Rapid tumorigenicity screening system

Abstract

The present invention provides a method for evaluating tumorigenicity of a test cell, including transplanting the cell into anterior chamber of an eye of an experimental animal, and observing the presence or absence of tumor formation. According to the present invention, tumorigenicity can be evaluated in a short period and conveniently.

Claims

1. A method for evaluating tumorigenicity of test cells, comprising transplanting at least 10.sup.5 of the test cells into the anterior chamber of an eye of an experimental, immunodeficient animal, and observing the presence or absence of tumor formation at least 4 weeks post transplantation.

2. The method according to claim 1, wherein the presence or absence of tumor formation is observed 4-8 weeks post transplantation.

3. The method according to claim 1, wherein 10.sup.5 cells-10.sup.7 test cells are transplanted.

4. The method according to claim 1, wherein the experimental, immunodeficient animal is a rat.

5. The method according to claim 1, wherein the test cells are stem cells or differentiated somatic cells induced therefrom.

6. The method according to claim 1, wherein the test cells are cancer cells.

7. The method according to claim 6, comprising further administering a test compound to the experimental, immunodeficient animal, and evaluating an antitumor activity of the compound by using tumor formation as an index.

8. The method according to claim 2, wherein 10.sup.5 cells-10.sup.7 test cells are transplanted.

9. The method according to claim 2, wherein the experimental, immunodeficient animal is a rat.

10. The method according to claim 9, wherein the test cells are stem cells or differentiated somatic cells induced therefrom.

11. The method according to claim 10, wherein the test cells are cancer cells.

12. The method according to claim 11, comprising further administering a test compound to the experimental, immunodeficient animal, and evaluating an antitumor activity of the compound by using tumor formation as an index.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the protocol of a tumorigenicity test using the anterior chamber of an eye.

(2) FIG. 2 shows teratoma formation (right) and cumulative survival rate (left) at one month after administration of human iPS cell 201B7 line within the anterior chamber of an eye of an immunodeficient rat.

(3) FIG. 3 shows teratoma formation (lower) and cumulative survival rate (upper) at one month after administration of human iPS cell FFI-01 line within the anterior chamber of an eye of an immunodeficient rat.

DESCRIPTION OF EMBODIMENTS

(4) The present invention is explained in more detail in the following. Unless particularly indicated, the terms used in the present specification have the meanings generally used in the pertinent field.

(5) The present invention is a method for evaluating tumorigenicity of a test cell, comprising transplanting the cell into anterior chamber of an eye of an experimental animal, and observing the presence or absence of tumor formation (hereinafter to be also referred to as “the evaluation method of the present invention”). As used herein, the “tumorigenicity” is used to encompass the ability to form teratomas of not only cancer cells but also undifferentiated cells.

(6) The experimental animal to be used in the present invention is not particularly limited as long as it is an experimental animal having an anterior chamber of an eye having a sufficient volume for receiving injection of about 1×10.sup.6 cells. For example, rat, rabbit, guinea pig, dog, cat and the like can be mentioned. Preferred are rat, rabbit, guinea pig, and more preferred is rat.

(7) The non-human animal to be used in the present invention is desirably genetically and microbiologically controlled such that it is suitable for undergoing the obtained evaluation experiment of the present invention using the animal model. That is, genetically, use of an inbred strain is preferable. For example, in the case of rat, Wistar strain, Sprague-Dawley strain and the like can be exemplified. Microbiologically, SPF or one of a gnotobiotic grade is preferably used.

(8) Sex, age and the like of the experimental animal is not particularly limited as long as the volume of the anterior chamber of an eye can meet the above-mentioned conditions. One with any sex, age can be used according to the animal species.

(9) The test cell to be the evaluation target of the evaluation method of the present invention is preferably a human cell in consideration of applicability to regenerative medicine and use as a screening system for anticancer drugs. Thus, in one embodiment, the experimental animal to be used for the evaluation method of the present invention is desirably an immunodeficient animal so that human cells are not rejected. For example, in the case of rat, for example, F344/NJcl-rnu/rnu rat lacking T cell function, SCID rat, X-SCID rat, FSG rat lacking both genes of SCID and XSCID and the like can be mentioned.

(10) However, eye is an immune-privileged site and is known to be less likely to reject transplanted cells. Thus, in another embodiment, a normal animal having a normal immune system can also be used as an experimental animal. Since immunodeficient animals are limited in species and expensive, the use of normal experimental animals is extremely significant in increasing the broad utility of the evaluation method of the present invention.

