Human lung adenocarcinoma cell line BCH-LUNK1 and application thereof

Abstract

The invention belongs to the technical field of gene engineering and cell engineering, particularly relates to an osimertinib-resistant human lung adenocarcinoma cell line BCH-LUNK1 fused with EGFR (epidermal growth factor receptor) mutation and TPM3-NTRK1 gene, and further discloses a construction method and application of the osimertinib-resistant human lung adenocarcinoma cell line BCH-LUNK1. The human lung adenocarcinoma cell line BCH-LUNK1 disclosed by the invention is a human lung adenocarcinoma cell line with EGFR (Epidermal Growth Factor Receptor) mutation and TPM3-NTRK1 gene fusion, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46021. According to the invention, a stable human lung adenocarcinoma cell line is successfully established, and the cell line has EGFR19 exon mutation and TPM3-NTRK1 gene fusion at the same time, and is a good model for researching EGFR targeted therapy drug resistance mechanism, and screening and drug resistance mechanism research of NTRK targeted inhibitors.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and cell engineering, and particularly relates to a human lung adenocarcinoma cell line BCH-LUNK1, which is a osimertinib-resistant human lung adenocarcinoma cell line with EGFR mutation and TPM3-NTRK1 gene fusion, and further discloses its application. Background Art

[0002] EGFR is the most common driver gene in lung adenocarcinoma, and the drug resistance of its inhibitors has become an important obstacle in the treatment of lung adenocarcinoma today. In addition to serving as a therapeutic target in lung adenocarcinoma, NTRK fusion may also be one of the drug resistance mechanisms of EGFR-targeted drugs. However, there is currently a lack of cell materials required for the study of treatment and drug resistance mechanisms.

[0003] Lung cancer is one of the most common malignant tumors in the world today, and its fatality rate ranks first among all cancers. In particular, non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Research shows that the most common driver gene alteration in NSCLC is epidermal growth factor receptor (EGFR) mutation. With the advent of corresponding target gene tyrosine kinase inhibitors (TKIs), it has brought revolutionary changes to the treatment of advanced NSCLC patients. However, the drug resistance caused by driver gene mutations has brought huge challenges to the treatment of NSCLC patients. In order to extend the lifespan of patients and improve the prognosis, clarifying the drug resistance mechanism has become an urgent problem to be solved, and the molecular biology research of tumor cells is a crucial part of elucidating the drug resistance mechanism.

[0004] Lung cancer cell lines have made great contributions to the translational research of lung cancer and biomedical discoveries. This is because establishing a tumor cell line with a clear genetic background can provide a practical model for the molecular biology research of tumors, the screening of anti-cancer drugs, diagnosis, and the discovery of biomarkers. Biomedical research on tumors largely depends on cell and animal models. Cell lines established from patient tissue or body fluid samples are a valuable cell resource. Currently, there are approximately hundreds of cell lines available for NSCLC research, such as NCI-H1299, NCI-H1975, A549, H460, PC9, etc. These cell lines show different morphological characteristics and biochemical markers, and only represent a part of the biological characteristics of NSCLC. Therefore, new cell line models derived from human primary tumors with a clear background are crucial for NSCLC biological research.

[0005] In addition, TKI resistance is currently a bottleneck in the treatment of NSCLC patients. Elucidating the mechanisms of TKI resistance helps to implement alternative treatment regimens for patients who no longer benefit, including adjusting dosages, developing and using new-generation TKIs, or using other bypass pathway inhibitors. With the rapid progress of genomic analysis techniques, whole-exome sequencing (WES) has identified some major resistance mechanisms, such as the T790M mutation in exon 20 of EGFR, MET amplification, and IGF1R activation. However, EGFR-TKI resistance due to NTRK1 fusion has only recently been reported. Due to the lack of corresponding experimental materials, there is currently a lack of research on NTRK1 fusion as a mechanism of resistance to osimertinib. At the same time, NTRK is a rare driver gene in lung cancer, and its TKI drug, entrectinib, has achieved good therapeutic effects in patients with primary NTRK-fused lung adenocarcinoma. However, the efficacy in patients with secondary mutations remains to be studied.

