Human lung adenocarcinoma EGFR-TKI-tolerant cell strain with high invasiveness and application of human lung adenocarcinoma EGFR-TKI-tolerant cell strain

Abstract

The invention belongs to the technical field of biological medicine. In-vivo and in-vitro experiments prove that ANXA6 + CAFs has the capability of promoting malignant biological behaviors of lung cancer cells, and can promote resistance of EGFR mutant lung adenocarcinoma cells to third-generation EGFR-TKI targeted drugs. ANXA6 + CAFs and HCC827 cells are jointly inoculated to the armpit of a nude mouse, a transplanted tumor is cultured to obtain HCC827-3 cells which are high in invasiveness and resistant to a third-generation EGFR-TKI targeted drug, and the lung cancer cell strain HCC827-D is established through three times of lung metastasis model screening and monoclonal screening methods. The IC50 (half maximal inhibitory concentration) of the HCC827-D cell within 48 hours is 870.81 times that of the HCC827 cell; the IC50 (half maximal inhibitory concentration) within 72 hours is 1411.54 times that of an HCC827 cell; the HCC827-D cell is prompted to generate obvious drug resistance to the third generation EGFR-TKI, and the drug resistance effect is more obvious along with the prolonging of time. The HCC827-D cells are inoculated into caudal veins of a mouse, and after the mouse grows for 1.5 months, the HCC827-D cells have obvious lung metastasis, brain metastasis and bone metastasis in the mouse body. The invention can provide a good cell model for studying lung cancer metastasis and EGFR-TKI drug resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line and application thereof. Background Art

[0002] Lung cancer is one of the malignant tumors that seriously threatens human health. In the past 50 years, its incidence and mortality have increased significantly. The incidence and mortality of men are both the first among all malignant tumors, and the incidence and mortality of women are the second. In clinical practice, more than two-thirds of lung cancer patients have metastasis when diagnosed and enter the terminal stage, which brings a heavy burden to the patient's body and significantly reduces the five-year survival rate. Tumor metastasis refers to the process in which tumor cells fall off from the primary site and reach discontinuous target tissues through various pathways to continue to proliferate and grow. Tumor metastasis is the main reason for patients to enter the late stage of cancer and lose the opportunity for surgery. The vast majority of cancer patients die from metastatic diseases rather than primary tumors. However, in the body, the metastatic ability of tumor cells is also different. Some tumor cells are not easy to fall off and metastasize, and some tumor cells may easily detach from the primary site and cause metastasis due to the activation of certain signal mechanisms. Therefore, it is necessary to deeply study the early metastasis mechanism of tumor cells and develop treatment strategies for their targets.

[0003] As a highly malignant solid tumor, lung tumors contain not only tumor cells, but also a large number of fibroblasts, immune cells, cytokines and blood vessels. These components constitute the tumor microenvironment (TME) and support the progression of the tumor. Among them, tumor-associated fibroblasts (CAFs) are an important component of the tumor microenvironment. They not only exist as stromal cells, but also affect other types of cells, such as immune cells and endothelial cells, in a paracrine manner. They play a vital role in the occurrence, development and metastasis of tumors, promote the malignant phenotypic ability of tumors, and are also related to tumor-mesenchymal transformation, matrix remodeling, angiogenesis and tumor resistance. CAFs have multiple sources, including resident fibroblasts, bone marrow mesenchymal stem cells, and epithelial cells (Zhao Qi, Mo Rigen, Fan Lifei. The formation of tumor-associated fibroblasts and their role in tumor occurrence and development [J]. Chinese Journal of Biochemistry and Molecular Biology, 2021, 37(8): 1005-1009. 2020.12.1501). These cells promote tumor proliferation, migration, and angiogenesis by secreting a variety of growth factors, cytokines, and metabolites, remodeling the extracellular matrix, and interacting in complex ways with tumor cells. CAFs are not a uniform cell population, but contain multiple subpopulations, each of which may have different functions and phenotypes. It can be seen that it is necessary to understand the functions and mechanisms of CAFs and to locate the cancer-promoting CAFs subpopulations through specific markers or secretions, so as to develop precision target therapy.

[0004] Drug resistance refers to the tolerance of tumor cells to anti-tumor drugs. Once tolerance occurs, the therapeutic effect will be significantly reduced, making the treatment difficult. At present, about 50% of Asian lung adenocarcinoma patients have EGFR sensitive mutations. There are a variety of targeted epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) approved for the treatment of patients with EGFR gene sensitive mutations, which can achieve good therapeutic effects, significantly prolong survival, and improve quality of life. The third-generation EGFR-TKI represented by osimertinib brings patients more than 3 years of long-term survival, but drug resistance is inevitable in the end.

