A method for constructing a breast cancer bone metastasis mouse model

Abstract

The present application relates to a breast cancer bone metastasis mouse model construction method, and belongs to the field of mouse tumor metastasis model construction. First, breast cancer cells are induced into a chemotherapy-resistant persistent state. Then, the culture supernatant of the breast cancer chemotherapy-resistant persistent state cells is continuously injected into the recipient mice for 14 days to create a pre-bone metastasis microenvironment of breast cancer. Finally, the breast cancer chemotherapy-resistant persistent state cell line is transplanted into the recipient mice by intra-iliac artery injection, and the formation of bone metastasis foci in the transplanted mice can be monitored about 14 days later. The modeling method of the present application has high tumor metastasis specificity, tumor formation rate and mouse survival rate. In addition, the present application is closer to the pathological occurrence process of clinical breast cancer bone metastasis, and has broad basic scientific research value.

Description

Technical Field

[0001] The invention belongs to the field of mouse tumor metastasis model construction and relates to a method for constructing a breast cancer bone metastasis mouse model. Background Art

[0002] Breast cancer is the most common malignant tumor in women. Bone metastasis is one of the most common metastases of breast cancer, with an incidence of approximately 70%. Among these, 27% to 50% present with bone metastasis as the first symptom. Patients with breast cancer bone metastasis often experience secondary bone metabolic disorders, leading to bone-related events such as bone pain, pathological fractures, and spinal cord compression, which severely impact their quality of life and prognosis, placing a long-term and significant burden on their families and society. Establishing a mouse model of breast cancer bone metastasis is an important approach to exploring the etiology, pathogenesis, and effective treatments of breast cancer bone metastasis.

[0003] Chinese patent CN112754717 A discloses a successful mouse model of breast cancer bone metastasis by disrupting the continuity of the mouse femur to form an incomplete fracture. However, this model cannot simulate the microenvironment of clinical breast cancer cell bone metastasis. Therefore, there is an urgent need to develop a mouse model that is closer to the process of breast cancer bone metastasis. Summary of the Invention

[0004] In response to the problems existing in the above-mentioned prior art, the present invention provides a method for simulating the pathological process of clinical breast cancer bone metastasis, and relates to a method for constructing a chemotherapy-resistant mouse breast cancer cell bone metastasis model.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a novel breast cancer bone metastasis mouse model, comprising the following steps:

[0007] 1) Chemotherapy drugs induce breast cancer cells in vitro into a drug-resistant state;

[0008] 2) Create a pre-bone metastatic microenvironment for breast cancer in recipient mice;

[0009] 3) Transplantation of in vitro chemotherapy-resistant breast cancer cells into recipient mice;

[0010] 4) Monitor the formation of bone metastases in mice.

[0011] Furthermore, the chemotherapy drug in step 1) is docetaxel.

[0012] Furthermore, the breast cancer cells in step 1) are mouse breast cancer cells.

[0013] Furthermore, the mouse breast cancer cells are of luminal B type.

[0014] Furthermore, the mouse breast cancer cells are luciferase-labeled mouse breast cancer cells.

[0015] Furthermore, the drug-resistant state in step 1) refers to a drug resistance index of 10, and the generation time of the drug-resistant cells is 3 times that of the parent cells.

[0016] Furthermore, in step 2), the pre-bone metastasis microenvironment of breast cancer in recipient mice is created by intraperitoneally injecting the culture supernatant of chemotherapy-resistant breast cancer cells in vitro into the mice. The mice can be 4-6 week old female C57BL / 6NCrl SPF mice. 20 μl / g of the culture supernatant of the chemotherapy-resistant cells is intraperitoneally injected into the mice once daily for 14 consecutive days.

[0017] Furthermore, in step 3), the in vitro chemotherapy-resistant breast cancer cells are transplanted into the recipient mice by injecting the in vitro chemotherapy-resistant breast cancer cells into the internal iliac artery of the mice. Specifically, the concentration of 2×10 6 A suspension of chemotherapy-resistant breast cancer cells (100 μl / ml) was injected into mice, 100 μl / mouse.

[0018] Furthermore, the monitoring of bone metastasis formation in mice in step 4) includes in vivo imaging of small animals, Micro-CT, H&E staining, and TRAP staining. The monitoring can be performed 14 days after transplanting chemotherapy-resistant breast cancer cells into recipient mice.

[0019] This also includes a mouse model established by a new method for constructing a breast cancer bone metastasis mouse model.

[0020] The beneficial effects of the present invention are as follows: the present invention creates a pre-bone metastasis microenvironment by continuously intraperitoneally injecting the culture supernatant of breast cancer cells in a persistent state of chemotherapy resistance into recipient mice; injects cells in a persistent state of chemotherapy resistance rather than ordinary tumor cells through the internal iliac artery to simulate the clinical breast cancer bone metastasis pathogenesis; the present invention simulates the entire process of breast cancer bone metastasis, and is an innovative high tumor formation rate model that is different from traditional models. The present invention is the first to establish a new breast cancer bone metastasis mouse model construction method with high survival rate, high metastasis specificity, easy tumor formation and consistent with clinical pathological characteristics.

[0021] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0023] Figure 1 This is an image of luciferase-labeled cells observed by a small animal living imaging system; Figure 1 A is EO771-Luc cell, Figure 1 B is EO771 cells;

[0024] Figure 2 The following is a 40-fold magnification of EO771-Luc parental cells and EO771-Luc cells in a state of persistent chemotherapy resistance; Figure 2 A is the EO771-Luc parent cell, Figure 2 B is EO771-Luc cells in a persistent state of chemotherapy resistance.

[0025] Figure 3 Construct a flow chart for the model of the present invention;

[0026] Figure 4 Detailed diagram of the procedure for injecting tumor cells into the internal iliac artery;

[0027] Figure 5 A graph monitoring bone metastasis formation in mice following transplantation of chemotherapy-resistant breast cancer cells. Figure 5 A, B, C, and D are the in vivo imaging monitoring images of small animals on days 3, 7, 10, and 14 after surgery, respectively;

[0028] Figure 6 This is an image of the small animal in vivo imaging system on the 14th day after tumor transplantation. Figure 6 A is control group 1: only EO771-Luc parental cells were injected into the internal iliac artery, Figure 6 B is control group 2: only the culture supernatant of EO771-Luc cells in the persistent state of chemotherapy resistance was injected intraperitoneally. Figure 6 C is control group 3: only EO771-Luc chemotherapy-resistant cells were injected into the internal iliac artery. Figure 6 D is control group 4: intraperitoneal injection of chemotherapy-resistant EO771-Luc cell culture supernatant, and internal iliac artery injection of EO771-Luc parental cells, Figure 6 E: A microenvironment was created and the EO771-Luc cells in a persistent state of chemotherapy resistance were injected into the internal iliac artery;

[0029] Figure 7 This is a Micro CT image of mouse bone tissue in vitro on day 14 after tumor transplantation. Figure 7 A group only created a microenvironment before bone metastasis, Figure 7 B. Group B: EO771-Luc chemotherapy-resistant persistent state cells injected into the internal iliac artery. Figure 7C. The microenvironment was created and the cells in the persistent state of EO771-Luc chemotherapy resistance were injected into the internal iliac artery;

[0030] Figure 8 This is the result of H&E staining of bone tissue sections. Figure 8 A only creates a microenvironment before bone metastasis, Figure 8 B. Group B: EO771-Luc chemotherapy-resistant persistent state cells injected into the internal iliac artery. Figure 8 C. Create a microenvironment and inject EO771-Luc cells into the internal iliac artery to maintain chemotherapy resistance;

