JP6436669B2 - Uterine sarcoma metastasis model animal - Google Patents

Description

  The present invention relates to a uterine sarcoma metastasis model animal, a production method and use thereof, and an isolated cell useful for the production.

  Uterine sarcomas are extremely rare (3-8% of endometrial malignant tumors) and have a poor prognosis. The 5-year survival rate is about 50% in stage I (the tumor is confined to the uterus), and half of those who have undergone complete resection have relapsed and metastasized to the lung. There are many outbreaks among unmarried people, obese people, and those taking hormonal drugs (especially tamoxifen). The number of patients is expected to increase in the future due to the declining birthrate and late marriage, an increase in breast cancer patients, and the spread of hormone therapy.

  On the other hand, the problems to be solved include the lack of useful diagnostic markers for uterine sarcoma, the lack of effective clinical research for rare cases, and the appropriate prevention of hematogenous metastasis / relapse to the lungs. For example, there is no treatment.

  As a method for producing a model animal having uterine sarcoma or its pulmonary metastasis, a uterine sarcoma is generated in the mouse uterus by manipulating a specific gene (eg, LMP2, CR-1, BRCA1) (non-patent document) Literature 1-3), in which uterine sarcoma was produced in the mouse uterus by directly injecting uterine sarcoma cells into the myometrium (Non-patent document 4), and uterine sarcoma cells were injected subcutaneously in the mouse, What formed the metastatic focus (nonpatent literature 5) is known.

Hayashi et al., Cancer Res. 2002, Jan 1; 62 (1): 24-7 Strizzi et al., J Pathology 2007, Jan; 211 (1): 36-44. Xing et al., Cancer Res. 2009, Nov 1; 69 (21): 8231-5. Ren et al., Clin. Cancer Res. 2008, Sep 1; 14 (17): 5466-75. Edris et al., Proc. Natl. Sci. Acad. USA 2012, Apr 24; 109 (17): 6656-61.

  The conventional method is limited to the formation of only the primary lesion or the formation of only the metastatic lesion. In addition, the maximum observation period for producing a model mouse is as long as 13 months.

  Accordingly, there is a need for a method that can more easily and easily produce a uterine sarcoma metastasis model animal that simultaneously reflects the clinical pathology of uterine sarcoma in humans and that has a primary uterine lesion and a metastatic lesion simultaneously. An object of the present invention is to provide such a method. The present invention is also useful for a model animal obtained by the method, use of the model animal for drug screening and evaluation of the effectiveness of the therapy, a method for establishing a highly metastatic strain derived from uterine sarcoma, and the production method described above. Also provided are isolated cells, and the like.

The inventors of the present invention have made extensive studies to solve the above problems, and after transplanting a uterine sarcoma-derived cell line to an immunodeficient animal to form a tumor, the tumor fragment obtained by fragmenting the tumor was immunized. We came up with a methodology for transplanting into the myometrium of deficient animals. Transplanted tumor pieces form primary tumor foci in the myometrium and metastatic foci in target organs such as the lungs in the animal.
Originally, metastasis of uterine sarcoma-derived cell line MES-SA cells is not observed by subcutaneous injection and injection directly into the myometrium of immunodeficient non-human mammals (Harker et al., Cancer Res. 43: 4943-4950, 1983. For the myometrium, see Ren et al., Clin Cancer Res. 14: 5466-5475, 2008.). One of the biggest reasons is that it was technically difficult to inject the amount of cells needed to form a metastatic tumor into the uterus of a very small immunodeficient non-human mammal. is there. In order to overcome this problem, the inventors have conceived of transplanting tumor pieces. Using this technique, we succeeded in creating a uterine sarcoma metastasis model animal that has both a primary uterine lesion and a metastatic lesion.
The present inventors have further studied based on the above findings, and have completed the present invention.