[0005] The human annexin A6 (ANXA6) gene consists of 26 exons, about 6000 bp, located on chromosome 5q32-q34. It is a highly conserved Ca2+-dependent membrane-bound protein, mainly present in the plasma membrane and endosomal compartments. It has multiple functions in cell development and differentiation. In addition to participating in cell proliferation, differentiation, inflammation, membrane repair and viral infection, studies have shown that ANXA6 is closely related to a variety of tumors, and the functions of ANXA6 in different types of tumors are also different. These functions are often related to changes in signal transduction activities such as Ras, Ras / MAPK and FAK / PI3K. Patent CN111388683B reports the use of an ANXA6 expression inhibitor in the preparation of a drug for treating lung cancer. The study found that after the expression of ANXA6 was inhibited, the proliferation of lung cancer cells was significantly inhibited, the colony-forming ability of lung cancer cells was inhibited, the subcutaneous tumor-forming ability of lung cancer cells was significantly reduced, and the growth rate of tumors was significantly inhibited. The specific action stage still needs further study.

[0006] ANXA6 was discovered in patent CN 117230200A + CAF promotes the malignant phenotype of HCC827 cells and increases the proliferation, migration and invasion of cancer cells; ANXA6 was found in patent CN 117230201 A + The three types of cells, HCC827, PC-9 and H1975, treated with CAF cell supernatant showed obvious resistance to osimertinib, but both resistant and sensitive types appeared in the treated cells, and the subsequent research on the mechanism of drug resistance is still insufficient. For the precise target treatment of lung cancer patients, cell models for studying lung cancer metastasis and EGFR-TKI resistance are urgently needed. Summary of the invention

[0007] The present invention finds that ANXA6 + CAFs and HCC827 cells were co-inoculated into the armpits of nude mice, and the metastatic ability of HCC827 cells increased. After continuous lung metastasis model screening and monoclonal screening, HCC827 cells sensitive to EGFR-TKI targeted drugs became resistant to the third-generation EGFR-TKI targeted drugs. Based on this, the present invention was completed.

[0008] In a first aspect, the present invention provides a highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line, the cell line name: HCC827-D human lung adenocarcinoma resistant EGFR-TKI cell line; preservation number: CGMCC No.46050; preservation unit: General Microbiology Center of China National Committee for the Collection of Microorganisms.

[0009] In a second aspect, the present invention provides a method for obtaining the highly invasive human lung adenocarcinoma EGFR-TKI-resistant cell line HCC827-D as described in the first aspect, wherein the method comprises the following steps:

[0010] S1. ANXA6 + CAFs cell suspension and HCC827 cell suspension were co-inoculated into the armpit of nude mice;

[0011] S2. After the mice were cultured and tumors formed, the mice were killed, the tumors were cut into pieces, washed and cultured;

[0012] S3. After the tumor cells adhered to the wall and grew, they were digested to obtain HCC827-3 cells with a malignant phenotype;

[0013] S4. HCC827-3 cells were injected into the tail vein to form lung metastases, and S2-S3 were repeated for the metastases to obtain HCC827-P1 cells;

[0014] S5. Repeat S4 with HCC827-P1 cells to obtain HCC827-P2 cells;

[0015] S6. Repeat S4 with HCC827-P2 cells to obtain HCC827-P3 cells;

[0016] S7. After three lung metastasis model screenings, HCC827-P3 cell monoclonal screening was performed to establish a highly invasive human lung adenocarcinoma EGFR-TKI-resistant cell line HCC827-D.

[0017] Further, in step S1, ANXA6 + The volume ratio of CAFs cell suspension and HCC827 cell suspension was 1:1.

[0018] Furthermore, the time for inoculating the nude mouse armpits in step S1 is 20-45 days, preferably 35-40 days.

[0019] Furthermore, the inoculation concentration in step S1 is 2 million cells / each site.

[0020] Furthermore, the nude mice in step S1 are BALB / c nude mice of 4 to 6 weeks old.

[0021] Furthermore, in step S2, the tumor-forming mice are cultured for 30-40 days, preferably 35-37 days.

[0022] Furthermore, the tumor mass culture time in step S2 is 10-20 days, preferably 15-18 days.

[0023] In a third aspect, the present invention provides a highly invasive human lung adenocarcinoma EGFR-TKI-resistant cell line as described in the first aspect for use in lung cancer research.

