Lung cancer cells, methods of making and uses thereof

Abstract

The application provides lung cancer cells and a method for establishing the same and application. The lung cancer cells highly express CD18, Strc, Cd79a, Amigo2, Plet1, Angptl4 and Plac1 molecules; do not express or lowly express Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y and Capn6 molecules. The lung cancer cells are obtained by inoculating common LLC cells into C57BL / 6 mice, culturing lung metastatic foci, inoculating the primary cultured metastatic foci cells into C57BL / 6 again, culturing different clones of lung metastatic foci again, and screening. After twice lung metastatic foci tumorigenesis and screening, compared with LLC cells, the lung cancer cells are shorter in use time for constructing a metastasis model, and higher in metastasis rate of main organs; and the lung cancer cells provide a high-efficiency cell model for better studying the mechanism of tumor invasion and metastasis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of lung cancer cell line model and its construction, and particularly relates to a lung cancer cell and a method for establishing the same and application thereof. BACKGROUND

[0002] Lung cancer is the most common malignant tumor in the world, and its incidence ranks second among malignant tumors, and its mortality ranks first among malignant tumors. Among them, 80%-85% are non-small cell lung cancer (NSCLC). In recent years, the treatment of NSCLC has made progress, but many patients still relapse and progress. The 5-year overall survival rate of NSCLC is about 19%. Tumor invasion and metastasis is the main cause of death of malignant tumors. Tumor cells from the primary lesion through a series of complex biological processes to reach the distant organs and form metastatic lesions, this process is called invasion and metastasis, and involves multiple steps. At present, the exact biological process and specific molecular mechanism of tumor invasion and metastasis are not very clear. Therefore, in order to better study the mechanism of tumor invasion and metastasis, efficient cell models and animal models are needed to simulate this process.

[0003] Lewis lung carcinoma (LLC) is a lung cancer that occurs in C57BL mice. In 1951, it was discovered by Dr. Margaret R. Lewis of Wistar Institute. The tumor tissue can be passed between immunocompetent mice and is widely used in the construction of lung cancer models. In 1980, John S. Bertram and Przemyslaw Janik established a cell line named LLC1 (hereinafter, unless otherwise specified, LLC cells are LLC1 cells). The cell line is widely used as a metastasis model and helps to study the mechanism of cancer chemotherapy drugs. Studies have reported that the cells have high tumorigenicity, but are weakly metastatic in mice. SUMMARY

[0004] The purpose of the present application is to provide a more efficient LLC cell with stronger invasion and metastasis ability for the construction of an immunocompetent mouse metastasis model.

[0005] The lung cancer cell of the present application highly expresses CD18 molecules.

[0006] The lung cancer cell has strong invasion and metastasis ability. After not expressing or lowly expressing CD18 molecules, the invasion and metastasis ability is weakened.

[0007] Further, the lung cancer cell highly expresses Strc, Cd79a, Amigo2, Plet1, Angptl4, Plac1 molecules; and does not express or lowly expresses Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y, Capn6 molecules.

[0008] The high expression of the lung cancer cell refers to that the expression ratio of the lung cancer cell to the wild type LLC cell is greater than or equal to 2, and the low expression refers to that the expression ratio of the lung cancer cell to the wild type LLC cell is less than or equal to 0.5. The expression amount is generally FPKM value, that is, the gene expression level of RNAseq of the second generation sequencing.

[0009] The expression of the application includes mRNA or protein expression.

[0010] The lung cancer cell strain LLC-ML2 of the application has a preservation number of CCTCC NO: C2022102, and is derived from a mouse.

[0011] The application further provides a construction method of the lung cancer cell, which is constructed by using any of the following methods:

[0012] (1) Directly screening the lung cancer cell with high expression, no expression or low expression of the molecules to obtain;

[0013] (2) Obtaining by overexpressing CD18 molecules in the lung cancer cell, or by overexpressing Strc, Cd79a, Amigo2, Plet1, Angptl4, Plac1 molecules, and interfering or knocking out the expression of Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y, Capn6 molecules.

