A mouse triple-negative breast cancer dual tumor model and a preparation method and application thereof

Abstract

The application relates to the field of biotechnology, and particularly discloses a preparation method of a mouse triple-negative breast cancer double-tumor model, which comprises the following steps: providing two kinds of triple-negative breast cancer cells; transplanting the two kinds of triple-negative breast cancer cells to two mammary pads of a mouse respectively; feeding the mouse for 7-10 days to obtain a mouse triple-negative breast cancer double-tumor model; wherein the two mammary pads are located on the same side of the mouse and are not adjacent. The application constructs a triple-negative breast cancer double-tumor model by transplanting different tumor cells to the same mouse, eliminates the problem that the drug reaction effect is different due to individual differences of tumors, greatly restores the heterogeneity and complexity of real tumors, and has great application prospect in the research on development characteristics of triple-negative breast cancer and novel treatment methods.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a mouse triple-negative breast cancer double tumor model and its preparation method and application. Background Technology

[0002] Breast cancer has surpassed lung cancer to become the most diagnosed cancer type globally, with 2.26 million new cases in 2020, accounting for 11.7% of all new cancer cases worldwide. As the most common cancer among women, breast cancer accounts for a staggering 24.5%. Based on molecular subtypes, breast cancer is broadly classified into three categories: hormone receptor-positive breast cancer, HER2-positive breast cancer, and triple-negative breast cancer. Triple-negative breast cancer (TNBC) refers to a type of breast cancer characterized by very low expression levels (<1%) of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2), accounting for 11%-20% of all breast cancers. Due to its rapid progression and high metastasis rate, triple-negative breast cancer is difficult to treat; and patients often experience recurrence after 3-5 years, resulting in a very poor prognosis.

[0003] Therefore, there is an urgent need for stable and representative tumor models to study the development characteristics and novel treatment methods of triple-negative breast cancer. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a mouse triple-negative breast cancer dual-tumor model and its preparation method and application, aiming to solve the problem of the lack of a stable and representative tumor model for studying triple-negative breast cancer.

[0005] The technical solution of this application is as follows:

[0006] The first aspect of this application provides a method for preparing a mouse triple-negative breast cancer double tumor model, comprising the steps of: providing two types of triple-negative breast cancer cells; transplanting the two types of triple-negative breast cancer cells onto two mammary pads of a mouse respectively; feeding the mouse for 7-10 days to obtain a mouse triple-negative breast cancer double tumor model; wherein the two mammary pads are located on the same side of the mouse and are not adjacent.

[0007] Preferably, the triple-negative breast cancer cells are selected from one of the following: mouse breast cancer cells EMT6, mouse breast cancer cells 4T1, and human breast cancer cells MD-MBA-231.

[0008] Preferably, the method for preparing triple-negative breast cancer cells includes the steps of: providing cryopreserved triple-negative breast cancer cells; reviving and expanding the cryopreserved triple-negative breast cancer cells to obtain the triple-negative breast cancer cells.

[0009] Preferably, the two mammary pads are located on the same side of the mouse, and are the second and fifth mammary pads starting from the head of the mouse.

[0010] Preferably, the step of transplanting the two types of triple-negative breast cancer cells onto two mammary pads of a mouse includes: anesthetizing the mouse and disinfecting the mouse's abdomen; and injecting the two types of triple-negative breast cancer cells into the two mammary pads respectively.

[0011] Preferably, the mice are selected from BALB / c or immunodeficient mice.

[0012] Preferably, the two types of triple-negative breast cancer cells are transplanted into two mammary pads of mice, respectively, and the number of transplanted triple-negative breast cancer cells is 5 × 10⁻⁶. 5 indivual.

[0013] A second aspect of this application provides a method for preparing a mouse triple-negative breast cancer double-tumor model as described in this application.

[0014] A third aspect of this application provides an application of the mouse triple-negative breast cancer dual-tumor model in this application for measuring the composition of the microenvironment within breast cancer tumor tissue.

