KR102576165B1 - An animal model for invasive brain cancer and a method for producing thereof - Google Patents

Abstract

The present invention relates to an animal model of invasive brain cancer and a method for preparing the same.
Since the invasive brain cancer animal model of the present invention exhibits strong invasiveness among brain cancers, such as glioblastoma, a candidate substance that inhibits invasion of brain cancer cells is screened or a candidate substance that suppresses metastasis of brain cancer caused by invasion of brain cancer cells. It is expected that it can be actively used for the development of therapeutic agents for invasive brain cancer, such as screening for .

Description

An animal model for invasive brain cancer and a method for producing it {An animal model for invasive brain cancer and a method for producing thereof}

The present invention relates to an animal model of invasive brain cancer and a method for preparing the same.

Among various cancers, brain cancer in particular occurs regardless of age, and has a higher incidence in children than other cancers. Brain cancer is divided into primary brain cancer that develops in the brain tissue and the meninges surrounding the brain, and secondary brain cancer that has metastasized from cancer that has developed in the skull or other parts of the body. Glioblastoma, the most common form of brain cancer, especially glioma ( glioma) is a tumor that accounts for 60% of primary brain tumors and has a high incidence. In addition, compared to other brain cancers, glioblastoma is the most malignant, highly invasive, and exhibits an aggressive variant, resulting in a very poor prognosis. If glioblastoma is not treated promptly, it can have fatal consequences within a few weeks. However, there is no specific treatment other than radiation therapy for brain cancer due to the inherent characteristic of the brain that it is difficult for a drug for treatment to be delivered to a target brain region by the Brain Blood Barrier. Therefore, the need for an invasive brain cancer animal model to be used for suppressing metastasis of glioblastoma or for prognosis has emerged.

The present invention was conceived to solve the above problems, and relates to an animal model for invasive brain cancer and a method for preparing the same. Since the animal model according to the present invention embodies strong invasiveness among brain cancers such as glioblastoma, it is expected to be actively used for the development of therapeutic agents for invasive brain cancer.
[Prior literature]
Prior Document 1: Korean Patent Publication No. KR 10-2015-0142928 A
Prior Document 2: Korean Patent Publication No. KR 10-2017-0105854 A
Prior Document 3: Korean Patent Publication No. KR 10-2017-0011984 A

One object of the present invention is to provide an animal model of invasive brain cancer.

Another object of the present invention is to provide a manufacturing method for preparing an animal model of invasive brain cancer.

Another object of the present invention is to provide invasive brain cancer cells.

Another object of the present invention is to provide a production method for the production of invasive brain cancer cells.

Another object of the present invention is to provide a composition for promoting invasion of brain cancer cells.

Another object of the present invention is to provide a method for screening a candidate substance that inhibits invasion of brain cancer cells.

Another object of the present invention is to provide a method for screening a candidate substance that inhibits brain cancer metastasis caused by invasion of brain cancer cells.

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

One embodiment of the present invention provides an animal model of invasive brain cancer, and a method for preparing the same.

The method of the present invention comprises mixing brain cancer cells and ventricular mesenchymal stem like cells (vMSLC); and transplanting the mixed cells into a non-human subject.

The "brain cancer" of the present invention refers to all tumors generated within the cranial cavity, and may be, for example, glioma, but is not limited thereto.

The "glioma" of the present invention is a tumor that accounts for 60% of primary brain tumors, is a malignant tumor that has a high incidence and is difficult to treat, and thus has no special treatment other than radiation therapy. For example, It may be astrocytoma, glioblastoma or oligodendroglioma, etc., preferably glioblastoma, but is not limited thereto.

The brain cancer of the present invention may be a cancer tumor cell, but is not limited thereto.

The "cancer tumor sphere" of the present invention refers to a cell aggregate in which some of the cells having characteristics similar to stem cells among cells constituting cancer tissue can be formed under three-dimensional culture conditions.

The "invasion" of the present invention refers to a phenomenon in which tumor cells generated in a primary organ acquire new genetic traits necessary for metastasis as cancer progresses, and then invade into blood vessels and lymph glands. In the case of high invasiveness, tumor cells infiltrated into blood vessels and lymphatics circulate along the lymph, settle in tissues existing in other organs from the primary organ, and then proliferate, so that cancer can ultimately easily metastasize to other organs.