[0024] Furthermore, the lung cancer research includes but is not limited to research on the proliferation ability, migration and invasion ability of lung cancer cells and the degree of EGFR-TKI resistance.

[0025] In a fourth aspect, the present invention provides a cell model for lung cancer research, wherein the cell model comprises the highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line as described in the first aspect.

[0026] Furthermore, the lung cancer research includes but is not limited to research on the proliferation ability, migration and invasion ability of lung cancer cells and the degree of EGFR-TKI resistance.

[0027] Beneficial Effects

[0028] The present invention proves that ANXA6 + CAFs have the ability to promote the malignant biological behavior of lung cancer cells and can promote the resistance of EGFR mutant lung adenocarcinoma cells to the third-generation EGFR-TKI targeted drugs. A highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line was screened and established. HCC827-D can provide a good cell model for studying lung cancer metastasis and EGFR-TKI resistance. Precision target therapy for lung cancer patients further contributes to the study of lung cancer cell proliferation ability, migration and invasion ability and EGFR-TKI resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The migration, invasion and metastasis abilities of HCC827 and HCC827-3 cells.

[0030] Note: A: Comparison of migration ability between HCC827 cells and HCC827-3 cells; B: Quantitative analysis of migration ability between HCC827 cells and HCC827-3 cells, *p<0.05; C: Comparison of invasion ability between HCC827 cells and HCC827-3 cells; D: Comparison of invasion ability between HCC827 cells and HCC827-3 cells, *p<0.05; E: Comparison of metastasis ability between HCC827 cells and HCC827-3 cells in NOD / SCID mice; F: Quantitative analysis of metastasis ability between HCC827 cells and HCC827-3 cells in NOD / SCID mice.

[0031] Figure 2 ANXA6 + Sensitivity of lung cancer cells to AZD9291 after treatment of EGFR mutant lung adenocarcinoma cells with CAFs supernatant.

[0032] Note: From left to right: ANXA6 + HCC827 cells were treated with CAFs supernatant, and the response of HCC827 cells to AZD9291; ANXA6 + PC-9 cells were treated with CAFs supernatant, and the response of PC-9 cells to AZD9291; ANXA6 + H1975 cells were treated with CAFs supernatant and the response of H1975 cells to AZD9291.

[0033] Figure 3 The process of obtaining HCC827-P1, HCC827-P2 and HCC827-P3 Note: A: The process of obtaining HCC827-3 cells; B: The response of HCC827-3 cell lung metastases to vometinib, *p < 0.05; C: The process of obtaining HCC827-P1, HCC827-P2 and HCC827-P3;

[0034] Figure 4 Comparison of the metastatic ability of HCC827, HCC827-3, and HCC827-D cells.

[0035] Figure 5 Drug sensitivity experiment of HCC827 and HCC827-D cells.

[0036] Note: A: HCC827 cells were treated with AZD9291 for 48 hours, and the drug sensitivity test of HCC827 cells was performed; B: HCC827-D cells were treated with AZD9291 for 48 hours, and the drug sensitivity test of HCC827-D cells was performed; C: HCC827 cells were treated with AZD9291 for 72 hours, and the drug sensitivity test of HCC827 cells was performed; D: HCC827-D cells were treated with AZD9291 for 72 hours, and the drug sensitivity test of HCC827-D cells was performed.

[0037] Figure 6 Effect of vometinib treatment after HCC827-D cells were inoculated into the femur. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] Test materials

[0041] Basal culture medium: serum-free RPMI-1640 culture medium was taken, and fetal bovine serum was added to a concentration of 10%, penicillin was added to a concentration of 1%, and streptomycin was added to a concentration of 1%.

[0042] 0.2% BSARPMI-1640 medium is: serum-free RPMI-1640 medium is taken, bovine serum albumin (BSA) is added to a concentration of 0.2%, penicillin is added to a concentration of 1%, and streptomycin is added to a concentration of 1%.

[0043] DMEM culture medium: Take serum-free DMEM culture medium and add fetal bovine serum to a concentration of 10%.

[0044] HCC827-3 cells: HCC827 cells and ANXA6 + CAFs cells were digested into single cells and counted. After that, HCC827 cell suspension and ANXA6 + CAFs cell suspension was mixed. The cell suspension was inoculated into the armpit of BALB / c nude mice. Tumors were formed subcutaneously in nude mice. The tumors were dissected out and cut into pieces. After washing with PBS, DMEM medium was added and cultured. Tumor cells adhered to the bottom of the culture flask and grew. They were digested and subcultured and named HCC827-3 cells.