[0014] Further, the application further provides a construction method of the lung cancer cell, which comprises inoculating common LLC cells into C57BL / 6 mice, taking lung metastatic foci for culture, inoculating the primary cultured metastatic foci cells into C57BL / 6 mice again, taking lung metastatic foci of different clones for culture again, and screening to obtain.

[0015] Specifically, the clone with the strongest metastasis ability is selected through in vitro cell matrix gel invasion experiment and in vivo mouse tumor metastasis experiment.

[0016] The construction method specifically comprises the following steps:

[0017] The first stage: after 2*10 6 LLC cells are inoculated subcutaneously into C57 mice, the mice are fed for 60 days, the lung metastatic foci are taken, the normal tissues are removed, washed, cut, digested with collagenase I for 1 hour, filtered through a 100 um filter screen, centrifuged at 300g horizontal rotor for 5 minutes, and cultured with DMEM+10%FBS+1% double-antibiotic medium to obtain LLC-ML1 cells;

[0018] The second stage: after 2*10 6After feeding 50 days after LLC-ML1 cells, different clones of lung metastasis were taken, normal tissues were removed, washed, cut, digested with collagenase I for 1h, filtered through a 100um filter screen, centrifuged at 300g horizontal rotor for 5 minutes, and cultured with DMEM+10%FBS+1% double antibody. Through in vitro cell matrix gel invasion experiment and in vivo mouse tumor metastasis experiment, the clone with the strongest metastasis ability was selected.

[0019] The construction method, and the screening criteria include high expression of CD18 molecules;

[0020] Further, the screening criteria also include high expression of Strc, Cd79a, Amigo2, Plet1, Angptl4, Plac1 molecules; no expression or low expression of Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y, Capn6 molecules.

[0021] Further, high expression refers to the ratio of expression amount of lung cancer cells to wild type LLC cells is greater than or equal to 2, and low expression refers to the ratio of expression amount of lung cancer cells to wild type LLC cells is less than or equal to 0.5.

[0022] Further, it is obtained by overexpressing, interfering or knocking out the expression molecules of the mouse lung cancer cells.

[0023] The application also provides the application of the lung cancer cells as a model tool for lung cancer research.

[0024] The application inoculates common LLC cells into C57BL / 6 mice, takes lung metastasis, and cultures the primary cultured metastasis cells. The LLC-ML2 cells are inoculated into C57BL / 6 mice again, and different clones of lung metastasis are taken for culture. After two times of lung metastasis and screening, the LLC-ML2 cells have shorter construction time of metastasis model compared with LLC cells, and have higher metastasis rate of main organs. The LLC-ML2 cells provide an efficient cell model for better studying the mechanism of tumor invasion and metastasis. The main molecular characteristics of LLC-ML2 cells are high expression of CD18 molecules. More detailed molecular characteristics of LLC-ML2 cells are high expression of CD18 (Itgb2), Strc, Cd79a, Amigo2, Plet1, Angptl4, Plac1 molecules; no expression or very low expression of Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y, Capn6 molecules (compared with wild type LLC cells).

[0025] The mouse lung cancer cells LLC-ML2 of the application were deposited at the China Center for Type Culture Collection on June 28, 2022, with a deposit number of CCTCC NO: C2022102, and an address of Wuhan University, China. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 6: Morphology of LLC-ML2 cells.

[0027] Figure 2 Figure 7: Growth curve comparison of LLC and LLC-ML2 cells.

[0028] Figure 3 Figure 8: Colony formation experiment comparison of LLC and LLC-ML2 cells.

[0029] Figure 4 Figure 9: Cell migration and invasion experiment comparison of LLC and LLC-ML2 cells.

[0030] Figure 5 Figure 10: Results of LLC-ML2 cells having higher metastasis ability in vivo in mice.