[0006] In addition, cellular senescence is an important issue that needs to be addressed in establishing a stable cell line. Currently, research on cellular immortalization usually uses telomerase reverse transcriptase (TERT), SV40 T antigen, and HPV E6 / E7 to immortalize cells. These methods can help us obtain unlimited experimental materials, but they always involve artificial components and inevitably introduce some confounding factors into the research. In rare cases, most commonly in cancer cells, cells can spontaneously immortalize. These cells are isolated from tissues or body fluids, cultured and passaged, and then their proliferative ability and characteristics are tested to obtain a cell line that conforms to natural conditions, providing more ideal materials for experimental research.

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

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

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

[0010] To solve the above 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 in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46021 and the deposit date of July 16, 2024.

[0011] The present invention also discloses a sub-cell line, which is obtained by subculturing the human lung adenocarcinoma cell line BCH-LUNK1 with EGFR mutation and TPM3-NTRK1 gene fusion.

[0012] 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 sub-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 sub-cell line in constructing a lung cancer drug-resistant cell model.

[0014] 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 sub-cell line in the field of studying the drug-resistant mechanism of EGFR-targeted therapy.

[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 sub-cell line in constructing a lung cancer animal model.

[0016] 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 sub-cell line in screening drugs for the treatment and / or prevention of lung cancer.

[0017] 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 sub-cell line in the field of preparing drugs for targeted therapy of lung cancer.

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

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

[0020] The present invention successfully established a stable human lung adenocarcinoma cell line, which simultaneously has an exon 19 mutation of EGFR and a TPM3-NTRK1 gene fusion, and is a good model for studying the drug resistance mechanism of EGFR-targeted therapy and the screening and drug resistance mechanism research of NTRK-targeted inhibitors.

[0021] The present invention established a new human lung adenocarcinoma cell line from the pleural effusion of a patient with advanced NSCLC. The patient was resistant to osimertinib treatment and secondary to TPM3-NTRK1 gene fusion, and this cell line was named BCH-LCNK1. The established cell line can provide new experimental materials in vitro and in vivo to study the drug resistance mechanism of NSCLC and screen new therapeutic drugs for NSCLC.

[0022] The cell line research of the present invention was obtained based on lung adenocarcinoma patients. Initial genetic testing found that tumor cells carried an exon 19 mutation of EGFR. After treatment with EGFR-TKI osimertinib, tumor progression occurred with drug resistance. Repeated genetic testing found that the exon 19 mutation of EGFR still existed, and at the same time, another driver gene, TPM3-NTRK1 gene fusion, was detected. The present invention extracted tumor cells from the pleural effusion of this patient and established a new primary lung adenocarcinoma cell line BCH-LCNK1, which carried an exon 19 mutation of EGFR and was secondary to TPM3-NTRK1 gene fusion, and could provide good experimental materials for subsequent research.

[0023] The present invention performed drug sensitivity tests on the newly established BCH-LCNK1 cell line with the third-generation EGFR-TKI osimertinib and NTRK-TKI entrectinib, respectively. The results showed that the IC50 of osimertinib for this cell line was 404.5 nM, which was nearly 10 times that of the other two EGFR-TKI-sensitive cell lines, indicating that the BCH-LCNK1 cell line was resistant to osimertinib; at the same time, the IC50 of entrectinib for this cell line was 53.49 nM, which was close to the IC50 of the NTRK-TKI-sensitive cell line reported in the literature, indicating that this cell line was currently sensitive to NTRK-TKI. This experimental result suggests that this cell line is a natural cell material for studying the drug resistance mechanism of EGFR-TKI, and also suggests that this patient will benefit from NTRK-TKI drugs, providing a basis for the selection of subsequent treatment regimens.