The present invention provides the following.
[1] A method for producing a uterine sarcoma metastasis model animal using a non-human mammal,
(A) transplanting metastatic and tumorigenic cells derived from uterine sarcoma into a recipient's immunodeficient non-human mammal, and forming a tumor in said recipient animal;
(B) excising the formed tumor to obtain a tumor fragment; and
(C) transplanting the tumor piece into the myometrium of an immunodeficient non-human mammal to be the model animal, wherein the transplanted tumor fragment comprises a primary tumor focus in the myometrium and Said method of forming a metastatic focus in a target organ.
[2] The method according to [1] above, wherein the cell transplanted in step (a) is integrated into the genome so that a gene encoding a fluorescent protein or a photoprotein can function in the cell.
[3] The method according to [2] above, wherein the fluorescent protein is green fluorescent protein.
[4] The method according to any one of [1] to [3] above, wherein the cells to be transplanted in step (a) are derived from a human.
[5] The method according to any one of [1] to [4] above, wherein the cells to be transplanted in the step (a) are derived from MES-SA cells, SKN cells, or SK-UT-1 cells.
[6] After step (c)
(D) The method according to any one of [1] to [5], further comprising the step of confirming whether or not a primary tumor foci in the myometrium and a metastatic foci in the target organ have been formed in the animal. .
[7] The method according to any one of [1] to [6] above, wherein the target organ is lung.
[8] The method according to any one of [1] to [7] above, wherein the model animal is a mouse or a rat.
[9] The method according to any one of [1] to [8] above, wherein the recipient animal is a mouse or a rat.
[10] A uterine sarcoma metastasis model non-human mammal obtained by the method according to any one of [1] to [9] above.
[11] The primary tumor foci formed in the myometrium and the metastatic foci in the target organ are compared with the corresponding sites in the same strain of animals having no tumor in the myometrium and the target organ. The animal according to [10] above, which shows enhanced expression of at least one of 1 and GLUT-3.
[12] A method for screening a substance having anticancer activity and / or metastasis-suppressing activity against uterine sarcoma,
(I) a step of providing a uterine sarcoma metastasis model non-human mammal according to [10] or [11] above,
(Ii) administering a test substance to the animal;
(Iii) after starting administration of the test substance, assessing the size of the primary tumor focus in the myometrium and / or the number and / or size of the metastatic focus in the target organ; and
(Iv) when the size of the primary tumor foci and / or the number and / or size of the metastases is reduced as compared to the case where the test substance is not applied, the test substance is treated with uterine sarcoma. The method comprising the step of selecting as a candidate for a substance having anticancer activity and / or metastasis-suppressing activity against.
[13] The method described in [12] above, wherein the animal is the animal described in [11] above, and the evaluation in step (iii) is performed using positron emission tomography.
[14] A method for determining the anticancer effect and / or metastasis-suppressing effect of a therapy against uterine sarcoma,
(I) a step of providing a uterine sarcoma metastasis model non-human mammal according to [10] or [11] above,
(Ii) applying the therapy to the animal;
(Iii) assessing the size of the primary tumor focus in the myometrium and / or the number and / or size of the metastatic focus in the target organ after initiation of the therapy; and
(Iv) The said method including the process of determining the anticancer effect and / or metastasis inhibitory effect with respect to the uterine sarcoma of this therapy based on the evaluation in a process (iii).
[15] The method described in [14] above, wherein the animal is the animal described in [11] above, and the evaluation in step (iii) is performed using positron emission tomography.
[16] A method for establishing a cell line derived from uterine sarcoma having improved metastatic potential,
(I) The parent cell line desired to be improved in metastatic ability is used as the cell to be transplanted in the step (a), and the method according to any one of [1] to [9] above is used. Creating a model animal,
(Ii) obtaining a tumor piece from a metastatic focus formed in the animal;
(Iii) Transplanting the tumor piece into the myometrium of another immunodeficient non-human mammal to form a primary tumor focus in the myometrium and a metastasis in the target organ in the animal, and the formed metastasis Obtaining a tumor piece from at least once, and
(Iv) separating and increasing cells in the obtained tumor piece in vitro, and establishing a cell line, wherein the established cell line has improved metastatic potential compared to the parent cell line, Said method.
[17] Gene expression analysis is performed on cells of the cell line established by the method described in [16] above and cells of a cell line having a lower ability to transfer to the target organ, and the gene expression of both cells is compared. A method for identifying a gene associated with metastasis of uterine sarcoma.
[18] The method according to [17] above, wherein the gene expression analysis is performed using a microarray method.
[19] A metastatic and tumorigenic isolated cell derived from a mammalian uterine sarcoma, wherein a gene encoding a fluorescent protein or photoprotein is integrated into the genome so that it can function in the cell Said cells.
[20] The cell according to [19] above, wherein the fluorescent protein is green fluorescent protein.
[21] The cell according to [19] or [20] above, wherein the mammal is a human.
[22] The cell according to any one of [19] to [21], which is derived from MES-SA cells, SKN cells, or SK-UT-1 cells.

  According to the method for producing a model animal of the present invention, a model animal having both a primary uterine lesion and a metastatic lesion such as a lung can be reproducibly (eg, 80% or more) in a short period (eg, about 10 weeks). ) Can be easily produced.

  As described in [2] and [3] above, if a method of forming a tumor using cells stably transfected with a fluorescent protein or photoprotein is used, the fluorescence or luminescence is used as an index, and cancer cells Behaviors such as proliferation, migration, invasion, and metastasis can be observed non-invasively, in real time, and in vivo.

As described in [11] above, in the model animal produced according to the present invention, the formed primary and metastatic tumors can highly express GLUT-1 and / or GLUT-3. Therefore, it may be possible to observe the primary lesion and the metastatic lesion non-invasively by a PET imaging experiment using a tracer compound such as ¹⁸ F-FDG.