[0031] A: Survival curve; B: LLC and LLC-ML2 cell invasion and metastasis incidence statistics in mice; C: LLC and LLC-ML2 cell lung metastasis in mice; D: statistics of C; E: LLC and LLC-ML2 cell liver metastasis in mice; F: statistics of E.

[0032] Figure 6 Figure 11: Heat map of LLC and LLC-ML2 cell characteristic molecule expression.

[0033] Figure 7 Figure 12: Statistics of LLC and LLC-ML2 cell characteristic molecule expression.

[0034] Figure 8 Figure 13: Western blot detection of CD18 protein expression in each cell.

[0035] Figure 9 Figure 14: Overexpression of CD18 in LLC cells can significantly increase cell migration and invasion ability.

[0036] A: Western blot detection of recombinant CD18 molecule expression in LLC cells, 1 is LLC transfected with empty vector, 2 is LLC transfected with recombinant CD18 expression plasmid with strepII tag; B: plate cloning experiment; C: scratch test; D: matrix gel invasion experiment; * indicates p<0.05, with significant.

[0037] Figure 10 Figure 15: Knockout of CD18 in LLC-ML2 cells can significantly reduce cell migration and invasion ability.

[0038] A: Western blot detection identified CD18 knockout positive clones, 1-D5 and 2-C4-2 were completely knocked out without CD18 expression; B: plate clone experiment; C: CCK8 cell proliferation experiment; D: Matrigel invasion experiment; E: C57 mouse tumor formation experiment; F: C57 mouse invasion and metastasis experiment, the upper left graph is lung metastasis, the lower right graph is liver metastasis, the upper right graph is the statistical graph of the number of metastatic foci and the number of metastatic organs, and the lower right graph is the survival curve of each group of mice; * indicates p<0.05, with significant. NTCG is a negative control cell transfected with an irrelevant sequence. DETAILED DESCRIPTION

[0039] The following examples are intended to further illustrate the present application, but not to limit the present application.

[0040] Example 1: Construction of mouse lung cancer cell LLC-ML2 of the present application

[0041] Specific steps:

[0042] First stage: 12 C57 mice were subcutaneously inoculated with 2x10 6 LLC cells and fed for 60 days (12 mice, 3 survived, 1 of which had liver and lung metastasis), and the lung metastatic tumor (mixed clone) was removed after normal tissue was removed, washed, and cut into pieces. Collagenase I digestion for 1 h, blow dispersion, filter through 100 um filter screen, centrifuge at 300g horizontal rotor for 5 minutes, culture with DMEM+10%FBS+1% double antibody, and obtain LLC-ML1 cells.

[0043] Second stage: 10 C57 mice were subcutaneously inoculated with 2x10 6 LLC-ML1 cells and fed for 50 days (5 mice had internal organ metastasis, including 1 lung metastasis, 2 lung and liver metastasis, and 3 lung, liver and kidney metastasis), and different clones of lung metastatic foci were taken, washed, cut into pieces after normal tissue was removed, collagenase I digestion for 1 h, blow dispersion, filter through 100 um filter screen, centrifuge at 300g horizontal rotor for 5 minutes, culture with DMEM+10%FBS+1% double antibody, and select the strongest clone with metastatic ability as LLC-ML2 cells through in vitro cell matrix gel invasion experiment and in vivo mouse tumor formation and metastasis experiment. Cell morphology is shown in Figure 1 .

[0044] Example 2: Culture conditions of LLC-ML2 cells of the present application

[0045] LLC-ML2 cells were cultured in a constant temperature incubator at 37°C with 5% CO2, using DMEM medium containing 10% fetal bovine serum (FBS) for culture. When the cells adherently grew to 80-90% density, trypsin digestion solution (containing 0.125% trypsin, 0.265 mM EDTA, half of the usual trypsin concentration) was used for digestion and subculture. The cells can be frozen using complete medium containing 10% DMSO and stored in liquid nitrogen.