[0015] The beneficial effects of this application are:

[0016] This application constructs a triple-negative breast cancer dual-tumor model by transplanting different tumor cells into the same mouse, eliminating the problem of differences in drug response due to individual differences in tumor location. At the same time, the different tumor cells can retain the heterogeneity of the tumor, greatly restoring the heterogeneity and complexity of real tumors. It has great application prospects in the research of the development characteristics of triple-negative breast cancer and new treatment methods. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0018] Figure 1 A schematic flowchart illustrating the method for preparing a mouse triple-negative breast cancer dual-tumor model provided in this application embodiment;

[0019] Figure 2 This is a schematic diagram of tumor volume provided in an embodiment of this application;

[0020] Figure 3 Tumor growth curves provided for embodiments of this application;

[0021] Figure 4 Tumor growth curves provided for embodiments of this application;

[0022] Figure 5 Flow cytometry plots for analyzing the intratumoral cell composition of the drug-treated group provided in the embodiments of this application;

[0023] Figure 6 Flow cytometry plot of intratumoral cell composition analysis of the control group provided in the embodiments of this application. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0025] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0026] Currently, the most common method for preparing triple-negative breast cancer tumor models is to construct murine breast cancer cell lines 4T1, EMT6, or MD-MBA-231 into Balb / c or immunodeficient mice to form in situ or subcutaneous tumors. However, almost all animal models are single-tumor-bearing mice, and tumor size and drug response are easily affected by individual mouse differences and cell line differences. Therefore, single-tumor models cannot accurately and effectively reflect the therapeutic effect of drugs on triple-negative breast cancer.

[0027] Based on this, a first aspect of the embodiments of this application provides a method for preparing a mouse triple-negative breast cancer dual-tumor model, comprising the steps of:

[0028] S1 provides two types of triple-negative breast cancer cells.

[0029] In some embodiments, the triple-negative breast cancer cells are selected from one of the following: murine breast cancer cells EMT6, murine breast cancer cells 4T1, and human breast cancer cells MD-MBA-231. For example, the two types of triple-negative breast cancer cells are murine breast cancer cells EMT6 and murine breast cancer cells 4T1, murine breast cancer cells EMT6 and human breast cancer cells MD-MBA-231, or murine breast cancer cells 4T1 and human breast cancer cells MD-MBA-231.

[0030] It should be noted that the choice of the two types of triple-negative breast cancer cell lines is related to the mice used. When using BALB / c mice, murine breast cancer cells EMT6 and 4T1 should be selected; when using immunodeficient mice, human breast cancer cells MD-MBA-231 can be selected.

[0031] In some embodiments, the method for preparing triple-negative breast cancer cells includes the steps of: providing cryopreserved triple-negative breast cancer cells; thawing and expanding the cryopreserved triple-negative breast cancer cells to obtain the triple-negative breast cancer cells.

[0032] Cells are usually stored by cryopreservation. Before use, cells need to be thawed. The specific steps include: seeding the cryopreserved cells in T75 cell culture flasks, digesting them when the confluence reaches 80-90%, centrifuging them at 1000 rpm for 5 min, discarding the supernatant, adding 5 mL of PBS, gently blowing and washing, and finally centrifuging. This process is repeated twice.

[0033] After resuscitation, the cells need to be expanded to meet the experimental requirements. The specific steps include: seeding the cells into a new T75 culture flask at a ratio of 1:4 to 1:6; when the confluence of cells reaches 80-90%, digesting them from the T75 culture flask, centrifuging at 1000 rpm for 5 min, discarding the supernatant, adding 5 mL of PBS, gently blowing and washing, and finally centrifuging. Repeat this process twice.

[0034] Typically, after cell expansion, the number of cells per unit volume in the cell suspension will exceed the experimental requirements. Therefore, in some implementations, it is necessary to adjust the cell density. Specific steps include: aspirating 10 μL of the revived cell suspension and calculating the cell concentration using a cell counting chamber under an optical microscope. The cell suspension is then diluted according to the required number of cells for the experiment to adjust the density to achieve the desired transplantation density.

[0035] S2. Two types of triple-negative breast cancer cells were transplanted into two mammary pads of mice, respectively.

[0036] In this case, the two mammary pads of the mouse are located on the same side of the mouse and are not adjacent.

[0037] Two types of triple-negative breast cancer cells were transplanted into the same-side, non-adjacent mammary pads of mice. The same-side ensured that the growth environment of the two types of triple-negative breast cancer cells was similar, while the non-adjacent nature reduced the burden on the mice and prevented the triple-negative breast cancer cells from affecting the mice's activity as they grew.