  As described in the above [12] to [15], the model animal produced according to the present invention is a method for screening a substance having anticancer activity and / or metastasis inhibitory activity against uterine sarcoma, and anti-uterine sarcoma anti-tumor activity. Applications such as a method for determining a cancer effect and / or a metastasis inhibitory effect can be provided.

  In addition, as described in [16] above, by applying the method for producing a model animal of the present invention, it becomes possible to establish a cell line derived from uterine sarcoma having improved metastatic potential. The high-metastasis strain thus established can be used to comprehensively analyze genes related to uterine sarcoma metastasis as described in [17] and [18] above.

  The present invention also provides an isolated cell useful for the method for producing a model animal of the present invention as described in [19] to [22] above.

Production of C9 cells, which are sub-strains of MES-SA GFP-J that constitutively express GFP, cloned by introducing the GFP gene into human uterine leiomyosarcoma-derived MES-SA cells (ATCC), It is a figure which shows the outline of inoculation to a mouse | mouth. Six weeks after orthotopic transplantation, before laparotomy (a left figure) and photograph of the primary tumor focus (a right figure), macroscopic image of uterine primary tumor focus (b) and lung metastases (c), GFP fluorescence Images and stained images of various markers (H & E, Vimentin, Ki-67, Glut-1) are shown. Six weeks after orthotopic transplantation, before laparotomy (a left figure) and photograph of the primary tumor focus (a right figure), macroscopic image of uterine primary tumor focus (b) and lung metastases (c), GFP fluorescence Images and stained images of various markers (H & E, Vimentin, Ki-67, Glut-1) are shown. An example of lung metastasis is shown. Tumor foci formed by metastasis are shown by fluorescence from GFP (a). (B) is a stained image of various markers (H & E, Glut-1, Ki-67), and (c) is an enlarged view (c) thereof. An example of lung metastasis is shown. Tumor foci formed by metastasis are shown by fluorescence from GFP (a). (B) is a stained image of various markers (H & E, Glut-1, Ki-67), and (c) is an enlarged view (c) thereof. The data of animal PET acquired about the produced model mouse are shown. A (side view) and B (top view) are normal mice, and C (side view) and D (top view) are PET images of the model mouse. The graph which investigated the correlation with primary tumor weight and primary tumor volume, the number of days after orthotopic transplantation, and the primary tumor volume, the number of lung metastases and the number of days after orthotopic transplantation, and the number of lung metastases and the primary tumor volume is shown. The graph which investigated the correlation with the number of lung metastases and lung FDG and the number of lung metastases and lung FLT is shown.

(Production method)
The present invention provides a method for producing a uterine sarcoma metastasis model animal (hereinafter also referred to as the production method of the present invention). The method comprises the steps of: (a) transplanting metastatic and tumorigenic cells derived from uterine sarcoma into a recipient immunodeficient non-human mammal and forming a tumor in the recipient animal; (b) the formation Removing a tumor obtained to obtain a tumor fragment, and (c) transplanting the tumor fragment into the myometrium of an immunodeficient non-human mammal to be the model animal. The transplanted tumor pieces form a primary tumor focus in the myometrium and a metastatic focus in the target organ in the animal.

  In the present specification, the “model animal for uterine sarcoma metastasis” means a model animal that can represent the pathological condition of metastatic uterine sarcoma in a mammal of interest. The model animal may be an animal that already has a primary tumor focus in the myometrium and a metastatic focus in the target organ, or has not yet formed a metastasis or primary tumor focus and metastasis, but the time It may also be an animal that is naturally expected to form these tumors. The mammal of interest is not particularly limited and may be any mammal, but is preferably a human from a clinical viewpoint.

The model animal may be any non-human mammal species as long as it can be produced according to the present invention. The animal is immunodeficient because it needs to be able to accept foreign tumor pieces, as described below. In the present specification, an “immune-deficient animal” has a significantly reduced immune function (eg, T cell function, B cell function, NK cell function, etc.) compared to an artificially or innately healthy animal. Means animals. Although it does not specifically limit as an animal species, From a viewpoint, such as preparation, acquisition, and ease of handling, Preferably it is a mouse | mouth and a rat, More preferably, it is a mouse | mouth. Specifically, for example, nude mice such as BALB / c Slc-nu / nu mice and KSN / Slc mice, rats lacking T cell functions such as F344 / NJcl-rnu / rnu rats, severe combined immunodeficiency (SCID) Mouse and rat, NOD.CBD17-Prkdc ^scid / J mouse (NOD-scid mouse), NOD / Shi-scid, IL2Rγ ^null mouse (NOG mouse), Rag2 ^null / IL2Rγ ^null mouse, and mice and rats that have improved these Can be mentioned.

