JP5875010B2 - Method for producing animal model for xenotransplantation - Google Patents

Description

  The present invention relates to a method for producing a xenotransplant model animal.

The treatment of malignant tumors is the world's largest medical problem in the 21st century, and cancer stem cells are the central issue in overcoming the “treatment resistance” of malignant tumors.
Technology for transplanting human cancer cells into experimental animals is essential for pre-clinical studies mainly in the development of anticancer drugs. It is very difficult to transplant cells between different species, and even cancer cells are rejected by an immune response on the host side when cells / tissues of other species are transplanted. Conventionally, immunodeficient animals such as NOD / SCID mice, and animals whose immune cell function is suppressed by administration of radiation or immunosuppressive drugs have been used. However, in human cancer transplanted animals using conventional immune inactivation technology, immunocompetent cell functions including T cells are suppressed, and research using human cancer cell transplanted animals is extremely difficult. It has become.
On the other hand, research on cancer stem cells has been conducted in response to the question of whether stem cells also exist in cancer as normal cells have. It is common knowledge in the world that the history of cancer stem cell research is the history of immunodeficient mouse development. However, in vivo exploratory research of selective human cancer stem cell therapeutics using immunodeficient mice has many difficulties due to its low throughput. In addition, animal experiments using cancer stem cells that contain very small amounts of cancer cells are very difficult in terms of labor and cost. There are several reports on transplanting human cancer cells into zebrafish, but there are reports on tumor angiogenesis and distant metastasis, and there are reports on transplanting cancer stem cells into zebrafish after irradiation (non-patent literature). 2) There was no novel anticancer drug screening method that constructed a human cancer stem cell transplantation model using a short immunodeficiency period in zebrafish larvae.
In cancer transplantation, it is considered that the clinical pathology of malignancy is reflected when tumor cells are three-dimensionally cultured and then transplanted as tumor tissue rather than transplanted with a cell suspension. However, tumor tissue transplantation was more difficult compared to cell suspensions. Furthermore, there has been no method for transplanting a tumor tissue in a living body such as a human as it is.

Southam CM.Immunologic tolerance to human cancer transplants in rats.Cancer Res. 1966 Dec; 26 (12): 2496-502. Marques, IJ., Weiss, FU., Vlecken, DH., Nitsche, C., Bakkers, J., Lagendijk, AK., Partecke, LI., Heidecke, CD., Lerch, MM. And Bagowski, CP. Metastatic behavior of primary human tumours in a zebrafish xenotransplantatio model.BMC Cancer. 28; 9: 128. 2009 Jordan CT. The leukemic stem cell. Best Pract Res Clin Haematol. 2007 20 (1): 13-8. Review.

  The present invention has been made in view of the above problems, and an object thereof is to provide a method for producing a model animal that can be transplanted into a heterologous animal without immunosuppression of human cancer cells, and the model animal. Is used to provide a pharmaceutical effect evaluation system for human cancer cells.

The inventor has found that rejection by an immune reaction can be avoided by transplanting human cancer cells that have undergone a specific treatment to small fish whose immune system is undifferentiated, and the present invention is basically completed. It came to do.
Thus, the method for producing a xenogeneic transplant model animal according to the present invention is characterized in that xenogeneic cells are transplanted at the first time when the immune cells of the model small fish are in an undifferentiated state.
The small fish means a small fish that grows quickly and is easy to breed. Examples thereof include medaka and zebrafish. When using small fish, it is preferable to use a transparent one that allows easy image processing. Such transparent small fish can be a commercial product or can be bred with appropriate equipment. Since fertilized eggs of small fish are transparent and the inside can be visually confirmed, heterogeneous cells can be easily transplanted. Among small fish, zebrafish hatch in about 3 to 4 days after fertilization. Medaka hatches about 10 days after fertilization. Since the period until hatching is short, it is preferable to use zebrafish as small fish.