[0038] In some embodiments, the two mammary pads are located on the same side of the mouse, specifically the second and fifth mammary pads starting from the mouse's head. The second mammary pad is located under the mouse's armpit, and the fifth mammary pad is located at the root of the mouse's thigh. The two mammary pads are located on the same side and are a certain distance apart. As triple-negative breast cancer cells grow, the growth environments of the two types of cancer cells are similar, so they will not affect each other or the mouse's daily activities.

[0039] Furthermore, the specific steps of the transplantation include:

[0040] (1) Mouse anesthesia

[0041] The mouse was placed in the anesthesia chamber of the anesthesia machine. The inhalation concentration of the anesthetic isoflurane was adjusted to 2.5, and the oxygen concentration to 0.5. The mouse was anesthetized after 2-5 minutes. The mouse was then placed on the operating table, and an inhalation mask was put on for continuous anesthesia. The limbs and chest were fixed with tape.

[0042] (2) Triple-negative breast cancer cell transplantation

[0043] Disinfect the mouse's abdomen with an alcohol swab. Gently shave the fur off the mouse's abdomen with a razor and disinfect again. Use tweezers to grasp the skin slightly to the left of the abdomen, cut open the skin and peritoneum with scissors, locate the mammary pad for transplanting triple-negative breast cancer cells, and remove it with tweezers. Draw the prepared triple-negative breast cancer cells from a 1.5mL centrifuge tube using an insulin syringe and slowly inject them into the mouse's mammary gland.

[0044] In some implementations, the number of triple-negative breast cancer cells transplanted is 5 × 10⁻⁶. 5 At this implantation density, two tumors can form almost simultaneously, with a size difference of approximately 100 mm. 3 Within a certain range, and with minimal physical burden on mice.

[0045] (3) Surgery

[0046] Take a No. 6 suture and hold the bottom of the suture hook with A-frame tweezers, ensuring the hook is facing you. With your left hand, hold the tweezers and pinch the skin on one side of the mouse. Insert the needle shallowly through the surface of the skin, then through the very tip of the skin on the other side. Remove the suture hook with the tweezers, discard the tweezers, and pull the suture with your left hand until only a few centimeters remain on the right side. With your right hand, use scissors to suture the wound once on each side. Trim any excess suture on both sides. Continue suturing slightly below the wound. Use the same method to suture the wounds created on the mouse's abdomen and chest.

[0047] S3. Mice were fed for 7-10 days to obtain a mouse triple-negative breast cancer double tumor model.

[0048] After transplanting two types of triple-negative breast cancer cells into mice, the mice were fed normally for 7-10 days. When both types of triple-negative breast cancer cells had grown to a certain extent, the mouse triple-negative breast cancer dual-tumor model was completed.

[0049] The second aspect of this application provides a method for preparing a mouse triple-negative breast cancer double-tumor model according to the embodiments of this application.

[0050] A third aspect of this application provides the application of the mouse triple-negative breast cancer dual-tumor model in this application in detecting the composition of the microenvironment within breast cancer tumor tissue.

[0051] The following will provide further explanation through specific embodiments.

[0052] Example 1: Preparation of a mouse triple-negative breast cancer dual-tumor model

[0053] I. Preparation of Triple-Negative Breast Cancer Cells

[0054] Experimental materials:

[0055] Mouse breast cancer cell line EMT6, mouse breast cancer cell line 4T1, 1640 medium, Waymouth's MB752 / 1 medium (Procell, catalog number: PM151215), bovine fetal serum FBS (Corning), penicillin-streptomycin mixture with double antibiotics (100×), trypsin-EDTA (0.25%), balanced salt buffered PBS (1×), T75 cell culture flasks, cell counting chambers, cell counters, pipettes of various sizes and sterile pipette tips, optical microscope, 37 ℃ constant temperature incubator, clean bench, centrifuge, etc.

[0056] Implementation plan:

[0057] (1) Cell resuscitation: Take one cell each of frozen EMT6 and 4T1 cells and resuscitate them in T75 cell culture flasks. When the confluence of the two types of tumor cells is 80%-90%, digest them from the T75 culture flasks, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL of PBS, gently pipette and wash, centrifuge, and repeat twice.

