KR20200068907A - Method of preparing animal models for drug screening using cancer cell line speroids and their use - Google Patents

Abstract

The present invention relates to an animal model using a cancer cell line spheroid. More specifically, the present invention relates to an animal model in which a cancer cell line spheroid and CAF are implanted, an anticancer drug screening method using the animal model, and the like. In the animal model according to the present invention, tumor vessels are well formed and there is no tumor necrosis. Therefore, the animal model of the present invention is expected to be useful for in vivo anti-cancer drug efficacy evaluation, research on the pathogenesis of tumors, and cancer therapy.

Description

Method of preparing animal models for drug screening using cancer cell line speroids and their use}

The present invention relates to an animal model using a cancer cell line spheroid, and more particularly, to a method for screening an anticancer agent using the animal model according to the present invention.

Even though cancer research has been conducted in more than 30 years, the incidence of cancer continues to increase due to environmental pollution and poor dietary habits, and more than 10 million patients occur worldwide every year, and the World Health Organization (WHO) dies. Cancer is one of the main causes of cancer. On the other hand, oral cancer among cancers is known to account for a very small proportion of about 2% of the incidence of cancer, but oral cancer is difficult to ingest normal foods due to deterioration of oral functions such as chewing or pronunciation even after treatment. It has been reported to cause chronic sequelae, such as deformation of the disease, which has a fatal effect on the patient's life. The survival rate of oral cancer is about 50%, which is known as malignant tumor. Squamous carcinoma is the most common, and in addition, salivary adenocarcinoma from small salivary glands of the oral mucosa, sarcoma from soft tissues such as jaw bones or facial muscles, and oral mucosa. Malignant melanoma in the palate, mucous membranes, gums, etc., rarely occurs in the form of lymphoma, and has recently been reported to increase the number of younger patients compared to the past. Has been reported as the cause.

Accordingly, new cancer treatment methods including oral cancer are steadily developed, and there are general cancer treatment methods such as surgical surgery, radiation therapy, and chemotherapy along with anti-cancer therapy, but it is known that there is a risk of recurrence along with serious side effects. In addition, in the case of general anticancer drugs, since they tend to act on specific cancers but do not act on other cancers and often develop resistance, development of a variety of new anticancer drugs is essential to enhance the cancer treatment effect. Development of a method for easily screening anticancer agents is also required, and thus, a tumor model for efficient screening is required.

Thus, in general, screening of anticancer agents has been carried out on tumor cells cultured in a two-dimensional (2-D) single layer. However, tumor cells growing in a 2-D single layer are not well formed in a tumor microenvironment (cell-substrate, cell-cell bioenvironment) that deeply affects tumor formation in vivo.

On the other hand, in the in vivo environment, since it has an elongated cell shape that is different from that in a 2D environment in vitro, in the study of actual cell morphology and function through cells cultured in 3D, and furthermore, in interaction studies with the microenvironment It can be a good alternative. In other words, 3D cultured cells have higher cell viability after transplantation into the body than normal single cell type cells, and drug screening can also provide results that can improve clinical efficiency. Is actively progressing. However, a lot of efforts have been made to create experimental animal models with 3-D spheroids, but in this case, tumors are often not formed.

On the other hand, the spheroid cultured in 3-D cancer cells has been applied to drug screening due to the above advantages that it can mimic an in vivo environment in various aspects. However, it is still difficult to overcome the limitations of in vitro assays. In particular, in the mouse animal model, various physical and chemical gradients that contribute to phenotypic heterogeneity in the tumor are caused, and a differential approach to the oxygenation gradient and nutrients is made. Due to this, necrosis often occurs inside a tumor in vivo, which is not only different from an actual in vivo tumor, but also has a problem in that it is difficult to accurately evaluate drug efficacy because it exhibits drug resistance.

Therefore, in order to confirm the well-formed tumor and the more accurate anticancer drug efficacy, an animal model in which the tumor microenvironment is well formed and without necrosis inside the tumor is required.

