WO2019203534A1 - Novel patient-derived xenograft model of glioblastoma and use thereof - Google Patents

Abstract

The present invention relates to a novel patient-derived xenograft model of a glioblastoma, a manufacturing method therefor and a use thereof and, more specifically, to: a patient-derived xenograft model of a glioblastoma, the model enabling the rapid selection of treatment options during the treatment of glioblastoma having a short average survival time; a manufacturing method therefor; and a method for selecting a patient-specific glioblastoma treatment by using same. A patient-derived xenograft animal model of a glioblastoma, according to the present invention, possesses the inherent characteristics of a tumor. In addition, even patient glioblastoma cells, which do not form intracranial tumors in orthotopic transplantation, form tumors within four weeks such that, even in consideration of the preparation period for patient-derived cells, treatment options can be selected within six weeks, which is much shorter than the median time from initial surgery up to recurrence. Furthermore, unlike a subcutaneously transplanted model of a glioblastoma, a patient-derived xenograft animal model of a glioblastoma, according to the present invention, can be an effective alternative by mimicking the microenvironment of the brain. Moreover, the present invention enables, without any additional equipment, easy monitoring with the naked eye, a simple indirect ophthalmoscope or the like, and requires relatively fewer glioblastoma cells for the manufacture of a glioblastoma model, so as to enable the simultaneous evaluation of several treatment options at the same time, thereby being useful.

Description

New xenograft model derived from glioblastoma patient and its use

The present invention relates to a novel glioblastoma patient-derived xenograft model and its preparation and use, and more particularly to a glioblastoma patient-derived xenograft model that enables a quick selection of treatment options for the treatment of glioblastomas with a short average survival. And a method for screening the patient-specific glioblastoma treatment using the same and a method thereof.

Glioblastoma is a tumor originating from glial cells that are abundant in brain tissue and is known to account for 12-15% of all brain tumors. Glial cells support the tissues of the central nervous system and are located between blood vessels and nerve cells to participate in the metabolism of nerve cells, and when they are injured or inflamed, they proliferate to help cells recover. The starting tumor is glioblastoma. Glioblastomas, however, are the most common and malignant cancers in the brain and have an average survival time of less than 15 months.

The development of glioblastoma patient-derived xenograft (PDX) models has been required to study the tumor characteristics of these glioblastomas and to investigate the potential therapeutic efficacy of various treatment options. A patient derived xenograft (PDX) model of known glioblastoma for this is based on subcutaneous or intracranial injection of tumor cells in immunocompromised mice. However, PDX tumors developed rapidly through subcutaneous injection, but were limited to subcutaneous spaces completely different from the brain microenvironment. Intracranial injection, on the other hand, tumors experience the microenvironment of the brain surrounding glioblastoma, but cells derived from some patients are often unable to form intracranial tumors and are often slow to form, resulting in lengthy treatments. Time was required Considering that the median survival of glioblastoma patients is less than 15 months, PDX of glioblastoma, which can be performed more quickly, can be formed as soon as possible, and simulates the microenvironment of the brain, is required.

Accordingly, the present inventors have made efforts to develop a new glioblastoma patient-derived xenograft model that can rapidly produce a glioblastoma model and simulate the brain microenvironment. The new glioblastoma PDX model, which is effective in simulating the microenvironment of the brain while screening treatment options and can be observed with the naked eye without separate or bioluminescent imaging, unlike the intracranial tumor, It was confirmed that the original tumor cells and immunohistochemical characteristics are the same and retain the characteristics of the original glioblastoma, and completed the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

An object of the present invention is to provide a patient-derived xenograft animal model of a new glioblastoma and its preparation.

Another object of the present invention is to provide information for patient-specific treatment using the animal model and to screen for glioblastoma therapeutics.

In order to achieve the above object, the present invention provides a patient-derived xenograft animal model of a new glioblastoma and its preparation and use.

Specifically, the present invention provides a method for producing a xenograft patient-derived xenograft animal model comprising the step of injecting glioblastoma cells isolated from a glioblastoma patient into the vitreous of an animal other than a human.

The present invention also provides a xenograft animal model derived from glioblastoma patient prepared by the above method.

The invention also provides a method of providing information for screening for a patient-specific glioblastoma treatment comprising the following steps:

(a) performing a candidate treatment method for glioblastoma in the xenograft animal model derived from glioblastoma patient; And

(b) confirming the therapeutic effect of the animal model in which the candidate treatment method was performed.

In this case, the genetic characteristics, immunochemical characteristics and tumor or immunoprotein expression characteristics of the glioblastoma of the glioblastoma patient-derived xenograft animal model may be further analyzed.