  The site of metastasis of the model animal, that is, the above-mentioned “target organ” is not particularly limited as long as a metastasis derived from uterine sarcoma can be formed. Examples of the target organ include lung, liver, heart, bone, skin, omentum, lymph node, mesentery, deep soft tissue, brain and the like, preferably lung and gut lymph node, more preferably The lungs.

  Hereinafter, each step will be described in detail.

(A) Transplanting metastatic and tumorigenic cells derived from uterine sarcoma into a recipient's immunodeficient non-human mammal and forming a tumor in said recipient animal. It is a cell derived from a uterine sarcoma (example: human). The cell may be newly isolated from a patient or animal suffering from uterine sarcoma according to a procedure known per se, or a commercially available cell line may be used. Commercially available cells include MES-SA cells derived from human anaplastic sarcoma (Harker WG, et al., Cancer Res. 43: 4943-4950 (1983); ATCC (American Type Culture Collection)), human uterine leiomyosarcoma SKN cells (Ishiwata et al., Cancer Res. 37: 658-664 (1977); RIKEN), human uterine leiomyosarcoma SK-UT-1 cells (Fogh et al., J Natl Cancer Inst 58: 209-214 (1977)); ATCC). The metastatic ability and tumorigenicity of cells can be confirmed using a method known per se. For example, for metastasis, a method using cell invasion ability measured using collagen gel as an index, for tumorigenicity, karyotype analysis, soft agar colony test, tumor formation ability test in immunodeficient animals, etc. may be used. it can.

  In a preferred embodiment, a cell that has been modified so that a gene encoding a fluorescent protein or photoprotein is integrated into the genome so that it can function in the cell and the protein is stably expressed is used. If such a cell is used to produce a model animal according to the production method of the present invention, the behavior of cancer cells such as proliferation, migration, invasion, and metastasis can be determined noninvasively in real time using the fluorescence or luminescence as an index. It becomes possible to observe in vivo. Such cells can be generated using common genetic engineering techniques, such as Chishima et al., Cancer Res. 1997; 57: 2042-2047, “Experimental Methods for Cancer Metastasis Research” ( Ed. Japanese Society for Cancer Metastasis, Kinyoshido). Any fluorescent protein and photoprotein that are commonly used in the art can be used. For example, examples of the fluorescent protein include green fluorescent protein (GFP), DsRed, and improved types thereof, and examples of the photoprotein include firefly luciferase, Renilla luciferase, jellyfish aequorin, and improved types thereof. . These are commercially available from suppliers well known to those skilled in the art.

  Any immunodeficient non-human mammal (recipient animal) can be used as long as it allows tumor formation in the animal. For example, the immunodeficient animals described above for the model animals of the present invention can be suitably used. For example, in the case of a mouse, an animal of about 6 weeks of age can be preferably used.

Tumor formation in recipient animals is described, for example, in the following literature: Fiebig et al., “Human Tumor Xenografts: Predictivity, Characterization and Discovery of New Anticancer Agents,” in Contributions to Oncology: Relevance of Tumor Models for Anticancer Drug Development, Fiebig & Burger, eds. (Basel, Kargerl999), vol. 54, pp. 29-50; Berger et al., "Establishment and Characterization of Human Tumor Xenografts in Thymus- Aplastic Nude Mice," in Immunodeficient Mice in Oncology, Fiebig & Berger, eds. (Basel, Karger 1992), pp. 23-46; Fiebig & Burger, "Human Tumor Xenografts and Explants," in Models in Cancer Research, Teicher, ed. (Humana Press 2002) pp. 113-137 Can be done according to. Specifically, for example, the following procedure can be followed. The cells can be separated from each other with a commercially available cell lysate, and then the cells are collected by centrifugation or the like, and then transplanted into the recipient animal. Transplantation can be performed by suspending the cells in physiological saline or PBS and injecting the cells into an appropriate site of the recipient animal using a syringe or the like. The site of inoculation is not particularly limited as long as it is a site that easily forms a tumor, but it is preferable to inoculate the animal subcutaneously because it is easy to inoculate and easily forms a tumor. Specifically, back subcutaneous and abdominal subcutaneous are suitable. For example, when inoculating subcutaneously into an immunodeficient mouse, about 1 × 10 ^{6 to} 1 × 10 ⁷ cells are suspended in about 100 μl of PBS or the like and injected into a syringe. Can be used to inoculate. A tumor can be formed in the body of the animal by inoculating the animal under aseptic conditions after the injection of the cells. Although it depends on the type of cells used and the number of transplanted cells, tumors can usually be formed in about 4 to 6 weeks.

(B) Step of excising the formed tumor to obtain a tumor piece In this step, the tumor formed in the step (a) is usually excised under aseptic conditions. The obtained tumor piece can be washed with PBS or the like. In the next step (c), a fresh tissue is cut into a size of about 2 mm square with a surgical scissors from the viewpoint that the tumor does not detach into the abdominal cavity at the time of attachment and has sufficient cells. It is preferable to use it. Moreover, it is preferable to use the tumor piece which has not caused necrosis in the following process (c).