Republic of Korea Patent Publication 10-2017-0131950

An object of the present invention is to provide a method for preparing an animal model for drug screening.

In addition, an object of the present invention is to provide a method of screening for an anticancer agent using the animal model.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the object of the present invention as described above, the present invention comprises: (a) preparing a cancer cell line spheroid; And (b) implanting the cancer cell line spheroid of step (a) and cancer-associated fibroblast (CAF) (hereinafter, CAF) together into an animal. to provide.

In one embodiment of the present invention, the drug may be an anti-cancer agent.

In addition, the present invention provides an anticancer drug screening method, wherein the method comprises using an animal model prepared by injecting a cancer cell line spheroid and CAF together.

In one embodiment of the present invention, the method comprises the steps of: (a) preparing a cancer cell line spheroid; (b) implanting the cancer cell line spheroid and CAF of step (a) together in an animal to form a tumor; (c) processing the candidate drug to the animal model on which the tumor was formed by the step (b); (d) after treating the candidate drug of step (c), confirming the size or metastasis of the tumor; And (e) when the size of the tumor is reduced or metastasis is suppressed as a result of the check in step (d), determining the candidate drug as an anti-cancer agent.

In addition, the present invention is an anticancer drug screening animal model, wherein the animal model is produced by transplanting a cancer cell line spheroid and a cancer-associated fibroblast (CAF) into an animal, and the animal model is a blood vessel inside a tumor. Provided is an anticancer drug screening animal model, characterized in that it is formed and free of tumor necrosis.

In one embodiment of the present invention, the animal may be a mouse.

In another embodiment of the present invention, the CAF may be derived from oral cancer patients.

In another embodiment of the present invention, the cancer may be oral cancer.

The animal model according to the present invention is an animal model prepared by transplanting a cancer cell line spheroid and cancer-associated fibroblast (CAF) together, in which the blood vessel is well formed inside the tumor, and tumor necrosis There was no confirmation. Unlike the existing animal model, which is difficult to accurately evaluate drug efficacy because necrosis occurs and not only differs from the actual in vivo tumor, but also drug resistance due to this necrosis, the animal model of the present invention is well formed with blood vessels inside the tumor. Since necrosis does not occur, it is possible to efficiently evaluate the anti-cancer drug efficacy. In addition, it is expected to be useful for research on the pathogenesis of tumors and the development of anticancer drugs.

FIG. 1 shows that tumors were formed in mice injected with the oral cancer cell line FaDu isolated cells (dissociated 2-D cells). In one case, it was confirmed that the tumor was not formed.
FIG. 2 shows that after transplantation of FaDu 2-D cells and CAF isolated from oral cancer patients, only tumors were extracted.
3A and 3B are diagrams confirming that the vascular distribution in tumors is insufficient in the animal model in which FaDu 2-D cells and CAF are transplanted together, and necrosis has occurred in the center of the tumor according to tumor growth.
Figure 4 shows that after transplantation of the FaDu 3-D spheroid and CAF isolated from oral cancer patients, only tumors were extracted.
5A and 5B are diagrams confirming that blood vessels are distributed not only around the tumor but also inside the tumor in an animal model in which FaDu 3-D spheroid and CAF are transplanted together, and tumor necrosis has not occurred.

The present inventors made a polite study to produce an animal model for drug screening. As a result, an animal model in which a cancer cell line (3-D) spheroid and CAF was implanted was produced, and blood vessels were inserted into the tumor from the animal model. It was confirmed that it was well formed and there was no tumor necrosis, and based on this, the present invention was completed.

Hereinafter, the present invention will be described in detail.

In an embodiment of the present invention, 3-D FaDu Spanish to see if they are well-formed tumors in Lloyd, the left cheek of the mouse, the separation FaDu cells (dissociated cell) according to the embodiment 1-1 1X10 ⁶ gae scanning, and The right cheek of the mouse was injected with 50 FaDu spheroids according to Examples 1-3 of about 400 micrometers in diameter, and it was confirmed that tumors were well formed in the FaDu spheroids (see Example 2).

