Preparation method of bone metastatic model animal of cancer

Abstract

To provide a preparation method of an animal model of bone metastasis of cancer and a use method thereof.SOLUTION: Provided is a preparation method of a bone metastatic model animal of cancer including a step of injecting a cancer cell into an abdominal aorta of a non-human animal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an animal model of cancer bone metastasis. [Background technology]

[0002] Bone metastasis of cancer refers to the spread of cancer cells via the bloodstream to bones such as the lumbar vertebrae, thoracic vertebrae, cervical vertebrae, pelvis, ribs, skull, humerus, femur, tibia, and fibula, where they grow and form lesions. Bone metastasis can cause severe pain, fractures, and paralysis in patients, significantly reducing their quality of life, so it is desirable to maintain that quality of life through prevention, early detection, and early treatment.

[0003] Research into the pathology and treatment of bone metastasis requires experimental animal models that reproduce bone metastasis that occurs in cancer patients. Conventional animal models of bone metastasis include the intraosseous orthotopic transplantation model of cancer cells (Non-Patent Documents 1 and 2), the intraventricular injection model (Non-Patent Document 3), and the tail artery injection model (Non-Patent Document 4). The intraosseous orthotopic transplantation model is created by directly injecting cancer cells into the bone marrow, where the intervening tissue from the epidermis is relatively thin, using a syringe needle. Although bone metastases form in this model, the traditional bone marrow structure is destroyed during the injection process, and bone marrow components leak from the injection site, causing fibrosis around the injection site. This makes it difficult to reproduce the microenvironment formed by cancer cells and surrounding non-cancer cells, which is important for the study and analysis of bone metastases. The intraventricular injection model is created by injecting cancer cells into the ventricles of the heart, which results in the formation of bone metastases, as well as brain and lung metastases, and significantly shortens survival time. The tail artery injection model is created by injecting cancer cells into the tail artery. While the tail artery injection model reduces metastasis to other organs compared to the intraventricular injection model, lung metastasis is still observed, making it unsuitable for long-term experiments such as investigating cancer progression in bone metastases or therapeutic effects. Furthermore, the tail artery injection model can vary the site of metastasis, such as the occurrence of lung metastasis, depending on the speed of tail artery injection. This makes it difficult to conduct quantitative experiments. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] S Miwa et al. J Cell Biochem. 2016, 117(11): 2533-2537 [Non-patent document 2] Y Sawada et al. Int J Cancer. 2020, 146(5): 1369-1382 [Non-patent document 3] JK Simmons et al. Vet Pathol. 2015, 52(5): 827-841 [Non-patent document 4] T Kuchimaru et al. Nat Commun. 2018, 9(1): 2981 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention relates to providing a method for producing an animal model of cancer bone metastasis and a method for using the same. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted extensive research and have found that when cancer cells are injected into the abdominal aorta of a non-human animal, metastatic lesions are formed specifically in the bones of the non-human animal, thereby completing the present invention.

[0007] That is, the present invention relates to the following 1) to 6). 1) A method for producing a cancer bone metastasis model animal, comprising the step of injecting cancer cells into the abdominal aorta of a non-human animal. 2) The method described in 1), wherein the cancer cells are prostate cancer cells, breast cancer cells, or ovarian cancer cells. 3) The method described in 1), wherein the injection of cancer cells into the abdominal aorta of a non-human animal is between the renal artery bifurcation and the abdominal aortic bifurcation of the abdominal aorta of the non-human animal. 4) A cancer bone metastasis model animal produced by the method described in any one of 1) to 3). 5) A method for evaluating or searching for a therapeutic agent for bone metastasis of cancer, comprising the steps of administering a test substance to the cancer bone metastasis model animal described in 4) and detecting the presence or extent of bone metastasis of cancer. 6) A method for evaluating or searching for a preventive or therapeutic agent for bone metastasis of cancer, comprising the following steps (a) to (c): (a) injecting cancer cells into the abdominal aorta of a non-human animal; (b) administering a test substance to the non-human animal before, simultaneously with, or after the injection of step (a). (c) after steps (a) and (b), further detecting the presence or extent of bone metastasis of cancer in the non-human animal. [Effects of the Invention]