(C) Transplanting the tumor piece into the myometrium of an immunodeficient non-human mammal to be the model animal In this step, the tumor piece obtained in step (b) is a non-immune deficient as described above. It is transplanted into the myometrium of a human mammal. For example, in the case of mice, animals of about 6 weeks of age can be used for transplantation. For transplantation, the animals can be given general anesthesia, for example by inhalation anesthesia. As the anesthetic, from the viewpoint of being widely used in animal experiments, a volatile anesthetic, more preferably isoflurane, sevoflurane, or the like can be used. After confirming the state of anesthesia, a midline abdominal incision is performed using a surgical scissors to expose the uterus. Next, for example, a small incision is made in the utero-cervical junction, and the tumor piece collected in the step (b) can be attached to the portion using a surgical suture such as nylon. For example, a single tumor piece cut into 2 mm squares can be attached to the site. Thereafter, the abdomen is closed using a surgical suture.

  After orthotopic transplantation according to the above, the primary tumor foci can be grown in the myometrium by normal rearing of the animals under aseptic conditions, and further, the metastatic foci can be formed in target organs such as the lungs. Can do. It usually takes about 6 weeks for the formation of primary tumor and metastases.

The production method of the present invention may further include, after step (c), (d) a step of confirming whether or not a primary tumor focus in the myometrium and a metastatic focus in the target organ have been formed in the animal. preferable. The confirmation of the tumor focus can be performed according to a method known to those skilled in the art. For example, in a model animal produced according to the present invention, since tumor cells normally express GLUT (glucose transporter) -1 and / or -3 at high levels, they are non-invasive using positron emission tomography (PET). It may be possible to observe primary and metastatic lesions. A tracer compound such as ¹⁸ F-FDG can be administered to the animal for PET observation. In addition, when cells modified in the step (a) so as to constantly express a fluorescent protein or photoprotein are used, formation of a tumor nest can be confirmed using the fluorescence or luminescence as an index.

(Screening method)
The present invention is also referred to as a method for screening a substance having anticancer activity (eg, tumor growth suppression, tumor regression, tumor disappearance, etc.) and / or metastasis suppression activity against uterine sarcoma (hereinafter also referred to as the screening method of the present invention). )I will provide a. The method comprises (i) a step of providing a model animal of the present invention, (ii) a step of administering a test substance to the animal, and (iii) a primary tumor focus in the myometrium after the start of administration of the test substance. Evaluating the size and / or the number and / or size of metastases in the target organ, and (iv) the size of the primary tumor focus compared to when the test substance is not applied And / or selecting the test substance as a candidate for a substance having anticancer activity and / or metastasis-suppressing activity against uterine sarcoma when the number and / or size of the metastasis decreases.

  As the model animal of the present invention provided in step (i), an animal of any age and any period after the orthotopic transplantation can be used depending on the purpose of the experiment. The animal may also have already grown primary tumors and metastases, or may not have grown primary tumors and / or metastases yet, but their formation is expected thereafter It may be a thing.

  In step (ii), a test substance is administered to the model animal. As the test substance, in addition to known synthetic compounds, peptides, proteins, DNA libraries, etc., for example, mammalian tissue extracts (eg, mice, rats, pigs, cows, sheep, monkeys, humans, etc.), cells A culture supernatant or the like is used. The administration method of the test substance is not particularly limited. For example, the test substance is administered orally or parenterally (eg, intravenous, intramuscular, intraperitoneal, intraarterial, subcutaneous, intradermal, intratracheal, etc.) in the form of solid, semi-solid, liquid, aerosol, etc. can do. The dose of the test substance varies depending on the type of compound, animal species, body weight, dosage form, etc., and can be appropriately selected from the range of 0.01 to 1000 mg / kg / day, for example. It can be administered in several or several divided doses. Although the administration period is not particularly limited, for example, it can be administered every day for 1 to 14 days or every 2 to 4 days. It is preferable to provide a negative control group (for example, administration of a solvent) in addition to the test substance administration group. Moreover, you may provide the positive control group which administers a known anticancer substance.

  In the step (iii), the evaluation may be performed any time after the start of administration of the test substance. It may be during the administration period of the test substance, at the end of the administration period, or after an arbitrary period has elapsed from the end of the administration period. The method for evaluating the size of the tumor nest is not particularly limited. For example, a method of calculating the tumor volume by measuring the length, width, and height of the tumor with a caliper or the like, a method of extracting the tumor and measuring its weight, and the like. Further, the method for evaluating the number or size of metastatic lesions in the target organ is not particularly limited. For example, a method of dissecting an animal to collect a target organ and measuring the number or size of metastatic lesions with the naked eye or under a microscope can be used. Examples of the target organ include those described above in the production method of the present invention, and the same applies to preferred embodiments. As described above, in the model animal of the present invention, the tumor can exhibit high GLUT-1 and / or GLUT-3 expression, and this can be used to evaluate the size and number of tumor foci by PET observation. . Further, in the embodiment in which the transplanted tumor-derived cells are modified to generate fluorescence or luminescence, the fluorescence or luminescence can also be used for the evaluation of this step.