In another embodiment of the present invention, in order to determine whether tumor formation, tumor angiogenesis, and necrosis occur in mice in which the FaDu isolation cells according to Example 1-1 and the CAF according to Example 1-2 are transplanted together, FaDu After extracting the tumor from the mouse transplanted with the separated cells and the CAF, the tumor was obtained through immunohistochemical analysis according to Example 1-6 and blood vessel imaging according to Example 1-5. In, it was confirmed whether tumor vessels were formed and whether necrosis occurred (see Example 3).

In another embodiment of the present invention, in order to determine whether tumor formation, tumor angiogenesis, and necrosis occur in mice transplanted with the FaDu spheroid according to Examples 1-3 and CAF according to Examples 1-2, After extracting the tumor from the mice implanted with the FaDu spheroid and CAF, through immunohistochemical analysis according to Example 1-6 and blood vessel imaging according to Example 1-5, It was confirmed whether tumor vessels formed in the tumor and necrosis occurred (see Example 4).

Accordingly, the present invention comprises the steps of (a) preparing a cancer cell line spheroid; And (b) transplanting the cancer cell line spheroid of step (a) and cancer-associated fibroblast (CAF) into the animal together, to provide an animal model preparation method for drug screening.

In the present invention, the drug refers to an unknown substance used in screening, and the drug may be, for example, a compound, a microbial culture solution or an extract, a natural product extract, and is preferably an anti-cancer agent, but is not limited thereto.

In addition, the present invention is an anticancer drug screening method, wherein the method comprises using an animal model prepared by injecting a cancer cell line spheroid and a cancer-associated fibroblast (CAF) together. to provide.

In the present invention, the method comprises the steps of (a) preparing a cancer cell line spheroid; (b) implanting the cancer cell line spheroid and CAF of step (a) together in an animal to form a tumor; (c) processing the candidate drug to the animal model on which the tumor was formed by the step (b); (d) after treating the candidate drug of step (c), confirming the size or metastasis of the tumor; And (e) if the tumor size is reduced or metastasis is suppressed as a result of the check in step (d), the candidate drug may be determined as an anti-cancer agent, but is not limited thereto.

In addition, the candidate drug means an unknown substance used in screening to measure tumor size or metastasis, and may be, but is not limited to, a compound, a microbial culture or extract, a natural product extract, and the like.

In addition, step (d) may be carried out through various methods known in the art that can confirm and measure the size or metastasis. Preferably, tumor weight measurement, blood vessel imaging, immunohistochemical analysis, polymerase chain reaction (PCR), microarray, northern blotting, western blotting It can be measured using lotion (western blotting), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation), or immunofluorescence staining (immunofluorescence), etc., but can be carried out very easily and easily by those skilled in the art. It is not limited to.

In addition, the present invention is an anticancer drug screening animal model, wherein the animal model is produced by transplanting a cancer cell line spheroid and a cancer-associated fibroblast (CAF) into an animal, and the animal model is a blood vessel inside a tumor. Provided is an anticancer drug screening animal model, characterized in that it is formed and free of tumor necrosis.

The term used in the present invention,'spheroid (spheroid)', refers to a collection of more than 1,000 single cells to form a three-dimensional spherical shape, in the present invention, the spheroid is a method known to those skilled in the art Accordingly, a spheroid can be prepared, and preferably, a cancer cell line can be produced in 3-D according to Examples 1-3. For example, an oral cancer, breast cancer, prostate cancer, lung cancer, liver cancer, ovarian cancer, or thyroid cancer cell line may be prepared, and preferably may be a Fadu 3-D spheroid made of 3-D oral cancer cell line, but is not limited thereto. Does not.

In addition, the spheroid may be a spheroid of various diameters, a small spheroid having a diameter of less than 200 μm may mostly include proliferating cells and normal oxygen (nostalgic) cells, and a spheroid having a diameter of about 500 μm is necrotic. You can show the part. Therefore, the diameter of the spheroid may be 200 ~ 500㎛, preferably about 400㎛, but is not limited thereto.