[0046] Example 3: Cell growth detection of the present application (CCK-8 assay)

[0047] (1) Prepare the cells to be tested for culture, and when the cells grow to the logarithmic phase, seed the plates (96-well plates), 5 repeated holes per day, detect for 6 days, 1 x 10 3 cells per well and evenly distributed in 100 μL of cell culture medium containing 10% FBS, and placed in a 5% CO2, 37°C incubator for culture;

[0048] (2) After the cells are completely adherent, add 10 μL of CCK-8 reagent to each well on the first day, and incubate in a 37°C incubator for 2 h (days 2-6, ensure the same incubation time every day);

[0049] (3) Use a multifunctional enzyme marker to detect the absorbance value of each well at a wavelength of 450 nm, take the absorbance value (average value) as the vertical coordinate and the incubation time as the horizontal coordinate to draw a growth curve.

[0050] In vitro cell growth experiments showed that there was no significant difference in growth rate between LLC (wild type) and LLC-ML2 cells, as shown in Figure 2 .

[0051] Example 4: Cell colony formation experiment of the present application

[0052] (1) Preparation of cell suspension: single-layer cultured cells in the logarithmic growth phase were dispersed into a single-cell suspension by general subculture method, and counted;

[0053] (2) Cell inoculation: prepare the cells to be tested for seeding (6-well plates), 3 repeated holes per group, 1 x 10 3 cells per well and evenly distributed in 2 mL of cell culture medium containing 10% FBS; placed in a 5% CO2, 37°C incubator for culture; the cells were washed 2-3 times every two days with D-Hanks solution and fresh culture medium was replaced;

[0054] (3) Culture: placed in a 5% CO2, 37°C incubator for culture, and left for 2 weeks;

[0055] (4) Fixation: When the cells grow into monoclonal that can be seen with the naked eye, the culture is terminated. The cells are carefully washed 3 times with 1x PBS buffer or D-Hanks solution, and 1 mL of 4% paraformaldehyde is added to fix the cells for 30 min;

[0056] (5) Staining: The paraformaldehyde is discarded, and 0.1% crystal violet dye is used to color for 10-30 min. The dye is slowly washed away with running water, and the cells are air-dried at room temperature;

[0057] (6) Observation with a microscope and photographing.

[0058] The colony formation experiment shows that there is no significant difference in growth rate between LLC and LLC-ML2 cells, as shown in Figure 3 .

[0059] Example 5: Cell migration and invasion experiment of the present application

[0060] (1) Before treating the cells to be detected, the required Transwell chamber is placed in a 24-well plate, 15 μL of Matrigel is then laid in the chamber (Matrigel: RPMI-1640 complete culture medium = 1:2), and then the chamber is solidified in a 37°C incubator for 1 h;

[0061] (2) The cells to be detected are digested, counted, and plated, 1x10 5 cells are laid in each chamber and evenly distributed in 200 μL of cell culture medium without FBS, 700 μL of culture medium containing 15% FBS is added to the lower chamber, and the experiment is divided into a migration group without the addition of Matrigel and an invasion group with the addition of Matrigel, and then the culture is placed in a 5% CO2, 37°C incubator for culture;

[0062] (3) Fixation: The Transwell chamber is carefully taken out with tweezers and placed in a new 24-well plate, and an appropriate amount of 4% paraformaldehyde is added to completely immerse the chamber, and the cells are fixed for 30 min;

[0063] (4) Staining: The chamber is slowly washed 3 times with 1x PBS buffer, and the chamber is inverted and air-dried at room temperature. After the chamber is inverted, an appropriate amount of crystal violet dye is added for staining for 5 min, and then the crystal violet dye is washed away with water, and then the dye on the inner side of the chamber membrane is slowly wiped clean with a cotton swab;

[0064] (6) The chamber is placed back in the 24-well plate, and it is filled with deionized water. Observation with a microscope and photographing are performed.

