Malignant phenotype large cell lung cancer cell line and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a malignant phenotype large cell lung cancer cell line and its application. The present invention provides a malignant phenotype large cell lung cancer cell line, the cell line comprising: any one of 801D-A10, 801D-F4, 801D-H10, 801D-E6, 801D-F10 and 801D-F11. The six human large cell lung cancer cell lines established by the present invention can all be successfully passaged to 60 generations, can be stably passaged, and grow rapidly. In vivo and in vitro experiments confirmed that the proliferation, migration and invasion abilities of 801D-A10, 801D-F4 and 801D-H10 cell lines are strong, while in vivo and in vitro experiments confirmed that the proliferation, migration and invasion abilities of 801D-E6, 801D-F10 and 801D-F11 cell lines are weak.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a malignant phenotype large cell lung cancer cell line and an application thereof. Background Art

[0002] Lung cancer is one of the malignant tumors that seriously threatens human health. Its morbidity and mortality rates have increased significantly in the past 50 years. The morbidity and mortality rates in men rank first among all malignant tumors, while the morbidity and mortality rates in women rank second. The cause of lung cancer is still not fully understood. Moreover, more than 50% of lung cancer patients are in the advanced stage when diagnosed, and the prognosis of patients with advanced lung cancer is extremely poor, with a 5-year survival rate of only 5%. It is estimated that in the next 20 years, lung cancer will become the disease with the heaviest social and economic burden in my country. Although targeted therapy and immunotherapy have significantly prolonged the survival time of patients in recent years, the frequent occurrence of targeted therapy resistance and adverse reactions of immunotherapy have made the treatment of lung cancer face difficulties. Therefore, exploring the molecular mechanisms of the occurrence and development of lung cancer and molecular therapeutic targets remains an urgent problem that needs to be solved.

[0003] Cells are the fundamental units of structure and function in organisms and form the basis of their individual and phylogenetic development. Tumor cells exhibit characteristics such as unlimited proliferation, loss of contact inhibition, and weakened intercellular adhesion. The in vitro culture and establishment of human lung cancer cell lines or strains are of great theoretical and clinical significance for studying the molecular mechanisms and biological characteristics of lung cancer carcinogenesis, invasion and metastasis, and multidrug resistance, as well as for developing new anti-lung cancer drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a large cell lung cancer cell line with a malignant phenotype that is easy to culture, stable in passage, easy to promote, and can be used in traditional cell biology laboratories.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a large cell lung cancer cell line with a malignant phenotype, the cell line comprising:

[0007] The accession number is CGMCC NO: 45140 801D-A10;

[0008] The accession number is CGMCC NO: 45141 801D-F4;

[0009] The accession number is CGMCC NO: 45139 801D-H10;

[0010] The accession number is CGMCC NO: 45138 801D-E6;

[0011] The accession number is CGMCC NO: 45142 801D-F10

[0012] And the deposit number is CGMCC NO: 45143 Any one of 801D-F11.

[0013] In a specific embodiment, among the cell lines, 801D-A10, 801D-F4 and 801D-H10 cell lines have strong proliferation, migration and invasion abilities, while 801D-E6, 801D-F10 and 801D-F11 cell lines have weak proliferation, migration and invasion abilities.

[0014] In a second aspect, the present invention provides a method for obtaining a large cell lung cancer cell line with a malignant phenotype, the method comprising:

[0015] Human giant cell lung cancer cells 801D were seeded on the upper layer of a Transwell chamber. After culture, the cancer cells were separated by a polycarbonate membrane into three types: cancer cells 801D attached to the polycarbonate membrane, cancer cells 801D below the membrane that did not adhere to the membrane and fell to the bottom of the well plate, and cancer cells 801D above the membrane that did not pass through the polycarbonate membrane.

[0016] The three cell types were cultured separately, digested, resuspended, and then cultured and expanded into single cells. Monoclonal clones 801D-A10, 801D-F4, 801D-H10, 801D-E6, 801D-F10, and 801D-F11 were obtained through screening.

[0017] In a specific embodiment, in the cell line:

[0018] 801D-A10 and 801D-F4 are single clones of 801D that adhered to the polycarbonate membrane;

[0019] 801D-E6 and 801D-H10 are monoclonal clones of 801D that did not pass through the polycarbonate membrane;

[0020] 801D-F10 and 801D-F11 are single clones of 801D that passed through the polycarbonate membrane and landed on the bottom of the well plate.

