Method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer individual

Abstract

The present invention relates to a method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from a brain cancer individual. It has been confirmed that two-dimensional brain cancer cells can be obtained by extracting cancer tissue from a brain cancer individual, and three-dimensional brain cancer spheroids can be produced from brain tissue discarded in this process. That is, according to the present invention, since both two-dimensional brain cancer cells and three-dimensional brain cancer spheroids, which are two screening agents, can be produced from one sample, the economic effect is excellent, and all advantages of respective drug screening can be utilized. Therefore, it is expected that an effect of increasing the accuracy of drug screening can be achieved by minimizing the disadvantages of each method.

Description

Simultaneous production of two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer cells

[0001] The present invention relates to a method for simultaneously producing two-dimensional brain cancer cells derived from a brain cancer entity and three-dimensional brain cancer spheroids.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0036278, filed March 15, 2024, and Korean Patent Application No. 10-2025-0027031, filed February 28, 2025, the entire disclosures of which are incorporated herein by reference.

[0003] Primary cell models currently used in laboratories involve artificially immortalizing cells derived from organs or tissues or culturing them on a two-dimensional plane to test the efficacy and toxicity of candidate substances. However, these artificially cultured cells in two dimensions suffer from critical drawbacks: they undergo changes in their original morphology and loss of cell function during the culture process after being isolated from the tissue, and they lose connection with the extracellular environment (ECM, microenvironment), failing to reflect phenomena observed in the body.

[0004] In addition, in cases where the cells are not human, the drug's action and mechanism of action are often not the same as in humans due to interspecies differences, which hinders the development of efficient drugs and requires a lot of time and money, making drug screening and predicting responsiveness to drugs very difficult due to various problems.

[0005] To address this issue, the development of stem cell organoid production technology has recently attracted attention as a method of producing patient-derived organoids for use in disease modeling, pathological research, drug screening, toxicity assessment, and genetic manipulation. It has been confirmed that when stem cells are differentiated into a 3D state using their pluripotency and intrinsic self-organization, 3D organoid (organoid) structures with structures similar to body organs are formed. Based on this, it has been reported that various mini-organoids such as brain organoids, heart organoids, liver organoids, lung organoids, and small intestine organoids can be produced.

[0006] These organoids contain various specific cell populations that constitute organs or tissues, have a morphological and structural organization similar to actual tissues or organs, and can reproduce the specialized functions of each organ. Organoids are formed through a common series of processes: cells with similar functions cluster together and are positioned appropriately. After the cells are compartmentalized, further differentiation occurs. This process, called lineage specification, involves the differentiation of precursor cells into fully functioning adult cells.

[0007] The development of these organoids has the advantage of being able to replicate the effects seen in actual human organs by creating a 3D environment close to the body, allowing for experiments in which drugs act as if they were “inside the body” even in experimental situations “outside the body,” and enabling new drug screening and drug toxicity testing.

[0008] In particular, among the organs of the body, the brain, which is fully developed during the fetal stage and early postnatal period, is virtually impossible to access directly, which poses many practical limitations for research, making it difficult to predict drug responsiveness as well as drug screening.

[0009] To address these issues, research is underway to develop a technology that differentiates stem cells into brain cells, including nerve cells. However, the success rate of producing patient-derived brain organoids is significantly low, and the development of an effective production method is necessary.

[0010] The purpose of the present invention is to provide a method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities, comprising the following steps:

[0011] (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and

[0012] (S2) A step of producing a three-dimensional brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

[0013] Another object of the present invention is to provide a method for screening a substance for preventing or treating brain cancer, comprising the following steps:

[0014] (S1) a step of treating a candidate substance to at least one of the two-dimensional brain cancer cells or three-dimensional brain cancer spheroids manufactured by the above simultaneous manufacturing method; and

[0015] (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one of two-dimensional brain cancer cells or three-dimensional brain cancer spheroids treated with the above candidate substance.

[0016]

[0017] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0018] The present invention provides a method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities, comprising the following steps:

[0019] (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and

[0020] (S2) A step of producing a three-dimensional brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

[0021] In one embodiment of the present invention, the two-dimensional brain cancer cells may be cells that have settled and attached to the brain cancer tissue culture medium in step (S1), but are not limited thereto.