The term used in the present invention,'cancer-associated fibroblasts (CAF)', is also referred to as cancer-related fibroblasts, tumor-associated fibroblasts, carcinogenic fibroblasts, and activated fibroblasts. The tumor microenvironment plays a pivotal role in tumor cell neoplastic (tumor) cell interaction or tumor formation, progression and metastatic spread. At this time, CAF is one of the complex and abundant cell types that promote the tumor characteristic when the extracellular matrix initiates the remodeling process or secretes cytokines within the tumor microenvironment. In addition, CAF participates in tumor progression, including invasion and metastasis, serves as an indicator of the patient's prognosis and promotes cancer progression in vivo when co-injected with tumor cells or recruited to tumor sites. CAF does not undergo cell death, so its number is not reduced, it is abundant in the tumor matrix, and is composed of myofibroblasts and fibroblasts.

In the present invention, according to Example 1-2, CAF can be isolated and cultured from a cancer patient, and preferably, CAF can be cultured according to Example 1-2. In addition, various CAFs known in the art can be used separately, for example, oral cancer, breast cancer, prostate cancer, lung cancer, liver cancer, ovarian cancer, or CAF derived from thyroid cancer patients, preferably, from oral cancer patients It may be isolated oral cancer-associated fibroblasts, but is not limited thereto.

In the present invention, the oral cancer-associated fibroblasts isolated from the oral cancer patient can be separated from the tissue of the oral cancer patient, the oral cancer tissue, etc. can be obtained from oral cancer epithelial tissue, oral cancer lymphoid tissue, etc., and the oral cancer epithelial tissue is Epithelium covering the surface of the oral mucosa, gingival (gingiva), oral canine (single-mouthed ceiling), rag dog (smooth-mouthed ceiling), nasal (lip) mucosal, narrow (ball) mucosa, tongue (lingual) mucosa, etc. , The base layer, the visible layer granular layer, and may be made of a total of four layers of stratum corneum. If the tissue is originated from oral cancer epithelial tissue or the like, the type is not limited, and for example, oral cancer tissue obtained from the oral epithelium of the mouse or the like can be used, and those skilled in the art can appropriately select and use it.

In the present invention, when transplanting cancer cell lines spheroids and CAFs, transplantation methods known in the art may be used, and preferably implantation may be performed by injection, but is not limited thereto.

In addition, in the present invention, instead of transplanting cancer cell lines spheroids and CAFs, the cells can be transplanted after co-culture, but are not limited thereto.

The cancer may be oral cancer, breast cancer, prostate cancer, lung cancer, liver cancer, ovarian cancer, or thyroid cancer, and is preferably oral cancer, but is not limited thereto.

The animal model may be manufactured using mammals other than humans, and mammals other than humans may be monkeys, rats, mice, rabbits, dogs, primates, and the like, preferably muridae animals. However, it is not limited thereto.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experiment preparation and experiment method

1-1. Patients, tissue specimens and cell lines

The sample was used after obtaining the patient's prior written consent after obtaining approval from the Institutional Research Ethics Committee of Kyungpook National University. Two major OSCC tissues were collected by surgical resection at Kyungpook National University Hospital. Before surgery, none of the patients received adjuvant treatment. FaDu was obtained from ATCC and maintained in Dulbecco's modified eagle medium (DMEM) medium containing 10% Fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. Cells were grown at 37° C. in a 5% CO ₂ humidity environment.

1-2. Primary culture of fibroblasts from patient tissue

Cancer tissues and paired normal oral tissues were cut into the smallest possible pieces in sterile DMEM medium, and then plated in 10-cm tissue culture dishes in 10 mL of DMEM supplemented with 10% FBS and incubated at 37°C for 30 minutes. Then, non-adherent cells (mainly tumor cells) were removed and adherent fibroblasts were cultured for further study.