[0024] The cell line of the present invention obtained a primary cell sample with characteristic mutations, did not perform any genetic modification on the cells, survived after 30 - 40 passages, and maintained stable proliferation activity, establishing a lung adenocarcinoma cell line that can be cultured for a long time and used for clinical research. Brief Description of the Drawings

[0025] To make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in combination with the accompanying drawings, wherein,

[0026] Figure 1 It is the detection result of cell proliferation characteristics in Example 3; wherein, A: showing the morphology of primary cells; B: showing that all cells are in epithelial-like morphology after more than 20 passages; C: the cell morphology remains unchanged after cryopreservation and resuscitation in liquid nitrogen; D: cell growth curve;

[0027] Figure 2 It is the drug sensitivity test results of osimertinib and entrectinib in Example 4; wherein, A: the drug sensitivity test result of osimertinib shows that the IC50 of the BCH-LCNK1 cell line is significantly higher than that of the other two EGFR-TKI sensitive cell lines; B: the drug sensitivity test of entrectinib shows that the IC50 of BCH-LCNK1 is about 53.49 nM;

[0028] Figure 3 It is the tumorigenesis experiment results of SCID mice in Example 5; wherein, A-B: showing that the tumor formation rate is 100% after inoculation into SCID mice; C: under low magnification, the tumor cells grow solidly (H&E); D: under high magnification, the tumor tissue mainly shows cribriform and micropapillary structures; E: intravascular tumor thrombus can be seen around the tumor;

[0029] Figure 4 It is the pathological feature results of xenograft tumors in Example 6; wherein, A1-A2: CK7; B1-B2: TTF-1; C1-C2: Napsin A; D1-D2: pan-TRK; E1-E2: EGFR;

[0030] Figure 5 It is the gene mutation detection result in Example 7; wherein, A: gene detection shows that the main base changes in this cell line are C>T and T>C; B: showing that there are a large number of unknown base change types in this cell line, and SBS5 is the main type in the known classification;

[0031] Figure 6 It is the gene mutation detection result in Example 8; wherein, A: showing the EGFR exon 19 mutation curve; B: showing the NTRK fusion curve;

[0032] Figure 7 It is the cell line quality control result in Example 9. Detailed implementation manners

[0033] Example 1 Sample collection

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

[0035] After resistance occurred after osimertinib treatment, circulating tumor cell gene detection was performed, and it was detected that the patient had both an exon 19 mutation of EGFR and a TPM3-NTRK1 gene fusion. Primary lung adenocarcinoma cells were obtained from the patient's pleural effusion for culture. This study has been approved by the Ethics Committee of Peking University Cancer Hospital, and the patient's informed consent has been obtained.

[0036] Example 2 Cell culture

[0037] In this example, the pleural effusion cells of the sample in Example 1 were separated into a suspension for primary culture, and fibroblasts were removed with G418.

[0038] Collect the patient's pleural effusion sample into a 50 ml centrifuge tube, transfer it to the laboratory within 30 minutes, centrifuge at 1000 - 1500 rmp for 4 - 5 minutes, and discard the supernatant; add 6 - 10 times the volume of red blood cell lysate based on the cell pellet volume to remove red blood cells, gently mix, let it stand at room temperature, centrifuge at 1000 - 1500 rmp for 4 - 5 minutes, and discard the supernatant; add 10 - 15 ml of sterile phosphate-buffered saline (PBS) containing 2X penicillin-streptomycin (PS, Gibco, 15140122) and wash 1 - 2 times, centrifuge at 1000 - 1500 rmp for 4 - 5 minutes, and discard the supernatant; resuspend the pellet with 1 ml of DMEM / F12 medium (Gibco) containing 10% fetal bovine serum (FBS) + 1X insulin-transferrin-selenium (ITS) + 1X PS, and inoculate the cells into a 25 cm 2 culture flask, supplement the medium to 7 ml per flask, and place it in an incubator at 37°C and 5% CO2 for culture. Change the medium the next day, wash with PBS buffer to remove non-adherent cells. Purification was performed using a method combining 0.05% trypsin-EDTA digestive solution and differential adherence. When the primary cultured cells reached sufficient growth, they were passaged at a ratio of 1:3 - 1:6 every 5 - 7 days, and samples were taken regularly and stored in liquid nitrogen.

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

[0040] The cell line was designated as BCH-LUNK1, and has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46021, the deposit date of July 16, 2024, and the deposit address being 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 exponentially growing cells, and inoculate them into 8 parallel wells on each plate of a 96-well plate, and culture them with growth medium.