[0021] In a third aspect, the present invention provides the use of a large cell lung cancer cell line with a malignant phenotype in lung cancer research.

[0022] In a fourth aspect, the present invention provides the use of a large cell lung cancer cell line with a malignant phenotype in the study of the proliferation, migration and invasion capabilities of lung cancer cells.

[0023] In a fifth aspect, the present invention provides a cell model for studying lung cancer cell proliferation and metastasis, wherein the cell model comprises any one of the 801D-A10, 801D-F4, 801D-H10, 801D-E6, 801D-F10 and 801D-F11 cell lines.

[0024] In a specific embodiment, the cell lines 801D-A10, 801D-F4 and 801D-H10 have strong proliferation, migration and invasion abilities.

[0025] The proliferation, migration and invasion abilities of 801D-E6, 801D-F10 and 801D-F11 cell lines were weak.

[0026] The six human large cell lung cancer cell lines established by the present invention are 801D-A10, 801D-F4, 801D-H10, 801D-E6, 801D-F10, and 801D-F11 cell lines. All six cell lines can be stably passaged and grow rapidly. In vitro and in vivo experiments confirmed that the 801D-A10, 801D-F4, and 801D-H10 cell lines have strong proliferation, migration, and invasion abilities, while in vitro and in vivo experiments confirmed that the 801D-E6, 801D-F10, and 801D-F11 cell lines have weak proliferation, migration, and invasion abilities.

[0027] The six human large cell lung cancer cell lines provided by the present invention provide good cell models for research related to lung cancer proliferation and metastasis.

[0028] Compared with the prior art, the advantages of the malignant phenotype large cell lung cancer cell line provided by the present invention are:

[0029] (1) Easy to cultivate and promote.

[0030] (2) Ability to be stably propagated.

[0031] Cell deposit information:

[0032] The malignant phenotype large cell lung cancer cell lines provided by the present invention were obtained by screening by the inventors of the present invention and deposited in the China General Microbiological Culture Collection Center. The deposited names of the malignant phenotype large cell lung cancer cell lines are: human lung cancer cell line 801D-E6, human lung cancer cell line 801D-A10, human lung cancer cell line 801D-F4, human lung cancer cell line 801D-H10, human lung cancer cell line 801D-F10, and human lung cancer cell line 801D-F11, and the deposit numbers are CGMCC NO.45138, CGMCC NO.45140, CGMCC NO.45141, CGMCC NO.45139, CGMCC NO.45142, and CGMCC NO.45143. The deposit date is March 30, 2022, and the depositor is the China General Microbiological Culture Collection Center, and the depositor's address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The malignant phenotype large cell lung cancer cell line was identified as alive by the General Microbiology Center of the China Culture Collection Administration on March 30, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention provides six types of human large cell lung cancer cell lines with cell morphology.

[0034] Figure 2 These are the growth curves of six types of human large cell lung cancer cells provided by the present invention.

[0035] Figure 3 The colony formation status of 6 human large cell lung cancer cell lines provided by the present invention.

[0036] Figure 4 These are the general pictures of nude mouse transplanted tumors of the six human large cell lung cancer cell lines provided by the present invention.

[0037] Figure 5 The six human large cell lung cancer cell lines provided by the present invention and the weight of nude mouse transplanted tumors of different cell lines were compared.

[0038] Figure 6 The results are a comparison of the metastatic ability of the 801D-A10 cells provided by the present invention and other cell lines.

[0039] Figure 7 The results are a comparison of the metastatic ability of the 801D-F4 cells provided by the present invention and other cell lines.

[0040] Figure 8 The results are compared between the metastasis ability of 801D-H10 cells provided by the present invention and other cell lines.

[0041] Figure 9The results are a comparison of the metastatic ability of the 801D-E6 cells provided by the present invention and other cell lines.

[0042] Figure 10 The results are a comparison of the metastatic ability of the 801D-F10 cells provided by the present invention and other cell lines.

[0043] Figure 11 This is the comparison result of the metastatic ability of 801D-F11 cells provided by the present invention and other cell lines.

[0044] Figures 6-11 In the figure, Figures 1, 2, and 3 on the left are pictures of lung metastasis of different cell lines; Figures 1, 2, and 3 on the right are pictures of metastatic foci in lung tissue sections of different cell lines; the arrows in Figures 1, 2, and 3 on the left point to mice with cachexia, and the arrows in Figures 1, 2, and 3 on the right point to metastatic foci. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail in the following examples. The following examples are only used to illustrate the invention but are not intended to limit the scope of the invention.