[0031] Figure 9 This is the result of TRAP staining of bone tissue sections. Figure 9 A group only created a microenvironment before bone metastasis, Figure 9 B. Group B: EO771-Luc chemotherapy-resistant persistent state cells injected into internal iliac artery. Figure 9 C. Create a microenvironment and inject EO771-Luc cells into the internal iliac artery to maintain chemotherapy resistance;

[0032] Figure 10 This is a multi-level pie chart of the survival rate, tumor formation rate, and specific bone metastasis rate of mice on day 14 after surgery. Figure 10 A is control group 1: only EO771-Luc parental cells were injected into the internal iliac artery, Figure 10 B is control group 2: only the culture supernatant of EO771-Luc cells in the persistent state of chemotherapy resistance was injected intraperitoneally. Figure 10 C is control group 3: only EO771-Luc chemotherapy-resistant cells were injected into the internal iliac artery. Figure 10 D is control group 4: intraperitoneal injection of chemotherapy-resistant EO771-Luc cell culture supernatant, and internal iliac artery injection of EO771-Luc parental cells, Figure 10 E. Create a microenvironment and inject the EO771-Luc cells in the internal iliac artery into the chemotherapy-resistant persistent group. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0034] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0035] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0036] Chemoresistance index (RI): half inhibition rate of resistant cell line / half inhibition rate of parental cell line

[0037] Generation time: refers to the time from the end of one cell division to the next division (i.e. the time it takes to form a generation).

[0038] Example 1

[0039] 1. Construction of Luciferase-labeled luminal B mouse breast cancer cell line EO771

[0040] (1) Take 6×10 5 Luminal B mouse breast cancer cell line EO771 (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-003-0098) in the logarithmic growth phase was inoculated into a 6-well plate containing 250 μl serum-free and double-antibody-free DMEM (Gibco, Cat. No. 11965092) medium. 50 μl of the medium was diluted to a concentration of 1.5*10 8TU / mL of HBLV-LUC-PURO virus (Hanbio Biotech (Shanghai) Co., Ltd., Cat. No. LV62121001) was added, followed by the addition of 50 μl of Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668027). The cells were incubated at room temperature for 5 minutes, gently mixed, and allowed to stand at room temperature for 20 minutes. 24 hours after transfection, the cells were digested with trypsin (Gibco, Cat. No. 25200056) and G418 (Geneticin, Cat. No. 10131035) was added at a concentration of 300 μg / mL. The culture medium was changed every two days until a single resistant clone emerged.

[0041] (2) Select resistant clones and transfer them to 96-well plates. When they reach the fifth generation, the cells are plated at a rate of 5 × 10 5 The clone and the control group (untreated EO771 cells) were inoculated into six-well plates and cultured for 8 hours. D-luciferin potassium salt luminescent substrate (D-Luciferin) (PerkinElmer, Cat.No.122799) was diluted with sterile PBS (Gibco, Cat.No.10010023) to a concentration of 30 mg / ml. The old culture medium was discarded, and D-Luciferin and DMEM culture medium containing 10% fetal bovine serum (Gibco, Cat.No.12484028) and 0.1 mg / ml penicillin-streptomycin (Gibco, Cat.No.15070063) were added to a final working concentration of 150 ug / ml of the substrate. The cells were placed in an incubator at 37°C, 5% CO2, and 95% humidity for 10 minutes. The cells were monitored using a small animal in vivo imaging system. If the fluorescence of the cells to be tested is obvious compared to the control group, the EO771 cells were successfully labeled. Figure 1 A shows the successfully recorded EO771 cells, which are designated as EO771-Luc cells.

[0042] 2. Construction of EO771-Luc cell line with persistent chemotherapy resistance

[0043] The mouse adenocarcinoma cell line EO771-Luc in the logarithmic growth cycle was inoculated into DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin and placed in an incubator at 37°C, 5% CO2, and 95% humidity. When the growth density reached 70-80%, a concentration of 3.9×10 -9mol / L docetaxel (MedChemExpress, Cat.No.HY-B0011), after culturing for 8 hours, replace with fresh DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin, continue to culture until the cell saturation density is above 90%, passage once, repeat the above operation, measure the IC50 of the cell line in docetaxel and calculate the chemotherapy resistance index (RI) every 2 passages, until the chemotherapy resistance index is greater than 10 and the cell proliferation rate is less than 3 times that of the primary EO771-Luc cells, that is, EO771-Luc cells with persistent chemotherapy resistance are obtained. Figure 2 .in Figure 2 A and B show the same amount of cells cultured for the same time period for EO771-Luc and EO771-Luc cells in a state of persistent chemotherapy resistance. It can be seen that the proliferation rate of the latter is 3 times lower than that of the former.

[0044] 3. Intraperitoneal injection of EO771-Luc cell culture supernatant in the persistent state of chemotherapy resistance

[0045] 5~7×10 6 A chemotherapy-resistant persistent cell line EO771-Luc established by docetaxel induction was routinely cultured in 5 ml of DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin, and placed in an incubator at 37°C, 5% CO2, and 95% humidity for 24 hours. The cell culture supernatant was collected, centrifuged at 1000 rpm, 4°C, for 5 minutes, and the precipitate was discarded, and the supernatant was collected. (Note: The supernatant was aliquoted and frozen at -80°C for later use. It should be used within 2 to 3 days after thawing to avoid repeated freezing and thawing.) 20 ul / g of the chemotherapy-resistant cell culture supernatant was intraperitoneally injected into 4-6 week old female C57BL / 6NCrl SPF grade (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Cat. No. C57BL / 6NCrl mouse SPF grade) mice (about 20 g) once a day for 14 consecutive days. For specific reference Figure 3 .

[0046] 4. Injection of EO771-Luc chemotherapy-resistant cell lines into the internal iliac artery of mice

[0047] (1) Take EO771-Luc cells in the logarithmic growth phase and discard the original culture medium. Add 5 mL of PBS to rinse. Add 1.5 mL of trypsin and place in a 37°C, 5% CO2, 95% humidity incubator for 2 minutes. Add 3 mL of complete culture medium to terminate digestion. Centrifuge at 4°C, 1000 rpm for 5 minutes. After centrifugation, discard the supernatant. Resuspend in sterile pre-cooled PBS to a single cell suspension concentration of 2 x 10 6 / ml. (Note: Single cell suspension must be placed on ice)

[0048] (2) 4- to 6-week-old female C57BL / 6NCrl SPF mice (approximately 20 g) in a pre-bone metastatic microenvironment were anesthetized with isoflurane. Hair was removed from the right side of the mouse from the lower edge of the ribs to the knee. The mouse was tilted approximately 45° clockwise with its legs naturally spread out, and the toes were fixed to a removable dissecting cardboard with tape. The right inguinal area was wiped with 70% ethanol, and the surgical area was disinfected again with iodine.

[0049] (3) Make an oblique skin incision about 1.0 cm long between the 4th and 5th pairs of nipples. Use a sterile surgical sheet to cover the animal's body except for the incision site and move the mouse to a standard desktop dissecting microscope. Under a four-fold magnifying glass, the internal iliac blood vessels and nerve bundles can be seen located outside and below the abdominal fat pad. Insert blunt separation forceps between the fat tissue and the peritoneum, push the tissue outward to both sides, and expose the internal iliac blood vessels and nerve clusters. With the help of straight forceps, take a 4-0 suture and pass it under the blood vessels. Gently pull back and forth to further bluntly separate the connective tissue between the blood vessels and nerves. For details, refer to Figure 4 .