[0065] The cell migration and invasion experiment shows that the migration and invasion ability of LLC-ML2 cells is significantly enhanced, as shown in Figure 4 .

[0066] Example 6: Detection of the in vivo metastasis ability of LLC-ML2 cells of the present application

[0067] 1. Take wild type LLC, LLC-ML2 cells, respectively, subcutaneously inoculate 15 C57 mice, each with 2x10 6 number, measure the mouse body weight and tumor size, ulceration, death time and other conditions regularly.

[0068] 2. Inoculation for 27 days, among which 8 mice in the metastasis group and 13 mice in the primary tumor group. After the mice are decapitated, the tumor, lung, liver and kidney tissues are taken respectively, the metastasis of each group of mice is observed and the results are counted.

[0069] 3. LLC-ML2 mouse model is more likely to die than wild type LLC mouse model, with shorter survival time Figure 5 A) The incidence of distant organ metastasis in LLC-ML2 group was 69%, and that in LLC group was 6.67%, with significant difference Figure 5 B) Among them, the number of visible metastatic foci on the surface of LLC-ML2 group was 2.25±2.435, which was significantly higher than that of LLC group 0 Figure 5 C, D) At the same time, the number of visible metastatic foci on the surface of LLC-ML2 group was 4.5±3.7, which was significantly higher than that of LLC group 0.2±0.599 Figure 5 E, F) This shows that LLC-ML2 cells are shorter than LLC cells in constructing metastasis model, and the incidence of metastasis in major organs is higher.

[0070] Example 7: Molecular characteristics of LLC-ML2 cells

[0071] 1. 2x10 6 LLC-ML2 cells were inoculated in a 60mm culture dish and cultured for 24 hours.

[0072] 2. Add 2ml TRIzol to the culture dish. Extract the RNA of LLC-ML2 cells according to the TRIzol instruction steps.

[0073] 3. Send the RNA to a sequencing company to use second-generation sequencing technology to determine the gene expression level of RNAseq.

[0074] 4. Use reverse transcription kit to reverse transcribe RNA into cDNA, design and synthesize gene-specific primers for fluorescent quantitative PCR detection to detect the expression level of the gene.

[0075] 5. The determination of CD18 gene protein level is to inoculate 2x10 6LLC-ML2 cells were seeded in 60mm culture dish for 24 hours, then cell lysate was added to extract cell protein, after electrophoresis, western blot was used to detect the expression of CD18 protein.

[0076] The main molecular characteristics of LLC-ML2 cells are high expression of Itgb2 (CD18), Strc, Cd79a, Amigo2, Plet1, Angptl4 and Plac1 molecules; no expression or very low expression of Ddx3y, Cbr3, Sox11, Pid1, Kdm5d, Eif2s3y and Capn6 molecules; Figure 6 The heat map of the expression amount of characteristic molecules of LLC cells and LLC-ML2 cells is shown in the following table: Figure 7 The expression amount of characteristic molecules of LLC cells and LLC-ML2 cells is shown in the following table. In the present application, high expression is LLC-ML2 / LLC at least ≥ 2, and low expression is LLC-ML2 / LLC at least ≤ 0.5 (the expression amount is generally FPKM value, i.e. gene expression level of RNAseq of second generation sequencing). The research focuses on CD18 molecule, western blot is used to detect the expression of CD18 at protein level in two kinds of cells, and it is found that the results are consistent with the mRNA level: LLC-ML2 cells highly express CD18 molecule, while LLC-WT cells (i.e. wild type) hardly express CD18 molecule. Figure 8 ).

[0077] Example 8: Overexpression of CD18 in LLC cells significantly increases the migration and invasion ability

[0078] 1. Construct overexpression CD18 plasmid: insert CD18 gene CDS sequence and Twin-Strep sequence into PB510b plasmid (piggybac system), wherein the Twin-Strep sequence is located at the C terminal of the CD18 gene, and the inserted gene is successfully verified by sequencing, and the PB510b-CD18-c-Twin-Strep plasmid is extracted for standby.

