Murine triple-negative breast cancer high-metastasis cell line and application thereof

Abstract

The invention relates to a murine triple-negative breast cancer high-metastasis cell line and application thereof, and belongs to the technical field of biological medicine. According to the invention, a breast cancer high-metastasis cell E0771 M-8 is screened by using a murine triple negative breast cancer cell line E0771, and is preserved in the China General Microbiological Culture Collection Center on March 12, 2025, and the preservation number is CGMCC NO.46331. The invention further discloses a preparation method of the breast cancer high-metastasis cell E0771. A cell function experiment shows that the in-vitro growth capability, the multiplication capability and the migration capability of the E0771 M-8 cell are enhanced; a mouse tumor-bearing experiment shows that the lung metastasis capability of the E0771 M-8 cell is enhanced. The highly metastatic breast cancer cell E0771 M-8 obtained by the invention provides a usable biological material for deeply researching a molecular mechanism of breast cancer metastasis and developing related antitumor drugs, and has relatively high scientific research and clinical application values.

Description

Technical Field

[0001] The present invention relates to a murine triple-negative breast cancer highly metastatic cell line and its application, belonging to the field of biomedical technology. Background Art

[0002] Currently, the incidence of breast cancer continues to rise globally, and the prevalence in young women has increased significantly. Metastasis is the process by which cancer cells spread from the primary tumor to distant organs, which is a decisive feature of malignant tumor cells and the main cause of death in cancer patients. Despite the progress in early screening techniques (such as mammography, ultrasound, MRI) and targeted therapies (such as trastuzumab), triple-negative breast cancer still lacks effective targets, and the 5-year survival rate of advanced patients is less than 50%.

[0003] Establishing a highly metastatic cell line is of great significance for studying the occurrence, development, invasion, metastasis and recurrence mechanisms of breast cancer. Chinese patent document CN116496990A (application number 202310496522.4) discloses a murine triple-negative breast cancer cell line and its application. A murine triple-negative breast cancer cell line was established through primary culture of excised specimens of murine mammary tumors in vitro, named MCMT-2021. This cell line has an abnormal chromosomal karyotype, can be continuously passaged in vitro while maintaining its characteristics unchanged, and its proliferation doubling time is significantly lower than that of existing murine triple-negative breast cancer cell lines, making it suitable as cell material for developing immunotherapies, diagnostics and drugs related to human triple-negative breast cancer. Chinese patent document CN110079501A (application number 201910396716.0) provides a murine breast cancer circulating tumor cell line 4T1-CTC, which is a good experimental object for studying tumor metastasis in the laboratory, and also provides a good research model for clinically predicting breast cancer metastasis and detecting the sensitivity of chemotherapy drugs, and provides a very good research tool for studying the characteristics of circulating tumor cells and tumor metastasis mechanisms in the laboratory.

[0004] Clinical studies have shown that distant metastasis has always been the main cause of treatment failure and death in breast cancer patients. Autopsy data show that 60% - 80% of patients who died of breast cancer had lung metastases. Therefore, strengthening the research on the metastasis mechanism of breast cancer cells is of great significance, and establishing a reliable lung highly metastatic cell line and animal model is a top priority. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a murine triple-negative breast cancer highly metastatic cell line and its application. This breast cancer highly metastatic cell line can be applied to construct a breast cancer highly metastatic cell model or animal model.

[0006] The technical solution of the present invention is as follows: A highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells was deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 12, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 46331.

[0007] The highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells was obtained by screening the murine triple-negative breast cancer cell line E0771.

[0008] The highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells has stronger lung metastasis ability and cell migration ability than the murine triple-negative breast cancer cell line E0771.

[0009] The progeny cells of the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells described above.

[0010] The application of the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells or its progeny cells in constructing a high-metastasis model of breast cancer.

[0011] A high-metastasis model of breast cancer was obtained by constructing with the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells or its progeny cells described above.

[0012] Preferably according to the present invention, the model includes a cell model and an animal model.

[0013] More preferably, the animal model includes a mouse model.

[0014] More preferably, the breed of the mouse includes C57BL / 6.

[0015] A method for constructing a high-metastasis mouse model of breast cancer includes the step of inoculating the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells or its progeny cells into the mammary fat pad under the fourth pair of nipples of a mouse.

[0016] The application of the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells or its progeny cells or the high-metastasis model of breast cancer in studying the pathogenesis and metastasis mechanism of breast cancer.

[0017] The application of the highly metastatic cell line E0771 M-8 of murine triple-negative breast cancer cells or its progeny cells or the high-metastasis model of breast cancer in screening therapeutic drugs for high-metastasis breast cancer.