[0050] (4) Hold the fine toothless forceps with your left hand, and make the horizontal lines of the toothless forceps parallel to the blood vessel. Hold the syringe with your right hand, with the cone of the needle facing upwards, and insert it into the arterial cavity along the indentation of the toothless forceps. Slowly inject the syringe with a concentration of 2x 10 6 Inject 100 μl of cell suspension into mice at a rate of 100 μl per mouse, from the proximal end to the distal end. (Note: Mix the cell suspension thoroughly between injections to separate the cells and prevent aggregation and embolism.)

[0051] (5) After the injection, remove the forceps and 4-0 sutures and quickly apply pressure to the arterial incision area with a cotton swab for 10 minutes to stop bleeding. After the bleeding stops, wipe the remaining area with alcohol to kill any remaining tumor cells. Suture the skin and place the mouse on a 27°C heating pad until awake.

[0052] 5. Monitoring of breast cancer bone metastasis formation

[0053] (1) In vivo imaging of small animals to monitor the formation of breast cancer bone metastasis

[0054] For mice in the group where tumor cell bone metastasis microenvironment was established and EO771-Luc cells were injected into the internal iliac artery to maintain chemotherapy resistance, the mice in the group where tumor cell bone metastasis microenvironment was established and EO771-Luc cells were injected into the internal iliac artery to maintain chemotherapy resistance were imaged using a small animal in vivo imaging system on days 3, 7, 10, and 14 after internal iliac artery injection (Note: If only low signal areas appear on day 14, the observation time can be appropriately extended to 21 days). D-Luciferin diluted solution 15 mg / ml was injected into the mice by intraperitoneal injection, with 200 μL injected into each mouse. If tumors have formed, high signal areas can be observed in the leg bones of the mice. For details, refer to Figure 5 . Figure 5 To create a microenvironment and inject EO771-Luc cells into the internal iliac artery to maintain chemotherapy resistance, the following images were taken by the small animal in vivo imaging system on days 3, 7, 10, and 14 after surgery. On day 14 after surgery, mice in control groups 1, 2, 3, and 4 were photographed using the small animal in vivo imaging system, and the 14-day survival rate, tumor formation rate, and specific bone metastasis rate were calculated. Figure 6 , Figure 10 . Figure 6 E is an image of the small animal in vivo imaging system on day 14 in the group where the microenvironment was created and the EO771-Luc cells in the iliac artery were injected into the chemotherapy-resistant state. Figure 6 The fluorescence signal of E was the strongest, indicating that the tumorigenic effect of the group in which the microenvironment was created and the EO771-Luc cells were injected into the internal iliac artery with persistent chemotherapy resistance was stronger than that of the control group 1, control group 2, control group 3 and control group 4. Figure 10 As shown in pie chart E, the 14-day survival rate, tumor formation rate, and specific bone metastasis rate of mice in the present application were all higher than those in control group 1, control group 2, control group 3, and control group 4.

[0055] (2) Micro CT evaluation of bone fusion after breast cancer bone metastasis

[0056] On the 14th day after surgery, the mice were killed by cervical dislocation, and bone tissue from the tumor invasion site was taken. After removing muscles and other tissues, bone damage was observed using a Micro CT instrument. The results were as follows: Figure 7 . Figure 7 A, B, and C are images of mouse bone tissue isolated from the body on day 14 under Micro CT observation. Figure 7 The osteolysis phenomenon in C was the most significant, indicating that the microenvironment before tumor metastasis was shaped. At the same time, the tumorigenic effect of cells injected with EO771-Luc chemotherapy resistance and persistence was stronger than that of the other groups.

[0057] (3) H&E staining to observe histological changes in bone metastasis sites

[0058] Tumor-bearing mice were sacrificed by cervical dislocation. The leg bone on the side of the bone metastasis was removed and other tissues were removed. The bone was then fixed, decalcified, dehydrated, transparentized, waxed, and embedded. The sectioning site was determined based on the high signal area in the imaging image of step (1) and the osteolytic area in the imaging image of step (2). The bone tissue in the target area was sliced ​​into slices with a thickness of 4 to 6 microns using a microtome.

[0059] Paraffin sections of bone metastasis sites were stained with hematoxylin and eosin (H&E) using a hematoxylin and eosin staining kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0105S). The required solution sequence and time are as follows: hematoxylin staining solution for 1 minute, tap water for 10 seconds, 75% alcohol-1% hydrochloric acid solution for 10 seconds, tap water for 5 minutes, 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, eosin staining solution for 1 minute, 95% alcohol for 10 seconds, 95% alcohol for 45 seconds, 95% alcohol for 1 minute, 100% alcohol for 2 minutes, 50% alcohol and 50% xylene for 4 minutes, xylene I for 6 minutes, xylene II for 6 minutes, and finally, resin mounting. After drying, photographs were taken under a microscope. The H&E staining results are shown in the figure below. Figure 8 Compared to Figure 8 A, B, Figure 8 C often shows irregularly scattered, misshapen cancer cells with enlarged, misshapen, hyperchromatic nuclei, an imbalanced nuclear-cytoplasmic ratio, altered cytoplasmic staining, or the presence of vacuoles. Furthermore, the background often contains necrosis. These results demonstrate that the method of the present invention successfully establishes breast cancer bone metastases.

[0060] 4. TRAP staining to observe the number of osteoclasts

[0061] TRAP staining kit (Solarbio, Cat. No. G1492) was used to perform TRAP staining on paraffin sections of mouse bone tissue. The sections were added with staining solution and placed in a humidified box at 37°C for staining for 1 hour. The staining solution formula is as follows (500uL system): FastGBC 5uL, Sodium Nitrate 5uL, ultrapure water 445uL, NAB5uL, Acetrate20ul, Tartrate10uL. The required solution sequence time is as follows: 2 minutes in ultrapure water, repeated 3 times; methyl green staining solution 1 minute; 1xPBS 10 seconds. After the sections were dried, they were mounted, photographed under a microscope, and statistical analysis of the TRAP staining results was performed, focusing on observing the number of osteoclasts at the tumor / bone interface. The results are as follows. Figure 9 , Figure 9 A, B, and C are images of bone tissue sections observed under TRAP staining microscope. Figure 9 A, B, Figure 9 C. A significant increase in the number of osteoclasts was observed. The above results indicate that the method of the present invention successfully established osteolytic bone metastases of breast cancer.

[0062] Control group 1

[0063] 1. Construction of Luciferase-labeled luminal B mouse breast cancer cell line EO771

[0064] (1) Take 6×10 5Luminal B mouse breast cancer cell line EO771 (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-003-0098) in the logarithmic growth phase was inoculated into a 6-well plate containing 250 μl serum-free, double-antibody-free DMEM (Gibco, Cat. No. 11965092) medium, and then 50 μl of 1.5*10 8 HBLV-LUC-PURO virus (Hanbio Biotech (Shanghai) Co., Ltd., Cat. No. LV62121001) at a concentration of 1 tb / mL was added to 50 μl of Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668027), incubated at room temperature for 5 minutes, gently mixed, and allowed to stand at room temperature for 20 minutes. 24 hours after transfection, cells were digested with trypsin (Gibco, Cat. No. 25200056) and G418 (Geneticin, Cat. No. 10131035) was added at a concentration of 300 μg / ml. The culture medium was changed every two days until a single resistant clone emerged.

[0065] (2) Select resistant clones and transfer them to 96-well plates. When they reach the fifth generation, the cells are plated at a rate of 5 × 10 5 The clone and the control group (untreated EO771 cells) were inoculated into six-well plates and cultured for 8 hours. D-luciferin potassium salt luminescent substrate (D-Luciferin) (PerkinElmer, Cat.No.122799) was diluted with sterile PBS (Gibco, Cat.No.10010023) to a concentration of 30 mg / ml. The old culture medium was discarded and D-Luciferin and DMEM culture medium containing 10% fetal bovine serum (Gibco, Cat.No.12484028) and 0.1 mg / ml penicillin-streptomycin (Gibco, Cat.No.15070063) were added to a final working concentration of 150 ug / ml of the substrate. The cells were placed in an incubator at 37°C, 5% CO2, and 95% humidity for 10 minutes. The cells were monitored by a small animal in vivo imaging system. If the fluorescence of the cells to be tested is obvious compared to the control group, the EO771 cells were successfully labeled. For details, refer to Figure 1 .