The immune system of small fish is not formed immediately after fertilization. For this reason, small fish transplanted with heterologous cells in the first period when immune cells are in an undifferentiated state cannot recognize self and nonself sufficiently, and therefore can recognize heterologous cells as nonself. It was found that it cannot be excluded. Thus, when a xenogeneic cell transplant is received in the first period, if the xenogeneic cell is transplanted again after maturation, it is not excluded by the immune system, and can be used as a xenotransplant model animal. Such first time varies depending on small fish, but in zebrafish, it is after about 30 hours to 72 hours (preferably after 36 hours to 60 hours) after fertilization.
In the present invention, the heterologous cell is preferably a human cancer cell. Further, it is preferable that the heterologous cell is a tumor tissue of a human cancer cell. Transplantation of xenogeneic cells is a difficult technique, but it becomes even more difficult when the xenogeneic cells are tumor tissue. According to the present invention, it is possible to directly transplant a tumor tissue derived from another living body as a heterogeneous cell.

Although there are various discussions about human cancer stem cells, “human cancer stem cells” used in this specification are those fractionated from human cancer cells by the surface antigen method. , Cells having ALDH (+) activity (ie, cells having stem cell-like characteristics fractionated from human cancer cells).
Further, the heterologous cell is a human cancer cell, (1) produced by irradiating the human cancer cell with gamma rays, or (2) the human cancer cell is fractionated by a surface antigen method, Preferably, the ratio of ALDH (+) in the cell population is 50% or more (preferably 70% or more).

At this time, if the heterologous cells are those produced by irradiating human cancer cells with gamma rays, the heterologous cells transplanted in the first period are almost eliminated from the animal. For this reason, xenogeneic cell transplantation model animals can be produced by transplanting again the transplanted cells that maintained the proliferative function in the second period. At this time, it is preferable that the second period is one month to six months after fertilization.
The surface antigen is preferably at least one selected from the group consisting of CD44 ⁺ , CD133 ⁺ , Sca-1 ⁺ , CD34 ⁺ CD38 ⁻ and CD44 ⁺ CD24 ^{− / Low} ESA ⁺ .
According to the above invention, a xenogeneic cell transplantation model animal can be provided.
The system for evaluating pharmaceutical effects on human cancer cells is the above-mentioned animal model for xenotransplantation, wherein the model animal is a transparent zebrafish, and the transparent zebrafish is classified into two groups in advance, and one group is a control group. The other group is a test group to which the test compound is administered, and the distribution and concentration of the human cancer cells in each zebrafish body after a predetermined period of time while keeping the zebrafish in any of the multi-well plates. Is imaged after being imaged with a fully automatic microscope photographing apparatus, and the anticancer action of the test compound is evaluated based on the result obtained by comparing the image data.
At this time, the image data is preferably luminance image data of a specific wavelength from the labeling compound.

Thus, according to the present invention, a method for producing a model animal that can be transplanted into a heterologous animal without immunosuppression of human cancer cells can be provided. By using this model animal, it is expected to be applied to elucidation of the functionality of tissues surrounding cancer in cancer and research on cancer stem cells. Furthermore, it can be used for screening of new antineoplastic drugs by a host that is more normal.
In addition, human cancer stem cells contain only 0.1 to 5% of all cancer cells, and in the existing transplant model system using mice and rats, the number of transplanted cells is ensured and comprehensive gene expression analysis of a small amount of sample is performed. There are still many technical difficulties. By using zebrafish, the burden of animal experiments, which is a bottleneck in the development of anticancer agents, can be reduced, and at the same time, a high-throughput screening system that is difficult for other vertebrates can be achieved.
Moreover, not only cancer cells but also tumor tissues can be transplanted. According to this invention, it becomes possible to bring the nature of cancer in the zebrafish body closer to clinical malignancy. In addition, it becomes possible to transplant a tumor tissue extracted from a cancer patient into a plurality of zebrafish and select a therapeutic agent effective for treatment at an early stage (for example, about one week). In this case, when a mouse is used, it takes about two months, and the amount of tumor tissue necessary for transplantation also increases. Therefore, the effect of the present invention (that is, a small amount of tumor tissue is sufficient, and a therapeutic agent is selected early. Can) is very useful.