[0058] (2) Cell expansion: Cells were seeded into new T75 culture flasks at a ratio of 1:4 to 1:6, and the number of cells cultured was determined according to experimental requirements. 5 × 10⁶ cells were seeded per mouse. 5 4T1 cells and 5×10 5Using EMT6 cells as a reference, reserve enough cells for 3-5 mice. When the cells reach 80%-90% confluence, digest them from the T75 culture flask, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1-2 mL of PBS, resuspend, and count.

[0059] (3) Adjusting cell density: Take 10 μL of cell suspension and calculate the cell concentration using a cell counting chamber under a light microscope. Adjust the density to the seeding density (e.g., the seeding density for EMT6 is 5 × 10⁻⁶). 5 Each vial contains 60-80 µL of injection volume, therefore the volume needs to be adjusted to 8.3 × 10⁻⁶. 6 (cell / mL). Aliquot 70 μL of cell suspension into 1.5 mL centrifuge tubes (reserve 10 μL for cell loss during transfer), and store the prepared centrifuge tubes containing cell suspension on ice.

[0060] (4) Preparation before injection: After arriving at the mouse operating room, centrifuge the centrifuge tube and shake off the cells on the tube wall. Before inoculation, resuspend the cells with a pipette, draw the corresponding volume of cell suspension with a 1mL insulin syringe, tighten the syringe cap and place it on ice to maintain cell viability.

[0061] II- and Triple-negative breast cancer transplantation

[0062] Experimental materials:

[0063] No. 6 sutures, sterilized forceps, hemostats and scissors, isoflurane anesthetic, dual-channel anesthesia machine, surgical pad, tape, alcohol swabs, razor and depilatory cream.

[0064] Implementation plan:

[0065] (1) Anesthesia of mice:

[0066] The mouse was placed in the anesthesia chamber of the anesthesia machine. The inhalation concentration of the anesthetic isoflurane was adjusted to 2.5, and the oxygen concentration to 0.5. The mouse was anesthetized after 2-5 minutes. The mouse was then placed on the operating table, and an inhalation mask was put on for continuous anesthesia. The limbs and chest were fixed with tape.

[0067] (2) Orthotopic transplantation of EMT6 tumor cells:

[0068] Disinfect the mouse abdomen with an alcohol swab. Transplant EMT6 cells into the fourth mammary pad of the mouse. Gently shave the fur off the mouse's abdomen with a razor and disinfect again. Use tweezers to grasp the skin slightly to the left of the abdomen, cut open the skin and peritoneum with scissors, locate the fifth mammary pad on the left side of the mouse, and remove it with tweezers. Draw the prepared EMT6 cells from a 1.5mL centrifuge tube using an insulin syringe and slowly inject them into the mouse's mammary gland. Note that during injection, try to ensure that the syringe is inside the mammary pad and that no liquid leaks out; otherwise, tumor cells may form tumors around the mammary gland, leading to peritoneal adhesions and poor model establishment.

[0069] (3) Orthotopic transplantation of 4T1 tumor cells:

[0070] 4T1 cells were transplanted into the second mammary pad on the same side of the mouse. The fur on the mouse's chest was gently shaved off with a razor, and the area was disinfected again. Using tweezers, the skin on the left side of the chest (axillary region) was grasped and cut open with scissors to locate the second mammary pad on the left side of the mouse, which was then removed with tweezers. The prepared 4T1 cells were drawn from a 1.5mL centrifuge tube using an insulin syringe and slowly injected into the mouse's mammary gland. Care was taken to ensure the syringe remained inside the mammary pad and that no fluid leaked out during injection; otherwise, tumor cells might form around the mammary gland, leading to abdominal adhesions and poor model establishment.

[0071] (4) Surgery

[0072] Take a No. 6 suture thread and hold the bottom of the suture hook with A-frame tweezers, ensuring the hook is facing you. With your left hand, hold the tweezers and pinch the skin on one side of the mouse. Insert the needle shallowly through the surface of the skin, then through the very tip of the skin on the other side. Remove the suture hook with the tweezers, discard the tweezers, and pull the thread with your left hand until only a few centimeters remain on the right side. With your right hand, use scissors to suture the wound once on each side. Trim any excess thread on both sides. Continue suturing slightly below the wound. Use the same method to suture the wounds created on the mouse's abdomen and chest. A 1-2 cm wound requires 4-5 stitches. Keep your wrist suspended during suturing to avoid touching other wounds.