The present invention also provides a method for screening a glioblastoma therapeutic agent comprising the following steps:

(a) treating a candidate substance for treatment with a xenograft animal model derived from glioblastoma patient or glioblastoma cells derived therefrom; And

(b) after treatment with the candidate substance, the animal model or glioblastoma cells are compared with the control group without treatment with the candidate substance, and the size of the tumor tissue of the animal model is reduced or metastasis is inhibited or the glioblastoma cells are inhibited. Determining the candidate as a glioblastoma therapeutic agent if proliferation of the inhibitor is inhibited or killed.

In this case, the genetic characteristics, immunochemical characteristics and tumor or immunoprotein expression characteristics of the glioblastoma of the glioblastoma patient-derived xenograft animal model may be further analyzed.

The present invention provides a patient-derived xenograft animal model of a new glioblastoma, and its preparation and use, which enables a fast treatment option selection in the treatment of glioblastoma with a short average survival.

A patient-derived xenograft animal model of glioblastoma according to the present invention inherently retains the characteristics of a tumor. In addition, even glioblastoma cells that do not form intracranial tumors by orthotopic transplantation form tumors within 4 weeks, considering the preparation period of patient-derived cells. Treatment options can be selected within a much shorter six weeks. In addition, the patient-derived xenograft animal model of glioblastoma according to the present invention is an effective alternative by simulating the microenvironment of the brain, unlike the glioblastoma model transplanted subcutaneously. In addition, it can be easily monitored by using the naked eye or simple indirect ophthalmoscope, without any additional equipment, and requires a relatively small number of glioblastoma cells for the production of glioblastoma model, which enables the simultaneous evaluation of several treatment options. Do.

1 is for tumor cell isolation and culture of primary and recurrent glioblastoma patients, 1A is a picture of H & E sections (circle magnification, x50) of primary and recurrent tumors, and 1B is a stereotactic biopy. , Schematic schedule for the preparation of craniotomy, chemoradiocombination (CCRT), and patient-derived cells (GBL-28 and GBL-37), where 1C represents the week after initial diagnosis and stereotactic biopsy. Pictures of GBL-28 and GBL-37 cells (scale bar, 100 μm).

FIG. 2 shows the uncertainty in orthotopic transplantation of glioblastoma cells derived from patients, 2A shows Kaplan-Meier survival curves of mice (n = 12) after intracranial injection of U-87 MG, GBL-28, and GBL-37 2B represents H & E sections of the brain of 4-6 weeks mice (U-87 MG (4 weeks), GBL-28 (6 weeks), GBL-37 (6 weeks)) after intracranial injection of each cell line. (Yellow dotted line indicates intracranial tumor, scale bar 2 mm), 2C shows 4-6 weeks after intracranial injection of each cell line (U-87 MG (4 weeks), GBL-28 (6 weeks), GBL-37 (6 weeks) shows photographs of brain sections stained with antibodies to DAPI and GFAP (yellow dotted line indicates intracranial tumor, scale bar 2 mm).

3 is for the development of a new patient-derived xenograft model of glioblastoma via intravitreal injection, 3A is a schematic illustrating intravitreal injection of tumor cells, and 3B is GBL-28 and GBL-37 undergoing normal culture conditions Relative proportion of mice with grade 2-5 tumors after intravitreal injection of 3C relative to mice with grade 3 and 4 tumors after intravitreal injection of GBL-28 and GBL-37 hypoxia treated for 4 hours before injection Indicates a ratio. 2D is a photograph of H & E sections within 4 weeks of intravitreal injection of GBL-28 and GBL-37 hypoxia treated for 4 hours prior to injection (yellow dashed lines represent the lens and retina, scale bar, 1 mm).

Figure 4 is a photograph showing the immunohistochemical characteristics of PDX tumors in the vitreous cavity, 4A is a photograph of eye sections stained using DAPI and antibodies to human mitochondria, GFAP, vimentin and nestin (scale bar, 1 mm 4B is an enlarged photograph (scale bar, 20 μm) of eye sections stained using antibodies against human mitochondria, GFAP, bimentin and nestin.

FIG. 5 is a photograph showing OLIG2-immune positive of PDX tumor in the vitreous cavity of mouse, which is an enlarged photograph of eye sections stained using an antibody against human OLIG2 antibody (scale bar, 10 μm).

Figure 6 is a photograph showing the separation and characteristics of tumor cells from the vitreous cavity of mice, 6A is the form of GBL-28, GBL-37, GBL-28N, GBL-37N, GBL-28H and GBL-37H glioblastoma cells 6B is a photograph of glioblastoma cells stained using DAPI and GFAP and antibodies to human mitochondria (scale bar, 100 μm).

Figure 7 is a photograph showing the non-mentin-immunopositive of tumor cells isolated from the vitreous cavity of mice (scale bar, 100 μm).