[0066] 2. Injection of EO771-Luc Parental Cells into the Internal Iliac Artery of Mice

[0067] (1) Take the parental EO771-Luc cells in the logarithmic growth phase, discard the original culture medium, and add 5 mL of PBS to rinse. Add 1.5 mL of trypsin and place in a 37°C, 5% CO2, 95% humidity incubator for 2 minutes; add 3 mL of complete culture medium to terminate the digestion. Centrifuge at 4°C, 1000 rpm for 5 minutes. After centrifugation, discard the supernatant; resuspend in sterile pre-cooled PBS to a single cell suspension concentration of 2 x 10 6 / ml. (Note: Single cell suspension must be placed on ice)

[0068] (2) 4- to 6-week-old female C57BL / 6NCrl SPF mice (approximately 20 g) without a pre-bone metastatic microenvironment were anesthetized with isoflurane. Hair was removed from the right side of the mouse from the lower edge of the ribs to the knee. The mouse was tilted approximately 45° clockwise with its legs naturally spread out, and the toes were fixed to a removable dissecting cardboard with tape. The right inguinal area was wiped with 70% ethanol, and the surgical area was disinfected again with iodine.

[0069] (3) Make an oblique skin incision about 1.0 cm long between the 4th and 5th pairs of nipples. Use a sterile surgical sheet to cover the animal's body except for the incision site and move the mouse to a standard desktop dissecting microscope. Under a four-fold magnifying glass, the internal iliac blood vessels and nerve bundles can be seen located outside and below the abdominal fat pad. Insert blunt separation forceps between the fat tissue and the peritoneum, push the tissue outward to both sides, and expose the internal iliac blood vessels and nerve clusters. With the help of straight forceps, take a 4-0 suture and pass it under the blood vessels. Gently pull back and forth to further bluntly separate the connective tissue between the blood vessels and nerves. For details, refer to Figure 4 .

[0070] (4) Hold the fine toothless forceps with your left hand, and make the horizontal lines of the toothless forceps parallel to the blood vessel. Hold the syringe with your right hand, with the cone of the needle facing upwards, and insert it into the arterial cavity along the indentation of the toothless forceps. Slowly inject the syringe with a concentration of 2x 10 6 Inject 100 μl of cell suspension into mice at a rate of 100 μl per mouse, from the proximal end to the distal end. (Note: Mix the cell suspension thoroughly between injections to separate the cells and prevent aggregation and embolism.)

[0071] (5) After the injection, remove the forceps and 4-0 sutures and quickly apply pressure to the arterial incision area with a cotton swab for 10 minutes to stop bleeding. After the bleeding stops, wipe the remaining area with alcohol to kill any remaining tumor cells. Suture the skin and place the mouse on a 27°C heating pad until awake.

[0072] (4) Hold the fine toothless forceps with your left hand, and make the horizontal lines of the toothless forceps parallel to the blood vessel. Hold the syringe with your right hand, with the cone of the needle facing upwards, and insert it into the arterial cavity along the indentation of the toothless forceps. Slowly inject the syringe with a concentration of 2x 10 6Inject 100 μl of cell suspension into mice at a rate of 100 μl per mouse, from the proximal end to the distal end. (Note: Mix the cell suspension thoroughly between injections to separate the cells and prevent aggregation and embolism.)

[0073] (5) After the injection, remove the forceps and 4-0 sutures and quickly apply pressure to the arterial incision area with a cotton swab for 10 minutes to stop bleeding. After the bleeding stops, wipe the remaining area with alcohol to kill any remaining tumor cells. Suture the skin and place the mouse on a 37°C heating pad until awake.

[0074] 3. Monitoring of breast cancer bone metastasis formation

[0075] (1) In vivo imaging of small animals to monitor the formation of breast cancer bone metastasis

[0076] On the 3rd, 7th, 10th and 14th days after the internal iliac artery injection, mice in the EO771-Luc cell group with persistent chemotherapy resistance and microenvironment were imaged under a small animal in vivo imaging system (Note: If only a low signal area appears on the 14th day, the observation time can be appropriately extended to 21 days). 15 mg / ml of D-Luciferin dilution was injected into the mice by intraperitoneal injection, with 200 μL injected into each mouse. If a tumor has formed, a high signal area can be observed in the leg bone of the mouse. In addition, the 14-day survival rate, tumor formation rate, and specific bone metastasis rate were statistically analyzed. For details, refer to Figure 6 , Figure 10 . Figure 6 A is the image of the small animal in vivo imaging system in the control group 1 on the 14th day. Figure 6 The fluorescence signal of E is the strongest. Figure 10 Pie chart A shows that the 14-day survival rate, tumor formation rate, and specific bone metastasis rate of mice in control group 1 were all lower than those in the present application group. The above two figures indicate that the tumor formation effect of the group with microenvironment creation and internal iliac artery injection of EO771-Luc cells with persistent chemotherapy resistance was stronger than that of the group with only internal iliac artery injection of EO771-Luc parental cells.

[0077] (2) Micro CT evaluation of bone fusion after breast cancer bone metastasis

[0078] On the 14th day after surgery, the mice were killed by cervical dislocation, and bone tissue from the tumor invasion site was obtained. After removing muscles and other tissues, bone scans were performed using a Micro CT instrument to observe bone damage.

[0079] (3) H&E staining to observe histological changes in bone metastasis sites

[0080] Tumor-bearing mice were sacrificed by cervical dislocation. The leg bone on the side of the bone metastasis was removed and other tissues were removed. The bone was then fixed, decalcified, dehydrated, transparentized, waxed, and embedded. The sectioning site was determined based on the high signal area in the imaging image of step (1) and the osteolytic area in the imaging image of step (2). The bone tissue in the target area was sliced ​​into slices with a thickness of 4 to 6 microns using a microtome.

[0081] Paraffin sections of bone metastases were stained with a hematoxylin and eosin (H&E) staining kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0105S). The required solutions and durations were as follows: hematoxylin for 1 minute, tap water for 10 seconds, 75% ethanol-1% hydrochloric acid solution for 10 seconds, tap water for 5 minutes, 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, eosin for 1 minute, 95% ethanol for 10 seconds, 95% ethanol for 45 seconds, 95% ethanol for 1 minute, 100% ethanol for 2 minutes, 50% ethanol and 50% xylene for 4 minutes, xylene I for 6 minutes, and xylene II for 6 minutes. Finally, the sections were mounted with resin. After drying, the sections were photographed under a microscope.

[0082] (4) TRAP staining to observe the number of osteoclasts

[0083] Mouse bone tissue paraffin sections were stained using the TRAP staining kit (Solarbio, Cat. No. G1492). Sections were added to the staining solution and placed in a humidified oven at 37°C for 1 hour. The staining solution (500 μL system) consists of 5 μL FastGBC, 5 μL Sodium Nitrate, 445 μL ultrapure water, 5 μL NAB, 20 μL Acetrate, and 10 μL Tartrate. The required incubation times were as follows: 2 minutes in ultrapure water, repeated three times; 1 minute in methyl green solution; and 10 seconds in 1x PBS. After sections were air-dried and mounted, images were taken under a microscope, and statistical analysis of the TRAP staining results was performed, focusing on the number of osteoclasts at the tumor / bone interface.