[0018] Use of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 or its progeny cells or breast cancer highly metastatic model in studying the drug resistance mechanism of breast cancer.

[0019] Beneficial effects: In the present invention, a breast cancer highly metastatic cell line E0771 M-8 is screened from the murine triple-negative breast cancer cell line E0771. Through cell functional experiments, it is found that the in vitro growth ability, proliferation ability, and migration ability of E0771 M-8 cells are enhanced; through the mouse tumor-bearing experiment, it is found that the lung metastasis ability of E0771 M-8 cells is enhanced. The breast cancer highly metastatic cell E0771 M-8 obtained in the present invention provides a biological material that can be used for in-depth study of the molecular mechanism of breast cancer metastasis and the development of related anti-tumor drugs, and has high scientific research and clinical application value. Description of the drawings

[0020] Figure 1 Flow chart of the operation for screening a breast cancer highly metastatic cell line from the breast cancer cell line E0771; Figure 2 In vivo imaging diagram of C57BL / 6 tumor-bearing mice; Figure 3 Picture of the Transwell migration of breast cancer cells; Figure 4 Bar chart of the number of migrating breast cancer cells; Figure 5 Pictures of breast cancer cells cultured for 0 h, 24 h, and 48 h after scratching; Figure 6 Bar chart of the 24 h cell migration rate of breast cancer cells; Figure 7 Bar chart of the 48 h cell migration rate of breast cancer cells; Figure 8 Colony picture of breast cancer cell clones; Figure 9 Bar chart of the number of breast cancer cell clones formed; Figure 10 Line chart of the proliferation rate of breast cancer cells. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments, but the protection scope of the present invention is not limited thereby. In the following embodiments, unless otherwise specified, all are conventional methods. The experimental materials or reagents used in the following embodiments, unless otherwise specified, are all conventional materials or reagents that can be purchased from the market.

[0022] In the following specific examples, the murine triple-negative breast cancer cell line E0771 was purchased from the American Type Culture Collection (ATCC), and C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0023] In the following specific examples, the cell culture medium used was DMEM medium, and the complete DMEM medium was DMEM medium containing 10% fetal bovine serum; the PBS buffer used was 1×PBS buffer (pH 7.4).

[0024] Example 1 A mammary gland orthotopic tumor model was constructed using the murine triple-negative breast cancer cell line E0771 and C57BL / 6 mice. After the mice developed lung metastases, tumor cells were isolated from the lungs for culture and passage. Then, the cultured and passaged tumor cells were injected into the mice through the mammary fat pad to construct a murine mammary gland orthotopic tumor model. This was repeated eight times to obtain the highly metastatic breast cancer cells E0771M-8. The operation process is as Figure 1 shown, and the specific steps are as follows: (1) Culture breast cancer E0771 cells under sterile conditions. Transfect the luciferase expression plasmid into the cells and screen and culture them in DMEM medium containing g418 (geneticin, 500 μg / mL) for 2 weeks to obtain successfully transfected E0771 Luci cells. (2) Culture the transfected E0771 Luci cells under sterile conditions. Collect the cells in the logarithmic growth phase according to the method of cell passage. Resuspend the cells with sterile PBS buffer and count them. Adjust the cell concentration to 1×10 7 cells / mL. Use a 1 mL syringe to inoculate 100 μL of the cell suspension into the mammary fat pad under the fourth pair of nipples of C57BL / 6 mice to construct a mammary gland orthotopic tumor model. (3) Perform in vivo imaging of the mice weekly to observe whether lung metastases form and screen the mice with earlier lung metastases. (4) Sacrifice the mice by cervical dislocation, immerse them in alcohol for 1 min, isolate the lung tumor tissue of the mice in a sterile operating table, transfer the tumor tissue to a 6-well cell culture plate, add 1 mL of medium, and cut the tumor tissue into small pieces with a volume of about 1 mm 3 using a sterile scissors. Then add 1 mL of tissue digestion solution (the tissue digestion solution is DMEM medium containing 2 mg / mL collagenase IV and 2 mg / mL DNase) to each well, place it in an incubator at 37°C for digestion for 30 min, shake and mix it once every 10 minutes. After digestion, add 1 mL of complete DMEM medium to terminate the digestion. (5) Filter the tumor tissue suspension obtained in the previous step through a sterile filter membrane with a pore size of 70 μm into a 50 mL sterile centrifuge tube. Gently grind the tissue mass remaining on the filter membrane with a grinding rod, and add 1 mL of complete DMEM medium to rinse to obtain a tumor cell suspension. (6) Centrifuge the tumor cell suspension obtained in step (5) at 4 °C and 1000 rpm for 5 minutes. Discard the supernatant, wash the cell pellet twice with sterile PBS buffer, discard the supernatant, resuspend the cells with 10 mL of complete DMEM medium, then add penicillin-streptomycin, and place it in a 37 °C, 5% CO 2 incubator for primary culture and subculture to obtain breast cancer cells E0771M-1. Collect the E0771 M-1 cells in the logarithmic growth phase of subculture, and repeat the above screening process according to steps (2)-(6); (7) Repeat the above screening process 8 times to obtain a total of breast cancer cells E0771 M-1, E0771 M-2, E0771 M-3, E0771 M-4, E0771 M-5, E0771 M-6, E0771 M-7, E0771 M-8.