1-3. Spheroid culture and mouse injection

FaDu spheroids were cultured in a 96-well plate format with one spheroid per well. Specifically, FaDu cells were dispensed into a 96-well U-bottom ultra-low attachment plate (4000 cells per well) and cultured for 3 days to form a spheroid (about 400 in diameter). Μm). In addition, in order to investigate the effect of patient-derived CAF on FaDu tumor formation in vivo, 10 ⁶ CAFs were injected with FaDu cells or FaDu spheroids isolated under the same experimental conditions. Specifically, the separation 10 ⁶ FaDu cells with 50 FaDu spheroid were each injected into the right and left view of a nude mouse. After 6 weeks, mice were sacrificed and tumorigenesis compared.

1-4. Blood vessel staining and tissue clearing

Animals were deeply anesthetized using a rodent inhalation anaesthesia apparatus (VETEQUIP, USA) equipped with an isoflavan evaporator. As carrier gas, 100% oxygen was used. For vascular staining, mice were injected intracardiacally with 100 ul Lycopersicon Esculentum Lectin (Lection 488), followed by rinsing through the left ventricular aorta and perfusion with a fixed solution. Rinse with about 30 mL of PBS for 3-5 minutes, and each mouse was fixed with 30-50 mL of 4% paraformaldehyde for 20-30 minutes. Tumor tissue was removed and placed in 4% paraformaldehyde for 12 hours. Tumor tissue was cleared using the Binaree Tissue Clearing Kit (Cat. SHBC-001, TiBinaree, Korea). Specifically, the fixed tissue was immersed in the fixed solution for 24 hours, and incubated with a tissue clearing solution in a shaking incubator at 35° C. for 18 hours. After rinsing the washing solution three times, the tissue was placed in a mounting and storage solution for 24 hours for further clearing (Cat. SHMS-050, Binaree, Korea). Photo-sheet imaging of cleared samples was performed using a Mounting and Storage solution (Cat. SHMS-050, Binaree, Korea) as mounting medium.

1-5. Blood vessel imaging using light-sheet microscopy

The vascular structure of the tumor tissue is 5x/0.1 dual side illumination optics and a light-sheet Z.1 fluorescence microscope with Plan-neofluar 5x/0.16 objective microscope) (Zeiss Corporation, Jena, Germany). Fluorescence was excited with 488nm and 561nm lasers, and emission was detected with 505-545nm and 575-615nm band-pass filters. All image data from LSFM was stored in the czi file type of zen software (Zeiss), the images were reconstructed using Arivis vison 4D software (arivis-AG, Rostock, Germany) and Imaris software (Bitplane, USA) and 3- D was rendered. Brain Research Institute's Brain Research Core Facility (BRCF) collected full LSFM images and image rendering data.

1-6. Immunohistochemical analysis

Paraffin-embedded tissue blocks were prepared from nude mice. This study was approved by the Kyungpook National University Hospital Ethics Committee. Specifically, after removing the wax, the sections (5 μm) were blocked for 5 minutes, and then cultured for 2 hours at room temperature with EGR1 antibody (1:50). IHC staining was performed using an UltraTek HRP Anti-Polyvalent kit (ScyTek Laboratories, Logan, UT, USA). The pigment source used was 3,3-diaminobenzidine (Dako, Carpinteria, CA, USA).

Example 2. Confirmation of tumor formation in 3-D spheroid

When the isolated cells according to Example 1-1 and the FaDu 3-D spheroid according to Example 1-3 were transplanted to the right and left cheeks of the same mouse, respectively, and cultured for 6 weeks, FaDu 3-D No tumor was formed in the left ear of the spheroid implant, and as shown in FIG. 1, it was confirmed that the tumor was formed in FaDu 2-D isolated cells.

Example 3. Confirmation of necrosis in mice transplanted with dissociated cells and CAF

Tumors were extracted from the mice transplanted with the FaDu isolation cells according to Example 1-1 and the CAF according to Example 1-2 (see FIG. 2), followed by blood vessel imaging according to Examples 1-5. And Immunohistochemical analysis according to Example 1-6. As a result, tumor vessels appeared around the tumor, but there was almost no blood vessel distribution in the tumor. It was confirmed that necrosis occurred in the center of the tumor with growth (see FIGS. 3A and 3B ).