Figure 8 is a photograph showing the nestin-immunopositive of tumor cells isolated from the vitreous cavity of mice (scale bar, 100 μm).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

The term "disease animal model" in the present invention refers to an animal having a form of disease very similar to that of humans. The significance of disease model animals in human disease research is due to physiological or genetic similarities between humans and animals. In disease research, biomedical disease model animals provide research materials for various causes, pathogenesis, and diagnosis of disease, and research on disease model animals allows for genetic, immunochemical, tumor and immune protein expression characteristics associated with disease. In addition, basic data can be obtained to identify prognostic factors, to understand the interactions between expression genes and expression proteins, and to determine whether they are viable through the actual efficacy and toxicity tests of new drug candidates.

The term “patient-derived xenograft (PDX)” in the present invention refers to a customized animal model for cancer patients produced by xenografting patient-derived cancer cells or cancer tissues to an immunodeficiency animal. And the morphological environment is the same or similar, the genetic environment is the same or similar, the expression properties of the marker protein of cancer is the same, it can provide conditions reflecting the genetic, physiological and environmental characteristics of cancer patients. Therefore, when an anticancer drug candidate, a radiation therapy (sensitizer), an immunotherapy candidate or the like which is determined to have an anticancer effect in a patient-derived xenograft animal model is treated to a cancer cell or a cancer tissue providing cancer patient, the anticancer agent Since the same effects as those treated with the candidate substance can be confirmed to the patient, the use of the patient-derived xenotransplantation animal model has the advantage that the anticancer drug can actually confirm the proper effect on the patient.

The term "animal" or "experimental animal" in the present invention means any mammalian animal other than human. The animals include animals of all ages including embryos, fetuses, newborns and adults. Animals for use in the present invention can be used, for example, from commercial sources. Such animals may be laboratory or other animals, rabbits, rodents (e.g. mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (e.g. Chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a mouse.

The term "treatment" in the present invention means an approach for obtaining beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (which may be partial or total), detectable or not detected. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders. By "palliating" a disease, the extent to which the disease state and / or undesirable clinical signs and / or the time course of progression is slowed or lengthened, as compared to the case without treatment.

Throughout this specification, the phrases “comprises” and “comprises”, unless stated otherwise in the context, include a given step or group of steps, but do not exclude any other step or group of steps. It must be understood.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel xenograft patient-derived xenograft animal model and a method of manufacturing the same.

Intraocular tumors of a xenograft animal model derived from glioblastoma patient according to the present invention are positive for GEAP, non-mentin and nestin markers, indicating the tissue and cellular characteristics of glioblastoma, and also of the glial cell lineage. Characterized by OLIG2 positive.

Glioblastoma patient-derived xenograft animal models according to the present invention are produced by injecting glioblastoma cells isolated from glioblastoma patients into the vitreous of animals other than humans. At this time, the "vitreous" is a colorless transparent gel-like structure filling the lens and the retina in the eye, also called a vitreous body. In the present invention, the "animal" refers to any mammalian animal other than human. In this case, the animal may be characterized as an immunodeficiency animal, and an "immune deficient animal" may artificially damage some components of the immune system at the genetic level so that glioblastoma can develop, thereby implementing a normal immune system. It means an animal produced by manipulation so as not to. The immunodeficiency animal may be an animal in which a nervous system is formed. Preferably, the immunodeficiency animal may be a mouse that is engineered to be immunodeficient. For example, Balb / c nude mice can be used.

The previously reported glioblastoma patient-derived xenograft model is to produce a patient-derived xenograft model of glioblastoma through subcutaneous and intracranial injection. The glioblastoma model, which is administered subcutaneously, is a tumor microstructure. It is not possible to identify environmental features. In addition, in this case, unlike the brain tumors protected by the blood brain barrier, the tumors are relatively easily exposed to therapeutic agents through systemic administration, making it difficult to be an alternative to the actual treatment options. In addition, the intracranial glioblastoma model has the advantage of providing a similar microenvironment (brain) in real patients, but it is often slower to form tumors and it takes more than 45 days to evaluate the efficacy of PDX therapeutics. Moreover, cells derived from some patients did not form intracranial tumors as in the control of the present invention. Moreover, in this case, although the intracranial tumor develops efficiently, imaging or bioluminescence imaging is required to investigate tumor formation.

In contrast, glioblastoma patient-derived xenograft animal models (PDXs) through intravitreal injection prepared according to the method of the present invention have several advantages.

First, the production of rapid glioblastoma PDX enables the selection of a fast treatment option for treating glioblastomas with short average survival. Since the average survival of glioblastoma patients is less than 15 months, efficient and rapid screening systems are needed to screen for secondary treatment options, particularly if there are tumors resistant to conventional therapies. Since plaque-like tumor formation is observed within 2 weeks upon intravitreal injection of patient-derived glioblastoma cells, treatment options can be screened through systemic or direct intraocular administration from 2 weeks after injection. In addition, as can be seen in the embodiment of the present invention, even glioblastoma cells of patients who did not form intracranial tumors even after 6 weeks when injected into the skull, tumor formation is observed within 2 weeks when injected into the vitreous and effectively It is worth noting that it will form intracranial tumors.