[0022] In one embodiment of the present invention, in the step (S1), the brain cancer tissue may be cultured in a culture medium containing amino acids, vitamins, inorganic salts, glucose, and lipids, but is not limited thereto.

[0023] In one embodiment of the present invention, the brain cancer tissue collected and cultured in step (S2) may be brain cancer tissue floating in the brain cancer tissue culture medium in step (S1), but is not limited thereto.

[0024] In one embodiment of the present invention, in the step (S2), the brain cancer tissue may be cultured in a culture medium containing, but not limited to, amino acids, vitamins, inorganic salts, glucose, lipids, N2 supplements, insulin, L-glutamine, penicillin-streptomycin, antioxidant enzymes, proteins, vitamins, fatty acids, and 2-mercaptoethanol.

[0025] In one embodiment of the present invention, the step (S1) may be performed for 5 to 15 days, but is not limited thereto.

[0026] In one embodiment of the present invention, the step (S2) may be performed for 10 to 30 days, but is not limited thereto.

[0027] In one embodiment of the present invention, the two-dimensional brain cancer cells and the three-dimensional brain cancer spheroids may be derived from brain cancer tissue of the same individual, but are not limited thereto.

[0028] In one embodiment of the present invention, steps (S1) and (S2) may be performed semi-continuously, but are not limited thereto.

[0029] In one embodiment of the present invention, the two-dimensional brain cancer cells may be one or more selected from the group consisting of, but are not limited to:

[0030] a) Contains homogeneous brain cancer cells; and

[0031] b) No drug resistance.

[0032] In one embodiment of the present invention, the brain cancer may be any one selected from the group consisting of meningocele meningioma, glioblastoma, pituitary adenoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor, but is not limited thereto.

[0033] In one embodiment of the present invention, the metastatic brain cancer may be any one selected from the group consisting of metastasized lung cancer, metastasized breast cancer, metastasized systemic lymphoma, metastasized melanoma, metastasized sarcoma, and metastasized gastrointestinal cancer, but is not limited thereto.

[0034]

[0035] The present invention provides a two-dimensional brain cancer cell or a three-dimensional brain cancer spheroid manufactured by the above method.

[0036] The present invention provides a method for producing an in vitro model of brain cancer, comprising the following steps:

[0037] (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and

[0038] (S2) A step of producing a 3D brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

[0039] In one embodiment of the present invention, the in vitro model of brain cancer may be, but is not limited to, a two-dimensional brain cancer cell and a three-dimensional brain cancer spheroid.

[0040] The present invention provides an in vitro model of brain cancer prepared by the above method.

[0041] The present invention provides a method for screening a substance for preventing or treating brain cancer, comprising the following steps:

[0042] (S1) a step of treating a candidate substance to at least one of the two-dimensional brain cancer cells or three-dimensional brain cancer spheroids manufactured by the above simultaneous manufacturing method; and

[0043] (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one of two-dimensional brain cancer cells or three-dimensional brain cancer spheroids treated with the above candidate substance.

[0044] In one embodiment of the present invention, the screening method comprises:

[0045] (S3) If the effect of preventing or treating brain cancer is confirmed, an additional step of determining the candidate substance as a substance for preventing or treating brain cancer may be included, but is not limited thereto.

[0046] In one embodiment of the present invention, the brain cancer may be any one selected from the group consisting of meningocele meningioma, glioblastoma, pituitary adenoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor, but is not limited thereto.

[0047] In one embodiment of the present invention, the metastatic brain cancer may be any one selected from the group consisting of metastasized lung cancer, metastasized breast cancer, metastasized systemic lymphoma, metastasized melanoma, metastasized sarcoma, and metastasized gastrointestinal cancer, but is not limited thereto.

[0048]

[0049] In addition, the present invention provides a kit for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer individuals, comprising a composition for culturing brain cancer cells and a composition for culturing brain cancer spheroids.

[0050] In one embodiment of the present invention, the kit may additionally include instructions. Furthermore, the instructions may include, but are not limited to, instructions for the manufacturing method.