In Example 1, it is a graph which shows the result of having investigated the cell growth ability when changing intensity | strength to the human chronic myeloid leukemia cell K562 and irradiating with radiation. It is a graph which shows the result of having investigated change of the number of dead cells when changing intensity to K562 and irradiating with radiation. It is the photograph figure which confirmed the state 24 hours after transplanting the K562 and K-562-Ir cell to the yolk sac site | part of a zebrafish fry. It is the photograph figure which confirmed the state seven days after transplanting K562 and K562-Ir cell to the yolk sac site | part of a zebrafish fry. It is a graph which shows the result of having investigated the survival curve after transplanting K562 and K562-Ir cells to the yolk sac site | part of a zebrafish fry. It is a photograph figure which shows the result of having transplanted K562 cell into the abdominal cavity again when it became 3 months old about the zebrafish transplanted with K562-Ir. FIG. 7 is a photograph of the zebrafish transplanted with the second round of K562 cells shown in FIG. 6, observing the transplant site and tail fin state on the 21st day after re-transplant. Regarding the zebrafish transplanted with K562-Ir, the relationship between the number of days after re-transplantation and the area of cancer cells is shown for zebrafish transplanted with K562 cells or PC-3 cells at the age of 3 months It is a graph. It is a photograph figure which shows the result of the 7th day and 14th day when a PC-3 cell is transplanted intraperitoneally about 3 months old about zebrafish transplanted with K562-Ir. In Example 2, it is a graph which shows the result of having investigated the change of the cell number of the 1st day after irradiation when changing intensity | strength to the human liver cancer cell HepG2 and irradiating. It is a photograph figure which shows the result of having transplanted HepG2 cell in the abdominal cavity again when it became 3 months old about the zebrafish transplanted with HepG2-Ir. It is a photograph figure when the zebrafish of FIG. 11 is dissected and observed on the 14th day after the second HepG2 cell transplantation. In Example 3, it is the photograph figure which investigated the mode of the zebrafish which transplanted the ALDH (+) cell fractionated from K562 cell. It is a graph which shows the result of having compared the proliferation ability of ALDH (+) and ALDH (-) cell. In Example 4, it is a photograph figure which shows the mode of distribution of the cancer cell after transplanting ALDH (+) and ALDH (-) cell fractionated from K562 cell to zebrafish. It is the graph which investigated the mode of (A) distant metastasis after transplanting ALDH (+) and ALDH (-) cells, and (B) tumor angiogenesis. “*” In the figure indicates that a significant difference was observed at p <0.05. In Example 5, ALDH (+) cells (cancer stem cells) and ALDH (-) cells (normal cancer cells) fractionated from K562 cells were administered to zebrafish and then selective for leukemia cancer stem cells. It is a fluorescence micrograph showing the state of cancer cell distribution when TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), an inhibitor, is administered. . It is the graph which compared the magnitude | size of the tumor when TDZD-8 is administered after administering ALDH (+) and ALDH (-) cells fractionated from K562 cells to zebrafish. The left side (A) shows negative control (DMSO) data, and the right side (B) shows TDZD-8 data. It is a photograph figure which shows the result in Example 6. It is a photograph figure which shows the result of having transplanted MIAPaCa-2 which made the tumor tissue again about the zebrafish which tolerates MIAPaCa-2 in the abdominal cavity. (A) is a photograph showing the tumor tissue before transplantation, (B) is a fluorescence photograph after transplanting the tumor tissue, (C) is a micrograph showing the state 3 days after transplantation, (D) is ( It is a microscope picture figure which expands and shows the abdominal part of C). It is a photograph figure which shows the result in Example 7. (A) is a photograph showing a KLM1-KOr tumor mass excised 4 weeks after subcutaneous implantation in nude mice, (I) is in bright field, (II) is under fluorescence, (B) Is a photographic diagram showing the tumor mass after shredding, and (C) is a photographic diagram showing the state 3 days after transplanting the tumor mass after shredding into zebrafish. The arrow G (green) in (C) indicates the vascular tissue, and the arrow Y (yellow) indicates the transplanted tumor.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.
<Example 1> Tolerance model for human chronic myeloid leukemia Human chronic myeloid leukemia cell K562 was cultured in a 25 cm ² flask using RPMI1640 medium supplemented with 10% FBS, and the number of cells was 4 × 10 ⁵ cells. When the concentration reached / ml, 0-40 Gy gamma irradiation was performed using a CAX-150-20 radiation irradiation apparatus (manufactured by Chubu Medical). Before and after the irradiation and for 13 days after the irradiation, the number of viable cells and the number of dead cells were counted using Trypan blue staining and Countes automatic cell counting device (manufactured by Life Technologies).
The results are shown in FIG. 1 and FIG. The cell growth ability was almost lost by irradiation of 6 Gy or more (Fig. 1). On the other hand, as shown in FIG. 2, it was found that the number of dead cells was increased by irradiation with gamma rays of 20 Gy or more. It was determined that 6-10 Gy was appropriate as a dose that did not kill cells while losing cell proliferation ability. Irradiated K562 cells were designated as K562-Ir.