In step (iv), the size of the primary tumor focus and / or the number and / or size of the metastatic focus when the test substance is administered and when the test substance is not administered are compared. The comparison may be performed for the animal before and after administration of the test substance, or may be a comparison with an animal not administered with the test substance (usually a negative control group (for example, a solvent administration group)).
If the tumor when the test substance is administered is smaller than the size of the tumor when the test substance is not administered, the test substance has anticancer activity (eg, tumor growth inhibition, tumor regression, tumor disappearance) Etc.). Preferably, a test substance that reduces the tumor size to 50% or less, more preferably a test substance that reduces the tumor size to 10% or less, is determined as an anticancer substance.
If the number of metastases when the test substance is administered is reduced compared to the number of metastases in the target organ when the test substance is not administered, the test substance has cancer metastasis inhibitory activity. Can be determined. In addition, if the size of the metastasis when the test substance is administered is small compared to the size of the metastasis in the target organ when the test substance is not administered, the test substance has cancer metastasis inhibitory activity. Can be determined. Preferably, a test substance that reduces the number or size of metastases to 20% or less, more preferably a test substance that decreases to 5% or less, is determined as a cancer metastasis inhibitor.

(Evaluation method)
The present invention also provides a method for determining the anticancer effect (eg, tumor growth suppression, tumor regression, tumor disappearance, etc.) and / or metastasis suppression effect of a therapy against uterine sarcoma. The method comprises (i) a step of providing a model animal of the present invention, (ii) a step of applying the therapy to the animal, and (iii) a size of a primary tumor focus in the myometrium after the start of the therapy. And / or assessing the number and / or size of metastases in the target organ, and (iv) based on the assessment in step (iii), the anti-cancer effect of the therapy on uterine sarcoma and And / or determining the effect of inhibiting metastasis. Steps (i), (iii) and (iv) can be carried out in the same manner as steps (i), (iii) and (iv) of the screening method of the present invention, and preferred embodiments are also the same.

  The therapy administered to the animal in step (ii) may be any therapy for which it is desired to determine the anticancer effect and / or metastasis suppression effect against uterine sarcoma. Examples of the therapy include surgical therapy, drug therapy, radiation therapy, and hyperthermia. Those therapies may be administered to the animal according to the mode for which it is desired to determine efficacy.

(Establishment method)
The present invention also provides a method for establishing a cell line derived from uterine sarcoma having improved metastatic potential (hereinafter also referred to as the establishment method of the present invention). The method includes the step of (i) producing the model animal using the production method of the present invention, using a cell of a parent cell line desired to improve metastatic ability as the cell to be transplanted in the step (a). (Ii) obtaining a tumor piece from a metastasis formed in the animal; (iii) transplanting the tumor piece into the myometrium of another immunodeficient non-human mammal; Forming a primary tumor focus and a metastasis in a target organ and obtaining a tumor piece from the formed metastasis at least once, and (iv) cells in the obtained tumor piece in vitro Separating and increasing and establishing a cell line. The established cell line may have improved metastatic potential compared to the parent cell line.

  The cell line used in step (i) is the starting cell line where it is desired to improve the metastatic potential. The cells of the cell line have, for example, low metastatic potential, that is, for example, in the production of a model animal according to the production method of the present invention, all the animals transplanted with the cell have primary tumor foci and metastatic foci. The model animal of the invention cannot be produced, and the model animal can be produced only from some animals. In step (i), a model animal is produced using such cells according to the production method of the present invention. Its preparation and preferred embodiments are as described above.

  In step (ii), when formation of a metastatic focus in the target organ is confirmed in the animal, the metastatic focus is excised from the animal to obtain a tumor piece. The “target organ” is as described above in the production method of the present invention, and is preferably the lung. The excision of the metastatic lesion can be performed in the same manner as in the step (b) of the production method of the present invention, and preferably a tumor piece cut into a size of about 2 mm square is obtained.

  In step (iii), the tumor piece obtained in step (ii) is transplanted into the myometrium of another immunodeficient non-human mammal. The animal as described above in the production method of the present invention can also be used. The transplantation can be performed in the same manner as in step (c) of the production method of the present invention. Subsequently, when it is confirmed that the primary tumor foci in the myometrium and the metastasis in the target organ have been formed in the animal, tumor pieces are obtained from the formed metastases in the same manner as in step (ii). Using the new animal each time, the above procedure is performed at least once, preferably about 3 to 5 times.