[0073] (5) The mice were fed for 10 days to obtain a mouse triple-negative breast cancer double tumor model.

[0074] Comparative Example 1

[0075] The transplant 10 were prepared using the same method as in Example 1. 6 4T1 cells and 10 6 A mouse triple-negative breast cancer dual-tumor model using EMT6 cells.

[0076] Comparative Example 2

[0077] 5×10 grafts were prepared using the same method as in Example 1. 5 A mouse triple-negative breast cancer monotumor model with 4T1 cells.

[0078] Comparative Example 3

[0079] 5×10 grafts were prepared using the same method as in Example 1. 5 A mouse triple-negative breast cancer monotumor model using EMT6 cells.

[0080] Test Example 1: Tumor growth curves and intratumoral composition differences in a mouse triple-negative breast cancer dual-tumor model.

[0081] Test materials

[0082] The mouse triple-negative breast cancer double-tumor model prepared in Example 1 and Comparative Example 1, the mouse triple-negative breast cancer single-flow model prepared in Comparative Example 2 and Comparative Example 3, vernier calipers, weight scale, scissors, forceps, PBS buffer, cell digestion solution, flow cytometer, fluorescent antibody, etc.

[0083] Implementation Plan

[0084] (1) Tumor and volume measurement: Tumor volume and mouse weight can be measured 10 days after tumor transplantation, every 3 days. Hold the mouse by the back of the neck with your left hand and fix the tail. Moisten the tumor skin with an alcohol swab to reveal the tumor outline and measure the length and width of the tumor with calipers. The tumor volume is calculated using the following formula: Tumor volume (mm) 3 = 0.5 × major axis × minor axis 2 The mice were then weighed three times on a scale, and their average weight was recorded.

[0085] (2) Tumor Removal: On day 13 after tumor transplantation, the mice were euthanized. The abdominal cavity was incised along the midline / suture line, the peritoneum and pleural cavity were separated, and the skin was turned back. Near the fourth and second mammary pads, two solid tumors formed by EMT6 and 4T1 were observed. The fur was parted with forceps, and the complete mammary tumor was gently cut off with scissors. The cleaned tumor was placed on a board and photographed with a ruler. The horizontal and vertical measurements were taken once each, and the tumor volume was calculated. The results are as follows. Figure 2 As shown. From Figure 2 From this, we can see that in 5×10 5 At the cell / mouse seeding density, two tumors can form almost simultaneously, with a size difference of 100 mm. 3 Within a certain range, and with minimal physical burden on mice. Furthermore, there was no significant difference in tumor volume between the mouse triple-negative breast cancer double-tumor model and the mouse triple-negative breast cancer single-tumor model; the original growth characteristics of the tumor were preserved, and two heterogeneous tumors could form in the same mouse. This demonstrates that the mouse triple-negative breast cancer double-tumor model prepared in this application embodiment retains tumor heterogeneity, greatly replicating the heterogeneity and complexity of real tumors.

[0086] After measurement, the tumor was placed in a culture dish containing PBS buffer to prepare for subsequent experiments.

[0087] (3) Tumor microenvironment detection:

[0088] 1) Remove the tumor tissue, remove the skin, adhered connective tissue and necrotic areas with scissors, put it into a 2mL centrifuge tube and record the weight;

[0089] 2) Cut the tissue into small pieces using scissors or a scalpel. Transfer the tissue pieces to 12-well plates. Add a mixture of 1 mg / mL collagenase and DNase, 1 mL per 0.1 g of tissue. Incubate at 37 ℃ in a CO2 incubator for 1 hour for digestion.

[0090] 3) Transfer the digested cell suspension to a 70 μm filter screen, and gently grind any undigested tissue fragments using a 2 mL syringe rubber stopper. Rinse the cells from the filter screen into a 50 mL centrifuge tube with room temperature 1×PBS buffer.