Transplantation of K562-Ir cells into zebrafish fry
After labeling K562 and K562-Ir cells with fluorescent dyes (forced expression of PKH26 or Kusabira-Orange fluorescent protein), about 200 cells were mixed with Matrigel (Japan BD) and zebrafish fry (48 hours after fertilization) Transplanted to the yolk sac site. Thereafter, the animals were reared in a 32 ° C. environment, and images were acquired using an MZ16F fluorescence inverted microscope (Leica) 24 hours and 7 days later. Survival numbers were also analyzed.
The results are shown in FIGS. At 24 hours after transplantation, there was no difference in the size of cancer between the groups transplanted with K562 / K562-Ir cells (Fig. 3), but on the 7th day, K562-Ir cells were transplanted with K562 cells. Compared to the transplanted group, the cancer was smaller (Fig. 4). The results of survival curve analysis (Fig. 5) almost died within 16 days, whereas the group transplanted with K562-Ir cells had the same survival rate as the control group (groups transplanted only with Mock and Matrigel). Was confirmed.

Second transfer of K562 cells to zebrafish The above-mentioned K562-Ir transplanted zebrafish was reared, and about 5000 K562 cells were transplanted into the abdominal cavity again at the age of 3 months. As a comparative control, K562 cells were transplanted intraperitoneally into zebrafish that had not been transplanted with K562 cells. One hour after transplantation, it was observed using an MZ16F fluorescence inverted microscope (manufactured by Leica), and it was confirmed that the transfer was successful (FIG. 6). Thereafter, the mice were reared at 32 ° C., and images were acquired over time.
The results are shown in FIGS. On the 21st day after transplantation, the cancer in the control group almost disappeared, whereas in the second transplant group, the cancer size increased, and cancer also appeared in the tail fin area (Fig. 7, arrow). ). FIG. 8 shows a graph in which the size of cancer is quantified from the acquired image using Image J (NIH).
Transplantation of PC-3 cells into K562-Ir cell-grafted zebrafish To prove that the K562-Ir cell-grafted zebrafish used above retains tolerance for transplantation only in syngeneic cells Lineage cancer cells PC-3 (human prostate cancer cells) were transplanted into K562-Ir cell transplant zebrafish. After labeling PC-3 cells with a fluorescent dye (forced expression of PKH26 or Kusabira-Orange fluorescent protein), about 5000 cells were transplanted intraperitoneally. Thereafter, images were acquired over time, and the size of the cancer was quantitatively analyzed using Image J.
The results are shown in FIG. Transplanted PC-3 cells were present at the transplant site until day 7, but then disappeared on day 14. The quantified results are shown in FIG.