In particular, in the present invention, glioblastoma cells isolated from the patient may be characterized in that the hypoxic treatment before injection into the vitreous of the animal. This resulted in a stable and consistent mass formation compared to normal culture group without hypoxic treatment (FIG. 3C). It is also noteworthy that all hypoxic treatments form tumors that extend out of the retina in the vitreous cavity. That is, the hypoxic treatment was confirmed invasive characteristics of glioblastoma cells during intravitreal injection. In the present invention, "hypoxia treatment" means the presence of less oxygen than the amount of oxygen in general cell culture conditions. In the present invention, the hypoxic condition may be characterized by less than 5%, preferably characterized in that the cells are cultured at an oxygen concentration of 0.01 to 3%, more preferably at an oxygen concentration of 1% or less. At this time, the low oxygen treatment time may be characterized in that 1 to 48 hours, preferably 4 to 24 hours, more preferably about 6 to 12 hours.

Second, the retina that makes up the central nervous system mimics the microenvironment of the original tumor. The stacked neurons with dynamic synapses and the blood-retinal barrier system of the retinal vascular structure make the retina an effective alternative to the brain. The retina and brain of the eye share many neurovascular features. The retina is composed of layers of nerve cells in which several synapses are formed between various neuronal cell types. In addition, microvascular endothelial cells of the brain and retina form a blood nerve barrier with peripheral cells, including perivascular and astrocytic cells. In addition, other cellular components of the tumor microenvironment of glioblastoma, including microglia and immune cells, are very similar between the brain and the retina.

Third, in comparison to the case of glioblastoma grafts (intracranial tumors), intraocular tumors can be directly observed with the naked eye or easily monitored through an indirect ophthalmoscope using a simple optical lens (such as a 78 diopter lens). Can be. It is easier to perform tumorigenicity testing and monitoring because no additional imaging system is needed. In addition, the visual grading system provides semi-quantitative scale data for quantitative analysis of tumor formation.

Fourth, glioblastoma PDX through intravitreal injection prepared according to the method of the present invention requires fewer glioblastoma cells than intracranial injection. That is, glioblastoma cells of the patient injected into the vitreous may be characterized in that 5 ~ 10 x 10 ⁴ cells. Therefore, given that the tumor tissue of the patient obtained during tumor removal surgery is limited, the advantage of requiring a small number of cells for PDX production is that the limited tissue allows for the production of more PDX, which has several options simultaneously. Enable evaluation Considering the preparation time of the patient-derived cells, all operations can be completed in 6 weeks, much shorter than the average time from initial surgery to relapse.

Due to these advantages, the novel glioblastoma patient-derived xenograft animal model according to the present invention can be used for various purposes.

In another aspect, the present invention, (a) performing a candidate treatment method for glioblastoma in the glioblastoma patient-derived xenograft animal model; And (b) to provide a method for providing information for the selection of patient-specific glioblastoma treatment comprising the step of confirming the therapeutic effect of the animal model in which the candidate treatment method was performed.

Even in patients with the same glioblastoma, the characteristics are all different individually, so the treatment effect may be different and the prognosis may be different for each patient. Therefore, when the same treatment is selected for all patients, the effects may be different, and thus, it is necessary to select a specific and more effective treatment method or screen a new treatment method among the various glioblastoma treatments. . The glioblastoma PDX of the present invention reflects the characteristics of the patient from which the transplanted glioblastoma cells are derived, and thus can be used to select a treatment method suitable for the patient.

The candidate treatment means a variety of treatments that can be used for the selection of a customized treatment for treating glioblastoma onset in a patient of interest, and may include all conventionally known possible treatments for glioblastoma. For example, it may be chemotherapy, radiation therapy, surgical therapy, immune cell therapy, or a combination thereof.

The chemotherapy refers to a method for treating glioblastoma by administering to a patient a therapeutic candidate having a commonly known anticancer activity. In addition, the radiation therapy is a method of treating glioblastoma by treating radiation to the patient, and the surgical therapy is a method of treating glioblastoma by extracting the site where glioblastoma develops as a surgical operation. The immunocytotherapy method is characterized by isolating immune cells exhibiting aggression against glioblastoma from peripheral blood mononuclear cells extracted from the patient's blood, fusing them with the glioblastoma cells isolated from the patient, and then administering them back to the patient in the form of an anticancer vaccine. To treat glioblastoma of the patient.

In addition, for selection of patient-specific glioblastoma treatment, further analysis of any one or more of the genetic characteristics, immunochemical characteristics and tumor or immunoprotein expression characteristics of glioblastoma of the glioblastoma patient-derived xenograft animal model The prognosis may be characterized.

Confirmation of the therapeutic effect is to confirm whether tumor size of the animal model is reduced or metastasis is inhibited, for example, but there is no limitation. Preferably, the eye of the animal model is directly observed by the naked eye or through an indirect ophthalmoscope. It may be characterized by.