The "mesenchymal stem like cell (MSLC)" of the present invention is a mesenchymal stem-like cell. Mesenchymal stem cells (MSC) are non-hematopoietic precursors with pluripotency originally isolated from bone marrow, but mesenchymal stem-like cells (MSLC) are detected in various tissues including brain tumors and have pluripotency. As a stromal cell, it means a cell having characteristics similar to mesenchymal stem cells. Depending on the location of acquisition, tMSLCs (tumor-derived mesenchymal stem-like cells; tumor MSLCs) are cells acquired from the tumor tissue itself, and vMSLCs (ventricular mesenchymal stem-like cells; ventricle MSLCs) are distant from the site of tumor origin, but can be found in glioblastoma. It is a mesenchymal stem-like cell isolated from the subventricular zone (SVZ) region located at the place of origin.

The animal model of the present invention refers to an individual transplanted with brain cancer cells and vMSLC (ventricular mesenchymal stem-like cells).

Another embodiment of the present invention provides invasive brain cancer cells, and a method for preparing the same.

The method of the present invention comprises the steps of obtaining a culture medium in which vMSLC (ventricular mesenchymal stem-like cells) are cultured; And, culturing the brain cancer cells in the culture medium; includes.

In the invasive brain cancer cells of the present invention, and the method for producing the same, "brain cancer", "glioma", "cancer tumor sphere", "invasive", and "vMSLC (ventricular mesenchymal stem-like cells)" are animals of the invasive brain cancer It is redundant with what was described in the model and its manufacturing method, and is omitted in order to avoid excessive complexity of the present specification.

Another embodiment of the present invention provides a composition for promoting invasion of brain cancer cells, comprising vMSLC (ventricular mesenchymal stem-like cells) or a culture product thereof as an active ingredient.

The "culture product" of the present invention is meant to include a cell, a cell lysate, or a cell culture, and the culture includes a culture medium used for cell culture, and by-products in the culture medium.

In the composition for promoting invasion of brain cancer cells of the present invention, "brain cancer", "glioma", "cancer tumor sphere", "invasive" corresponding to infiltration, and "vMSLC (ventricular mesenchymal stem-like cells)" It overlaps with what was described in the animal model of invasive brain cancer, and its preparation method, and is therefore omitted to avoid excessive complexity in the present specification.

In the animal model of invasive brain cancer, invasive brain cancer cells, or a composition for promoting invasion of brain cancer cells, invasiveness in a meaning corresponding to invasiveness is measured by measuring the expression level of a gene or protein encoding a protein related to invasiveness, or by invasive area. You can check.

Measuring the expression level of the gene is to check the expression level of the gene to be measured included in the sample, and to measure the level of a gene transcribed from the gene to be measured, for example, mRNA transcribed from the gene. can be implemented Specifically, the method is RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR (Competitive RT-PCR), real time RT-PCR (real time quantitative RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blot analysis (Northern blot assay), can be implemented using a primer pair or probe that can specifically bind to the mRNA to be measured used in methods such as DNA chip analysis.

The "primer" is a base sequence having a short free 3'hydroxyl group, which can form a base pair with a complementary template strand, and functions as a starting point for copying the template strand. It means a short nucleotide sequence that does. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

The "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a base capable of specifically binding to the gene or mRNA transcribed from the gene, and such a probe determines the presence or absence of a specific mRNA, expression It may be labeled so that the level (expression level) can be identified. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe, but is not limited thereto.

The primers or probes can be easily prepared by a known method by a person skilled in the art by referring to a known nucleotide sequence, for example, the known nucleotide sequence of Zeb1, an invasion marker.

The measurement of the expression level of the protein is to check the expression level of the protein encoded by the gene to be measured included in the sample, and is a method using an antibody or an aptamer capable of specifically binding to the protein to be measured. can be implemented Specifically, the method includes Western blot assay, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket Rocket immunoelectrophoresis, Immunohistochemical staining, Immunoprecipitation Assay, Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS It can be implemented using antibodies or aptamers used in methods such as (fluorescence activated cell sorter analysis) and protein chip technology assay.