<Example 2> Immune tolerance model for human liver cancer cells Human liver cancer cells HepG2 were cultured in a 25 cm ² flask using a medium obtained by adding 10% FBS to DMEM medium, and 4 × 10 ⁵ cells / When the concentration reached ml, 0-40 Gy gamma irradiation was performed using a CAX-150-20 irradiation apparatus (manufactured by Chubu Medical). CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) was used to count the number of cells before and after irradiation and on the first and fifth days after irradiation.
The results are shown in FIG. The ability of HepG2 to proliferate was lost by irradiation with gamma rays of 10 Gy or more. This cell was designated as HepG2-Ir.
Transplantation of HepG2-Ir cells into juvenile zebrafish
After labeling HepG2 and HepG2-Ir cells with a fluorescent dye (forced expression of PKH26 or Kusabira-Orange fluorescent protein), about 200 cells were mixed with Matrigel (Japan BD), and zebrafish fry (48 hours after fertilization) Transplanted to the yolk sac site. Thereafter, the animals were reared in a 32 ° C environment.

Second transplantation of HepG2 cells to zebrafish The zebrafish described above were bred until 3 months after fertilization, and about 5000 HepG2 cells were transplanted into the abdominal cavity again. As a comparative control, HepG2 cells were transplanted intraperitoneally into zebrafish that had not been transplanted with HepG2 cells before. Thereafter, the animals were reared continuously and observed using an MZ16F fluorescence inverted microscope (manufactured by Leica).
The results are shown in FIG. On the 7th day after transplantation, the cancer in the control group almost disappeared, but the presence of cancer cells persisted in the second transplantation group, suggesting that immune tolerance was established.
Next, these fish were dissected on the 14th day, and the cancer tissue was observed using an MZ16F fluorescence inverted microscope (manufactured by Leica). As shown in FIG. 12, no cancer was observed in the control group, but the presence of cancer was confirmed in the second transplant group.

  In this way, when transplanted with human cancer cells that have lost (weakened) proliferative function in zebrafish that is still in an undifferentiated immune system, the transplanted cancer cells disappear as the animal grows. To do. Then, by transplanting human cancer cells that have not lost their ability to grow the same strain to the same animal that has grown, the human cancer cells can be engrafted without immunosuppression and proliferate in vivo. Was able to develop distant metastases. Conventionally, there are reports that human cancer cells can be transplanted without immunosuppression to rats immediately after birth, but they died of cancer within one month and could not be used in subsequent experiments (non-patented) Reference 1). The inventor of the present invention has developed a cancer of the same strain of the second time when it becomes adult by transplanting the first cancer cell in the early stage of development by adding a modification that eliminates (weakens) the growth function. Cell transplantation was successful. It can be used for experiments as a cancer-bearing model using this adult.

<Example 3> Investigation of tumorigenic performance of cancer stem cells Human chronic myeloid leukemia cells K562 were pre-cultured in RPMI1640 medium supplemented with 10% FBS, ALDEFLUOR reagent (Veritas) and FACSAria flow cytometry (Japan) Becton Dickinson) was used to extract a fraction with high activity of ALDEHYDE DEHYDROGENASE and CD44 surface antigen (+) cells, that is, containing a large amount of cancer stem cells (over 80%). For the extraction of CD44 ⁺ cells, Monoclonal Anti-CD44-PE antibody (SIGMA-ALDRICH) was used.
After labeling each with a fluorescent dye, 1 cell was transplanted into a zebrafish juvenile (a transparent, observable body, 2 days after fertilization). From the first day after transplantation, fluorescence images of the transplanted cells were obtained using an MZ16F fluorescent stereomicroscope (Leica Microsystems).
The results are shown in FIG. 13 and FIG. In the experimental group transplanted with ALDH (+) 1 cells, which are the fraction of cancer stem cells, tumors that could not be confirmed in the group transplanted with ALDH (-) 1 cells were formed, and it was established as a cancer stem cell transplant model. Confirmed that.

<Example 4> Transplantation of cancer stem cells into zebrafish Human chronic myeloid leukemia cells K562 were pre-cultured in RPMI1640 medium supplemented with 10% FBS, ALDEFLUOR reagent (Veritas) and FACSAria flow cytometry ( Nippon Becton Dickinson) was used to extract a fraction containing more than 80% of cancer stem cells. Each was labeled with a fluorescent dye and then transplanted into a zebrafish juvenile (transparent and observable in the body, 2 days after fertilization). From the first day after transplantation, fluorescence images of the transplanted cells were obtained using an MZ16F fluorescent stereomicroscope (Leica Microsystems).
The results are shown in FIG. 15 and FIG. Compared with the ALDH (-) group, the experimental group transplanted with cancer stem cell fraction ALDH (+) cells has a higher incidence of distant metastasis and tumor angiogenesis, and is established as a cancer stem cell transplant model It was confirmed.