In another aspect, the present invention provides a method for treating a glioblastoma cell derived from a glioblastoma patient-derived xenograft animal model or glioblastoma cell derived therefrom ; And (b) after treating the candidate substance, comparing the animal model or the glioblastoma cells with a control group not treated with the candidate substance, where the size of the tumor tissue of the animal model is reduced or metastasis is inhibited or the glioblastoma The present invention relates to a method for screening a glioblastoma therapeutic agent comprising determining a candidate as a glioblastoma therapeutic agent when proliferation of cells is inhibited or killed.

In the present invention, the eye of the animal model can be observed directly with the naked eye or through an indirect ophthalmoscope to confirm the reduction in the size of the tumor tissue.

In addition, in order to determine a glioblastoma therapeutic agent, preferably, one or more genetic, immunochemical and tumor or immunoprotein expression characteristics of the glioblastoma of the glioblastoma patient-derived xenograft animal model are further analyzed. It may be characterized by predicting.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, in the following examples, experiments were performed using a mouse as an animal, but the present invention is not limited thereto, and any mammal can be applied regardless of the type, which will be apparent to those skilled in the art.

Example 1

Preparation of xenograft animal model (PDX) derived from glioblastoma patient

1-1: Isolation and Primary Culture of Glioblastoma Cells

Primary samples were taken from a 39-year-old female glioblastoma patient approved by the Seoul National University Hospital Clinical Trial Committee (IRB No. H-1009-025-331). A 39-year-old female patient whose initial symptom was a short-term memory deficit of 2 weeks, glioblastoma diagnosed with stereotactic brain biopsy (FIG. 1A). The patient was treated with chemoradiotherapy with surgical tumor removal (primary collection, primary tumor) and temozolamide. Three months after the initial operation, another surgical tumor removal (secondary collection, recurred tumor) was performed to control the recurrent tumor. Tumor tissue was prepared for primary culture in each surgery and glioblastoma cells were designated as GBL-28 (primary tumor) and GBL-37 (recurrent tumor), respectively (FIG. 1B).

Each sample collected during the second tumor resection was placed in a Hanks Balanced Salt Solution (HBSS) containing calcium and magnesium and then chopped with a surgical knife. Tissues were centrifuged for 4 min at 1,100 rpm, rinsed with PIPES buffer and resuspended in phosphate buffered saline (PBS) with trypsin-EDTA at 37 ° C. The tissue was then digested with DNase I (20 U / mL) in a rocking shaker at 37 ° C. for 90 minutes and resuspended in DMEM (Dulbecco's Modified Eagle's media) containing 10% fetal bovine serum (FBS). And centrifuged at 1,100 rpm for 4 minutes. The resuspended cells were then filtered with a 40-μm cell strainer and plated in culture flasks. The cells of primary and recurrent tumors were named GBL-28 and GBL-37, respectively. In the following examples, glioblastoma cells obtained from tumors formed after intravitreal injection were also isolated according to the same protocol.

On the other hand, the isolated cells and cells used in the following examples were maintained as follows. That is, U-87 MG cells (catalog no. HTB-14, ATCC), GBL-28 and GBL-37 cells were prepared in DMEM containing 37 ° C. 10% FBS in humidified air of 95% air and 5% CO ₂ . Maintained.

1-2: Preparation of Laboratory Animals

Six-week-old male Balb / c nude mice were purchased from Central Laboratory Animals and maintained under a 12 hour dark cycle. All animal experiments were conducted in accordance with the statement of the Association of Vision and Ophthalmology for Animal Use in Ophthalmology and Vision Research and approved by the Institutional Animal Care and Use Committee of Seoul National University and Seoul National University Hospital.

1-3: (Comparative) Preparation of an Animal Model of Glioblastoma According to the Conventional Method (Alloplastic Transplantation of Glioblastoma Cells) and Uncertainty of Alloplastic Transplantation of Patient-Derived Glioblastoma Cells

After deep anesthesia, the mice of Examples 1-2 were placed in the stereotactic frame (David Kopf Instruments). Small-scale craniotomy was performed 2-3 mm in the midline and 1 mm in the coronary sutures. Glioblastoma cells of Example 1-1 (U-87 MG, GBL-28, GBL-37, 5 μL of 3 × 10 ⁵ cells) were injected stereotactically into the brain parenchyma 3 mm deep. Four to six weeks after tumor cell injection, thin sections of mouse brain (10 μm) were treated for H & E (hematoxylin and eosin) staining and immunofluorescence staining of GFAP. That is, thin slices of mouse brain were washed with PBS, infiltrated with PBS containing 0.05% (v / v) saponin and 5% (v / v) normal goat serum for 3 minutes, and then to block nonspecific binding 1 Treated with PBS containing 1.5% normal goat serum for an hour. The slice was then overnight at 4 ° C. anti-GFAP antibody (1: 100, catalog number M0761 or Z0334, Dako), anti-human mitochondrial antibody (1: 100, catalog number MAB1273, Millipore or cat.no.PA5 -29550, Life Technologies), anti-nonmentin antibody (1: 100, catalog number ab11256, abcam), anti-estin antibody (1: 100, catalog number MAB5326, Millipore) and anti-oligodendrocyte transcription factor 2 ( OLIG2; 1: 100; cat.no.sc-293163, Santa Cruz) and correspond to the corresponding Alexa Fluor 488 or 594 IgG (1: 500, cat.no.A11008, A11029, A11032, A11037, A11055 and A21207, Life Technologies) for 1 hour. Nuclear staining was performed using 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma). The slides were then observed under a fluorescence microscope (Leica).