[0084] Control group 2

[0085] 1. Construction of Luciferase-labeled luminal B mouse breast cancer cell line EO771

[0086] (1) Take 6×10 5 Luminal B mouse breast cancer cell line EO771 (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-003-0098) in the logarithmic growth phase was inoculated into a 6-well plate containing 250 μl serum-free, double-antibody-free DMEM (Gibco, Cat. No. 11965092) medium, and then 50 μl of 1.5*10 8HBLV-LUC-PURO virus (Hanbio Biotech (Shanghai) Co., Ltd., Cat. No. LV62121001) at a concentration of 1 tb / mL was added to 50 μl of Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668027), incubated at room temperature for 5 minutes, gently mixed, and allowed to stand at room temperature for 20 minutes. 24 hours after transfection, cells were digested with trypsin (Gibco, Cat. No. 25200056) and G418 (Geneticin, Cat. No. 10131035) was added at a concentration of 300 μg / ml. The culture medium was changed every two days until a single resistant clone emerged.

[0087] (2) Select resistant clones and transfer them to 96-well plates. When they reach the fifth generation, the cells are plated at a rate of 5 × 10 5 The clone and the control group (untreated EO771 cells) were inoculated into six-well plates and cultured for 8 hours. D-luciferin potassium salt luminescent substrate (D-Luciferin) (PerkinElmer, Cat.No.122799) was diluted with sterile PBS (Gibco, Cat.No.10010023) to a concentration of 30 mg / ml. The old culture medium was discarded and D-Luciferin and DMEM culture medium containing 10% fetal bovine serum (Gibco, Cat.No.12484028) and 0.1 mg / ml penicillin-streptomycin (Gibco, Cat.No.15070063) were added to a final working concentration of 150 ug / ml of the substrate. The cells were placed in an incubator at 37°C, 5% CO2, and 95% humidity for 10 minutes. The cells were monitored by a small animal in vivo imaging system. If the fluorescence of the cells to be tested is obvious compared to the control group, the EO771 cells were successfully labeled. For details, refer to Figure 1 .

[0088] 2. Construction of EO771-Luc cell line with persistent chemotherapy resistance

[0089] The mouse breast cancer cell line EO771-Luc in the logarithmic growth cycle was inoculated into a DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin and placed in a 37°C, 5% CO2, 95% humidity incubator. When the growth density was 70-80%, docetaxel was added to a final concentration of docetaxel that was the IC50 concentration of the EO771-Luc cell line in the docetaxel. After culturing for 8 hours, fresh DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin was added and cultured until the cell saturation density was above 90%. The cell line was passaged once and the above operation was repeated. The IC50 of the cell line in docetaxel was determined every 2 passages and the chemotherapy resistance index (RI) was calculated until the chemotherapy resistance index was >10 and the cell proliferation rate was less than 3 times that of the primary EO771-Luc cell. For details, refer to Figure 2 .in Figure 2 A and B show EO771-Luc cells and EO771-Luc cells in a persistent state of chemotherapy resistance after culture for the same cell amount and time. It can be seen that the proliferation rate of the latter is 3 times lower than that of the former.

[0090] 3. Intraperitoneal injection of EO771-Luc cell culture supernatant in the persistent state of chemotherapy resistance

[0091] 5~7×10 6 A docetaxel-induced chemoresistant persistent cell line, EO771-Luc, was routinely cultured in 5 ml of DMEM supplemented with 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin in an incubator at 37°C, 5% CO2, and 95% humidity for 24 hours. The cell culture supernatant was collected and centrifuged at 1000 rpm at 4°C for 5 minutes. The pellet was discarded and the supernatant was collected. (Note: The supernatant was aliquoted and frozen at -80°C until use. Use within 2-3 days after thawing to avoid repeated freeze-thaw cycles.) 20 μl / g of the chemoresistant cell culture supernatant was intraperitoneally injected into 4- to 6-week-old female C57BL / 6NCrl SPF mice once daily for 14 days.

[0092] 4. Monitoring of breast cancer bone metastasis formation

[0093] (1) In vivo imaging of small animals to monitor the formation of breast cancer bone metastasis

[0094] On days 3, 7, 10, and 14 after intraperitoneal injection, mice in the group receiving only the chemoresistant persistent state of EO771-Luc cell culture supernatant were imaged using a small animal in vivo imaging system (Note: If only a low signal area appears on day 14, the observation time can be appropriately extended to 21 days). 15 mg / ml of D-Luciferin diluted solution was injected into the mice via intraperitoneal injection, with 200 μL injected into each mouse. If a tumor has formed, a high signal area can be observed in the leg bone of the mouse. The 14-day survival rate, tumor formation rate, and specific bone metastasis rate were calculated. Figure 6 , Figure 10 . Figure 6 B is the image of the small animal in vivo imaging system in the control group 2 on the 14th day. Figure 6 The fluorescence signal of E is the strongest. Figure 10 Pie chart B shows that the 14-day survival rate, tumor formation rate, and specific bone metastasis rate of mice in control group 2 were all lower than those in the present application group. The above two figures indicate that the tumor formation effect of the group in which a microenvironment was created and EO771-Luc cells with persistent chemotherapy resistance were injected into the internal iliac artery was stronger than that of the group in which only the microenvironment was created.

[0095] (2) Micro CT evaluation of bone fusion after breast cancer bone metastasis

[0096] On the 14th day after surgery, the mice were killed by cervical dislocation, and bone tissue from the tumor invasion site was taken. After removing muscles and other tissues, bone damage was observed using a Micro CT instrument. The results were as follows: Figure 7 . Figure 7 A is the image of mouse bone tissue in vitro observed by Micro CT on day 14. Figure 7 The osteolysis phenomenon in C was the most significant, indicating that the microenvironment before tumor metastasis was shaped. At the same time, the tumorigenic effect of cells injected with EO771-Luc chemotherapy resistance and persistence was stronger than that of the other groups.

[0097] (3) H&E staining to observe histological changes in bone metastasis sites

[0098] Tumor-bearing mice were sacrificed by cervical dislocation. The leg bone on the side of the bone metastasis was removed and other tissues were removed. The bone was then fixed, decalcified, dehydrated, transparentized, waxed, and embedded. The sectioning site was determined based on the high signal area in the imaging image of step (1) and the osteolytic area in the imaging image of step (2). The bone tissue in the target area was sliced ​​into slices with a thickness of 4 to 6 microns using a microtome.

[0099] Paraffin sections of bone metastasis sites were stained with hematoxylin and eosin (H&E) using a hematoxylin and eosin staining kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0105S). The required solution sequence and time are as follows: hematoxylin staining solution for 1 minute, tap water for 10 seconds, 75% alcohol-1% hydrochloric acid solution for 10 seconds, tap water for 5 minutes, 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, eosin staining solution for 1 minute, 95% alcohol for 10 seconds, 95% alcohol for 45 seconds, 95% alcohol for 1 minute, 100% alcohol for 2 minutes, 50% alcohol and 50% xylene for 4 minutes, xylene I for 6 minutes, xylene II for 6 minutes, and finally, resin sealing. After drying, photos were taken under a microscope. The H&E staining results are shown in the figure below. Figure 8 Compared to Figure 8 A, Figure 8 C often shows irregularly scattered, misshapen cancer cells with enlarged, misshapen, hyperchromatic nuclei, an imbalanced nuclear-cytoplasmic ratio, altered cytoplasmic staining, or the presence of vacuoles. Furthermore, the background often contains necrosis. These results demonstrate that the method of this application successfully establishes breast cancer bone metastases.