The "antibody" refers to a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and may include polyclonal antibodies, monoclonal antibodies, or even parts of antibodies as long as they have antigen-binding properties, and may include all types of immunoglobulin antibodies. In addition, special antibodies such as humanized antibodies may be included, and the antibodies include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

The "aptamer" refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures depending on its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. An aptamer can inhibit the activity of a given target molecule by binding to the given target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or cyclic in shape.

The antibody can be prepared by a conventional method after cloning a known nucleotide sequence into an expression vector according to a conventional method to obtain a protein encoded by the gene, and the aptamer can be prepared by reference to a known nucleotide sequence. It can be easily manufactured by a person skilled in the art according to a known method.

Another embodiment of the present invention provides a method for screening a candidate substance that inhibits invasion of brain cancer cells.

The screening method of the present invention comprises the steps of treating the animal model of invasive brain cancer of the present invention or the invasive brain cancer cells of the present invention with a candidate substance that inhibits brain cancer cell invasion; and determining the candidate material as a brain cancer cell invasion inhibitor when invasion of brain cancer cells is inhibited by the candidate material.

In the method of screening for a candidate substance that inhibits brain cancer cell invasion of the present invention, "brain cancer", "glioma", "cancer tumor sphere", "invasive", and "vMSLC (ventricular mesenchymal stem-like cell)" It overlaps with what was described in the animal model of invasive brain cancer, and its preparation method, and is therefore omitted to avoid excessive complexity in the present specification.

Another embodiment of the present invention provides a method for screening a candidate substance that inhibits brain cancer metastasis caused by invasion of brain cancer cells.

The screening method of the present invention comprises the steps of treating the animal model of invasive brain cancer of the present invention or the invasive brain cancer cells of the present invention with a candidate substance that inhibits brain cancer cell invasion; and, when invasion of brain cancer cells is suppressed by the candidate substance, determining the candidate substance as a substance that suppresses brain cancer metastasis caused by brain cancer cell invasion.

In the method of screening a candidate substance that inhibits brain cancer metastasis caused by the invasion of brain cancer cells of the present invention, "brain cancer", "glioma", "cancer tumor sphere", "invasive", and "vMSLC (ventricular mesenchyme) Stem-like cells)" is redundant with what was described in the animal model of invasive brain cancer and the preparation method thereof, and is therefore omitted to avoid excessive complexity in the present specification.

Since the invasive brain cancer animal model of the present invention exhibits strong invasiveness among brain cancers, such as glioblastoma, a candidate substance that inhibits invasion of brain cancer cells is screened or a candidate substance that suppresses metastasis of brain cancer caused by invasion of brain cancer cells. It is expected that it can be actively used for the development of therapeutic agents for invasive brain cancer, such as screening for .

1 is a diagram showing the positions of a tumor sphere (TS), a ventricle sphere (VS), a tumor mesenchymal stem like cell (tMSLC), and a ventricle mesenchymal stem like cell (vMSLC) in the brain according to an embodiment of the present invention. .
2 is a diagram showing the results of confirming whether cells extracted from the brain are tMSLC or vMSLC according to an embodiment of the present invention through morphology, differentiation, and MSLC (mesenchymal stem like cell) surface markers.
Figure 3 is a diagram showing the results of confirming the difference in invasiveness of TS by tMSLC and vMSLC in terms of invasive area and expression level of invasive proteins according to an embodiment of the present invention.
4 is a diagram showing the results of analyzing the difference in gene expression between tMSLC and vMSLC by gene expression profiles of tMSLC and vMSLC according to an embodiment of the present invention.
Figure 5 is a diagram showing the results of confirming the difference in invasiveness of VS according to the culture medium in terms of the invasive area and the expression level of invasive proteins, according to an embodiment of the present invention.
6 is a diagram showing the results of confirming the difference in invasiveness of cancer cells in a mouse animal model transplanted with TS, TS+tMSLC, or TS+vMSLC according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1. Cell extraction and culture

Tissues from newly diagnosed brain cancer patients without surgical intervention, chemotherapy, or radiotherapy were used in this study. MR images were taken using an Achieva 3.0T system (Philips Medical Systems) within 7 days before performing surgery to remove cancer tissue in each patient. Axial images were taken parallel to the anterior and posterior edges of the corpus callosum. The positions of tumor sphere (TS), ventricle sphere (VS), tumor mesenchymal stem like cell (tMSLC), and ventricle mesenchymal stem like cell (vMSLC) are indicated in the image and shown in FIG. 1 .