As a result, as shown in Figure 2, unlike the U-87 MG group in the group of transplanted primary and recurrent tumor-derived cells isolated from glioblastoma patients did not form intracranial tumor even after 6 weeks. Previously, orthotopic transplantation was repeatedly performed by several researchers, including the inventors, but some patient-derived cells slowly formed intracranial tumors or did not develop tumors at all. Figure 2 shows low reliability during orthotopic transplantation of glioblastoma cells derived from patients, 2A shows Kaplan-Meier survival of mice (n = 12) after intracranial injection of U-87 MG, GBL-28, and GBL-37. It shows a curve. In the control example of the present invention, when U-87 MG, GBL-28 and GBL-37 were injected 3 x 10 ⁵ cells into the striatum of Balb / c nude mice, the U-87 MG group and the patient-derived glioblastoma Survival patterns were significantly different between groups of cells (P value <0.0001; FIG. 2A). At this time, statistical analysis was performed using GraphPad Prism (GraphPad Software), and the log rank was used to find statistically significant differences between groups undergoing orthotopic transplantation of GBL-28, GBL-37, and U-87 MG. The test was performed. P values of <0.05 were considered statistically significant.

Figure 2B is representative of H & E sections of the brain of 4-6 weeks mice (U-87 MG (4 weeks), GBL-28 (6 weeks), GBL-37 (6 weeks)) after intracranial injection of each cell line. Photograph (yellow dotted line indicates intracranial tumor, scale bar 2 mm), 2C 4-6 weeks after intracranial injection of each cell line (U-87 MG (4 weeks), GBL-28 (6 weeks), GBL) Representative photographs of brain sections stained with antibodies to DAPI and GFAP at −37 (week 6) are shown (yellow dashed lines indicate intracranial tumors, scale bars 2 mm). In histological and immunohistochemical studies, U-87 MG cells had well formed intracranial tumors (FIGS. 2B and 2C, left), while GBL-28 and GBL-37 cells formed 6 weeks after intracranial injection. It wasn't. (2B and 2C, middle and right).

1-4: (Experimental) Intravitreal injection of glioblastoma cells and preparation of new glioblastoma animal model

GBL-28 and GBL-37 cells obtained in Example 1-1 were each injected with 1 × 10 ⁵ cells into the vitreous cavity of 6-week-old male Balb / c nude mice of Examples 1-2. Further, the 6-week-old males of Example 1-2 after treating 1 × 10 ⁵ cells each of GBL-28 and GBL-37 cells obtained in Example 1-1 in a hypoxic state (1% O ₂ ) for 4 hours. Injected into the vitreous cavity of Balb / c nude mice. From 2 weeks after injection, the eye was examined daily to observe tumor formation.

Intravitreal injection is a method of establishing an orthotopic model of retinoblastoma and delivering a therapeutic agent to retinal nerve tissue. Intravitreal administered cells primarily form ocular tumors in the vitreous cavity between the lens and the retina (FIG. 3A). The tumor can then expand into the anterior chamber (between the cornea and the lens) and occupy the entire eye. The degree of tumor formation can be graded with a simple visual grading system, because the intravitreal cavity and the retina of Balb / c nude mice can be observed with the naked eye or through an indirect ophthalmoscope. The visual grading system used grades of tumor formation from 0 to 5: grade 0 (no tumor formation), grade 1 (striped tumor), grade 2 (plaque-like tumor), grade 3 (clear mass formation), grade 4 (tumor-filled tumor), grade 5 (with spherical enlargement or eye rupture).

As a result, as shown in Examples 1-3, transplantation of patient-derived glioblastoma cells according to the conventional method (orthotopic transplantation) failed to form intracranial tumors up to 6 weeks after tumor cell injection. In the method according to the intravitreal injection, the patient-derived glioblastoma cells effectively formed plaque-like tumors from 2 weeks after intravitreal injection, and formed intracranial tumors by 4 weeks.

Interestingly, there was another pattern in tumor formation after injection of cells that were treated in normal state (FIG. 3B) and hypoxic (1% O ₂ ) for 4 hours (FIG. 3C). Cells injected after incubation at steady state formed various grades of tumor (FIG. 3B). In contrast, hypoxic treatment resulted in stable and consistent lump formation (FIG. 3C). In Figure 3, 3B is the relative proportion of mice with grades 2-5 tumors after intravitreal injection of GBL-28 and GBL-37 undergoing normal culture conditions, 3C is GBL-28 treated with hypoxia for 4 hours before injection and Relative proportions of mice with grade 3 and 4 tumors after intravitreal injection of GBL-37 are shown.