[0045] Example 1 CAFs promote the malignant phenotype of HCC827 cells

[0046] 1.1 Comparison of migration and invasion abilities of HCC827 cells and HCC827-3 cells

[0047] A. Test Methods

[0048] (1) The culture medium of HCC827 cells and HCC827-3 cells was replaced with 0.2% BSAR PMI-1640 culture medium and cultured;

[0049] (2) Add RPMI-1640 medium to the lower chamber of the Transwell chamber and add 0.2% BSA RPMI-1640 to the upper chamber.

[0050] culture medium, hydration;

[0051] (3) Add trypsin digestion solution to HCC827 cells and HCC827-3 cells until they are completely digested into single cells.

[0052] After neutralization with RPMI-1640 medium, centrifuge, discard the supernatant, resuspend, and count;

[0053] (4) Resuspend the above two cells with 0.2% BSAR PMI-1640, with the ratio of basement gel to cell suspension being 1:5;

[0054] (5) Aspirate the rehydrated culture medium in the Transwell chamber, add the cell suspension in (4), and culture;

[0055] (6) Take out the small chamber without mixed glue, wash it and put it into gentian violet solution. After a period of time, wash off the gentian violet, observe and take pictures; (7) Treat the small chamber with mixed glue in the same way as in (6), observe and take pictures.

[0056] B. Test Results

[0057] like Figure 1 A and 1B show that in the non-mixed gel chamber, the number of HCC827-3 cells that passed through the membrane was much greater than that of HCC827 cells. After counting the number of cells that passed through the membrane and performing statistical analysis, it was found that the migration ability of HCC827-3 cells was stronger than that of HCC827 cells, and there was a statistical difference (P<0.05).

[0058] like Figure 1 As shown in C and 1D, in the mixed gel chamber, the number of HCC827-3 cells that passed through the membrane was much greater than that of HCC827 cells. After counting the number of cells that passed through the membrane and performing statistical analysis, it was found that the invasion ability of HCC827-3 cells was stronger than that of HCC827 cells, and the difference was statistically significant (P<0.05).

[0059] 1.2 Comparison of metastatic ability between HCC827 cells and HCC827-3 cells

[0060] A. Test Methods

[0061] (1) Cultivating HCC827 cells and HCC827-3 cells;

[0062] (2) After digestion, add 10% RPMI-1640 medium to neutralize, mix well, centrifuge and discard the supernatant;

[0063] (3) Resuspend in 10% RPMI-1640 medium and count;

[0064] (4) Washing and resuspending to 10 million / ml;

[0065] (5) HCC827 cells and HCC827-3 cell suspensions were mixed and inoculated into NOD / SCID mice via the tail vein at a density of 2 million per mouse;

[0066] (6) Regularly inject luciferase and observe tumor metastasis in mice.

[0067] B. Test Results

[0068] like Figure 1 E shows that after the same growth period, mice inoculated with HCC827-3 cells developed bone metastasis and brain metastasis, and the volume of lung metastasis was much larger than that of mice inoculated with HCC827 cells; mice inoculated with HCC827 cells only developed lung metastasis, and the volume of metastasis was smaller than that of mice inoculated with HCC827-3 cells. This shows that the metastatic ability of HCC827-3 cells is stronger than that of HCC827 cells.

[0069] Example 2 ANXA6 + Sensitivity of lung cancer cells to EGFR-TKI after CAFs treatment

[0070] A. Test Methods

[0071] (1) ANXA6 + CAFs cells were cultured in DMEM medium, and the cell culture supernatant was collected;

[0072] (2) HCC827, PC-9, and H1975 cells were inoculated in 10% RPMI-1640 medium and passaged;

[0073] (3) Digest to single cells, add complete medium, mix well, and count;

[0074] (4) Inoculate HCC827, PC-9, and H1975 cells in a well plate;

[0075] (5) ANXA6 + The CAFs cell culture supernatant was mixed with 10% RPMI-1640 medium, and different concentrations of AZD9291 of 6 nM, 12 nM, 120 nM, 250 nM, 500 nM, 1200 nM, 6000 nM, 12000 nM, and 24000 nM were prepared therewith, and the supernatant was discarded, and different concentrations of AZD9291 were added to the well plate inoculated with HCC827 cells;

[0076] (6) ANXA6 + The CAFs cell culture supernatant was mixed with 10% RPMI-1640 medium, and different concentrations of AZD9291, 1 nM, 2 nM, 4 nM, 40 nM, 400 nM, 4000 nM, 6000 nM, 8000 nM, and 16000 nM, were prepared therewith.