[0008] The method of the present invention can provide an animal model that forms bone metastases of cancer. The animal is useful for studying the pathology of bone metastasis of cancer and for developing foods and pharmaceuticals that prevent or ameliorate bone metastasis. [Brief explanation of the drawings]

[0009] [Figure 1] A bone metastasis model was created by injecting cancer cells (4T1) into the abdominal aorta of a mouse. (A) Imaging of cancer cells one week after injection; the gray areas indicate the accumulation of cancer cells. (B) Pathological specimen of femoral metastatic tissue. DETAILED DESCRIPTION OF THE INVENTION

[0010] As used herein, "bone metastasis of cancer" refers to cancer cells traveling through the bloodstream to reach bone, where they proliferate and form lesions. Bone metastases can cause fibrosis, necrosis, osteoid formation, and changes in hematopoietic activity in the surrounding bone marrow. Bone metastases are classified into osteoblastic metastasis, osteolytic metastasis, trabecular metastasis, and mixed metastasis based on differences in bone reactions in bone metastases, and all of these are encompassed in the term "bone metastasis" in this specification.

[0011] The method of the present invention for producing a non-human animal model of cancer bone metastasis (hereinafter also referred to as the method of the present invention for producing a bone metastasis model) comprises the step of injecting cancer cells into the abdominal aorta of a non-human animal.

[0012] The non-human animal used may be any animal other than a human, but is preferably a non-human mammal. Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats. However, from the perspective of creating a pathological animal model system, rodents, particularly mice, are preferred because they have a relatively short ontogeny and biological cycle and are easy to breed. The sex and age of the non-human animal are not particularly limited, but for example, when the non-human animal is a rodent, its age is preferably 5 weeks or older, and more preferably 7 weeks or older.

[0013] The cancer cells used may be any cancer-derived cells capable of causing bone metastasis. Examples of such cancers include prostate cancer, breast cancer, lung cancer, thyroid cancer, kidney cancer, head and neck cancer, uterine cancer, esophageal cancer, ovarian cancer, colon cancer, gastric cancer, pancreatic cancer, biliary tract cancer, liver cancer, and bladder cancer. Of these, prostate cancer, breast cancer, and ovarian cancer are preferred. The bone in which bone metastases form varies depending on the type of cancer cells used, but examples include the lumbar vertebrae, thoracic vertebrae, cervical vertebrae, pelvis, ribs, skull, humerus, femur, tibia, and fibula. Preferably, the bone is one or more bones selected from the femur, tibia, and fibula, and more preferably the femur.

[0014] In the method for producing a bone metastasis model of the present invention, cancer cells are injected into the abdominal aorta of a non-human animal. The injection site in the abdominal aorta is not particularly limited, but from the viewpoint of the efficiency of producing the model, it is preferably between the renal artery bifurcation of the abdominal aorta and the abdominal aortic bifurcation, and more preferably between the mesenteric artery of the abdominal aorta and the abdominal aortic bifurcation. The amount of cancer cells injected may be determined appropriately depending on the type of cancer cells, the type, body weight, age in weeks, etc. of the non-human animal to be treated, but is preferably 2.5 × 10 4 ~2.5×10 7 cells / kg is preferred, 2.5 × 10 5 ~2.5×10 6 When the non-human animal is a mouse, the amount of cancer cells injected is preferably 5×10 2 ~5×10 5 cells / individual is preferred, 5 x 10 3 ~5×10 4 More preferably, 100 cells / individual is injected. The cancer cells are preferably injected in a form suitable for cell survival and injection into the blood vessels of non-human animals, for example, in the form of a suspension in various buffer solutions such as physiological saline or PBS. The number of injections of cancer cells into non-human animals is usually one, but may be divided into 2 to 4 injections.

[0015] In a preferred embodiment, a non-human animal subjected to the method for producing a bone metastasis model of the present invention is subjected to a pretreatment of exposing the abdominal aorta prior to injection of cancer cells into the abdominal aorta, and is also subjected to a posttreatment of stopping bleeding and closing the abdomen after injection of the cancer cells into the abdominal aorta. The pretreatment and posttreatment steps can each be carried out by known methods. A specific example of the method for producing a bone metastasis model of the present invention, including the pretreatment and posttreatment steps, is shown below, but the method for producing a bone metastasis model of the present invention is not limited thereto.