[0091] 4) Centrifuge at 500g on a horizontal rotor for 10 minutes at room temperature;

[0092] 5) Discard the supernatant, add 2 mL of erythrocyte lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA dissolved in ddH2O, and adjust the pH to 7.2-7.4), mix by pipetting, and incubate at room temperature for 5 min. After lysis, add 5 mL of 1×PBS buffer directly, mix by inversion, and centrifuge.

[0093] 6) Collect cells (usually 1×10⁻⁶) 6 / tube), resuspend cells in 30µL CD16 / 32 PBA dilution buffer for blocking, and incubate at 4℃ for 15 min;

[0094] 7) Add 30µL of a mixture containing fluorescent antibodies with specific surface labels, and incubate at 4°C in the dark for 15-20 minutes. Epithelial cells, T cells, B cells, macrophages, and neutrophils can be detected here as needed. The corresponding antibodies are: Krt8 / Krt18 antibody, CD45+CD3 antibody, CD19 antibody, F4 / 80 antibody, and Ly6C / Ly6G antibody (Note that Epcam antibody cannot be used for detecting epithelial cells or tumor cells in EMT6 tumors because the expression level of EPCAM in the EMT6 cell line is very low; Krt8 / Krt18 antibody can be used instead).

[0095] 8) Add 1×PBS and wash once (invert to mix well) (6000rpm×2min);

[0096] 9) Resuspend cells in 200-300 µL of 1×PBS;

[0097] 10) The cell suspension was filtered through a 70mm filter / gauze / nylon mesh to remove impurities, then transferred to centrifuge tubes for flow cytometry analysis.

[0098] Test results as follows Figure 5 and Figure 6 As shown. Among them is the mouse triple-negative breast cancer double-tumor model prepared in Example 1, treated with the drug (…). Figure 5 ) and control group ( Figure 6 Flow cytometry analysis of intratumoral cellular composition. Figure 5 and Figure 6 As can be seen, after drug treatment, the number of T cells and B cells in the mouse triple-negative breast cancer double-tumor model prepared in Example 1 decreased, indicating that the drug's effect may mainly depend on innate immune cells; the decrease in the number of tumor cells indicates that the drug can kill or reduce tumor cells. This indirectly suggests that the mouse triple-negative breast cancer double-tumor model in this application can be used for the analysis of the drug's effect on tumors.

[0099] Test Example 2: Mouse Triple-Negative Breast Cancer Dual-Tumor Model and Response to Therapeutic Bacteria

[0100] Taking engineered Salmonella DB1 as an example, this bacterium can treat both solid and metastatic tumors, but the treatment effect may vary in different tumor models.

[0101] (1) Preparation of engineered Salmonella DB1

[0102] 1) Take out the frozen Salmonella DB1 culture from the -80°C freezer, streak it on a plate, and then place it in a 37°C bacterial incubator to revive and grow.

[0103] 2) After 36 hours, pick a single colony and put it into 3 ml of LB medium containing 50 μg / mL chloramphenicol and 50 μg / mL DAP, and incubate overnight at 37°C in a shaker.

[0104] 3) Transfer the bacteria that have reached the plateau phase to fresh LB medium containing chloramphenicol and DAP at a ratio of 1:100, and incubate at 37°C in a shaker for 2.5 h to bring the bacteria into the exponential growth phase (OD600 value between 0.2 and 0.5).

[0105] 4) Centrifuge at 5000 rpm / min for 5 min at room temperature to collect bacterial cells, and wash twice with an equal volume of 1×PBS buffer;

[0106] 5) Resuspend the bacterial stock solution in an appropriate amount of 1×PBS and place it on ice for later use. After serial dilution, stain with DAPI and count the bacteria using flow cytometry. Dilute the stock solution to 10⁻⁶ according to experimental requirements. 8 CFU / mL.

[0107] (2) Salmonella DB1 treatment in mice to create a double-tumor model of triple-negative breast cancer

[0108] 1) Mice with the mouse triple-negative breast cancer double tumor model prepared in Example 1 and Comparative Example 1, and mice with the mouse triple-negative breast cancer single-flow model prepared in Comparative Examples 2 and 3 were selected. The mice were kept in the tumor-bearing state until the tumors reached 200 mm. 3 After size observation, mice with similar tumor sizes were selected and divided into groups of 6-8 mice each for tumor size observation experiments.