Cells were then isolated from glioblastoma (GBM) patient-derived tissues and TS medium containing DMEM/F-12 (Corning), 1x B27 (Invitrogen), 20 ng/mL bFGF, and 20 ng/mL EGF (Novoprotein) (tumor sphere complete media). From this, MEMα, 10% FBS (Lonza), 2 mM L-glutamine (Mediatech), and 100x antibiotic-antimycotic solution (Gibco) were used to culture tMSLC (tumor mesenchymal stem like cell) and vMSLC (ventricle mesenchymal stem like cell). Cells isolated from tissues were cultured in the included MSC medium (MSC complete media). tMSLC or vMSLC was seeded in a culture dish by adding 70% or more MSC medium, and after the cells were attached, the MSC medium was removed and TS medium was added to further culture for 24 hours, and the medium supernatant from which foreign substances were removed was obtained, respectively. It was used as tMSLC conditioned media (tMSLC conditioned media) and vMSLC conditioned media (vMSLC conditioned media).

Example 2. Cell Identification

Whether the cells extracted in Example 1 were suitable for tMSLC or vMSLC was confirmed through morphology, differentiation, and MSLC (mesenchymal stem like cell) surface markers, which are shown in FIG. 2 .

First, as a result of observing the cells under an optical microscope, it was confirmed that both tMSLC and vMSLC were typical cell types of MSLC.

Differentiation characteristics were identified by dividing them into adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation. To confirm fat differentiation, tMSLC or vMSLC was cultured in MSC medium at a density of 4×10 ⁴ cells/well in a 6-well culture dish. After the cells were attached, adipogenic differentiation BulletKit (Lonza Walkersville, Walkersville, MD, USA) medium was added, and the medium was replaced every 3 to 4 days for 3 weeks. After 3 weeks, the medium was removed, the cells were washed with PBS, fixed in 10% formalin (Fisher Scientific, Fair Lawn, NJ, USA) for 30 minutes, and treated with 60% isopropanol (Sigma-Aldrich) for 5 minutes after washing with PBS. and stained with Oil Red O solution (Sigma-Aldrich) for 10 minutes. After washing with PBS, they were stained with hematoxylin (Sigma-Aldrich) for 1 minute, washed, and observed. To confirm bone differentiation, tMSLC or vMSLC was cultured in MSC medium at a density of 3×10 ⁴ cells/well in a 6-well culture dish. After the cells were attached, osteogenic differentiation BulletKit (Lonza Walkersville) medium was added, and the medium was replaced every 3 to 4 days for 3 weeks. After 3 weeks, the medium was removed, and the cells were washed with PBS, fixed with 70% ethanol (Sigma-Aldrich) at refrigerated temperature for 1 hour, washed with PBS, and then washed with 40 mM Alizarin Red (pH 4.2; Sigma-Aldrich) for 10 minutes. Stained at room temperature. This was observed after washing 5 times with distilled water. To confirm chondrogenic differentiation, 2.5 × 10 ⁵ tMSLC or vMSLC were centrifuged at 150 × g for 5 minutes in a 15 ml tube (SPL, Pocheon, Gyeonggi, Republic of Korea) to collect the cells, which were mixed with 20 μg/ml of TGF-β3. (Ontogeny Research Products, Cambridge, MA, USA) was added to the chondrogenic differentiation Bullet Kit (Lonza Walkersville). The medium was replaced every 3 to 4 days for 3 weeks, and after 3 weeks, the cell pellet was fixed in 10% formalin for 1 hour and stained with toluidine blue (Sigma-Aldrich). All three stainings were performed in the same way as the control group MSC medium. As a result of confirming the fat differentiation, bone differentiation, and cartilage differentiation, it was confirmed that both tMSLC and vMSLC were well differentiated into adipocytes, osteocytes, and chondrocytes.