[0079] 2. 1×10 6 LLC- cells were seeded in 30mm culture dish for 24 hours.

[0080] 3. 5ug PB510b-CD18-c-Twin-Strep plasmid and 1ug PBT transposase plasmid were co-transfected into LLC cells using Lipofectamine3000.

[0081] 4. After transfection, normal culture for 48 hours, then add 2.5ug / ml puromycin medium for continuous culture and screening for 7 days.

[0082] 5. Extract cell protein, western blot detection.

[0083] In LLC cells, overexpression of artificially recombined CD18 molecules makes LLC have high levels of CD18 molecules Figure 9 A). CD18 high expression has little effect on cell proliferation, and plate cloning experiments show no significant difference Figure 9 B). However, CD18 high expression can significantly improve the migration and invasion ability of LLC cells Figure 9 C, D). It is proved that CD18 high expression is the key molecule for LLC cells to transform into high-metastatic LLC-ML2 cells.

[0084] Example 9: Knockout of CD18 in LLC-ML2 cells significantly reduces cell growth and migration and invasion ability

[0085] Construct a plasmid to knock out CD18: design 3 pairs of sgRNA according to the CDS sequence of CD18 gene, the specific sequence is as follows: Px458-CD18-1:

[0086] 5-CACCGACACTCACTGCTGCTTGCCC-3; see SEQ ID NO. 1; Px458-CD18-2:

[0087] 5-CACCGGGACTGTTCTTCCTGGGATC-3; see SEQ ID NO. 2;

[0088] Px458-CD18-3: 5-CACCGGACTGTATCCAGTCGGGGCC-3; see SEQ ID NO. 3.

[0089] Synthesize 3 sequences and insert them into Px458 plasmid (containing GFP gene) respectively, and the inserted fragments are successfully sequenced and aligned. Px458-CD18-1 / 2 / 3 plasmids are extracted respectively for standby.

[0090] 2. 2x10 6 LLC-ML2 cells were inoculated in a 60mm culture dish and cultured for 24 hours.

[0091] 3. According to the steps, 5ug of Px458-CD18-1 / 2 / 3 plasmid was added to LLC-ML2 cells respectively for transfection. After normal culture for 48 hours, the LLC-ML2 cells with green fluorescence were sorted by flow cytometry and cultured in a 96-well plate.

[0092] 4. After the monoclonal cells are formed, the culture is gradually expanded, seeded in a 60 mm culture dish until the cells cover the dish, cell lysate is added and cell protein is extracted, after electrophoresis, western blot is used to detect the expression of CD18 protein. The LLC-ML2 cell line with no CD18 protein expression is identified as a complete knockout success.

[0093] In LLC-ML2 cells, CD18 in LLC-ML2 is knocked out using CRISPR / Case9 technology, so that LLC-ML2 cells completely lack the expression of CD18 Figure 10 A). In vitro cell experiments show that after knocking out CD18, the growth and proliferation of cells can be significantly inhibited Figure 10 B C), while the migration and invasion ability of LLC-ML2 cells can be greatly inhibited Figure 10 D). In vivo mouse model experiments also show that after LLC-ML2 cells completely lack CD18, the growth of transplanted tumors is significantly inhibited Figure 10 E), while the distant metastasis ability of LLC-ML2 cells in mice is significantly inhibited Figure 10 F). This fully proves that CD18 high expression is the key molecule for LLC-ML2 cells to have high invasion and metastasis ability.

Claims

1. A method for enhancing the invasive and metastatic ability of lung cancer cells, characterized in that, High expression of CD18 molecules.

2. The method according to claim 1, characterized in that, High expression refers to a ratio of expression levels ≥2 between lung cancer cells and wild-type LLC cells.

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