[0043] From the next day, according to the instructions of Cell Counting Kit-8 (CCK-8, Dojindo, CK04), detect cell proliferation every two days for 5 consecutive times to obtain the cell growth curve. The doubling time is calculated using the online algorithm software provided at http: / / www.doubling-time.com / compute.php. The calculation formula is as follows:

[0044]

[0045] As shown in the Figure 1 results, under an inverted microscope, some cells in the primary culture showed an epithelial-like morphology, and some cells had an atypical morphology ( Figure 1 A in Figure 1 ), after more than 20 passages, the cells firmly adhered to the culture flask and grew in islands, and all cells showed an epithelial cell-like morphology ( Figure 1 B in Figure 1 ). It can be seen that the entire cell line grew relatively slowly in vitro, and the cell doubling time was 5.6 - 7.4 days (

[0046] D in

[0047] ). After recovery from liquid nitrogen, the cryopreserved cells could proliferate in culture, and there were no obvious changes in growth and morphology (

[0048] C in

[0049] ). It can be seen that the cell line established in the present invention did not show signs of senescence and was therefore considered immortal. And in this example, no signs of fungal or bacterial contamination were observed, and the culture method showed that there was no mycoplasma in the cell line.

[0050] Cells were seeded in 96-well plates at a density of 2000 - 6000 cells / well and continuously exposed to each drug for 96 hours. Data analysis was performed using the non-linear regression method 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 drug sensitivity test.

[0051] As shown in the Figure 2 results, 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 in A); while the IC50 of entrectinib was approximately 53.49 nM ( Figure 2 in B), which was close to the IC50 value of the NTRK-TKI sensitive cell line reported in the literature.

[0052] Example 5 SCID mouse tumorigenesis experiment

[0053] In this example, the tumorigenicity of the cell line was evaluated by the ability to form tumors at the subcutaneous injection site in SCID mice. Cells in the exponential growth phase were collected, suspended in PBS, and a cell suspension of 6×10 7 cells / ml was prepared. 0.1 ml of the cell suspension was subcutaneously injected into the right axilla of five 6-week-old female SCID mice (Beijing Hanfukang Biotechnology Co., Ltd.), and the mice were examined and measured once a week during tumor formation. When the tumor diameter approached 1.5 cm, the mice were sacrificed. The tumorigenesis experiment was repeated using five-week-old male SCID mice.

[0054] As shown in the Figure 3 results, two months after inoculating the cells in female SCID mice, a tumor mass could be palpated subcutaneously at the inoculation site. One month later, the mass diameter approached 1.5 cm, and the tumor formation rate was 100% ( Figure 3 in A, B). The same results were repeated in male SCID mice. H&E staining showed that the tumor was a poorly differentiated adenocarcinoma, growing in a solid, cribriform, and micropapillary pattern, and mucus secretion was visible in some cells ( Figure 3 in C, D), and intravascular tumor thrombi were seen around the tumor ( Figure 3 in E).

[0055] Example 6 Pathological characteristics of xenograft tumors

[0056] In this example, the tumor tissues formed after transplantation were fixed with 4% formaldehyde, embedded in paraffin, and subjected to H&E staining. The tissue sections were stained with characteristic markers of lung adenocarcinoma: CK7, TTF-1, Napsin A, neuroendocrine markers: Syn, CgA, CD56, and gene abnormally expressed proteins: EGFR, pan-TRK (EPR17341, Roche), ALK, ROS1, HER2, and c-MET were performed according to the instructions of the automatic immunohistochemistry instrument manufacturers of Dako (Link 48, USA) and Leica (BOND-III, Germany).

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

[0058] Example 7 Gene Mutation Detection

[0059] In this example, gene alterations in the cell line were detected based on NGS.

[0060] The genomic DNA samples of the cell line were screened for 618 specific gene mutations on the Illumina sequencing platform (AccBio, Beijing, China) by NGS technology. Among the 618 genes, 232 genes were related to molecular targeted therapy, such as EGFR, KRAS, NRAS, ALK, ROS1, and MET, etc.; 439 genes were related to signal pathways and immunotherapy, 53 genes were related to chemotherapy; in addition, 123 cancer-related genes were also detected.

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

[0062] Example 8 Gene Mutation Detection

[0063] In this example, the RT-PCR method was used to detect the exon 19 mutation of EGFR and the TPM3-NTRK1 gene fusion in mouse xenograft tumor tissues.