It is also noteworthy that when hypoxic treatment (FIG. 3D), both GBL-28 and GBL-37 cells, which explain the invasive characteristics of glioblastoma cells, form tumors that extend out of the retina in the vitreous cavity. 3D is a representative photograph of H & E sections within 4 weeks after intravitreal injection of GBL-28 and GBL-37 hypoxia treated for 4 hours prior to injection (yellow dashed lines represent the lens and retina. Scale bar, 1 mm) . At this time, thin flakes (10 μm) of the mouse eye for the experiment was prepared and tested as in Example 1-3.

Example 2

Immunohistochemical Characterization of Novel Glioblastoma PDX Tumors

In the novel glioblastoma PDX according to the present invention, the immunohistochemical characteristics of PDX tumors in the vitreous cavity are examined to confirm their similarity to the original tumors, and whether these cells are caused by injected tumor cells rather than mice. In order to confirm, the following experiment was performed.

First, before examining glioblastoma PDX tumor tissue according to the present invention, the characteristics of the original tumors, that is, the original tumors of GBL-28 and GBL-37 were examined. On histological examination of glioblastoma patients, both primary and recurrent tumors were WHO grade IV glioblastoma (Table 1). In addition, both tumors were positive for GFAP, vimentin and nestin in immunohistochemical analysis (Table 1). Both cells showed morphological characteristics of glial cells (FIG. 1C).

Histological and Immunohistochemical Characteristics of Primary and Recurrent Glioblastoma Tumors.

characteristic	Primary tumor	Recurrent tumor
WHO rating	IV / IV	IV / IV
Increased cellularity	U	U
Nuclear polymorphism	U	U
Mitosis (pHH3)	25/10 HPF	27/10 HPF
Vascular endothelial hyperplasia	U	radish
Necrosis	U	U
Immunohistochemistry
GFAP	positivity	positivity
Vimentin	positivity	positivity
Nestin	positivity	positivity

HPF, high-power field; pHH3, phosphohistone H3

On the other hand, thin flakes of the mouse eye of Examples 1-4 were washed with PBS, and allowed to infiltrate PBS containing 0.05% (v / v) saponin and 5% (v / v) normal goat serum for 3 minutes, and then nonspecific To block binding, the cells were treated with PBS containing 1.5% normal goat serum for 1 hour. The slice was then overnight at 4 ° C. anti-GFAP antibody (1: 100, catalog number M0761 or Z0334, Dako), anti-human mitochondrial antibody (1: 100, catalog number MAB1273, Millipore or cat.no.PA5 -29550, Life Technologies), anti-nonmentin antibody (1: 100, cat.no.ab11256, abcam), anti-nestine antibody (1: 100, catalog number MAB5326, Millipore) and anti-oligodendrocyte transcription factors 2 (OLIG2; 1: 100; cat.no.sc-293163, Santa Cruz) and correspond to the corresponding Alexa Fluor 488 or 594 IgG (1: 500, cat.no.A11008, A11029, A11032, A11037, A11055 and A21207, Life Technologies) for 1 hour. Nuclear staining was performed using DAPI (4 ', 6-diamidino-2-phenylindole dihydrochloride, Sigma). The slides were then observed under a fluorescence microscope (Leica).

As a result, as shown in Fig. 4A, tumors in the vitreous cavity and outside the retina of the glioblastoma animal model of Example 1-4 were positive for GFAP, bimentin, nestin and human mitochondria. In addition, all markers showed cytoplasmic patterns indicating their intracellular location (FIG. 4B). As shown in Table 1, the original tumors of GBL-28 and GBL-37 are positive for GFAP, bimentin, and nestin, and thus, the tumors of glioblastoma PDX according to the present invention retain their original tumor characteristics.

In addition, as shown in Figure 5, the tumors in the vitreous cavity and outside the retina of the glioblastoma animal model of Example 1-4 showed a positive response to OLIG2, one of the specific markers of glial cell lineage.

This shows that the immunohistochemical characteristics of the tumors of the glioblastoma xenograft model according to the present invention retain the characteristics of the tumor of the original patient. It can be used to

Example 3

Tumor Cell Isolation and Characterization of Novel Glioblastoma PDX Tumors

Tumor cells were isolated from novel glioblastoma PDX according to the present invention and confirmed similarity with original tumor cells (GBL-28 and GBL-37), and GBL having normal culture conditions in the mouse model of Examples 1-4. In order to compare the characteristics of the cells isolated from tumors of PDX injected with -28 and GBL-37 and those from PDX injected after hypoxic treatment for 4 hours before injection, the following experiment was performed.