In addition, it was confirmed that both tMSLC and vMSLC did not form tumors in vivo through hematoxylin staining and morphological observation of brain tissue after transplanting the cells into mice.

To confirm the MSLC marker marker, the tumor sphere was treated with trypsin-EDTA to collect cell pellets, and then prepared in an EP tube at a concentration of 1x10 ⁶ cells / 100ul using FACS buffer (10% FBS + 90% PBS) did Afterwards, 2~5ul of CD16 (DB pharmingen) was applied to all EP tubes at 4℃ for 15~20 minutes, left cell petlets through centrifugation, washed once with FACS buffer, and pelleted using 100ul FACS buffer. released It was treated with the primary antibody at 5-10ul/100ul at 4°C for 30 minutes, centrifuged and cells washed, and then treated with the secondary antibody at 1-20ul/100ul at 4°C for 30 minutes. Collect the cells by centrifugation again, wash twice, fix with 100ul of 1% paraformaldehyde for 10 minutes, put 100ul of the sample into a FACS tube, add 400ul or more of PBS, and then use FACS to express the marker. was measured. As a result of the experiment, it was confirmed that representative surface markers of MSLC were expressed.

Example 3. Confirmation of differences in invasiveness between tMSLC and vMSLC

A 96-well culture dish was filled with Matrigel, collagen type I (Corning Incorporated), and TS medium, and one tumor sphere was seeded in each well. When the matrix was properly hardened, TS medium, tMSLC conditioned medium, or vMSLC conditioned medium was added and cultured for 72 hours and observed. After 72 hours, the invasion area of the tumor sphere was measured and compared with day 0. This is shown in Figure 3.

As a result of the experiment, it was confirmed that the infiltration of tumor cells was increased by placing the tMSLC-conditioned medium or the vMSLC-conditioned medium in the matrix. The infiltration area increased in the order of control < tMSLC < vMSLC.

Example 4. Confirmation of difference in gene expression between tMSLC and vMSLC

Total RNA was extracted from glioblastoma tumor spheres and tissues of the patient using the Qiagen RNeasy Plus Mini kit (Qiagen, USA) according to the method provided by the manufacturer. The extracted total RNA was loaded onto an Illumina HumanHT-12 v4 Expression BeadChip (Illumina, USA). For stabilization and standardization of data transformation, a quantile normalization method using the R/Bioconductor lumi package was used. Average linkage hierarchical clustering was performed using Pearson's correlation as a distance metric using GENE-E software, and the expression level of each gene was represented as a heat map. Genes were functionally annotated by over-representation analysis (ORA) using GO gene sets and then visualized as enrichment maps using Cytoscape with the ClueGO plug-in. . Enriched GO terms were classified according to kappa scores (> 0.4). Statistical significance was determined using a two-sided hypergeometric test and only parts with a P-value < 0.01 were indicated. The results are shown in FIG. 4 .

As a result of the experiment, tMSLC and vMSLC were similar in whole gene expression profiles, but the presence of 643 differentially expressed genes (DEGs) was confirmed. Specifically, in the case of vMSLC, the expression of genes related to invasion-related cell migration, chemokine production, and chemotaxis was high.

Example 5. Verification of difference in invasiveness of VS (ventricle sphere) according to culture medium

Using the matrix of Example 3, VS was cultured in TS medium, tMSLC conditioned medium, or vMSLC conditioned medium, cell lysates were performed, and SDS- PAGE was run. Electrophoresed proteins were transferred to nitrocellulose membranes, and β-catenin (BD Biosciences), N-cadherin (R&D Systems), Zeb1 (Sigma-Aldrich), CD44 (Cell Signaling Technology), or GAPDH (Santa Cruz Biotechnology) was treated with the primary antibody. Thereafter, the secondary antibody was treated, and horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnology) with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Waltham) was treated. Experimental result images were taken with ImageQuant LAS 4000 mini (GE Healthcare Life Sciences), and bands were quantified using imageJ software. The results are shown in FIG. 5 .