<Example 5> Experiment for administration of selective inhibitor of cancer stem cells Human chronic myeloid leukemia cells K562 were pre-cultured in RPMI1640 medium supplemented with 10% FBS, ALDEFLUOR reagent (Veritas) and FACSAria flow cytometry ( Nippon Becton Dickinson) was used to extract a fraction containing more than 80% of cancer stem cells. Each was labeled with a fluorescent dye and then transplanted into a zebrafish juvenile (transparent and observable in the body, 2 days after fertilization). From the first day after transplantation, a known cancer stem cell selective inhibitor (TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione): Non-patent document 3 And fluorescence images of the transplanted cells were acquired over time using an MZ16F fluorescent stereomicroscope (Leica Microsystems).
The results are shown in FIGS. In control (DMSO), cancer stem cells (ALDH (+)) proliferated significantly compared to normal cancer cells (ALDH (-)). On the other hand, administration of the cancer stem cell selective inhibitor TDZD-8 significantly suppressed the proliferation of cancer stem cells compared to normal cancer cells. Thus, it was confirmed that the cancer stem cell selective inhibitor TDZD-8 significantly reduced the number of transplanted cancer stem cells.

  Thus, according to the above example, cancer stem cells are extracted from the cancer cell population by the surface antigen method and transplanted into zebrafish. A cancer transplanted zebrafish is raised in a multi-well plate (96-well plate) and a test compound is administered. Thereafter, after a certain period of time, cancer cells in the zebrafish body were photographed using a fully automatic microscope photographing apparatus, and the anticancer activity of the test compound could be evaluated. That is, using a short immunodeficiency period in zebrafish larvae, a human cancer stem cell transplantation model was established, and fish that express GFP in vascular endothelial cells and various dye-deficient fishes can be crossed, and the body can be observed transparently Human cancer stem cells that construct lineages and express KusabiraOrange are purified, and human cancer stem cell growth by high-throughput in vivo imaging of a 96-well fully automated microscope imaging device, and quantitative analysis of tumor angiogenesis and metastasis by human cancer stem cells Established and established an anti-neoplastic drug screening system.

<Example 6> Tumor tissue transfer to zebrafish (1)
Human pancreatic cancer cells MIAPaCa-2 were cultured in a three-dimensional manner in a low-adsorption plastic petri dish Ultra-Low Attachment plate (Corning) using a medium in which 10% FBS was added to a DMEM medium until fluorescence was exhibited. Labeled with dyes PKH26 (Sigma Aldrich) or CM-DiL (Life Technologies). Note that this fluorescent label can also be applied by a method of forced and constant expression of fluorescent proteins such as DsRed and Kusabila-Orange. Tumor tissue was transplanted to an immunotolerant zebrafish produced according to the method of Example 2 against MIAPaCa-2. Thereafter, the animals were reared in a 32 ° C environment. Immediately after transplantation and 3 days after transplantation, images were acquired using an MZ16F fluorescence inverted microscope (Leica).
The results are shown in FIG. The transplanted tumor tissue had a spherical shape (FIG. 19A), and was transplanted to the zebrafish as it was (FIG. 19B). Three days after transplantation, compared to the case of transplanting normal cancer cells, it shows prominent metastatic symptoms (FIGS. 19 (C) and (D)), more malignant evaluation of human cancer cells, And suitable as a model animal for compound screening.