[0109] 2) The 10 prepared above 8 CFU / mL bacterial suspension: 100 μL of DB1 bacterial suspension was injected into the tail vein of each mouse using an insulin syringe, i.e., 10 mice were injected with the same amount of DB1 bacterial suspension. 7 CFU bacteria were injected into the control group with an equal volume of 1×PBS buffer.

[0110] (3) Tumor volume measurement

[0111] The tumor volume was measured for 30 consecutive days. The specific steps were as follows: The mouse was held by the back of its neck and tail with the left hand. The tumor skin was moistened with an alcohol swab to reveal the tumor outline, and the length and width of the tumor were measured with calipers. The tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = 0.5 × major axis × minor axis 2 The mice were then weighed three times on a scale, and their average weight was recorded.

[0112] Measurement results as follows Figure 3 and Figure 4 As shown. Among them Figure 3 Normal curves of 4T1 tumors after treatment in the mouse triple-negative breast cancer dual-tumor model prepared in Example 1 and Comparative Example 1, and the mouse triple-negative breast cancer single-stream model prepared in Comparative Example 2. Figure 4 The images show normal curves of EMT6 tumors after treatment in the mouse triple-negative breast cancer dual-tumor model prepared in Example 1 and Comparative Example 1, and the mouse triple-negative breast cancer single-flow model prepared in Comparative Example 3. (From...) Figure 3 and Figure 4 As can be seen from the data, the mouse triple-negative breast cancer dual-tumor model in this application responds to the drug, and the changes in the two tumors after drug treatment are consistent. This further demonstrates that the mouse triple-negative breast cancer dual-tumor model in this application preserves tumor heterogeneity, greatly replicating the heterogeneity and complexity of real tumors, and can be used for drug-induced tumor analysis.

[0113] In summary, the mouse triple-negative breast cancer dual-tumor model in this application eliminates the problem of differences in drug response due to individual differences in tumor location. At the same time, different tumor cells can retain the heterogeneity of the tumor, greatly restoring the heterogeneity and complexity of real tumors. It has great application prospects in the research of the development characteristics of triple-negative breast cancer and new treatment methods.

[0114] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a mouse triple-negative breast cancer dual-tumor model, characterized in that, Including the following steps: Two types of triple-negative breast cancer cells were provided; The two types of triple-negative breast cancer cells were transplanted into two mammary pads of mice, respectively; The mice were fed for 7-10 days to obtain a mouse triple-negative breast cancer double tumor model. The two mammary pads are located on the same side of the mouse and are not adjacent to each other; The two types of triple-negative breast cancer cells are murine breast cancer cells EMT6 and murine breast cancer cells 4T1. The two mammary pads are located on the same side of the mouse, and are the second and fifth mammary pads starting from the head of the mouse; The two types of triple-negative breast cancer cells were transplanted into two mammary pads of mice, with the number of triple-negative breast cancer cells transplanted being 5 × 10⁻⁶. 5 indivual.

2. The method for preparing a mouse triple-negative breast cancer dual-tumor model according to claim 1, characterized in that, The method for preparing triple-negative breast cancer cells includes the following steps: Provide cryopreserved triple-negative breast cancer cells; The frozen triple-negative breast cancer cells were revived and expanded to obtain the triple-negative breast cancer cells.

3. The method for preparing a mouse triple-negative breast cancer dual-tumor model according to claim 1, characterized in that, The step of transplanting the two types of triple-negative breast cancer cells into two mammary pads of mice includes: The mouse was anesthetized and its abdomen was disinfected. The two types of triple-negative breast cancer cells were injected into the two breast pads, respectively.

4. The method for preparing a mouse triple-negative breast cancer dual-tumor model according to claim 1, characterized in that, The mice in question were immunodeficient mice.

5. The method for preparing a mouse triple-negative breast cancer dual-tumor model according to claim 4, characterized in that, The immunodeficient mice were BALB / c mice.

6. The application of a mouse triple-negative breast cancer dual-tumor model in detecting the composition of the microenvironment within breast cancer tumor tissue, characterized in that, The mouse triple-negative breast cancer double tumor model is prepared by the method described in any one of claims 1-5.

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