[0016] 1. Non-human animals are subjected to inhalation anesthesia and intravenous anesthesia, and sedatives and analgesics are also used. 2. An inhalation anesthesia mask is placed over the mouth of the non-human animal, and inhalation anesthesia is administered continuously. 3. The sedated and analgesic non-human animal is fixed in a position where the abdominal area of the surgical site can be observed under a stereomicroscope. 4. After confirming that sedation and analgesia are sufficient, a midline incision is made in the abdomen of the non-human animal. 5. Obtain a view of the abdomen of the non-human animal. 6. Expose the abdominal aorta of a non-human animal. 7. Locate the insertion site in the abdominal aorta of a non-human animal. 8. After determining the insertion site, peel off the capsule. 9.Cancer cells are injected into the insertion site using a syringe. 10. After insertion, apply pressure to the insertion site. When applying pressure, check the color of the tail artery to prevent arterial occlusion due to excessive pressure. 11. After applying pressure, check that the bleeding has stopped. If bleeding continues, apply more pressure. 12. After confirming that bleeding has stopped, place the hemostatic pad at the insertion site and apply pressure again. 13. After confirming hemostasis, suture the abdominal peritoneum and skin and close the abdomen. 14. After abdominal closure, the non-human animal is transferred to an animal cage and its movements after awakening from anesthesia are confirmed.

[0017] In the non-human animals obtained in this manner, the formation of bone-specific cancer metastases is observed approximately 1 to 4 weeks after cancer cell injection, as shown in the Examples below. However, metastasis to organs such as the lungs and brain, which would shorten survival time, is not observed. The presence or extent of bone metastasis can be assessed using conventional testing techniques. Testing techniques for bone metastasis include bone scintigraphy, PET, PET-CT, CT, X-ray, MRI, optical imaging, and histology. For example, the presence or extent of bone metastasis can be determined by bone accumulation of radioisotopes in bone using bone scintigraphy; by glucose derivatives in bone using PET and PET-CT; by bone destruction images, osteoblast images, and intraosseous tumor shadows using CT, X-ray, and MRI; by bioluminescence (luminescence imaging) or fluorescence (fluorescence imaging) visualization of cancer cells in optical imaging; and by tumor cells in collected bone tissue using histology.

[0018] Such non-human animals with bone metastases are considered to be potential animal models for cancer bone metastasis. The bone metastasis animal model of the present invention does not impair the bone microenvironment due to its production method, and has bone-specifically formed metastatic lesions. Therefore, it can solve the problems of conventional bone metastasis models, such as the destruction of the existing bone microenvironment and the difficulty of bone-specific metastasis. The bone metastasis animal model of the present invention can be used for research into the pathology of bone metastasis and the prevention or treatment of bone metastasis, for example, for the evaluation or discovery of preventive or therapeutic agents for bone metastasis.

[0019] Evaluation or screening of therapeutic agents for bone metastasis of cancer using the bone metastasis model animal of the present invention can be carried out, for example, by administering a test substance to the bone metastasis model animal of the present invention and detecting the presence or extent of bone metastasis of cancer.

[0020] The test substance may be any substance for which the therapeutic effect on bone metastasis is to be predicted, and is not particularly limited. The test substance may be a naturally occurring substance or a substance artificially synthesized by chemical or biological methods, or may be a compound, composition, or mixture. The method of administering the test substance is not particularly limited and may be selected appropriately depending on the animal species to be administered and the characteristics of the test substance. Examples include oral administration, intravenous administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, and intrarectal administration. The dose of the test substance may also be determined appropriately depending on the animal species to be administered and the characteristics of the test substance. The test substance may be administered once or multiple times, approximately 2 to 4 times.

[0021] The presence or extent of cancer bone metastasis can be detected by the bone metastasis testing method described above. In a preferred embodiment, the effect of the test substance on bone metastasis is evaluated by comparing the presence or extent of bone metastasis detected between the test group and the control group. If bone metastasis is not detected in the test group but is detected in the control group, the test substance can be evaluated or selected as a therapeutic agent for bone metastasis of cancer. Alternatively, if the extent of bone metastasis in the test group is lower than that in the control group, the test substance can be evaluated or selected as a therapeutic agent for bone metastasis of cancer. Comparison with the control group is preferably performed statistically. For example, if the extent of bone metastasis in the test group is statistically significantly lower than that in the control group, the test substance can be evaluated or selected as a therapeutic agent for bone metastasis of cancer. Here, the bone metastasis model animal of the present invention, which has not been administered the test substance, is preferably used as the control group.