[0051] In addition, the present invention provides a kit for screening a brain cancer prevention or treatment agent, comprising two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from the brain cancer entity.

[0052]

[0053] In addition, the present invention provides a use of a composition for culturing brain cancer cells and a composition for culturing brain cancer spheroids for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer individuals.

[0054] In addition, the present invention provides a composition for culturing brain cancer cells and a use of the composition for culturing brain cancer spheroids for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer individuals.

[0055] In addition, the present invention provides a use of the two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from the brain cancer entity for screening agents for preventing or treating brain cancer.

[0056] According to the method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities, it was confirmed that two-dimensional brain cancer cells can be obtained by extracting cancer tissue from a brain cancer entity, and three-dimensional brain cancer spheroids can be produced from brain tissue discarded in the process. That is, according to the present invention, since two screening agents, two-dimensional brain cancer cells and three-dimensional brain cancer spheroids, can both be produced from a single sample, it is expected that the disadvantages of each method can be minimized to create an effect of increasing the accuracy of drug screening in that it is economically effective while also being able to utilize the advantages of each drug screening.

[0057] Figures 1a and 1b illustrate the entire process of the method for simultaneously producing two-dimensional cancer cells derived from a brain cancer patient and three-dimensional cancer spheroids according to the present invention.

[0058] Figure 2a shows the results of two-dimensional culture of cancer cells derived from brain cancer patients according to the method of the present invention.

[0059] Figure 2b illustrates a three-dimensional cancer spheroid produced according to the method of the present invention.

[0060] FIGS. 3a to 3e are the results of comparative analysis of 3D cancer spheroids produced from 5 brain cancer individuals according to the method of the present invention and the pathological tissues of each brain cancer individual, respectively, showing that the 3D cancer spheroids produced according to the method of the present invention reflect the characteristics of the pathological tissues of each brain cancer individual from which they were derived.

[0061] The present invention provides a method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities, comprising the following steps:

[0062] (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and

[0063] (S2) A step of producing a three-dimensional brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

[0064] In the present invention, "brain cancer tissue culture" refers to brain cancer tissue cultured from a brain cancer subject. The brain cancer cell culture of the present invention may refer to a cluster of brain cancer cells that reflect all histopathological characteristics of the brain cancer subject, including the characteristics of brain cancer, such as the degree of resistance development.

[0065] In the present invention, the brain cancer tissue collected from a brain cancer subject may be derived from any part of the brain where the brain cancer tissue has spread. Furthermore, in one embodiment of the present invention, the experiments were conducted using tissue removed from the brain cancer subject within 24 hours, which may be due to the inhibition of brain cancer tissue growth activity 24 hours after collection.

[0066] In addition, in the present invention, “two-dimensional brain cancer cells” may refer to the lower layer of the brain cancer tissue culture medium that has sunk to the bottom of the culture vessel when culturing brain cancer tissue. More preferably, in one embodiment of the present invention, the lower layer may refer to, but is not limited to, cells or a cluster thereof that have sunk and attached to the brain cancer tissue culture medium in step (S1) as the two-dimensional brain cancer cells. At this time, since the attached cells or a cluster thereof form a brain cancer cell layer, the two-dimensional brain cancer cells may refer to, but is not limited to, a cell layer that has sunk to the brain cancer tissue culture medium or a part thereof.

[0067] The method of the present invention may additionally include a step of preprocessing brain cancer tissue isolated from a brain cancer subject. The preprocessing step may include, but is not limited to, cutting the isolated brain cancer tissue into an appropriate size and storing it under specific conditions for the production of brain cancer tissue culture medium and brain cancer spheroids. The preprocessing step may be broadly defined to encompass all processes, treatments, and methods commonly performed in the art.

[0068] In one embodiment of the present invention, the brain cancer tissue collected and cultured in step (S2) may be brain cancer tissue floating in the brain cancer tissue culture medium in step (S1), but is not limited thereto.