  Cells in metastatic foci contained in animals after one or repeated procedures in step (iii) can be expected to have a higher metastatic potential compared to the parent cell line. Therefore, by isolating such cells from metastases and establishing cell lines according to methods known per se, cell lines derived from uterine sarcomas having the desired improved metastatic potential (eg, always metastasizing in the target organ) Cell lines capable of forming nests) can be established. Using known means for confirming metastatic potential (for example, a method using cell invasion ability measured using the above-mentioned collagen gel as an index), the cell metastatic ability of the cell line is compared with that of the parent cell line. It is preferable to confirm that it is improved.

(Identification method)
The present invention also provides a method for identifying a gene associated with metastasis of uterine sarcoma (also referred to as the identification method of the present invention). The method comprises conducting gene expression analysis on cells of a cell line established by the establishment method of the present invention and cells of a cell line having a lower ability to transfer to a target organ, and comparing the gene expression of both cells. Including. The term “target organ” is as described above in the production method of the present invention.

  The above-mentioned “cell line having a lower ability to metastasize to a target organ” has more ability to metastasize to a target organ than a cell line established by the establishment method of the present invention (also referred to as a high metastasis line). There is no particular limitation as long as it is low. For example, as the cell group, a low metastasis strain or a parent cell strain in the establishment method of the present invention can be used.

  Comparison of gene expression can be performed using a method known per se. Preferably, the comparison involves comprehensive analysis of gene expression (eg, tens of thousands to hundreds of thousands of gene expressions). For such a comprehensive analysis, for example, a DNA microarray can be used. As the DNA microarray, a commercially available product such as Gene Chip of Affymetrix can be used. Genes whose expression is significantly increased or decreased in cells with high metastatic potential compared to cells with low metastatic potential can be identified as genes that may be associated with uterine sarcoma metastasis.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited at all by the following examples.

1. Preparation of human uterine sarcoma metastasis model mouse A human uterine sarcoma metastasis model mouse was prepared according to the following procedure.
C9 cells, which are sub-strains of MES-SA GFP-J that constitutively express GFP, were cloned by introducing the GFP gene into human uterine leiomyosarcoma-derived MES-SA cells (ATCC). The outline is shown in FIG. Briefly, PB513B-1 in the PiggyBac ^™ Transposon Vector System was introduced into MES-SA cells. The puromycin resistance gene was introduced at the same time as the GFP gene, and the GFP-introduced cells were cultured in a medium containing puromycin for selection. Thereafter, each cell was cloned by isolating and culturing one cell.
The cells were injected subcutaneously into 5-week-old KSN female mice (Japan SLC) with 5 × 10 ⁶ cells / 100 μl of PBS. After 4 weeks, the tumor formed subcutaneously was excised under aseptic conditions. The tumor pieces were washed with PBS, and the fresh tissue was cut into 2 mm squares with a surgical scissors. Next, inhalation anesthesia with laughing was performed on 5-week-old KSN female mice (Japan SLC). After confirming the state of anesthesia, a midline abdominal incision was made with a surgical scissors. After exposing the uterus, a small incision was made in the utero-cervical junction, and a 2 mm square fresh tissue previously collected was attached to the portion with 5-0 dexon. Thereafter, the abdomen was closed with 5-0 dexon thread. Animal PET was performed 6 weeks after the orthotopic transplantation experiment, and the degree of FDG accumulation in the tumor was confirmed, and the presence or absence of tumor formation was confirmed.

Six weeks after orthotopic transplantation, before laparotomy (a left figure) and photograph of the primary tumor focus (a right figure), macroscopic image of uterine primary tumor focus (b) and lung metastases (c), GFP fluorescence Images and stained images of various markers (H & E, Vimentin, Ki-67, Glut-1) are shown in FIG.
An example of lung metastasis is shown in FIG. Tumor foci formed by metastasis are shown by fluorescence from GFP (a). In addition, from the stained images (b) of various markers (H & E, Glut-1, Ki-67) and its enlarged view (c), a huge tumor embolism (Glut-1 strong positive), alveolar capillaries in the alveolar cavity It can be seen that the tumor cell embolism (Ki-67 strong positive), the alveolar minimal artery and the capillary lumen in the blood vessel were blocked with Glut-1 strong positive tumor cells.
Further, the data of the above animal PET is shown in FIG. A (side view) and B (top view) are normal mice, and C (side view) and D (top view) are PET images of the model mouse. Images were acquired 60 minutes after administration of 12.1-14.4 MBq ¹⁸ F-FDG. In model animals, stronger signals were observed in the uterus, lungs, and heart.
FIG. 5 is a graph showing the correlation between primary tumor weight and primary tumor volume, days after orthotopic transplantation and primary tumor volume, number of lung metastases and days after orthotopic transplantation, number of lung metastases and primary tumor volume. There was no correlation between lung metastases, days after orthotopic transplantation, and primary tumor volume.
FIG. 6 is a graph showing the correlation between the number of lung metastases and lung FDG, and the number of lung metastases and lung FLT. ¹⁸ F-FDG accumulation ability was correlated with the number of lung metastases, but ¹⁸ F-FLT accumulation ability was not correlated with the number of lung metastases.

