High-metastasis cell 4T1 M-8 of murine triple negative breast cancer cell line 4T1 and application of high-metastasis cell 4T1 M-8

Abstract

The invention relates to a mouse triple negative breast cancer cell line 4T1 highly-metastatic cell 4T1 M-8 and application thereof, and belongs to the technical field of biological medicine. The breast cancer high-metastasis cell 4T1 M-8 is screened by using a mouse source triple negative breast cancer cell line 4T1 and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2024, and the preservation number is CGMCC NO.46135. The breast cancer high-metastasis cell 4T1 M-8 has the advantages that the breast cancer high-metastasis cell 4T1 M-8 is obtained; cell functional experiments show that the in-vitro proliferation and migration capabilities of the 4T1 M-8 cells are enhanced; a mouse tumor-bearing experiment shows that the lung metastasis capability of the 4T1 M-8 cell is enhanced. The highly-metastatic breast cancer cell 4T1 M-8 obtained by the invention provides a usable biological material for deeply researching a molecular mechanism of breast cancer metastasis and developing related anti-tumor drugs, and has relatively high scientific research and clinical application values.

Description

Technical Field

[0001] The present invention relates to a highly metastatic cell 4T1 M-8 of a murine triple-negative breast cancer cell line 4T1 and its application, belonging to the technical field of biomedicine. Background Art

[0002] Worldwide, cancer is the leading cause of death, and the World Health Organization attributes more than 90% of cancer-related deaths to metastasis. Among the incidence rates of female cancers, breast cancer has a relatively high incidence rate and has become one of the common diseases that endanger the physical and mental health of women. Recurrence and metastasis are the main causes of death in breast cancer patients. However, cancer metastasis is a complex process involving many complex mechanisms such as the genetic basis of tumor metastasis, host dependence, and tissue affinity. Due to the wide range involved and the long relevant scientific research cycle, the current research on its metastasis mechanism has not been fully clarified.

[0003] Establishing highly metastatic and highly differentiated cell lines has practical significance for the research on the metastasis mechanism of various cancers. Chinese patent document CN103275933A (application number 201310164388.4) discloses a highly differentiated human liver cancer cell line HLCZ01, which can be used as a cell model and animal model to support hepatitis virus infection, and can also be used for the preparation, screening or evaluation of anti-hepatitis virus drugs and anti-tumor drugs. Chinese patent document CN114196617A (application number 202111578132.9) discloses an organ-specific breast cancer metastasis model and its establishment method and application. The metastatic breast cancer cells constructed in this invention are derived from the 4T07 cell line and have higher migration and metastasis abilities compared with the 4T07 cell line, and can be used to establish a breast cancer metastasis animal model.

[0004] The organ-specific highly metastatic cell line isolated from a low-metastatic cell line can establish a model that can better amplify the influencing factors of tumor self-specific metastasis. Moreover, establishing a highly metastatic breast cancer cell line can significantly shorten the research cycle of basic scientific research related to tumor metastasis and has important significance for the research on the metastasis mechanism of breast cancer. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a highly metastatic cell 4T1 M-8 of a murine triple-negative breast cancer cell line 4T1 and its application. This highly metastatic breast cancer cell 4T1 M-8 can be used to construct a highly metastatic breast cancer cell model or animal model.

[0006] The technical solution of the present invention is as follows: A highly metastatic cell 4T1 M-8 of a murine triple-negative breast cancer cell line 4T1 ( Mus musculus), was deposited with the China General Microbiological Culture Collection Center on November 14, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 46135.

[0007] The highly metastatic cell line 4T1 M-8 of murine triple-negative breast cancer cells was obtained by screening the murine triple-negative breast cancer cell line 4T1.

[0008] The lung metastasis ability, cell growth ability, and cell migration ability of the highly metastatic cell line 4T1 M-8 of murine triple-negative breast cancer cells are stronger than those of the murine triple-negative breast cancer cell line 4T1.

[0009] The progeny cells of the highly metastatic cell line 4T1 M-8 of murine triple-negative breast cancer cells described above.