<Example 7> Tumor tissue transfer to zebrafish (2)
Human pancreatic cancer cells KLM1-KOr were pre-cultured in RPMI1640 medium supplemented with 10% FBS, and the collected cells were concentrated to a PBS solution by centrifugation, and immunosuppressed mice (nude mice, BALB / c strain, Japan) (Claire) was implanted subcutaneously on the dorsal thigh using 1 × 10 ⁷ , 26G needles and syringes. Tumors that grew in the mouse subcutaneously 4 weeks after transplantation were excised under general anesthesia using isoflurane, and penetrated into a PBS solution containing 100 μg / ml kanamycin (Nacalai Tesque) for 10 minutes. The extracted tumor mass (see FIGS. 20 (A), (I), and (II)) is chopped to a size of 100 to 200 μm using a razor blade (see FIG. 20 (B)), and the resulting tumor mass is The zebrafish yolk sac was transplanted 72 hours after fertilization using Sertram oil (Eppendorf). After transplantation, zebrafish were bred in a 32 ° C. environment, and images were acquired using an MZ16F fluorescence inverted microscope (manufactured by Leica) immediately after transplanting and 3 days after transplanting.
The results are shown in FIG. The transplanted KLM1-KOr expresses Kusabira-orange fluorescent protein for the purpose of in vivo imaging. For this reason, as shown in FIG. 20 (C), KLM1-KOr was engrafted in the abdomen of zebrafish and was observed as yellow (arrow Y) larger than that at the time of transplantation.
According to this example, these tumor masses can be separated into cell bodies using collagenase (Life Technologies) and transplanted into zebrafish. In addition to Kusabira-orange fluorescent protein, KLM1-KOr can also be observed using fluorescent dyes for cell staining such as PKH26 (Sigma Aldrich) and CM-DiL (Life Technologies).
As described above, by using the zebrafish of this embodiment, it was possible to transplant a human tumor tissue which is a heterogeneous animal. For this reason, it has become possible to bring the nature of cancer in laboratory animals closer to clinical malignancy. In addition, it has become possible to transplant tumor tissues excised from cancer patients into a plurality of zebrafish, and to select an effective therapeutic agent at an early stage (for example, about one week). In particular, if zebrafish is used, a smaller amount of tumor tissue is required and a therapeutic agent can be selected at an early stage as compared with the case of using mice, so that it becomes a more effective model animal.

Thus, according to the present embodiment, a method for producing a model animal that can be transplanted into a heterologous animal without immunosuppression of human cancer cells can be provided. By using this model animal, it is expected to be applied to elucidation of the functionality of tissues surrounding cancer in cancer and research on cancer stem cells. Furthermore, it can be used for screening of new antineoplastic drugs by a host that is closer to normal.
In addition, human cancer stem cells contain only 0.1 to 5% of all cancer cells, and in the existing transplant model system using mice and rats, the number of transplanted cells is ensured and comprehensive gene expression analysis of a small amount of sample is performed. There are still many technical difficulties. By using zebrafish, the burden of animal experiments, which is a bottleneck in the development of anticancer agents, can be reduced, and at the same time, a high-throughput screening system that is difficult for other vertebrates has become possible.
Moreover, not only cancer cells but also tumor tissues could be transplanted. For this reason, it has become possible to bring the nature of cancer in the zebrafish body closer to clinical malignancy. In addition, it has become possible to transplant a tumor tissue removed from a cancer patient into a plurality of zebrafish and select an effective therapeutic agent at an early stage (for example, about one week).

Claims (4)

After transplantation of gamma-irradiated human cancer cells in the first period of 36 to 48 hours after fertilization in which zebrafish immune cells are in an undifferentiated state, the first one to six months after fertilization A method for producing a xenotransplant model animal, wherein the human cancer cells that maintain their proliferative function at the time of 2 are transplanted again . A xenotransplant model animal produced by the method according to claim 1 . The xenograft model animal according to claim 2 , wherein the model animal is a transparent zebrafish, and the transparent zebrafish is classified into two groups in advance, one group being a control group and the other group being a test compound. Each group of zebrafish is kept in a multi-well plate, and the distribution and concentration of the human cancer cells in each zebrafish body are imaged with a fully automatic microscopic imaging device. Then, based on the result obtained by converting the image data into image data and comparing the image data, the anti-cancer effect of the test compound is evaluated. The said image data is the brightness | luminance image data of the specific wavelength from a labeled compound, The pharmaceutical effect evaluation system with respect to the human cancer cell of Claim 3 characterized by the above-mentioned.