WO2019203534A1 - Nouveau modèle de xénogreffe dérivé de patient d'un glioblastome et son utilisation - Google Patents

Nouveau modèle de xénogreffe dérivé de patient d'un glioblastome et son utilisation Download PDF

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WO2019203534A1
WO2019203534A1 PCT/KR2019/004572 KR2019004572W WO2019203534A1 WO 2019203534 A1 WO2019203534 A1 WO 2019203534A1 KR 2019004572 W KR2019004572 W KR 2019004572W WO 2019203534 A1 WO2019203534 A1 WO 2019203534A1
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glioblastoma
patient
cells
animal model
derived
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Korean (ko)
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백선하
이주영
김정훈
조동현
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서울대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a patient-derived xenograft animal model of a new glioblastoma and its preparation and use.
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 1B is a stereotactic biopy.
  • Pictures of GBL-28 and GBL-37 cells (scale bar, 100 ⁇ m).
  • FIG. 2 shows the uncertainty in orthotopic transplantation of glioblastoma cells derived from patients
  • 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.
  • 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
  • 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 ⁇ m) 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 ⁇ m).
  • 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
  • 6B is a photograph of glioblastoma cells stained using DAPI and GFAP and antibodies to human mitochondria (scale bar, 100 ⁇ m).
  • Figure 7 is a photograph showing the non-mentin-immunopositive of tumor cells isolated from the vitreous cavity of mice (scale bar, 100 ⁇ m).
  • Figure 8 is a photograph showing the nestin-immunopositive of tumor cells isolated from the vitreous cavity of mice (scale bar, 100 ⁇ m).
  • 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.
  • 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.
  • 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.
  • patient-derived xenograft 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.
  • PDX patient-derived xenograft
  • 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.
  • 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.
  • 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.
  • treatment in the present invention means an approach for obtaining beneficial or desirable clinical results.
  • 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.
  • 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.
  • the "vitreous” is a colorless transparent gel-like structure filling the lens and the retina in the eye, also called a vitreous body.
  • 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.
  • the immunodeficiency animal may be an animal in which a nervous system is formed.
  • the immunodeficiency animal may be a mouse that is engineered to be immunodeficient.
  • 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.
  • the tumors are relatively easily exposed to therapeutic agents through systemic administration, making it difficult to be an alternative to the actual treatment options.
  • 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.
  • cells derived from some patients did not form intracranial tumors as in the control of the present invention.
  • imaging or bioluminescence imaging is required to investigate tumor formation.
  • glioblastoma patient-derived xenograft animal models through intravitreal injection prepared according to the method of the present invention have several advantages.
  • 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.
  • 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.
  • hypoxic treatment means the presence of less oxygen than the amount of oxygen in general cell culture conditions.
  • 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.
  • 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.
  • 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.
  • microvascular endothelial cells of the brain and retina form a blood nerve barrier with peripheral cells, including perivascular and astrocytic cells.
  • other cellular components of the tumor microenvironment of glioblastoma including microglia and immune cells, are very similar between the brain and the retina.
  • 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.
  • the visual grading system provides semi-quantitative scale data for quantitative analysis of tumor formation.
  • 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.
  • novel glioblastoma patient-derived xenograft animal model according to the present invention can be used for various purposes.
  • 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.
  • 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.
  • the radiation therapy is a method of treating glioblastoma by treating radiation to the patient
  • 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.
  • glioblastoma 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.
  • the eye of the animal model is directly observed by the naked eye or through an indirect ophthalmoscope. It may be characterized by.
  • 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.
  • 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.
  • 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.
  • PDX xenograft animal model
  • glioblastoma 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).
  • HBSS Hanks Balanced Salt Solution
  • 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.
  • PBS phosphate buffered saline
  • 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.
  • DNase I (20 U / mL
  • DMEM Dulbecco's Modified Eagle's media
  • FBS fetal bovine serum
  • the resuspended cells were then filtered with a 40- ⁇ m cell strainer and plated in culture flasks.
  • the cells of primary and recurrent tumors were named GBL-28 and GBL-37, respectively.
  • glioblastoma cells obtained from tumors formed after intravitreal injection were also isolated according to the same protocol.
  • 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.
  • mice 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.
  • 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 ⁇ L of 3 ⁇ 10 5 cells) were injected stereotactically into the brain parenchyma 3 mm deep.
  • thin sections of mouse brain (10 ⁇ m) were treated for H & E (hematoxylin and eosin) staining and immunofluorescence staining of GFAP.
  • 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).
  • 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
  • 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).
  • 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).
  • 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).
  • transplantation of patient-derived glioblastoma cells according to the conventional method failed to form intracranial tumors up to 6 weeks after tumor cell injection.
  • the patient-derived glioblastoma cells effectively formed plaque-like tumors from 2 weeks after intravitreal injection, and formed intracranial tumors by 4 weeks.
  • FIG. 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.
  • 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.
  • the characteristics of the original tumors that is, the original tumors of GBL-28 and GBL-37 were examined.
  • both primary and recurrent tumors were WHO grade IV glioblastoma (Table 1).
  • both tumors were positive for GFAP, vimentin and nestin in immunohistochemical analysis (Table 1). Both cells showed morphological characteristics of glial cells (FIG. 1C).
  • 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).
  • 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.
  • all markers showed cytoplasmic patterns indicating their intracellular location (FIG. 4B).
  • 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.
  • 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.
  • 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.
  • GBL-28 and GBL-37 original tumor cells
  • GBL normal culture conditions
  • Example 1-1 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).
  • 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.
  • 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.

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Abstract

La présente invention concerne un nouveau modèle de xénogreffe dérivé de patient d'un glioblastome, son procédé de fabrication et son utilisation et, plus précisément, un modèle de xénogreffe dérivé de patient d'un glioblastome, le modèle permettant la sélection rapide d'options de traitement pendant le traitement d'un glioblastome possédant un temps de survie moyen court ; un procédé de fabrication associé ; et un procédé de sélection d'un traitement de glioblastome spécifique à un patient à l'aide de ce dernier. Selon la présente invention, le modèle animal de xénogreffe dérivé de patient d'un glioblastome possède les caractéristiques inhérentes d'une tumeur. De plus, même des cellules de glioblastome de patient, qui ne forment pas de tumeurs intracrâniennes dans une transplantation orthotopique, forment des tumeurs en quatre semaines de sorte que, même en tenant compte de la période de préparation pour des cellules dérivées de patient, des options de traitement peuvent être sélectionnées en six semaines, ce qui est beaucoup plus court que le temps médian de la chirurgie initiale jusqu'à la récurrence. En outre, selon la présente invention, contrairement à un modèle de transplantation sous-cutanée d'un glioblastome, un modèle animal de xénogreffe dérivé de patient d'un glioblastome peut être un choix efficace en imitant le microenvironnement du cerveau. Qui plus est, la présente invention permet, sans équipement supplémentaire, une surveillance facile à l'œil nu, à l'aide d'un ophtalmoscope indirect simple ou similaire, et nécessite relativement moins de cellules de glioblastome pour la fabrication d'un modèle de glioblastome, de façon à permettre l'évaluation simultanée de plusieurs options de traitement en même temps, d'où son utilité.
PCT/KR2019/004572 2018-04-18 2019-04-16 Nouveau modèle de xénogreffe dérivé de patient d'un glioblastome et son utilisation WO2019203534A1 (fr)

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