[0010] The application of the highly metastatic cell line 4T1 M-8 of murine triple-negative breast cancer cells or its progeny cells in constructing a breast cancer cell model or an animal model.

[0011] Preferably according to the present invention, the cell model or the animal model can be used to study the pathogenesis and metastasis mechanism of breast cancer.

[0012] Preferably according to the present invention, the cell model or the animal model can be used to screen drugs for treating highly metastatic breast cancer.

[0013] Preferably according to the present invention, the cell model or the animal model can be used to study the drug resistance mechanism of breast cancer.

[0014] Preferably according to the present invention, the animal model includes a mouse model.

[0015] More preferably, the breeds of the mice include BALB / c and Nude.

[0016] A method for constructing a highly metastatic mouse model of breast cancer, comprising the following steps: inoculating the highly metastatic cell line 4T1 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.

[0017] Beneficial effects: The present invention uses the murine triple-negative breast cancer cell line 4T1 to screen out a highly metastatic breast cancer cell line 4T1 M-8. Through cell functional experiments, it is found that the in vitro proliferation and migration abilities of 4T1 M-8 cells are enhanced; through mouse tumor-bearing experiments, it is found that the lung metastasis ability of 4T1 M-8 cells is enhanced. The highly metastatic breast cancer cell line 4T1 M-8 obtained in the present invention provides a biological material that can be used for in-depth research on 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

[0018] Figure 1 It is a flowchart of the operation for screening a highly metastatic breast cancer cell line from the breast cancer cell line 4T1; Figure 2 It is a flowchart of the operation for detecting the metastasis ability of breast cancer cells; Figure 3 It is a survival curve of BALB / c tumor-bearing mice; Figure 4 It is an in vivo imaging diagram of BALB / c tumor-bearing mice; among them, (A) is the in vivo imaging diagram of the mice; (B) is the scatter plot of the photon flux in the lungs of breast cancer cells; Figure 5 It is a colony picture of breast cancer cell clones; Figure 6 It is a bar chart of the number of breast cancer cell clones; Figure 7 It is a picture of the Transwell migration of breast cancer cells; Figure 8 It is a bar chart of the number of breast cancer cell migrations; Figure 9 It is a picture of breast cancer cells after culturing for 24 hours after scratching; Figure 10 It is a bar chart of the migration rate of breast cancer cells; Figure 11 It is a flow cytometry result diagram of breast cancer cells; Figure 12 It is a bar chart of the apoptosis percentage of breast cancer cells; Figure 13 It is a line chart of the proliferation of breast cancer cells. Detailed Embodiments

[0019] The present invention will be further described below in conjunction with the accompanying drawings of 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.

[0020] In the following specific examples, the murine triple-negative breast cancer cell line 4T1 was purchased from the American Type Culture Collection (ATCC), and BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0021] 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 7.0).

[0022] Example 1 A murine orthotopic breast tumor model was constructed using the murine triple-negative breast cancer cell line 4T1 and BALB / c 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 orthotopic breast tumor model. This process was repeated eight times to obtain the highly metastatic breast cancer cells 4T1 M-8. The operation process is as Figure 1 shown, and the specific steps are as follows: (1) Culture breast cancer 4T1 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 4T1 Luci cells; (2) Culture the transfected 4T1 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, and 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 BALB / c mice to construct an orthotopic breast 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 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 is completed, add 1 mL of complete DMEM medium to terminate 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 4T1M-1. Collect the 4T1M-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 4T1M-1, 4T1M-2, 4T1M-3, 4T1M-4, 4T1M-5, 4T1M-6, 4T1M-7, 4T1M-8.

[0023] Among them, the above-mentioned mouse-derived triple-negative breast cancer cell line 4T1 highly metastatic cell 4T1M-8 ( Mus musculus ), was obtained from Jinan, Shandong Province, China in September 2024, and was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on November 14, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 46135.