[0100] (4) TRAP staining to observe the number of osteoclasts

[0101] TRAP staining kit (Solarbio, Cat. No. G1492) was used to perform TRAP staining on paraffin sections of mouse bone tissue. The sections were added with staining solution and placed in a humidified box at 37°C for staining for 1 hour. The staining solution formula is as follows (500uL system): FastGBC 5uL, Sodium Nitrate 5uL, ultrapure water 445uL, NAB5uL, Acetrate20ul, Tartrate10uL. The required solution sequence time is as follows: 2 minutes in ultrapure water, repeated 3 times; methyl green staining solution 1 minute; 1xPBS 10 seconds. After the sections were dried, they were mounted, photographed under a microscope, and statistical analysis of the TRAP staining results was performed, focusing on observing the number of osteoclasts at the tumor / bone interface. The results are as follows. Figure 9 , Figure 9 A is the TRAP staining microscopic observation of bone tissue sections in the group that only created a microenvironment before bone metastasis. Figure 9 A, Figure 9 C. A significant increase in the number of osteoclasts was observed. The above results indicate that the method of the present invention successfully established osteolytic bone metastases of breast cancer.

[0102] Control group 3

[0103] 1. Construction of Luciferase-labeled luminal B mouse breast cancer cell line EO771

[0104] (1) Take 6×10 5Luminal B mouse breast cancer cell line EO771 (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-003-0098) in the logarithmic growth phase was inoculated into a 6-well plate containing 250 μl serum-free, double-antibody-free DMEM (Gibco, Cat. No. 11965092) medium, and then 50 μl of 1.5*10 8 HBLV-LUC-PURO virus (Hanbio Biotech (Shanghai) Co., Ltd., Cat. No. LV62121001) at a concentration of 1 tb / mL was added to 50 μl of Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668027), incubated at room temperature for 5 minutes, gently mixed, and allowed to stand at room temperature for 20 minutes. 24 hours after transfection, cells were digested with trypsin (Gibco, Cat. No. 25200056) and G418 (Geneticin, Cat. No. 10131035) was added at a concentration of 300 μg / ml. The culture medium was changed every two days until a single resistant clone emerged.

[0105] (2) Select resistant clones and transfer them to 96-well plates. When they reach the fifth generation, the cells are plated at a rate of 5 × 10 5 The clone and the control group (untreated EO771 cells) were inoculated into six-well plates and cultured for 8 hours. D-luciferin potassium salt luminescent substrate (D-Luciferin) (PerkinElmer, Cat.No.122799) was diluted with sterile PBS (Gibco, Cat.No.10010023) to a concentration of 30 mg / ml. The old culture medium was discarded and D-Luciferin and DMEM culture medium containing 10% fetal bovine serum (Gibco, Cat.No.12484028) and 0.1 mg / ml penicillin-streptomycin (Gibco, Cat.No.15070063) were added to a final working concentration of 150 ug / ml of the substrate. The cells were placed in an incubator at 37°C, 5% CO2, and 95% humidity for 10 minutes. The cells were monitored by a small animal in vivo imaging system. If the fluorescence of the cells to be tested is obvious compared to the control group, the EO771 cells were successfully labeled. For details, refer to Figure 1 .

[0106] 2. Construction of EO771-Luc cell line with persistent chemotherapy resistance

[0107] The mouse breast cancer cell line EO771-Luc in the logarithmic growth cycle was inoculated into a DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin and placed in a 37°C, 5% CO2, 95% humidity incubator. When the growth density was 70-80%, docetaxel was added to a final concentration of docetaxel that was the IC50 concentration of the EO771-Luc cell line in the docetaxel. After 8 hours of culture, fresh DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin was added and cultured until the cell saturation density was above 90%. The cell line was passaged once and the above operation was repeated. The IC50 of the cell line in docetaxel was determined every 2 passages and the chemotherapy resistance index (RI) was calculated until the chemotherapy resistance index was >10 and the cell proliferation rate was less than that of the primary EO771-Luc cell. For details, refer to Figure 2 .in Figure 2 A and B show EO771-Luc cells and EO771-Luc cells in a persistent state of chemotherapy resistance with the same cell number and cultured for the same time. It can be seen that the proliferation rate of the latter is lower than that of the former.

[0108] 3. Injection of EO771-Luc chemotherapy-resistant cell lines into the internal iliac artery of mice

[0109] (1) Take EO771-Luc cells in the logarithmic growth phase, discard the original culture medium, and add 5 mL of PBS to rinse. Add 1.5 mL of trypsin and place in a 37°C, 5% CO2, 95% humidity incubator for 2 minutes; add 3 mL of complete culture medium to terminate digestion. Centrifuge at 4°C, 1000 rpm for 5 minutes. After centrifugation, discard the supernatant; resuspend in sterile pre-chilled PBS to a single cell suspension concentration of 2 x 10 6 / ml. (Note: Single cell suspension must be placed on ice)

[0110] (2) 4- to 6-week-old female C57BL / 6NCrl SPF mice (approximately 20 g) in a pre-bone metastatic microenvironment were anesthetized with isoflurane. Hair was removed from the right side of the mouse from the lower edge of the ribs to the knee. The mouse was tilted approximately 45° clockwise with its legs naturally spread out, and the toes were fixed to a removable dissecting cardboard with tape. The right inguinal area was wiped with 70% ethanol, and the surgical area was disinfected again with iodine.

[0111] (3) Make an oblique skin incision about 1.0 cm long between the 4th and 5th pairs of nipples. Use a sterile surgical sheet to cover the animal's body except for the incision site and move the mouse to a standard desktop dissecting microscope. Under a four-fold magnifying glass, the internal iliac blood vessels and nerve bundles can be seen located outside and below the abdominal fat pad. Insert blunt separation forceps between the fat tissue and the peritoneum, push the tissue outward to both sides, and expose the internal iliac blood vessels and nerve clusters. With the help of straight forceps, take a 4-0 suture and pass it under the blood vessels. Gently pull back and forth to further bluntly separate the connective tissue between the blood vessels and nerves. For details, refer to Figure 4 .

[0112] (4) Hold the fine toothless forceps with your left hand, and make the horizontal lines of the toothless forceps parallel to the blood vessel. Hold the syringe with your right hand, with the cone of the needle facing upwards, and insert it into the arterial cavity along the indentation of the toothless forceps. Slowly inject the syringe with a concentration of 2x 10 6 Inject 100 μl of cell suspension into mice at a rate of 100 μl per mouse, from the proximal end to the distal end. (Note: Mix the cell suspension thoroughly between injections to separate the cells and prevent aggregation and embolism.)

[0113] (5) After the injection, remove the forceps and 4-0 sutures and quickly apply pressure to the arterial incision area with a cotton swab for 10 minutes to stop bleeding. After the bleeding stops, wipe the remaining area with alcohol to kill any remaining tumor cells. Suture the skin and place the mouse on a 27°C heating pad until awake.

[0114] 4. Monitoring of breast cancer bone metastasis formation

[0115] (1) In vivo imaging of small animals to monitor the formation of breast cancer bone metastasis

[0116] On the 3rd, 7th, 10th and 14th days after the internal iliac artery injection, mice in the group that received only the EO771-Luc chemotherapy-resistant persistent state cells were imaged using a small animal in vivo imaging system (Note: If only a low signal area appears on the 14th day, the observation time can be appropriately extended to 21 days). 15 mg / ml of D-Luciferin dilution was injected into the mice by intraperitoneal injection, with 200 μL injected into each mouse. If a tumor has formed, a high signal area can be observed in the leg bone of the mouse. In addition, the 14-day survival rate, tumor formation rate, and specific bone metastasis rate were calculated. Figure 6 , Figure 10 . Figure 6 C is the image of the small animal in vivo imaging system in the control group 3 on the 14th day. Figure 6 The fluorescence signal of E is the strongest. Figure 10 As can be seen from the pie chart C, the 14-day survival rate, tumor formation rate, and specific bone metastasis rate of mice in the control group 3 were all lower than those in the present application group. The above two figures indicate that the tumor formation effect of the group in which a microenvironment was created and EO771-Luc cells in a persistent chemotherapy-resistant state were injected into the internal iliac artery was stronger than that of the group in which only EO771-Luc cells in a persistent chemotherapy-resistant state were injected into the internal iliac artery.