[0077] The supernatant was discarded, and different concentrations of AZD9291 were added to the well plates seeded with PC-9 cells and H1975 cells;

[0078] (7) Cultivation: The OD values ​​of the cells were measured after AZD9291 was applied to the cells for 48 hours, 72 hours, and 96 hours.

[0079] B. Test Results

[0080] Based on the data, a drug sensitivity curve of cells to AZD9291 was drawn (e.g. Figure 2 shown).

[0081] Compared with HCC827 (IC50 is 21.1nM), PC-9 (IC50 is 57.3nM), and H1975 (IC50 is 92.75nM), ANXA6 + After the CAFs cell culture supernatant was used to treat HCC827, PC-9, and H1975, the IC50 values ​​of the three cell types for AZD9291 were 854.3 nM, 1390.7 nM, and 8980.67 nM, respectively. The IC50 values ​​of the three cell types increased by 40.488 times, 24.271 times, and 96.827 times, respectively, indicating that ANXA6 + CAFs promote the resistance of EGFR mutant cells to EGFR-TKI.

[0082] Example 3 Screening of highly invasive cells in lung metastasis model

[0083] A. Test Methods

[0084] (1) Cultivating HCC827-3 cells;

[0085] (2) Digestion, neutralize with 10% RPMI-1640 medium, mix, centrifuge and discard the supernatant, resuspend the cells with 10% RPMI-1640 and count;

[0086] (3) Washing and resuspending to 10 million / ml;

[0087] (4) The HCC827-3 cell suspension was mixed and inoculated into NOD / SCID mice via the tail vein of mice;

[0088] (5) Regularly inject luciferase to observe lung metastasis in mice;

[0089] (6) After the fluorescent signal is observed in the lungs of the mice, the mice are killed, the lungs are dissected out, and the part with the fluorescent signal is cut off;

[0090] (7) Washing lung tumor tissue, chopping it into small pieces, centrifuging it and discarding the supernatant;

[0091] (8) Add serum-free medium, mix well, centrifuge and discard the supernatant, and repeat the operation;

[0092] (9) transferring the tumor tissue pieces into culture medium and culturing them;

[0093] (10) Cultivate until there are many cells attached to the bottom of the cell culture bottle, digest them into single cells, centrifuge after neutralization and discard the supernatant, and remove the precipitate.

[0094] After resuspending, the cells were cultured and allowed to adhere to the wall and grow, and were named HCC827-P1 cells;

[0095] (11) After culturing HCC827-P1 cells to 80% to 90% confluence, repeat steps 2 to 10 to obtain HCC827-P2 cells;

[0097] (12) After culturing HCC827-P2 cells to a confluence of 80% to 90%, repeat steps 2 to 10 to obtain HCC827-P3 cells.

[0098] B. Test Results

[0099] The obtained HCC827-3 cells ( Figure 3 A) Injected into NOD / SCID mice via tail vein, lung metastases were formed. Compared with the metastases formed by HCC827 cells, the cells showed tolerance to the third-generation EGFR-TKI targeted drugs ( Figure 3 B), HCC827-3 formed lung metastases and was named HCC827-P1. Figure 3 Step A was repeated twice to obtain HCC827-P3.

[0100] Example 4: Monoclonal screening of HCC827-P3 cells to obtain HCC827-D cells

[0101] A. Test Methods

[0102] (1) HCC827-P3 cells were digested into single cells, neutralized, centrifuged, the supernatant was discarded, and the pellet was resuspended;

[0103] (2) After counting, the cell suspension is diluted in series, inoculated, and cultured;

[0104] (3) After culturing for a period of time, observe the cell clone formation and mark the clones formed by single cells;

[0105] (4) After monoclonal growth, cells with better growth were selected, passaged, and the corresponding clones were expanded to obtain the desired clone cell line, which was named HCC827-D.

[0106] B. Test Results

[0107] like Figure 4It can be seen that after HCC827-D cells were inoculated into the tail vein of mice, HCC827-D cells showed obvious lung metastasis, brain metastasis and bone metastasis after the mice grew for 1.5 months; after HCC827 cells were inoculated into the tail vein of mice for 3 months, no lung metastasis appeared; after HCC827-3 cells were inoculated into the tail vein of mice for 2 months, small metastasis appeared in the lungs of mice. From this comparison, it can be seen that the metastatic ability of HCC827-D cells is stronger than that of HCC827 cells and HCC827-3 cells.