[0024] Example 2 Detection of cell metastasis ability Divide 5-week-old female BALB / c mice into 2 groups evenly, with 6 mice in each group. Culture 4T1 and 4T1M-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 4T1 and 4T1M-8 cell suspensions respectively at the mammary fat pad under the fourth pair of nipples of the mice to construct a mammary in situ tumor model. Inject 1×10 6 cells / 100 μL for each mouse, continuously observe the survival status of the mice, and perform in vivo imaging of the mice on the 21st day. The operation process is as Figure 2 shown.

[0025] The survival curves of the two groups of BALB / c tumor-bearing mice are as Figure 3 shown. The survival period of the mice in the group inoculated with 4T1 cells is longer, and there is a significant difference in the survival period compared with the mice in the group inoculated with 4T1M-8 cells.

[0026] The in vivo imaging of the two groups of BALB / c mice on the 21st day is as Figure 4As shown, the results indicate that almost no lung metastases were formed by 4T1 cells, while all 4T1 M-8 cells formed lung metastases, suggesting that 4T1 M-8 cells have a stronger ability to metastasize to the lungs.

[0027] Example 3 Detection of cell growth The growth of 4T1 and 4T1 M-8 cells was evaluated by a cell colony formation assay. 4T1 and 4T1 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 250 cells / mL. The two types of cells 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 for 2 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 were observed and photographed under the microscope, as Figure 5 shown. A clone was counted as having ≥50 cells.

[0028] The number of clones was calculated using Image J software, and a bar graph was plotted using GraphPad prism 10.1.2. The abscissa was the cell type, and the ordinate was the number of clones. The results were as Figure 6 shown. More clones were formed by 4T1 M-8 cells than by 4T1 cells, and there were significant biological differences.

[0029] Example 4 Detection of cell migration ability (1) A Transwell migration assay was performed using a Transwell chamber. 4T1, 4T1 M-1, 4T1 M-4, and 4T1 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, and 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 7 shown.

[0030] The number of cells in three random fields of view in each group was counted under a 40-fold magnifying microscope, which was the number of migrated or invaded cells. A bar chart was plotted using GraphPad prism 10.1.2, with the cell type on the abscissa and the number of migrated cells on the ordinate. The results are as Figure 8 shown. Among 4T1, 4T1 M-1, 4T1 M-4, and 4T1 M-8 cells, the number of migrated cells of 4T1 M-8 cells was the largest, and there were significant biological differences, indicating that the migration ability of 4T1 M-8 cells was stronger.

[0031] (2)The scratch assay was used to evaluate the cell migration ability. 4T1, 4T1 M-1, 4T1 M-4, and 4T1 M-8 cells were cultured respectively according to the method of subculture. Cells in the logarithmic growth phase were collected, resuspended with DMEM medium, and the cell concentration was adjusted. The cell concentration was adjusted to 5×10 5 cells / mL. The four groups of cells in the logarithmic growth phase were seeded in 6-well plates at 1×10 6 cells / well. 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 24 h. Photos were taken under an inverted microscope at 0 h and 24 h, as Figure 9 shown, and the migration rate was calculated according to the following formula: Migration rate % = (scratch area at 0 h - scratch area at 24 h) / scratch area at 0 h × 100% The migration rate results of different cells are as Figure 10 shown. The average migration rate of 4T1 cells at 24 h was 49.29%, the average migration rate of 4T1 M-1 cells at 24 h was 59.43%, the average migration rate of 4T1 M-4 cells at 24 h was 61.53%, and the average migration rate of 4T1 M-8 cells at 24 h was 77.23%. That is, the migration ability of 4T1 M-8 cells was significantly enhanced compared with 4T1, 4T1 M-1, and 4T1 M-4 cells.

[0032] After multiple subcultures, the migration ability of 4T1 M-8 cells did not degenerate.