[0117] (2) Micro CT evaluation of bone fusion after breast cancer bone metastasis

[0118] On the 14th day after surgery, the mice were killed by cervical dislocation, and bone tissue from the tumor invasion site was taken. After removing muscles and other tissues, bone damage was observed using a Micro CT instrument. The results were as follows: Figure 7 . Figure 7 B is the image of mouse bone tissue in vitro observed by Micro CT on day 14. Figure 7 The osteolysis phenomenon in C was the most significant, indicating that the microenvironment before tumor metastasis was shaped. At the same time, the tumorigenic effect of cells injected with EO771-Luc chemotherapy resistance and persistence was stronger than that of the other groups.

[0119] (3) H&E staining to observe histological changes in bone metastasis sites

[0120] Tumor-bearing mice were sacrificed by cervical dislocation. The leg bone on the side of the bone metastasis was removed and other tissues were removed. The bone was then fixed, decalcified, dehydrated, transparentized, waxed, and embedded. The sectioning site was determined based on the high signal area in the imaging image of step (1) and the osteolytic area in the imaging image of step (2). The bone tissue in the target area was sliced ​​into slices with a thickness of 4 to 6 microns using a microtome.

[0121] Paraffin sections of bone metastasis sites were stained with hematoxylin and eosin (H&E) using a hematoxylin and eosin staining kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0105S). The required solution sequence and time are as follows: hematoxylin staining solution for 1 minute, tap water for 10 seconds, 75% alcohol-1% hydrochloric acid solution for 10 seconds, tap water for 5 minutes, 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, eosin staining solution for 1 minute, 95% alcohol for 10 seconds, 95% alcohol for 45 seconds, 95% alcohol for 1 minute, 100% alcohol for 2 minutes, 50% alcohol and 50% xylene for 4 minutes, xylene I for 6 minutes, xylene II for 6 minutes, and finally, resin sealing. After drying, photos were taken under a microscope. The H&E staining results are shown in the figure below. Figure 8 Compared to Figure 8 B, Figure 8 C often shows irregularly scattered, misshapen cancer cells with enlarged, misshapen, hyperchromatic nuclei, an imbalanced nuclear-cytoplasmic ratio, altered cytoplasmic staining, or the presence of vacuoles. Furthermore, the background often contains necrosis. These results demonstrate that the method of this application successfully establishes breast cancer bone metastases.

[0122] (4) TRAP staining to observe the number of osteoclasts

[0123] TRAP staining kit (Solarbio, Cat. No. G1492) was used to perform TRAP staining on paraffin sections of mouse bone tissue. The sections were added with staining solution and placed in a humidified box at 37°C for staining for 1 hour. The staining solution formula is as follows (500uL system): FastGBC 5uL, Sodium Nitrate 5uL, ultrapure water 445uL, NAB5uL, Acetrate20ul, Tartrate10uL. The required solution sequence time is as follows: 2 minutes in ultrapure water, repeated 3 times; methyl green staining solution 1 minute; 1xPBS 10 seconds. After the sections were dried, they were mounted, photographed under a microscope, and statistical analysis of the TRAP staining results was performed, focusing on observing the number of osteoclasts at the tumor / bone interface. The results are as follows. Figure 9 , Figure 9 B is the TRAP staining microscopic observation of bone tissue sections in the group of cells injected with EO771-Luc chemotherapy resistance in the internal iliac artery. Figure 9 B, Figure 9 C. A significant increase in the number of osteoclasts was observed. The above results indicate that the method of the present invention successfully established osteolytic bone metastases of breast cancer.

[0124] Control group 4

[0125] 1. Construction of Luciferase-labeled luminal B mouse breast cancer cell line EO771

[0126] (1) Take 6×10 5 Luminal B mouse breast cancer cell line EO771 (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-003-0098) in the logarithmic growth phase was inoculated into a 6-well plate containing 250 μl serum-free, double-antibody-free DMEM (Gibco, Cat. No. 11965092) medium, and then 50 μl of 1.5*10 8 HBLV-LUC-PURO virus (Hanbio Biotech (Shanghai) Co., Ltd., Cat. No. LV62121001) at a concentration of 1 tb / mL was added to 50 μl of Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668027), incubated at room temperature for 5 minutes, gently mixed, and allowed to stand at room temperature for 20 minutes. 24 hours after transfection, cells were digested with trypsin (Gibco, Cat. No. 25200056) and G418 (Geneticin, Cat. No. 10131035) was added at a concentration of 300 μg / ml. The culture medium was changed every two days until a single resistant clone emerged.

[0127] (2) Select resistant clones and transfer them to 96-well plates. When they reach the fifth generation, the cells are plated at a rate of 5 × 105 The clone and the control group (untreated EO771 cells) were inoculated into six-well plates and cultured for 8 hours. D-luciferin potassium salt luminescent substrate (D-Luciferin) (PerkinElmer, Cat.No.122799) was diluted with sterile PBS (Gibco, Cat.No.10010023) to a concentration of 30 mg / ml. The old culture medium was discarded and D-Luciferin and DMEM culture medium containing 10% fetal bovine serum (Gibco, Cat.No.12484028) and 0.1 mg / ml penicillin-streptomycin (Gibco, Cat.No.15070063) were added to a final working concentration of 150 ug / ml of the substrate. The cells were placed in an incubator at 37°C, 5% CO2, and 95% humidity for 10 minutes. The cells were monitored by a small animal in vivo imaging system. If the fluorescence of the cells to be tested is obvious compared to the control group, the EO771 cells were successfully labeled. For details, refer to Figure 1 .

[0128] 2. Construction of EO771-Luc cell line with persistent chemotherapy resistance

[0129] The mouse breast cancer cell line EO771-Luc in the logarithmic growth cycle was inoculated into a DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin and placed in a 37°C, 5% CO2, 95% humidity incubator. When the growth density was 70-80%, docetaxel was added to a final concentration of docetaxel that was the IC50 concentration of the EO771-Luc cell line in the docetaxel. After 8 hours of culture, fresh DMEM culture medium containing 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin was added and cultured until the cell saturation density was above 90%. The cell line was passaged once and the above operation was repeated. The IC50 of the cell line in docetaxel was determined every 2 passages and the chemotherapy resistance index (RI) was calculated until the chemotherapy resistance index was >10 and the cell proliferation rate was much lower than that of the primary EO771-Luc cell. For details, refer to Figure 2 .in Figure 2 A and B show EO771-Luc cells and EO771-Luc cells in a persistent state of chemotherapy resistance after culture for the same amount of cells for the same time. It can be seen that the proliferation rate of the latter is much lower than that of the former.

[0130] 3. Intraperitoneal injection of EO771-Luc cell culture supernatant in the persistent state of chemotherapy resistance

[0131] 5~7×10 6A docetaxel-induced chemoresistant persistent cell line, EO771-Luc, was routinely cultured in 5 ml of DMEM supplemented with 10% fetal bovine serum and 0.1 mg / ml penicillin-streptomycin in an incubator at 37°C, 5% CO2, and 95% humidity for 24 hours. The cell culture supernatant was collected and centrifuged at 1000 rpm at 4°C for 5 minutes. The pellet was discarded and the supernatant was collected. (Note: The supernatant was aliquoted and frozen at -80°C until use. Use within 2-3 days after thawing to avoid repeated freeze-thaw cycles.) 20 μl / g of the chemoresistant cell culture supernatant was intraperitoneally injected into 4- to 6-week-old female C57BL / 6NCrl SPF mice once daily for 14 days.