[0025] Among them, the highly metastatic cells E0771 M-8 of the murine triple-negative breast cancer cell line E0771 were deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 12, 2025. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 46331.

[0026] Example 2 Detection of cell metastasis ability Divide 6-week-old female C57BL / 6 mice into 2 groups evenly, with 6 mice in each group. Culture E0771 and E0771 M-8 cells respectively according to the method of subculture. Collect the cells in the logarithmic growth phase, resuspend the cells with sterile PBS buffer and adjust the cell concentration. Inject E0771 and E0771 M-8 cell suspensions into the tail veins of mice respectively to construct a tumor metastasis model. Inject 1×10 6 cells per mouse / 100 μL, and perform in vivo imaging of mice on the 5th day.

[0027] The in vivo imaging of two groups of C57BL / 6 mice on the 5th day is as Figure 2 shown. The results show that almost no lung metastases were formed by E0771 cells, while all E0771 M-8 cells formed lung metastases, indicating that E0771 M-8 cells have stronger lung metastasis ability.

[0028] Example 3 Detection of cell migration ability (1)Transwell migration assay was performed using Transwell chambers. E0771, E0771 M-1, E0771 M-4, and E0771 M-8 cells were cultured separately according to the method of subculture. Cells in the logarithmic growth phase were collected, resuspended in DMEM medium, and the cell concentration was adjusted. The cell concentration was adjusted to 2×10 5 cells / mL. 4×10 4 cells were inoculated into the upper chamber of the chamber respectively. The bottom of the chamber contained DMEM medium with 20% FBS. After incubation for 24 h, the cells were fixed with methanol for 10 min and stained with 1% crystal violet for 15 min. Then, the cells on the top surface of the membrane were wiped off, and the cells on the lower surface were examined under an inverted optical microscope. As Figure 3 shown, the three horizontal groups of pictures represent three parallels in each group.

[0029] The number of cells in three random fields of view in each group was counted under a microscope with 40× magnification, which was the number of migrated or invaded cells. A bar chart was plotted using GraphPad prism 10.1.2. The abscissa was the cell type, and the ordinate was the number of migrated cells. As Figure 4 shown, the average number of migrated cells of E0771 cells was 5, the average number of migrated cells of E0771 M-1 cells was 22.3, the average number of migrated cells of E0771 M-4 cells was 37, and the average number of migrated cells of E0771 M-8 cells was 55. Among them, the number of migrated cells of E0771 M-8 cells was the largest, and there were significant biological differences, indicating that the migration ability of E0771 M-8 cells was stronger.

[0030] (2)The scratch assay was used to evaluate the cell migration ability. E0771, E0771M-1, E0771 M-4, and E0771 M-8 cells were cultured separately according to the method of subculture. Cells in the logarithmic growth phase were collected, resuspended in DMEM medium, and the cell concentration was adjusted. The cell concentration was adjusted to 5×10 5 cells / mL. 4 groups of cells in the logarithmic growth phase were seeded into 6-well plates at 1×10 6 cells / well respectively. After culturing for 6 h, a scratch was made with a 200 μL sterile pipette tip. The old medium was discarded, and the cells were washed 3 times with PBS buffer. Then, DMEM medium containing 2% FBS was added and cultured for 48 h. Photos were taken under an inverted microscope at 0 h, 24 h, and 48 h respectively. As Figure 5 shown, and the migration rate was calculated according to the following formula: Migration rate of cells at 24 h or 48 h % = (Scratch area at 0 h - Scratch area at 24 h or 48 h) / Scratch area at 0 h × 100% The migration rate results of the four types of cells at 24 h and 48 h are respectively as Figure 6 ,Figure 7 As shown, the cell migration ability of E0771 M-8 cells was significantly enhanced compared with that of E0771, E0771 M-1, and E0771 M-4 cells.

[0031] After multiple passages of culture, the cell migration ability of E0771 M-8 cells did not degenerate.