[0022] Furthermore, evaluation or screening of preventive or therapeutic agents for bone metastasis of cancer using the bone metastasis animal model of the present invention can be carried out, for example, by the following steps (a) to (c). (a) injecting cancer cells into the abdominal aorta of a non-human animal; (b) administering a test substance to the non-human animal before, simultaneously with, or after the injection of step (a). (c) after steps (a) and (b), further detecting the presence or extent of bone metastasis of cancer in the non-human animal.

[0023] In the above evaluation or screening method, the injection of cancer cells into the abdominal aorta of a non-human animal in step (a) is as described above. The administration of the test substance in step (b) is carried out before, simultaneously with, or after step (a). In one example, in evaluating or screening for a preventive agent for bone metastasis, it is preferable to carry out step (b) before step (a). The detection of the presence or extent of bone metastasis of cancer in step (c) is carried out after steps (a) and (b).

[0024] The test substance is not particularly limited as long as it is a substance for which the preventive or therapeutic effect on bone metastasis is to be predicted. The test substance may be a naturally occurring substance or a substance artificially synthesized by chemical or biological methods, or may be a compound, composition, or mixture. The method of administering the test substance is not particularly limited and may be appropriately selected depending on the animal species to be administered and the characteristics of the test substance. Examples include oral administration, intravenous administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, and intrarectal administration. The dose of the test substance may also be appropriately determined depending on the animal species to be administered and the characteristics of the test substance. The test substance may be administered once or multiple times, approximately 2 to 4 times.

[0025] The presence or extent of cancer bone metastasis can be detected using the bone metastasis testing method described above. In a preferred embodiment, the effect of the test substance on bone metastasis is evaluated by comparing the presence or extent of bone metastasis detected between the test group and the control group. If bone metastasis is not detected in the test group but is detected in the control group, the test substance can be evaluated or selected as a preventive or therapeutic agent for cancer bone metastasis. Alternatively, if the extent of bone metastasis in the test group is lower than that in the control group, the test substance can be evaluated or selected as a preventive or therapeutic agent for cancer bone metastasis. Comparison with the control group is preferably performed statistically. For example, if the extent of bone metastasis in the test group is statistically significantly lower than that in the control group, the test substance can be evaluated or selected as a preventive or therapeutic agent for cancer bone metastasis. Here, the bone metastasis model animal of the present invention, which has not been administered the test substance, is preferably used as the control group. Here, "prevention" refers to preventing or delaying the onset of bone metastasis in an individual, or reducing the risk of developing bone metastasis in an individual. "Treatment" refers to improving or alleviating bone metastasis, preventing or delaying the worsening of bone metastasis, or reversing, preventing, or delaying the progression of bone metastasis. [Example]