(11) The test cell to be the evaluation target of the evaluation method of the present invention is not particularly limited as long as it may have tumorigenicity and any cell can be used. In preferable one embodiment, since the evaluation method of the present invention is used for evaluating safety of cells for transplantation, the test cell is exemplified by pluripotent stem cells such as embryonic stem cell (ES cell), induced pluripotent stem cell (iPS cell), mesenchymal stem cell (MSC) and the like, somatic stem cell (e.g., neural stem cell etc.), somatic cell (e.g., corneal cell, hepatocyte, kidney cell, pancreatic β cell, cardiac muscle cell, blood cells etc.) obtained therefrom by differentiation induction, and tissues (e.g., cornea, retinal pigment epithelium, hepatic tissue, kidney tissue, pancreatic tissue, cardiac muscle etc.) formed therefrom. In consideration of the fact that the in vivo test is necessary for evaluating tumorigenicity under influence of the microenvironment of the transplantation site, preferably, the test cell is desirably a cell or tissue constituting the eye. Even cells constituting other tissues can be applied to the evaluation method of the present invention as a primary screening means prior to a test at the transplantation site.

(12) In another embodiment, the evaluation method of the present invention is used for evaluation of cancer malignancy or as a screening system for anticancer drugs. Thus, various cancer cells (e.g., cancer cell collected from patients by biopsy and the like, cancer cell line etc.) can also be used as the test cells.

(13) The test cell may be derived from any animal. Preferred are cells derived from warm-blooded animal such as human and pet animal (e.g., dog, cat etc.) or domestic animal (e.g., bovine, swine etc.) or poultry (e.g., chicken, duck etc.) and the like. More preferred is a human cell.

(14) The number of test cells necessary for the evaluation method of the present invention is not particularly limited as long as it is not less than 10.sup.5 cells. In consideration of the volume of the anterior chamber of an eye which is the transplantation site, it is preferably 10.sup.5 cells-10.sup.7 cells. International guidelines proposed by WHO indicate transplantation of 10.sup.7 cells. Using the evaluation method of the present invention, however, tumorigenicity can be detected with about 60% accuracy by using 10.sup.5 cells, and nearly 100% accuracy by using 10.sup.6 cells. More preferably, therefore, the number of the test cells is 10.sup.5 cells-10.sup.6 cells, particularly preferably about 10.sup.6 cells (e.g., not less than 5×10.sup.5 cells and less than 5×10.sup.6 cells).

(15) The test cells can be used for transplantation by suspending the above-mentioned amount in, for example, 5-10 μl of a suitable medium or isotonic solution (e.g., saline, PBS etc.). To increase the retentivity of the test cells in the anterior chamber of an eye, the cell suspension can be transplanted into the anterior chamber of an eye after mixing with an extracellular matrix known per se such as Matrigel and the like.

(16) The anterior chamber of an eye is a region inside the eye between the iris and the endothelial cells in the innermost layer of the cornea and is filled with aqueous humor. The anterior chamber of an eye allows easy monitoring of the state of the transplanted cells at any time through the cornea, and can detect tumor formation conveniently and early. The test cells can be transplanted into the anterior chamber of an eye by injecting the cell suspension prepared above from the corneal limbic area of the eye. Transplantation may be performed for one eye, and the other eye can be used as a control.

(17) After transplantation, experimental animals can be bred in the same way as before transplantation. The follow-up can be performed, for example, by visually observing tumor formation under a stereomicroscope at appropriate intervals. In the evaluation method of the present invention, for example, when the tumor formed occupies ¼ or more of the inner volume of the anterior chamber of an eye, (tumorigenic) death can be determined.

(18) In addition, after a certain period of time, the eyeballs may be removed and formation of tumor including teratomas can be confirmed by tissue staining (e.g., HE staining, immunostaining by tissue specific marker) and the like.

(19) In conventional evaluation by subcutaneous transplantation, a tumor cannot be determined until it grows to a size of about 20 mm, and during that time, observation is not possible since it is under the skin. In contrast, in the evaluation method of the present invention, tumor formation can be detected sharply since transplanted cells can be observed at any time while the experimental animals are alive. Therefore, nearly 100% of tumor formation can be detected at 4 weeks post-transplantation, depending on the kind of test cells and the number of transplanted cells. Even when the number of cells to be transplanted is as small as about 10.sup.5 cells, tumor formation can be detected with high accuracy within 8 weeks after transplantation.

(20) Therefore, the period of follow-up in the evaluation method of the present invention is preferably 4-8 weeks.

(21) In the evaluation method of the present invention, the presence or absence of tumorigenicity can be evaluated with sufficient accuracy when the number of experimental animals is 5. International guidelines proposed by WHO indicate testing using 10 individuals. Since the evaluation method of the present invention has less variation between individuals, it is also advantageous in that the number of individuals used for evaluation can be reduced from those used conventionally.

(22) In the evaluation method of the present invention, by using a cancer cell, for example, a cancer cell collected from a patient as a test cell, malignancy of the cancer cell can be evaluated. For example, first, the evaluation method of the present invention is performed on cancer cells known to have low clinical malignancy, the number of transplanted cells in which cancer cells do not proliferate is determined, after which the test cancer cells in the above number are transplanted into the anterior chamber of an eye of the experimental animal and follow-up is performed. When tumor formation is found, the test cancer cell can be judged to have high malignancy.