[0046] illustrate:

[0047] The cell culture conditions were: 37°C, 5% CO2 static culture.

[0048] The basal culture medium used in the examples was: serum-free RPMI-1640 medium supplemented with fetal bovine serum to a concentration of 10%, penicillin to a concentration of 1%, and streptomycin to a concentration of 1%. 0.2% BSA RPMI-1640 medium was: serum-free RPMI-1640 medium supplemented with bovine serum albumin (BSA) to a concentration of 0.2%, penicillin to a concentration of 1%, and streptomycin to a concentration of 1%.

[0049] Example 1

[0050] Cell culture and separation of different cell lines across Matrigel-coated polycarbonate membranes

[0051] 1. Cell culture, passaging, and inoculation

[0052] 1. 801D cells were seeded in basal culture medium.

[0053] 2. Subculture the cells when they have grown to 90% confluence, add trypsin digestion solution, and digest at 37°C for about 5 minutes until the cells are completely digested into single cells.

[0054] 3. Add complete culture medium and mix thoroughly by pipetting. After mixing, count the cells using a Muse cell counter.

[0055] 4. Inoculate 801D cells in a 6-well plate with 500,000 cells per well and inoculate 3 wells with each cell type.

[0056] 5. Incubate at 37°C, 5% CO2 overnight.

[0057] 2. Separation of different cell lines through Matrigel-coated Polycarbonate membrane

[0058] 1. Seed the cells in a 6-well plate and observe the next day. The cell confluence is about 30-40%.

[0059] 2. Aspirate the supernatant from each well and replace with 0.2% BSAR PMI-1640 culture medium and continue culturing at 37°C and 5% CO2 for 24 hours.

[0060] 3. Coat the upper chamber surface of the bottom membrane of a Transwell chamber with a pore size of 8.0 μm and a diameter of 6.5 mm with Matrigel gel and 0.2% BSA RPMI-1640 culture medium at a ratio of 1:5, and incubate at 37°C overnight to solidify.

[0061] 4. Add 100 μl of 0.2% BSA RPMI-1640 medium to each well of the upper chamber of the Transwell chamber and rehydrate for 30 minutes. Add 600 μl of basal medium to the lower chamber. Passage the cells and add trypsin digestion buffer. Digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Add complete medium and mix thoroughly by pipetting. After mixing, count the cells using a Muse cell counter.

[0062] 5. Resuspend 801D cells in 0.2% BSA RPMI-1640 to a cell concentration of 2 million cells / ml. Aspirate 100 μl of the rehydrated 0.2% BSA RPMI-1640 medium from the Transwell chamber and add 100 μl of the cell suspension to each well. Incubate at 37°C, 5% CO2 for 48–72 hours.

[0063] 6. At this point, the cells that have passed through the Matrigel-coated polycarbonate membrane will be divided into two groups: cells that adhere to the polycarbonate membrane and grow, and cells that have passed through the polycarbonate membrane but have not grown and have fallen to the bottom of the 24-well plate. The cells that adhered to the polycarbonate membrane and have grown are trypsinized and cultured; the cells that have fallen to the bottom of the 24-well plate are cultured. Additionally, the cells in the upper chamber of the Transwell that have not passed through the polycarbonate membrane are aspirated and cultured. This results in three cell types: cells in the upper chamber, cells adhered to the membrane, and cells that have fallen to the bottom of the 24-well plate. These cells are designated as 801D-Upper, 801D-Membrane, and 801D-Lower, respectively.

[0064] 3. Screening of three 801D cell monoclonal cell lines

[0065] 1. Culture 801D upper, 801D membrane, and 801D lower cells in basal medium until they are 70% to 80% confluent.

[0066] 2. Add trypsin digestion solution and digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Add basal culture medium, mix thoroughly, centrifuge, and discard the supernatant. Resuspend in basal culture medium and count using a Muse cell counter.

[0067] 3. Dilute the culture according to the counting results, and the final dilution is 10 cells / ml.

[0068] 4. After mixing, inoculate into 96-well plates with 100 μl of cell suspension in each well.

[0069] 5. Incubate at 37°C and 5% CO2 and observe after one week.

[0070] 6. Select wells with only one cell in each well and observe them continuously until they are ready for passage and expansion.

