture Collection (DSMZ) and the Biomedical Cell Resource Center (BMCR) (http: / / www.dsmz.de / fp / cgi-bin / str.html, http: / / cellresource.cn / str / default.aspx).

[0073] like Figure 7The results show that DNA samples from different generations (P0, P21, P40) underwent STR typing analysis, and a total of 18 STR loci and Amelogenin sex determination loci were amplified in each sample. Data analysis determined the alleles for each locus.

[0074] As shown in Table 1 below, the STR atlas of BCH-LCNK1 cells is identical to that of the original tumor compared to the data published on the DSMZ and BMCR STR databases, indicating that the BCH-LCNK1 cell line is authentic and reliable.

[0075] Table 1. STR maps of BCH-LCNK1 cells from generations 0, 21, and 40.

[0076]

[0077]

[0078] In summary, the newly cultured cell line of this invention is an epithelial cell line, passaged more than 40 times, with a population doubling time of 5.6-7.4 days in vitro, and the culture is mycoplasma negative. Identification showed that these cells have the same STR profile as patient tissues, and no interspecies or intraspecies cross-contamination was detected. IHC cytology showed that this cell line expresses TTF-1 and NapsinA, indicating lung adenocarcinoma cells, with diffuse strong cytoplasmic staining for EGFR and pan-TRK. NGS results showed a mutation in exon 19 of EGFR and a TPM3-NTRK1 gene fusion. The half-maximal inhibitory concentration (IC50) of osimertinib in this cell line was 404.5 nM, significantly higher than the other two EGFR-mutant human lung adenocarcinoma cell lines, where entrectinib's IC50 was 53.49 nM. The tumor formation rate in SCID mice was 100%. Histopathologically, the xenograft tumors were poorly differentiated lung adenocarcinomas, showing solid, cribriform, and micropapillary structures. IHC results showed that TTF-1 and Napsin A were still expressed after tumor formation. EGFR and pan-TRK showed diffuse strong cytoplasmic staining. RT-PCR analysis showed EGFR exon 19 mutation and TPM3-NTRK1 gene fusion in the tumor tissue.

[0079] In summary, this invention has successfully established a stable lung adenocarcinoma cell line that possesses both EGFR exon 19 mutation and TPM3-NTRK1 gene fusion. This cell line is a good model for studying EGFR-targeted therapy resistance mechanisms, as well as for screening NTRK-targeting inhibitors and studying resistance mechanisms.

[0080] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A human lung adenocarcinoma cell line BCH-LUNK1, characterized in that, The human lung adenocarcinoma cell line BCH-LUNK1 is a human lung adenocarcinoma cell line with EGFR mutation and TPM3-NTRK1 gene fusion. It has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46021.

2. The application of the human lung adenocarcinoma cell line BCH-LUNK1 as an experimental tool in the study of lung adenocarcinoma according to claim 1.

3. The application of the human lung adenocarcinoma cell line BCH-LUNK1 as described in claim 1 in constructing a drug-resistant lung adenocarcinoma cell model.

4. The application of the human lung adenocarcinoma cell line BCH-LUNK1 as described in claim 1 in the construction of an animal model of lung adenocarcinoma.

5. The use of the human lung adenocarcinoma cell line BCH-LUNK1 as described in claim 1 in screening drugs for the treatment and / or prevention of lung adenocarcinoma.

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