[0064] A human lung cancer 11 mutation gene detection kit (ADx, China) was used to detect the alteration status of EGFR and NTRK genes in mouse tumor-bearing tissues, and the operating procedures were carried out according to the instructions of the commercial kit.

[0065] As Figure 6 shown in the results, the RT-PCR results showed that the exon 19 mutation of EGFR and the TPM3-NTRK1 gene fusion still existed in the tumor tissues formed by xenotransplantation.

[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 type identification, STR analysis, and mycoplasma detection to ensure that there was no contamination by other cells and foreign microorganisms.

[0068] Mycoplasma detection

[0069] Referring to the Chinese Pharmacopoeia, we used arginine broth culture and semi-fluid culture methods to detect mycoplasma in the P20 cell culture supernatant within 2 hours. At the same time, all samples were detected by PCR.

[0070] In this example, PCR detection showed that there was 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 identification markers were amplified and analyzed by capillary electrophoresis. STR data were analyzed using the online STR databases of the German Collection of Microorganisms and Cell Cultures (DSMZ) and the Biomedical Cell Resource Center (BMCR) (http: / / www.dsmz.de / fp / cgi-bin / str.html, http: / / cellresource.cn / str / default.aspx).

[0073] As Figure 7The results shown are that DNA samples of different passages (P0, P21, P40) were analyzed by STR genotyping, and a total of 18 STR loci and the Amelogenin sex identification locus were amplified in each sample. After data analysis, the alleles of each locus were determined.

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

[0075] Table 1 STR profiles of BCH-LCNK1 cells at passages 0, 21, and 40

[0076]

[0077]

[0078] In summary, the newly cultured cell line of the present invention is an epithelial cell, with more than 40 passages, a population doubling time in vitro of 5.6 - 7.4 days, and the culture is mycoplasma negative. Identity verification shows that these cells have the same STR profile as the patient's tissue, and no interspecies and intraspecies cross-contamination is detected. Cytological IHC shows that this cell line expresses TTF-1 and Napsin A, is a lung adenocarcinoma cell, and both EGFR and pan-TRK show strong diffuse cytoplasmic staining. NGS results show that there is a mutation in exon 19 of EGRF and a TPM3-NTRK1 gene fusion. The half-maximal inhibitory concentration (IC50) of osimertinib for this cell line is 404.5 nM, which is significantly higher than the other two EGFR-mutated human lung adenocarcinoma cell lines, and the IC50 of entrectinib is 53.49 nM. The tumor formation rate in SCID mice is 100%. Histopathologically, the xenograft tumor is a poorly differentiated lung adenocarcinoma, showing solid, cribriform, and micropapillary structures. IHC results show that TTF-1 and Napsin A are still expressed after tumor formation, and both EGFR and pan-TRK show strong diffuse cytoplasmic staining. RT-PCR results show that exon 19 of EGFR is mutated and the TPM3-NTRK1 gene is fused in the tumor-forming tissue.

[0079] In summary, the present invention successfully established a stable lung adenocarcinoma cell line that simultaneously has a mutation in exon 19 of EGFR and a TPM3-NTRK1 gene fusion, and is a good model for studying the resistance mechanism of EGFR-targeted therapy and the screening and resistance mechanism research of NTRK-targeted inhibitors.

[0080] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to exhaustively list all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present 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, and has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.46021.

2. A progeny cell line, characterized in that The progeny cell line is obtained by subculturing the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1.

3. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 as an experimental tool in studying cancer.

4. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 in constructing a drug-resistant cell model of lung cancer.

5. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 in the field of studying the mechanism of drug resistance in EGFR targeted therapy.

6. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 in constructing an animal model of lung cancer.

7. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 in screening drugs for treating and / or preventing lung cancer.

8. Use of the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2 in the field of preparing drugs for targeted treatment of lung cancer.

9. The use according to any one of claims 3 to 8, characterized in that: The lung cancer includes lung adenocarcinoma.

10. A model of EGFR targeted therapy resistance mechanism, characterized in that: It comprises the human lung adenocarcinoma cell line BCH-LUNK1 according to claim 1 or the progeny cell line according to claim 2.

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