Specifically, as shown in FIG. 2, tumors were extracted from 5 mice transplanted with FaDu isolate cells and CAF, respectively, and tumors extracted from 2 of them were shown in FIGS. 3A and 3B. That is, as shown in FIGS. 3A and 3B, in the left blood vessel imaging, the green part was not well visible in the dotted part, and it was confirmed that the blood vessel was not well formed in the center of the tumor. As a result of examining the square area of the magnification at 100 times, many white or pale pink areas appeared, indicating that many necrosis occurred in the center of the tumor.

Example 4. Confirmation of necrosis in mice implanted with 3-D spheroid and CAF

Tumors were extracted from the mice transplanted with the FaDu spheroid according to Example 1-3 and the CAF according to Example 1-2 (see FIG. 4), followed by blood vessel imaging according to Examples 1-5. And Immunohistochemical analysis according to Example 1-6. As a result, the tumor cellularity increased due to the distribution of blood vessels around the tumor, and the blood vessel distribution inside the tumor as well as around the tumor showed that tumor necrosis did not occur (see FIGS. 5A and 5B ). ).

Specifically, as shown in FIG. 4, tumors were extracted from 5 mice transplanted with FaDu spheroid and CAF, respectively, and tumors extracted from 2 of them were shown in FIGS. 5A and 5B. That is, as shown in FIGS. 5A and 5B, in the left blood vessel imaging, the green part in the dotted line portion is better than in FIGS. 3A and 3B, and it can be confirmed that blood vessels are well formed in the center of the tumor, and the right immunity In the histochemical analysis, as a result of examining the square region portion of the 40-fold portion at a 100-fold magnification, it was confirmed that necrosis did not occur in the center of the tumor, as compared to FIG. 3A and FIG. .

The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.

Claims (14)

A method for preparing an animal model for drug screening comprising the following steps:
(a) preparing a cancer cell line spheroid; And
(b) transplanting the cancer cell line spheroid of step (a) and cancer-associated fibroblast (CAF) into an animal together.
According to claim 1,
The animal is characterized in that the mouse, the manufacturing method.
According to claim 1,
The CAF is characterized in that it is derived from oral cancer patients, the manufacturing method.
According to claim 1,
The drug is characterized in that it is an anti-cancer agent, the manufacturing method.
According to claim 1,
The cancer is characterized in that the oral cancer, the manufacturing method.
As an anticancer agent screening method, the method includes using an animal model prepared by injecting a cancer cell line spheroid and a cancer-associated fibroblast (CAF) together.
The method of claim 6,
The method comprises screening an anticancer agent comprising the following steps:
(a) preparing a cancer cell line spheroid;
(b) implanting the cancer cell line spheroid and CAF of step (a) together in an animal to form a tumor;
(c) processing the candidate drug to the animal model on which the tumor was formed by the step (b);
(d) after treating the candidate drug of step (c), confirming the size or metastasis of the tumor; And
(e) when the tumor size is reduced or metastasis is suppressed as a result of the check in step (d), determining the candidate drug as an anti-cancer agent.
The method of claim 6,
The animal is characterized in that the mouse, anti-cancer agent screening method.
The method of claim 6,
The CAF is characterized in that originated from oral cancer patients, anticancer agent screening method.
The method of claim 6,
The cancer is characterized in that the oral cancer, anti-cancer agent screening method.
As an anticancer drug screening animal model, the animal model is produced by transplanting a cancer cell line spheroid and a cancer-associated fibroblast (CAF) into an animal, and the animal model is formed with blood vessels inside the tumor, and the tumor An anticancer drug screening animal model characterized by having no necrosis.
The method of claim 11,
The animal is characterized in that the mouse, anticancer drug screening animal model.
The method of claim 11,
The CAF is characterized in that originated from oral cancer patients, anticancer drug screening animal model.
The method of claim 11,
The cancer is characterized in that the oral cancer, anticancer drug screening animal model.

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