Cells were isolated from the tumors of the mouse model of Example 1-4 in the same manner as in Example 1-1, inoculated into 4-well chamber slides (Nunc) and stabilized overnight. Cells were fixed at 4 ° C. for 10 minutes with 1% paraformaldehyde and infiltrated with 0.1% Triton X-100 solution (Cat. No. T8787, Sigma) for 3 minutes at room temperature. Label cells with anti-GFAP antibody, anti-human mitochondrial antibody, anti-mententin antibody, anti-nestine antibody overnight at 4 ° C. after treatment with 1% bovine serum albumin to minimize nonspecific binding and the corresponding Alexa Fluor 488 or 594 IgG (1: 500, Cat. No. A11008, A11029, A11032, A11037, A11055 and A21207, Life Technologies) was treated for 1 hour. Nuclear staining was performed using DAPI. The slides were then observed under a fluorescence microscope (Leica).

As a result, as shown in Figure 6A, all the cells isolated from the tumor of the glioblastoma model according to the present invention retained the morphological characteristics of their parent cells, that is, the glioblastoma cells originally isolated from the tumor. In addition, as shown in Figure 6B, the GFAP marker was also positively the same as the cells isolated from the original tumor. In addition, the cells isolated from the tumor of the glioblastoma model according to the present invention were all positively the same as the cells isolated from the non-mentin (FIG. 7) and nestin (FIG. 8) markers. 6 to 8, GBL-28 and GBL-37 are cells isolated from primary and recurrent tumors of glioblastoma patients, respectively, and GBL-28N and GBL-37N are normal when the glioblastoma model is produced in Examples 1-4. GBL-28 and GBL-37 cells having culture conditions were cells isolated from mice 4 weeks after intravitreal injection, and GBL-28H and GBL-37H were injected into mice when the glioblastoma model was prepared in Example 1-4. All GBL-28 and GBL-37 cells were hypoxic treated for 4 hours and then isolated from mice 4 weeks after intravitreal injection.

This shows that the glioblastoma xenograft model according to the present invention maintains the original tumor characteristics even through tumor cell separation experiments, suggesting that the glioblastoma xenograft model according to the present invention can be used to select treatment options of patients. do.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

1. A method for producing a xenograft patient-derived xenograft animal model comprising injecting glioblastoma cells isolated from a glioblastoma patient into the vitreous of an animal other than a human.
2. The method of claim 1, wherein the glioblastoma cells are hypoxic treated.
3. The method of claim 2, wherein the hypoxic treatment is characterized in that the glioblastoma cells are cultured at an oxygen concentration of 1% or less.
4. The method of claim 1, wherein the glioblastoma cells are injected with 5-10 ^{10 10} cells.
5. The method of claim 1, wherein said method is isolated from a patient with said primary or recurrent glioblastoma.
6. A xenograft transplant animal model derived from a glioblastoma patient prepared by the method of any one of claims 1 to 5.
7. The xenograft patient-derived xenograft animal model of claim 6, wherein the intraocular tumor of the animal model is positive for GEAP, non-mentin and nestin markers.
8. The xenograft patient derived xenograft animal model according to claim 7, wherein said animal model is further positive for an OLIG2 marker.
9. How to provide information for screening patient-specific glioblastoma treatment, which includes the following steps:

   (a) performing a candidate treatment method for glioblastoma in a xenograft animal model derived from glioblastoma patient of claim 6; And

   (b) confirming the therapeutic effect of the animal model in which the candidate treatment method was performed.
10. The method of claim 9, further comprising analyzing at least one of genetic, immunochemical, and tumor or immunoprotein expression characteristics of glioblastoma of the glioblastoma patient-derived xenograft animal model.
11. The method of claim 9, wherein the candidate treatment is chemotherapy, radiation therapy, surgical therapy, immune cell therapy, or a combination thereof.
12. The method of claim 9, wherein the confirmation of the therapeutic effect is characterized by observing the eye of the animal model directly with the naked eye or through an indirect ophthalmoscope.
13. A method for screening a glioblastoma therapeutic agent comprising the following steps.

    (a) treating a xenograft animal model derived from glioblastoma patient of claim 6 or a glioblastoma cell derived therefrom; And

    (b) after treatment with the candidate substance, the animal model or glioblastoma cells are compared with the control group without treatment with the candidate substance, and the size of the tumor tissue of the animal model is reduced or metastasis is inhibited or the glioblastoma cells are inhibited. Determining the candidate as a glioblastoma therapeutic agent if proliferation of the inhibitor is inhibited or killed.
14. The method of claim 13, further comprising analyzing at least one of genetic, immunochemical and tumor or immunoprotein expression characteristics of the glioblastoma of the glioblastoma patient-derived xenograft animal model.
15. The method of claim 13, wherein the eyeball of the animal model is directly observed with the naked eye or through an indirect ophthalmoscope to confirm the reduction in tumor tissue size.

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