[0032] Example 4 Detection of cell growth The growth of E0771 and E0771 M-8 cells was evaluated by cell colony formation assay. E0771 cells, E0771 M-4 cells, and E0771 M-8 cells were cultured according to the method of subculture. E0771 cells and E0771 M-4 cells in the logarithmic growth phase were collected, and E0771 M-8 cells were continuously subcultured to the 25th passage. E0771 M-8 cells in the logarithmic growth phase of the 5th and 25th passages were collected. The cells were resuspended with DMEM medium and the cell concentration was adjusted to 250 cells / mL. E0771 cells, E0771 M-4 cells, E0771 M-8 cells of the 5th passage, and E0771 M-8 cells of the 25th passage were inoculated into 6-cm culture dishes at a density of 500 cells / dish, with three replicates for each group of cells, and incubated in an incubator at 37 °C, 95% air and 5% CO 2 2. The culture medium was changed every two days. After 10 days, a large number of cell colonies were observed under the microscope. The culture medium was discarded, and the cells were gently washed once with PBS buffer. The PBS buffer was discarded, and the cells were fixed with methanol for 10 min and stained with 0.5% crystal violet for 15 min. After washing 10 times with PBS buffer, obvious cell colonies could be observed under the microscope and photographed, as Figure 8 shown. A cell colony with a cell number ≥50 was counted as 1 clone.

[0033] The number of clones was calculated using Image J software, and a bar chart was drawn using GraphPad prism 10.1.2. The results are shown in Figure 9 Figure [X], the number of clones formed by E0771 M-8 cells of the 5th and 25th passages was increased compared with that of E0771 cells and E0771 M-4 cells, and there were significant biological differences. There was no significant difference in the number of clones formed by E0771 M-8 cells of the 5th and 25th passages, indicating that the growth ability of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 did not change after multiple passages of culture.

[0034] Example 5 Cell proliferation detection Cell proliferation was evaluated using the CCK-8 assay. E0771, E0771 M-1, E0771 M-4, and E0771 M-8 cells were cultured separately according to the method of subculture. When the cells reached the logarithmic growth phase, they were resuspended in DMEM medium containing 10% FBS and the cell concentration was adjusted. The cell concentration was adjusted to 15,000 cells / mL. The four types of cells were seeded into 96-well plates at 1,500 cells / well and incubated at 37°C and 5% CO 2 ₂. On the 0th, 1st, 2nd, and 3rd days of incubation, the original medium was aspirated, and DMEM medium without cells containing 10% FBS was added. Then, 10 μL of CCK-8 reagent was added to each well and incubated for 3 h. The absorbance was measured using a microplate reader at 450 nm. Each experiment was repeated 3 times. The cell proliferation rate was calculated according to the following formula: Cell proliferation rate % = (Absorbance on the Nth day - Absorbance on the 0th day) / Absorbance on the 0th day × 100% The results are as Figure 10 shown. The proliferation ability of E0771 M-8 cells was significantly enhanced compared with that of E0771 cells, E0771 M-1 cells, and E0771 M-4 cells, and the results were statistically significant.

Claims

1. A murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 12, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.46331.

2. The progeny cells of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 according to claim 1.

3. Use of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 described in claim 1 or the progeny cell described in claim 2 in constructing a breast cancer highly metastatic model.

4. A breast cancer high metastasis model, characterized in that: The method is obtained by constructing the mouse triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 according to claim 1 or the progeny cell according to claim 2.

5. The breast cancer high metastasis model according to claim 4, characterized in that: Including cell models and animal models.

6. The breast cancer high metastasis model according to claim 5, characterized in that: The animal model includes a mouse model.

7. A method for constructing a mouse model of breast cancer with high metastasis, characterized in that: The method comprises the steps of: inoculating the mouse triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 described in claim 1 or the progeny cell described in claim 2 into the mammary fat pad below the fourth pair of nipples of mice.

8. Use of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 according to claim 1 or the progeny cell according to claim 2 or the breast cancer highly metastatic model according to any one of claims 4 to 6 in studying the pathogenesis and metastasis mechanism of breast cancer.

9. Use of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 according to claim 1 or the progeny cell according to claim 2 or the breast cancer highly metastatic model according to any one of claims 4 to 6 in screening therapeutic drugs for highly metastatic breast cancer.

10. Use of the murine triple-negative breast cancer cell line E0771 highly metastatic cell E0771 M-8 according to claim 1 or the progeny cell according to claim 2 or the breast cancer highly metastatic model according to any one of claims 4 to 6 in studying the drug resistance mechanism of breast cancer.

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