[0108] Example 5 HCC827-D cell drug sensitivity test

[0109] A. Test Methods

[0110] (1) HCC827-D cells were inoculated in 10% RPMI-1640 medium and passaged;

[0111] (2) Digest to single cells, add complete medium, mix well, and count;

[0112] (3) Inoculating HCC827-D cells in a well plate;

[0113] (4) Configuration: 6nM, 12nM, 120nM, 250nM, 500nM, 1200nM, 6000nM, 12000nM, 24000nM

[0114] Different concentrations of AZD9291 were added to the well plates seeded with HCC827 cells;

[0115] (5) The OD value of the cells was measured after AZD9291 was applied to the cells for 48 hours and 72 hours.

[0116] B. Test Results

[0117] like Figure 5 As shown, the IC50 of HCC827-D cells at 48 hours was 870.81 times that of HCC827 cells; and the IC50 of HCC827 cells at 72 hours was 1411.54 times that of HCC827 cells, indicating that HCC827-D cells have obvious resistance to the third-generation EGFR-TKI, and the resistance effect is more obvious as time goes by.

[0118] Example 6 Resistance of HCC827-D cells to vometinib in mouse transplanted tumors

[0119] A. Test Methods

[0120] (1) Cultivate HCC827-D cells to 80% to 90% confluence;

[0121] (2) Digestion, neutralization by adding RPMI-1640 medium, mixing, centrifugation and discarding the supernatant, resuspending and counting;

[0122] (3) washing and diluting;

[0123] (4) HCC827-D cell suspension was inoculated into the femoral bone marrow cavity of NOD / SCID mice;

[0124] (5) The mice were raised normally and the tumor on their legs grew to 1 cm 3 about;

[0125] (6) Vometinib was administered to mice by oral gavage;

[0126] (7) Luciferase was injected into mice at 0, 45, and 75 days after administration, and the size of the femoral transplanted tumors was observed.

[0127] B. Test Results

[0128] like Figure 6 As shown, at 45 and 75 days after administration, the tumor size in the femur of mice did not change significantly compared with that before treatment, indicating that the transplanted tumor formed by HCC827-D cells was resistant to vometinib.

Claims

1. A highly invasive human lung adenocarcinoma resistant to EGFR-TKI cell line, the cell line name: HCC827-D human lung adenocarcinoma resistant to EGFR-TKI cell line; deposit number: CGMCC No.46050; deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration.

2. A method for screening the highly invasive human lung adenocarcinoma EGFR-TKI resistant cell line HCC827-D as claimed in claim 1, wherein the method comprises the following steps: S1. ANXA6 + CAFs cell suspension and HCC827 cell suspension were co-inoculated into the armpit of nude mice; S2. After the mice were cultured and tumors formed, the mice were killed, the tumors were cut into pieces, washed, and cultured; S3. After the tumor cells adhere to the wall and grow, they are digested to obtain HCC827-3 cells with malignant phenotype; S4. HCC827-3 cells were injected into the tail vein to form lung metastases, and S2-S3 were repeated for the metastases to obtain HCC827-P1 cells; S5. Repeat S4 with HCC827-P1 cells to obtain HCC827-P2 cells; S6. Repeat S4 with HCC827-P2 cells to obtain HCC827-P3 cells; S7. After three lung metastasis model screenings, HCC827-P3 cell monoclonal screening was performed to establish a highly invasive human lung adenocarcinoma EGFR-TKI-resistant cell line HCC827-D.

3. ANXA6 as described in step S1 of claim 2 + CAFs cell suspension and HCC827 cell suspension, among which, The volume ratio of the suspension was 1:

1.

4. The inoculation suspension as described in step S1 of claim 2, wherein the inoculation concentration is 2 million cells / each site.

5. The nude mice described in step S1 of claim 2 are BALB / c nude mice of 4 to 6 weeks old.

6. The time for inoculating the nude mouse axilla as described in step S1 of claim 2 is 20-45 days, preferably 35-40 days.

7. Use of the highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line as claimed in claim 1 in lung cancer research.

8. A cell model for lung cancer research, the cell model comprising the highly invasive human lung adenocarcinoma resistant EGFR-TKI cell line as claimed in claim 1.

9. The lung cancer research as described in claim 7 or 8, including but not limited to the research on the proliferation ability, migration and invasion ability of lung cancer cells and the degree of EGFR-TKI resistance.

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