[0026] The present invention will now be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0027] Example 1: Creation of a cancer bone metastasis model (1) Method Three male or female BALB / c, C57BL6, nude, NSG, or NOD / SCID mice (Jackson Laboratory Japan, Inc.), aged 7–10 weeks, were anesthetized with isoflurane (Fujifilm Wako Pure Chemical Corporation) in combination with a triple anesthetic mixture (sedative, analgesic, and muscle substitute; Medetomin Injection (0.75 mg / kg; Meiji Animal Health Co., Ltd.; midazolam (4 mg / kg; Fujifilm Wako Pure Chemical Corporation; and Vetorfar (5 mg / kg; Meiji Animal Health Co., Ltd.)). An anesthesia mask was placed over the mouse's mouth, and isoflurane was continuously administered at a concentration of 3%. A sterile cloth was placed under a stereomicroscope (Nikon Corporation) to prepare the surgical environment. The sedated and analgesic mouse was placed on the sterile cloth, and the limbs of the mouse were fixed with tape at a location where the abdominal area could be observed under the stereomicroscope. After confirming that sedation and analgesia were sufficient, a midline abdominal incision was made. The midline incision, the abdominal skin and peritoneum resection edges of the mouse, and the sterile cloth were sutured with needled sutures (Surgisorb, Bear Medic Co., Ltd.), and tension was applied to ensure a clear view of the abdomen. The abdominal organs were moved laterally with a cotton swab (As One Corporation) to expose the abdominal aorta. The insertion site was located between the bifurcation of the renal arteries and the abdominal aorta bifurcation. The insertion site was located as close to the midline of the aorta as possible, below the mesenteric artery. After determining the injection site, the capsule was peeled off with a cotton swab, and a suspension of cancer cells (human breast cancer cell line: MDAMB231, mouse breast cancer cell line: 4T1, EO771, human ovarian cancer cell line: ES2, RMG1, or human prostate cancer cell line: PC3) was transplanted using a 30G needle syringe (NIPRO). The number of transplanted cancer cells was adjusted according to the degree of cancer cell proliferation, and was set at 5 × 10 per individual. 3 ~5×10 4The injection volume was in the range of 100-150 cells, and 50-100 μl. The suspension was prepared in PBS or physiological saline. After insertion, the insertion site was compressed with a cotton swab for at least 5 minutes. The color of the tail artery was checked to prevent arterial occlusion due to excessive compression. After 5 minutes of compression, the cotton swab was removed and hemostasis was confirmed. If bleeding continued, compression with the cotton swab was continued. After hemostasis was confirmed, Surgicel (registered trademark) Fibrillar (Johnson & Johnson KK) was placed at the insertion site and pressure was applied again for approximately 1 minute. After hemostasis was confirmed, the abdominal peritoneum and skin were sutured and the abdomen was closed. The mouse was transferred to an animal cage equipped with a heater and its body movements after waking from anesthesia were checked.

[0028] (2) Results All mice injected with cancer cells into the abdominal aorta using this method developed bone metastases in the femur and / or tibia / fibula within 1 to 4 weeks after injection, with most developing at least femur metastases. Bone metastases were confirmed by cancer cell imaging (luminescence imaging using luciferase and luciferin) and pathological specimens. Figure 1 shows an example of cancer cells (4T1) injected into the abdominal aorta of a mouse, with accumulation in the femur (gray area in Figure 1(A) and red, yellow, and green areas in the color image, Figure 1(B)). No metastases to major organs such as the brain, lungs, or liver were observed in any of the mice. Furthermore, all mice survived for more than 4 weeks after injection. Non-Patent Documents 3 and 4 describe intraventricular injection and tail artery injection models as animal models of bone metastasis. Both models induce lung metastasis along with bone metastasis, making it impossible to perform long-term observation of mice for more than four weeks to assess the therapeutic efficacy and resistance of drugs to bone metastasis. Furthermore, in the tail artery injection model, cancer cells are refluxed from the artery during preparation, and the organs to which they are seeded vary depending on the pressure of the cancer cells injected during administration. Similarly, in the intraventricular injection model, the type of metastatic organ and the size of the metastatic lesions are largely determined by chance. Thus, existing bone metastasis models have poor long-term survival and reproducibility, making this model advantageous in this respect.

Claims

1. A method for producing a cancer bone metastasis model animal, comprising the step of injecting cancer cells into the abdominal aorta of a non-human animal.

2. The method of claim 1, wherein the cancer cells are prostate cancer cells, breast cancer cells, or ovarian cancer cells.

3. The method according to claim 1, wherein the injection of cancer cells into the abdominal aorta of a non-human animal is between the renal artery bifurcation and the abdominal aortic bifurcation of the abdominal aorta of a non-human animal.

4. A cancer bone metastasis model animal produced by the method according to any one of claims 1 to 3.

5. A method for evaluating or searching for a therapeutic agent for bone metastasis of cancer, comprising the steps of administering a test substance to the cancer bone metastasis model animal of claim 4 and detecting the presence or extent of bone metastasis of cancer.

6. A method for evaluating or searching for a preventive or therapeutic agent for bone metastasis of cancer, comprising the following steps (a) to (c): (a) injecting cancer cells into the abdominal aorta of a non-human animal (b) administering a test substance to the non-human animal before, simultaneously with, or after the injection of step (a). (c) after steps (a) and (b), further detecting the presence or extent of bone metastasis of cancer in the non-human animal.

Images