[0071] 7. Six clones were obtained from the single clones screened from 801D upper, 801D membrane and 801D lower, namely:

[0072] 801D: 801D-E6 and 801D-H10;

[0073] 801D membrane: 801D-A10 and 801D-F4;

[0074] 801D: 801D-F10 and 801D-F11.

[0075] Example 2

[0076] Comparison of cell proliferation ability among different cell lines

[0077] 1. Cell Growth Curve Experiment

[0078] 1. All cells were cultured in basal medium to 70%-80% confluence.

[0079] 2. Add trypsin digestion solution and digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Neutralize with basal culture medium, mix thoroughly, centrifuge, and discard the supernatant. Resuspend in basal culture medium and count using a Muse cell counter.

[0080] 3. Dilute the culture according to the counting results, and the final dilution is 30,000 cells / ml.

[0081] 4. After mixing, inoculate into 96-well plates, with 5 plates for each group of cells and 5 wells for each cell type at each time point. The cell suspension should be 3000 cells / 100 μl in each well and cultured at 37°C and 5% CO2.

[0082] 5. Cell viability was detected with CCK8 after 0 h, 24 h, 48 h, 72 h, and 96 h, respectively: 10 μl of CCK8 was directly added to each well at 0 h, incubated at 37°C for two h, and the absorbance at 450 nm was measured with a microplate reader; in the other four time periods, 10 μl of CCK8 was added to 100 μl of basal culture medium to prepare the working solution, and the absorbance at 450 nm was measured with a microplate reader after incubation at 37°C for two h.

[0083] 6. Draw the cell growth curve based on the absorbance values ​​at 5 time points. The results are as follows: Figure 2 shown.

[0084] Compared with 801D-E6 and 801D-F11 cells, the growth rates of 801D-A10 cells and 801D-F4 cells were similar, and the growth rates were significantly increased, with statistical differences (p<0.05).

[0085] Compared with 801D-E6 and 801D-F11 cells, the growth rates of 801D-F4 cells and 801D-H10 cells were similar, and the growth rates of both cells were significantly increased, with statistical differences (p<0.05).

[0086] Compared with 801D-F4 and 801D-H10 cells, the growth rates of 801D-E6 cells were similar to those of 801D-F10 cells, but the growth rates of both cells were significantly slower, with statistical differences (p<0.05).

[0087] Compared with 801D-E6 and 801D-F11 cells, the growth rates of 801D-H10 cells and 801D-A10 cells were similar, and the growth rates of both cells were significantly increased, with statistical differences (p<0.05).

[0088] Compared with 801D-F4 and 801D-H10 cells, the growth rates of 801D-F10 and 801D-F11 cells were similar, and the growth rates were significantly slower, with statistical differences (p<0.05).

[0089] Compared with 801D-F4 and 801D-A10 cells, the growth rates of 801D-F11 cells were similar to those of 801D-F10 cells, but the growth rates of both cells were significantly slower, with statistical differences (p<0.05).

[0090] 2. Colony Formation Assay

[0091] 1. All cells were cultured in basal medium to 70% to 80% confluence.

[0092] 2. Add trypsin digestion solution and digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Add basal culture medium, mix thoroughly, centrifuge, and discard the supernatant. Resuspend in basal culture medium and count using a Muse cell counter.

[0093] 3. Dilute the culture according to the counting results, and the final dilution is 3000 cells / ml.

[0094] 4. After mixing, inoculate into 6-well plates, with three wells for each cell type, containing 300 cells / 2 ml / well of basal culture medium.

[0095] 5. Incubate at 37°C and 5% CO2 and observe after 7 to 9 days.

[0096] 6. Discard the cell culture supernatant, wash twice with PBS, add 2 ml of 2% gentian violet staining solution, and stain for 30 minutes.

[0097] 7. Rinse slowly with tap water to remove excess gentian violet stain and air dry. Figure 3 shown.

[0098] 8. Count colonies larger than 50 cells under a microscope. The results show:

[0099] The colony numbers of 801D-A10 and 801D-F4 cells were significantly higher than those of 801D-E6 and 801D-F11 cells (P<0.05).

[0100] The colony numbers of 801D-F4 and 801D-H10 cells were significantly higher than those of 801D-E6 and 801D-F11 cells (P<0.05).

[0101] The colony numbers of 801D-F4 and 801D-H10 cells were significantly higher than those of 801D-E6 and 801D-F10 cells (P<0.05).