[0033] Example 5 Cell apoptosis detection 4T1 and 4T1 M-8 cells were cultured in 6-well plates respectively according to the method of subculture. When cultured to the logarithmic growth phase, adherent cells were digested with trypsin (400 μL per well), centrifuged at 4 °C and 300 G for 5 min, and the supernatant was discarded; after resuspension, 1 - 5×10 5Transfer cells to an EP tube, set three replicate groups for each group. Suspend the cells and centrifuge at 4°C and 300G for 5 min, discard the supernatant, wash the cells twice with pre-cooled PBS buffer, each time centrifuge at 300G and 4°C for 5 min. Resuspend each tube with 1 mL of pre-cooled absolute ethanol, incubate at 4°C for 2 h to fix the cells, and discard the absolute ethanol after centrifugation. Wash the cells with pre-cooled PBS buffer, centrifuge at 300G and 4°C for 5 min, and discard the supernatant. Add 5 μL of Annexin V-FITC and 5 μL of PI Staining Solution, gently blow to mix evenly; incubate in the dark at room temperature (20 - 25°C) for 10 min; add 400 μL of 1×Binding Buffer, gently mix well. Detect the stained samples with a flow cytometer within 1 h, and the results are as Figure 11 . Typical cells can be divided into three subpopulations. Viable cells are negative for both V-FITC and PI, early apoptotic cells are positive for V-FITC alone, and late apoptotic cells are positive for both V-FITC and PI. Calculate the percentage of apoptotic cells according to the following formula: Percentage of apoptotic cells = (Number of early apoptotic cells + Number of late apoptotic cells) / Total number of cells × 100% The calculated results are as Figure 12 shown. The average percentage of apoptotic 4T1 cells is 2.5%, and the average percentage of apoptotic 4T1 M-8 cells is 1.23%. 4T1 M-8 cells are less likely to undergo apoptosis compared to 4T1 cells.

[0034] Example 6 Cell proliferation detection Evaluate cell proliferation using the CCK-8 assay. Culture 4T1, 4T1 M-1, 4T1 M-4, and 4T1 M-8 cells respectively according to the method of subculture. When cultured to the logarithmic growth phase, resuspend the cells with DMEM medium containing 10% FBS and adjust the cell concentration to 15000 cells / mL. Seed 4 types of cells at 1500 cells / well in a 96-well plate and incubate at 37°C and 5% CO 2 conditions. On the 0th, 1st, 2nd, and 3rd days of incubation, aspirate the original medium, add cell-free DMEM medium containing 10% FBS, and add 10 μL of CCK-8 reagent to each well. Incubate for 3 h, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Each experiment is repeated 3 times. Calculate the cell proliferation rate 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 13 shown. The 4T1 M-8 cells have the strongest proliferation ability, followed by 4T1 M-4, and the 4T1 cells have the weakest proliferation ability. The results are statistically significant.

Claims

1. A murine triple-negative breast cancer cell line 4T1 highly metastatic cell 4T1 M-8 ( Mus musculus ), characterized in that, It was deposited in the General Microbiology Center of China Culture Collection Administration on November 14, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.46135.

2. The progeny cells of the murine triple-negative breast cancer cell line 4T1 highly metastatic cell 4T1 M-8 according to claim 1.

3. Use of the murine triple-negative breast cancer cell line 4T1 highly metastatic cell 4T1 M-8 according to claim 1 or the progeny cell according to claim 2 in constructing a breast cancer cell model or an animal model.

4. The use according to claim 3, characterized in that The cell model or animal model can be used to study the pathogenesis and metastasis mechanism of breast cancer.

5. The use according to claim 3, characterized in that The cell model or animal model can be used to screen drugs for treating highly metastatic breast cancer.

6. The use according to claim 3, characterized in that The cell model or animal model can be used to study the drug resistance mechanism of breast cancer.

7. The use according to claim 3, characterized in that The animal model includes a mouse model.

8. The use according to claim 7, characterized in that The mouse strains include BALB / c and Nude.

9. A method for constructing a mouse model of breast cancer with high metastasis, characterized in that: The method comprises the following steps: inoculating the mouse triple-negative breast cancer cell line 4T1 highly metastatic cell 4T1 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.

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