As a result of the experiment, it was confirmed that the invasiveness of VS on the collagen-Matrigel matrix was increased by the tMSLC-conditioned medium or the vMSLC-conditioned medium. The invasive capacity of the cells increased in the order of control, TS medium < tMSLC conditioned medium < vMSLC conditioned medium. In addition, it was confirmed that the expression of invasion-related proteins and the area of invasion increased in the order of control, TS medium < tMSLC condition medium < vMSLC condition medium.

Example 6. Preparation of mouse orthotopic xenograft model

Male athymic nude mice (6 weeks old, Central Lab. Animal Inc.) were used for the experiment after acclimatization period in a sterilized state under controlled light, temperature, and humidity for one week before the experiment. For the preparation of a mouse stereotactic xenograft model, glioblastoma tumorspheres (GBM TSs, 5x10 ⁵ per mouse) and the same number of tMSLCs or vMSLCs were placed in the right frontal lobe of the mouse with a guide-screw system. were transplanted to a depth of 4.5 mm. Euthanasia was performed when the body weight of the animal decreased by more than 15% from the maximum value during the test period. In order to obtain brain tissue from mice and proceed with immunostaining, the tissue was prepared as a paraffin block and cut into 5-μm thick using a microtome to prepare slides. Antigen retrieval and antibody attachment were performed using an automated device (Discovery XT), and Zeb1, an invasion marker, was observed by peroxidase/DAB staining. The results are shown in FIG. 6 . As a result of confirming the survival rate and tumor formation of mice in vivo, significant brain cancer was generated in the TS+vMSLC experimental group, and the expression of Zeb1, an invasion marker, increased in the order of control group (TS) < tMSLC < vMSLC.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (15)

(a) mixing brain cancer cells and ventricle mesenchymal stem like cells; and,
(b) transplanting the mixed cells of (a) to a subject other than a human;
According to claim 1,
Wherein the brain cancer is glioblastoma (Glioblastoma), a method for producing an invasive brain cancer animal model.
According to claim 1,
Wherein the brain cancer is a cancer tumor sphere, a method for producing an invasive brain cancer animal model.
As an invasive brain cancer animal model transplanted with brain cancer cells and ventricular mesenchymal stem like cells,
The animal is an animal model of invasive brain cancer that is a non-human subject.
According to claim 4,
The brain cancer is glioblastoma (Glioblastoma), an invasive brain cancer animal model.
According to claim 4,
The brain cancer is a cancer tumor cell, an invasive brain cancer animal model.
(a) obtaining a culture medium in which ventricle mesenchymal stem like cells are cultured; and,
(b) culturing the brain cancer cells in the culture medium of (a); a method for producing invasive brain cancer cells, comprising:
According to claim 7,
Wherein the brain cancer is glioblastoma (Glioblastoma), a method for producing invasive brain cancer cells.
According to claim 7,
Wherein the brain cancer is a cancer tumor sphere, a method for producing invasive brain cancer cells.
Invasive brain cancer cells, which are brain cancer cells cultured in a culture medium in which ventricular mesenchymal stem like cells are cultured.
According to claim 10,
The brain cancer is glioblastoma (Glioblastoma), invasive brain cancer cells.
According to claim 10,
The brain cancer is a cancer tumor sphere, invasive brain cancer cells.
A composition for promoting invasion of brain cancer cells, comprising ventricular mesenchymal stem like cells or a culture product thereof as an active ingredient.
(a) treating the animal model of claim 4 or the cells of claim 10 with a candidate substance that inhibits invasion of brain cancer cells; and,
(b) a screening method for a candidate substance for inhibiting invasion of brain cancer cells, comprising the step of determining the candidate substance as a brain cancer cell invasion inhibitor when invasion of brain cancer cells is inhibited by the candidate substance.
(a) treating the animal model of claim 4 or the cells of claim 10 with a candidate substance that inhibits invasion of brain cancer cells; and,
(b) a candidate substance for inhibiting brain cancer metastasis, comprising the step of determining the candidate substance as a substance that inhibits brain cancer metastasis caused by brain cancer cell invasion when invasion of brain cancer cells is inhibited by the candidate substance screening method.