[0102] The colony numbers of 801D-H10 and 801D-A10 cells were significantly higher than those of 801D-E6 and 801D-F11 cells (P<0.05).

[0103] The colony numbers of 801D-F4 and 801D-H10 cells were significantly higher than those of 801D-F10 and 801D-F11 cells (P<0.05).

[0104] The colony numbers of 801D-F4 and 801D-A10 cells were significantly higher than those of 801D-F11 and 801D-F10 cells (P<0.05).

[0105] 3. Subcutaneous tumor formation experiment in BALB / c nude mice

[0106] 1. Culture different cells in basal medium to 70% to 80% confluence.

[0107] 2. Add trypsin digestion solution and digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Add basal culture medium, mix thoroughly, centrifuge, and discard the supernatant. Resuspend in basal culture medium and count using a Muse cell counter.

[0108] 3. Dilution was performed according to the counting results, and the final dilution was 1.0×10 7 / ml.

[0109] 4. Prepare a corresponding number of BALB / c female nude mice, aged 4-6 weeks.

[0110] 5. After the cell suspension is mixed, it is inoculated into the armpits of nude mice. Each site is injected with 2 million cells / 200ul of cell suspension, and 5 to 6 mice are inoculated with each cell type.

[0111] 6. Observe the tumor formation in the nude mouse armpit every 3 or 4 days, measure its long and short diameters, and calculate its volume.

[0112] 7. When the volume of the subcutaneous tumor is close to 1000 mm 3 The mice were killed by cervical dislocation, and the tumors were dissected out, weighed, and photographed.

[0113] 8. Analyze data based on tumor volume and weight.

[0114] Figure 4 and Figure 5 As shown in the results, compared with 801D-E6 and 801D-F11 cells, the growth rate of transplanted tumors of 801D-A10 and 801D-F4 cells was significantly increased (P<0.05), and their subcutaneous tumor formation ability was significantly enhanced (P<0.05).

[0115] Compared with 801D-E6 and 801D-F11 cells, the growth rate of transplanted tumors of 801D-F4 and 801D-H10 cells was significantly increased (P<0.05), and their subcutaneous tumor formation ability was significantly enhanced (P<0.05).

[0116] Compared with 801D-H10 and 801D-F4 cells, the growth rate of transplanted tumors of 801D-F10 and 801D-E6 cells was significantly slower (P<0.05), and their subcutaneous tumorigenicity was significantly reduced (P<0.05).

[0117] Compared with 801D-E6 and 801D-F11 cells, the growth rate of transplanted tumors of 801D-H10 and 801D-A10 cells was significantly increased (P<0.05), and their subcutaneous tumor formation ability was significantly enhanced (P<0.05).

[0118] Compared with 801D-H10 and 801D-F4 cells, the growth rate of transplanted tumors of 801D-F10 and 801D-F11 cells was significantly slower (P<0.05), and their subcutaneous tumor-forming ability was significantly reduced (P<0.05).

[0119] Compared with 801D-A10 and 801D-F4 cells, the growth rate of transplanted tumors of 801D-F11 and 801D-F10 cells was significantly slower (P<0.05), and their subcutaneous tumorigenicity was significantly reduced (P<0.05).

[0120] Example 3

[0121] Evaluation of the metastatic ability of six human large cell lung cancer cell lines

[0122] 1. Culture different cells in basal medium to 70% to 80% confluence.

[0123] 2. Add trypsin digestion solution and digest at 37°C for approximately 5 minutes until the cells are completely digested into single cells. Add basal culture medium, mix thoroughly, centrifuge, and discard the supernatant. Resuspend in basal culture medium and count using a Muse cell counter.

[0124] 3. Wash the cells 3 times with PBS and dilute according to the counting results. The final dilution is 1.0×10 7 / ml.

[0125] 4. Prepare a corresponding number of NOD / SCID female mice, aged 4 to 6 weeks.

[0126] 5. After the cell suspension is mixed, lung cancer cells are inoculated through the tail vein. Each mouse is injected with 0.2 ml (2 million) of cell suspension, and 5 mice are inoculated in each group.

[0127] 6. Every 7 days, luciferase was injected intraperitoneally at a dose of 150 mg / kg. Fifteen minutes later, lung metastasis of the mice was observed using a small animal in vivo imaging system.