(23) In the evaluation method of the present invention, a cancer cell, for example, various cancer cell lines conventionally used for the evaluation of anticancer drugs, is used as a test cell, a candidate compound for an anticancer drug is administered as a test compound to an experimental animal, tumor formation in the anterior chamber of an eye of the experimental animal is examined, whereby a test compound having an antitumor activity can be screened for. For example, the number of transplanted cells in which a test cancer cell line can form a tumor is first determined, after which the test cancer cells in the above number are transplanted and the test compound is administered into the anterior chamber of an eye of the experimental animal and follow-up is performed. When suppression of tumor formation is observed, the test compound can be judged to have an antitumor activity. The test compound can be administered to the experimental animal by any administration route and dose. In preferable one embodiment, it can be administered topically to the eye. Topical administration to the eye may be performed by injection into the anterior chamber of the eye as in the cell transplantation, or may be performed by instillation. In this case, cancer cells may be transplanted into both eyes, the drug may be administered to only one eye, and the suppressive effect on tumor formation can also be evaluated by comparing the both eyes.

(24) By practicing the evaluation method of the present invention for a compound selected as having an antitumor activity as a result of primary screening using a cancer cell line, or an existing anticancer drug, and by changing the test cells to a cancer cell derived from a patient, an anticancer drug effective for the patient can be selected, and a personalized cancer treatment can be performed.

(25) While the present invention is more specifically explained by referring to the following Examples, it is needless to say that the present invention is not limited thereto.

EXAMPLE

(1) Method

(26) As an experimental animal, an immunodeficient rat F344/NJcl-rnu/rnu (5-week-old, CLEA Japan, Inc.) was used. A mixed anesthesia solution of three types of Domitor (medetomidine hydrochloride) (75 μg/ml), Dormicum (Midazolam) (0.4 mg/ml), Betorphal (butorphanol tartrate) (0.5 mg/ml) was intraperitoneally injected at 500 μl/100 g to perform systemic anesthesia. In addition, topical anesthesia was performed with Benoxil (0.4%, Oxybuprocaine Hydrochloride eye drop) instillation. A mixed solution of a cell suspension containing 5 μl human iPS cell line 201B7 (cell.brc.riken.jp/ja/hps/hps0063_info) or FFI-01 (cira.kyoto-u.ac.jp/ciRA Center for iPS Research, Kyoto University) (1×10.sup.4 cells-1×10.sup.7 cells), and a solution containing Matrigel (5 μl) was injected into one eye of rats (5 rats in each group) placed under a stereomicroscope. As an antibacterial agent, a cravit instillation solution (0.5%, levofloxacin hydrate) was instilled. After transplantation, rats were followed-up for a certain period (immediately thereafter to several months). Thereafter, for more detailed analysis, the rats were euthanized by intraperitoneal overdose of pentobarbital (120 mg/kg). The eyeballs were collected, HE staining etc. was performed, and teratoma formation (differentiation into lineages of three germ layers) was confirmed. The protocol of this test is shown in FIG. 1.

2) Results

(27) Tumor formation occupying ¼ or more of the anterior chamber inner volume was determined to mean dead, and Kaplan-Meier survival curve was generated. In the case of 201B7 line, when 1×10.sup.6 cells were transplanted, all five rats were judged dead (with teratoma formation ability) at 4 weeks after transplantation (FIG. 2, upper left). As a result of HE staining, differentiation into lineages of three germ layers was confirmed (FIG. 2, right). Even when 1×10.sup.5 cells were transplanted, 60% (⅗ rats) were judged dead within 8 weeks (FIG. 2, lower left). Even when FFI-01 line was transplanted, similar results were obtained (FIG. 3). When 1×10.sup.7 cells were transplanted, all rats were judged dead at 4 weeks post-transplantation for any iPS cell line. On the other hand, when 1×10.sup.4 cells were transplanted, tumor formation was not found in any cell line.

(28) In conventional evaluation by subcutaneous transplantation, a tumor cannot be determined until it grows to a size of about 20 mm, and during that time, observation is not possible since it is under the skin. In contrast, in the evaluation method of the present invention, tumor formation can be detected sharply since transplanted cells can be observed at any time while the rats are alive. Thus, tumor formation was detectable with 100% accuracy at 4 weeks post-transplantation.

INDUSTRIAL APPLICABILITY

(29) According to the present invention, tumorigenicity evaluation, which previously required several months of observation period and a large number of animals, can be shortened to about 4 to 8 weeks and the number of animals can be decreased. Thus, the present invention is useful for in vivo screening for tumorigenicity of cells for transplantation. In addition, the present invention is extremely useful since it can be utilized for evaluation of cancer malignancy or as a screening system for anticancer drugs.

(30) This application is based on a patent application No. 2017-148605 filed in Japan (filing date: Jul. 31, 2017), the contents of which are hereby incorporated by reference in full herein.