[0132] 4. Injection of EO771-Luc Parental Cells into the Internal Iliac Artery of Mice

[0133] (1) Take the parental EO771-Luc cells in the logarithmic growth phase, discard the original culture medium, and add 5mL PBS to rinse. Add 1.5ml of trypsin and place in a 37℃, 5% CO2, 95% humidity incubator for 2 minutes; add 3ml of complete culture medium to terminate the digestion. Centrifuge at 4℃, 1000r for 5 minutes. After centrifugation, discard the supernatant; resuspend in sterile pre-cooled PBS to a single cell suspension concentration of 2x 106 cells / ml. (Note: The single cell suspension must be placed on ice)

[0134] (2) 4- to 6-week-old female C57BL / 6NCrl SPF mice (approximately 20 g) in a pre-bone metastatic microenvironment were anesthetized with isoflurane. Hair was removed from the right side of the mouse from the lower edge of the ribs to the knee. The mouse was tilted approximately 45° clockwise with its legs naturally spread out, and the toes were fixed to a removable dissecting cardboard with tape. The right inguinal area was wiped with 70% ethanol, and the surgical area was disinfected again with iodine.

[0135] (3) Make an oblique skin incision about 1.0 cm long between the 4th and 5th pairs of nipples. Use a sterile surgical sheet to cover the animal's body except for the incision site and move the mouse to a standard desktop dissecting microscope. Under a four-fold magnifying glass, the internal iliac blood vessels and nerve bundles can be seen located outside and below the abdominal fat pad. Insert blunt separation forceps between the fat tissue and the peritoneum, push the tissue outward to both sides, and expose the internal iliac blood vessels and nerve clusters. With the help of straight forceps, take a 4-0 suture and pass it under the blood vessels. Gently pull back and forth to further bluntly separate the connective tissue between the blood vessels and nerves. For details, refer to Figure 4 .

[0136] 5. Monitoring of breast cancer bone metastasis formation

[0137] (1) In vivo imaging of small animals to monitor the formation of breast cancer bone metastasis

[0138] On the 3rd, 7th, 10th and 14th days after the internal iliac artery injection, mice in the microenvironment-created and internal iliac artery-injected EO771-Luc parental cell group were photographed under a small animal in vivo imaging system (Note: If only a low signal area appears on the 14th day, the observation time can be appropriately extended to 21 days). D-Luciferin dilution 15 mg / ml was injected into the mice by intraperitoneal injection, with 200 μL injected into each mouse. If a tumor has formed, a high signal area can be observed in the mouse leg bone. In addition, the 14-day survival rate, tumor formation rate, and specific bone metastasis rate were calculated. For details, refer to Figure 6 , Figure 10 . Figure 6 D is the image of the small animal in vivo imaging system of the control group 4 on the 14th day. Figure 6 The fluorescence signal of E is the strongest. Figure 10 Pie chart D shows that the 14-day survival rate, tumor formation rate, and specific bone metastasis rate of mice in control group 4 were all lower than those in the present application group. The above two figures indicate that the tumor formation effect of the group in which a microenvironment was created and EO771-Luc cells in a persistent chemotherapy-resistant state were injected into the internal iliac artery was stronger than that of the group in which EO771-Luc cell culture supernatant in a persistent chemotherapy-resistant state was injected into the abdominal cavity and EO771-Luc parental cells were injected into the internal iliac artery.

[0139] (2) Micro CT evaluation of bone fusion after breast cancer bone metastasis

[0140] On the 14th day after surgery, the mice were killed by cervical dislocation, and bone tissue from the tumor invasion site was obtained. After removing muscles and other tissues, bone scans were performed using a Micro CT instrument to observe bone damage.

[0141] (3) H&E staining to observe histological changes in bone metastasis sites

[0142] Tumor-bearing mice were sacrificed by cervical dislocation. The leg bone on the side of the bone metastasis was removed and other tissues were removed. The bone was then fixed, decalcified, dehydrated, transparentized, waxed, and embedded. The sectioning site was determined based on the high signal area in the imaging image of step (1) and the osteolytic area in the imaging image of step (2). The bone tissue in the target area was sliced ​​into slices with a thickness of 4 to 6 microns using a microtome.

[0143] Paraffin sections of bone metastases were stained with a hematoxylin and eosin (H&E) staining kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0105S). The required solutions and durations were as follows: hematoxylin for 1 minute, tap water for 10 seconds, 75% ethanol-1% hydrochloric acid solution for 10 seconds, tap water for 5 minutes, 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, eosin for 1 minute, 95% ethanol for 10 seconds, 95% ethanol for 45 seconds, 95% ethanol for 1 minute, 100% ethanol for 2 minutes, 50% ethanol and 50% xylene for 4 minutes, xylene I for 6 minutes, and xylene II for 6 minutes. Finally, the sections were mounted with resin. After drying, the sections were photographed under a microscope.

[0144] (4) TRAP staining to observe the number of osteoclasts

[0145] Mouse bone tissue paraffin sections were stained using the TRAP staining kit (Solarbio, Cat. No. G1492). Sections were added to the staining solution and placed in a humidified oven at 37°C for 1 hour. The staining solution (500 μL system) consists of 5 μL FastGBC, 5 μL Sodium Nitrate, 445 μL ultrapure water, 5 μL NAB, 20 μL Acetrate, and 10 μL Tartrate. The required incubation times were as follows: 2 minutes in ultrapure water, repeated three times; 1 minute in methyl green solution; and 10 seconds in 1x PBS. After sections were air-dried and mounted, images were taken under a microscope, and statistical analysis of the TRAP staining results was performed, focusing on the number of osteoclasts at the tumor / bone interface.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for constructing a new breast cancer bone metastasis mouse model, characterized in that: The following steps are involved: 1) Chemotherapy drugs induce in vitro breast cancer cells to enter a drug-resistant state; the breast cancer cells are mouse breast cancer cells of luminal B type; the chemotherapy drug is docetaxel; the drug-resistant state refers to a drug resistance index of 10 and a generation time of resistant cells that is three times that of the parental cells; 2) Create a pre-bone metastatic microenvironment for breast cancer in recipient mice; 3) Transplantation of in vitro chemotherapy-resistant breast cancer cells and injection of the cell culture supernatant of the resistant breast cancer cells into the internal iliac artery of recipient mice; 4) Monitor the formation of bone metastases in mice.

2. The method for constructing a novel breast cancer bone metastasis mouse model according to claim 1, characterized in that: The mouse breast cancer cells are luciferase-labeled mouse breast cancer cells.

3. The method for constructing a novel breast cancer bone metastasis mouse model according to claim 1, characterized in that: The microenvironment before breast cancer bone metastasis in recipient mice is created as described in step 2) by intraperitoneally injecting the culture supernatant of in vitro chemotherapy-resistant breast cancer cells into the mice.

4. The method for constructing a novel breast cancer bone metastasis mouse model according to claim 1, characterized in that: Monitoring the formation of bone metastases in mice as described in step 4) includes observing the presence of tumor metastases in the mouse bones using a small animal in vivo imaging system, observing the tumor metastasis sites in the mouse bone tissue ex vivo using Micro-CT, and observing paraffin sections of the mouse breast cancer bone metastasis sites using H&E staining and TRAP staining.

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