[0128] 7. At 1.5 to 2 months of age, the mice were killed by cervical dislocation, and the lung tissues were dissected, photographed, fixed with formalin, and stained with HE to observe lung metastasis.

[0129] like Figure 6 As shown, in vivo imaging of small animals revealed metastatic lesions in four mice in both the 801D-A10 and 801D-F4 groups, while no metastases were observed in the 801D-E6 or 801D-F11 groups. On day 28, one mouse in the 801D-F4 group developed cachexia and a tumor in the left thigh. HE staining revealed multiple metastatic lesions in the lungs of both the 801D-A10 and 801D-F4 groups, while no metastases were observed in the 801D-E6 or 801D-F11 groups. This suggests that compared with 801D-E6 and 801D-F11 cells, both 801D-A10 and 801D-F4 cells have enhanced metastatic capacity.

[0130] like Figure 7 As shown, in vivo imaging revealed metastatic lesions in four mice in both the 801D-F4 and 801D-H10 groups, while no metastases were observed in either the 801D-E6 or 801D-F11 groups. On day 28, one mouse in the 801D-F4 group developed cachexia and a tumor in the left thigh. HE staining revealed multiple metastatic lesions in the lungs of both the 801D-F4 and 801D-H10 groups, while no metastases were observed in either the 801D-E6 or 801D-F11 groups. This suggests that compared to 801D-E6 and 801D-F11 cells, the metastatic capacity of 801D-F4 and 801D-F11 cells is weakened.

[0131] like Figure 8 As shown, in vivo imaging of small animals revealed metastatic lesions in four mice in both the 801D-F4 and 801D-H10 groups. In the 801D-E6 and 801D-F10 groups, one mouse developed metastases, while none of the other mice did. On day 28, one mouse in the 801D-F4 group developed cachexia and a tumor in the left thigh. HE staining revealed multiple lung metastases in both the 801D-F4 and 801D-H10 groups. In the 801D-E6 and 801D-F10 groups, one mouse developed metastases, while none of the other mice did. This suggests that compared with 801D-H10 and 801D-F4 cells, the metastatic capacity of 801D-E6 and 801D-F10 cells is reduced.

[0132] like Figure 9As shown, in vivo imaging revealed metastatic lesions in four mice in both the 801D-H10 and 801D-A10 groups, while no metastases were observed in the 801D-E6 and 801D-F11 groups. HE staining revealed multiple metastatic lesions in the lungs of both the 801D-H10 and 801D-A10 groups. This suggests that both 801D-H10 and 801D-A10 cells have enhanced metastatic capacity compared to 801D-E6 and 801D-F11 cells.

[0133] like Figure 10 As shown, in vivo imaging of small animals revealed metastatic lesions in four mice in both the 801D-F4 and 801D-H10 groups. In the 801D-F10 and 801D-F11 groups, one mouse in the 801D-F10 group developed metastases, while none of the other mice developed metastases. On day 28, one mouse in the 801D-F4 group developed cachexia and a tumor in the left thigh. HE staining revealed multiple metastatic lesions in the lungs of both the 801D-F4 and 801D-H10 groups. In the 801D-F10 and 801D-F11 groups, one mouse in the 801D-F10 group developed metastases, while none of the other mice developed metastases. This suggests that compared with 801D-H10 and 801D-F4 cells, the metastatic capacity of 801D-F10 and 801D-F11 cells is reduced.

[0134] like Figure 11 As shown, in vivo imaging of small animals revealed metastatic lesions in four mice in both the 801D-F4 and 801D-A10 groups, while no metastases were observed in the 801D-E6 and 801D-F11 groups. On day 28, one mouse in the 801D-F4 group developed cachexia and a tumor in the left thigh. HE staining revealed multiple metastatic lesions in the lungs of the 801D-F4 and 801D-A10 groups, while no metastases were observed in the 801D-F11 or 801D-F10 groups. This suggests that compared to 801D-H10 and 801D-F4 cells, the metastatic capacity of 801D-F11 and 801D-F10 cells is reduced.

[0135] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various changes can be made to the technical solutions of the present invention. These simple variations all fall within the scope of protection of the present invention.

[0136] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will no longer separately describe various possible combinations.

[0137] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A large cell lung cancer cell line with a malignant phenotype, the cell line having the accession number of CGMCC NO: 45140.

2. Use of the cell line according to claim 1 in preparing a lung cancer cell model used in in vitro experiments.

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