Claims (18)

A method for producing a uterine sarcoma metastasis model animal using a non-human mammal,
(A) transplanting metastatic and tumorigenic cells derived from uterine sarcoma into a recipient's immunodeficient non-human mammal, and forming a tumor in said recipient animal;
(B) excising the formed tumor to obtain a tumor fragment; and
(C) transplanting the tumor piece into the myometrium of an immunodeficient non-human mammal to be the model animal, wherein the transplanted tumor fragment comprises a primary tumor focus in the myometrium and Said method of forming a metastatic focus in a target organ.   The method according to claim 1, wherein the cell transplanted in step (a) is integrated into the genome so that a gene encoding a fluorescent protein or photoprotein can function in the cell.   The method of claim 2, wherein the fluorescent protein is a green fluorescent protein.   The method according to any one of claims 1 to 3, wherein the cells to be transplanted in the step (a) are derived from a human.   The method according to any one of claims 1 to 4, wherein the cells to be transplanted in the step (a) are derived from MES-SA cells, SKN cells or SK-UT-1 cells. After step (c)
(D) The method of any one of Claims 1-5 which further includes the process of confirming whether the primary tumor nest in the myometrium and the metastatic nest in the target organ were formed in this animal.   The method according to claim 1, wherein the target organ is a lung.   The method according to any one of claims 1 to 7, wherein the model animal is a mouse or a rat.   The method according to any one of claims 1 to 8, wherein the recipient animal is a mouse or a rat.   A uterine sarcoma metastasis model non-human mammal obtained by the method according to any one of claims 1 to 9.   The primary tumor foci formed in the myometrium and the metastatic foci in the target organ are compared to the corresponding sites in the same strain of animals that do not have a tumor in the myometrium and the target organ. 12. The animal of claim 10, which exhibits enhanced expression of at least one of -3. A method for screening a substance having anticancer activity and / or metastasis-suppressing activity against uterine sarcoma,
(I) providing a uterine sarcoma metastasis model non-human mammal according to claim 10 or 11,
(Ii) administering a test substance to the animal;
(Iii) after starting administration of the test substance, assessing the size of the primary tumor focus in the myometrium and / or the number and / or size of the metastatic focus in the target organ; and
(Iv) when the size of the primary tumor foci and / or the number and / or size of the metastases is reduced as compared to the case where the test substance is not applied, the test substance is treated with uterine sarcoma. The method comprising the step of selecting as a candidate for a substance having anticancer activity and / or metastasis-suppressing activity against.   The method according to claim 12, wherein the animal is the animal according to claim 11 and the evaluation in step (iii) is performed using positron emission tomography. A method for determining the anticancer effect and / or metastasis-suppressing effect of therapy on uterine sarcoma,
(I) providing a uterine sarcoma metastasis model non-human mammal according to claim 10 or 11,
(Ii) applying the therapy to the animal;
(Iii) assessing the size of the primary tumor focus in the myometrium and / or the number and / or size of the metastatic focus in the target organ after initiation of the therapy; and
(Iv) The said method including the process of determining the anticancer effect and / or metastasis inhibitory effect with respect to the uterine sarcoma of this therapy based on the evaluation in a process (iii).   15. The method of claim 14, wherein the animal is the animal of claim 11 and the evaluation in step (iii) is performed using positron emission tomography. A method of establishing a cell line derived from uterine sarcoma with improved metastatic potential,
(I) The model using the method according to any one of claims 1 to 9, wherein cells of a parent cell line for which improvement of metastatic potential is desired are used as cells to be transplanted in the step (a). Producing animals,
(Ii) obtaining a tumor piece from a metastatic focus formed in the animal;
(Iii) Transplanting the tumor piece into the myometrium of another immunodeficient non-human mammal to form a primary tumor focus in the myometrium and a metastasis in the target organ in the animal, and the formed metastasis Obtaining a tumor piece from at least once, and
(Iv) separating and increasing cells in the obtained tumor piece in vitro, and establishing a cell line, wherein the established cell line has improved metastatic potential compared to the parent cell line, Said method.   Carrying out gene expression analysis on cells of a cell line established by the method of claim 16 and cells of a cell line having a lower ability to metastasize to a target organ, and comparing the gene expression of both cells, Methods for identifying genes associated with metastasis of uterine sarcoma.   The method according to claim 17, wherein the gene expression analysis is performed using a microarray method.