[0069] In the above step (S2), the process of collecting brain cancer tissue from the brain cancer tissue culture medium in step (S1) requires selecting and collecting brain cancer tissue that is not attached but floating in the brain cancer tissue culture medium. Therefore, it may be preferable to perform step (S1) after a two-dimensional brain cancer cell layer that attaches and proliferates to the bottom of the brain cancer tissue culture medium is formed after step (S1) has been performed for 5 to 15 days. Accordingly, in one embodiment of the present invention, three-dimensional brain cancer spheroids are manufactured by collecting brain cancer tissue floating in the brain cancer tissue culture medium after step (S1) has been cultured for about 10 days, but the present invention is not limited thereto.

[0070] In the present invention, the brain cancer tissue culture medium used in the three-dimensional brain cancer spheroid may refer to a portion of the upper layer of the brain cancer tissue culture medium that is not attached to the culture vessel but floats.

[0071] In one embodiment of the present invention, the step (S1) may be performed for 5 to 15 days, for example, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, or 10 days, but is not limited thereto.

[0072] In one embodiment of the present invention, the step (S2) may be performed for 10 to 30 days, for example, 10 to 25 days, 10 to 20 days, 10 to 18 days, 10 to 15 days, 11 to 30 days, 11 to 25 days, 11 to 20 days, 11 to 18 days, 11 to 15 days, 12 to 30 days, 12 to 25 days, 12 to 20 days, 12 to 18 days, 12 to 15 days, 13 to 30 days, 13 to 25 days, 13 to 20 days, 13 to 18 days, 13 to 15 days, 14 to 30 days, 14 to 25 days, It may be performed for, but is not limited to, 14 to 20 days, 14 to 18 days, 14 to 15 days, or 15 days.

[0073] In the present invention, "spheroid" refers to a three-dimensional cell aggregate capable of self-organization. Spheroids are miniature, simplified, in vitro three-dimensional organs that mimic the anatomical structure of actual tissues and can be used for various drug screening and disease models.

[0074] In the present invention, “three-dimensional brain cancer spheroid” may mean a three-dimensional aggregate of brain cancer cells produced by culturing brain cancer tissue obtained from the same brain cancer individual by the simultaneous production method of the present invention, and then culturing brain cancer tissue that floats without attaching in the brain cancer tissue culture medium after two-dimensional brain cancer cells are formed, in a separate culture medium.

[0075] Both organoids and spheroids are three-dimensionally cultured cells, and the term "organoid" broadly encompasses spheroids. Organoids are generally known to be highly complex and more biologically similar than spheroids. However, organoid production is extremely complex and challenging, requires a very long production period (at least 40 days), and has the significant drawback of not being able to produce functional organoids due to a problem in any one of the various steps. In contrast, the simultaneous production method of the present invention allows for the rapid and simple production of two-dimensional brain cancer cells and three-dimensional brain cancer spheroids, while simultaneously leveraging the advantages of therapeutic screening for cell lines, spheroids, and organoids. Furthermore, the two-dimensional brain cancer cells and three-dimensional brain cancer spheroids can be produced separately from the same brain cancer cell population. Therefore, the simultaneous production method of the present invention can be more usefully utilized in the production of customized pharmaceutical preparations that overcome the shortcomings of organoids by simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids.

[0076] In the present invention, 3D brain cancer spheroids were produced with reference to “Lancaster, M., Knoblich, J. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc 9, 2329-2340 (2014). https: / doi.org / 10.1038 / nprot.2014.158.” In one embodiment of the present invention, the same culture medium as that used in the above paper was used to produce 3D brain cancer spheroids, but the present invention is not limited thereto, and additional compositions that can be generally included in the art may be further included.

[0077] At this time, DMEM medium (Gibco 11965092) containing 10% FBS and 5% penicillin-streptomycin was used for culturing brain cancer cells. Therefore, in one embodiment of the present invention, in step (S1), brain cancer tissue may be cultured in a culture medium containing amino acids, vitamins, inorganic salts, glucose, and lipids, but is not limited thereto.

[0078] Additionally, a medium containing the following composition was prepared for culturing 3D brain cancer spheroids:

[0079] (1) ∼250ml of medium;

[0080] (2) the medium combines 125ml of DMEM-F12 (Dulbecco's Modified Eagle Medium (DMEM) Gibco 11320033);

[0081] (3) 125ml of basal medium (Neurobasal™ Medium (Gibco 21103049);

[0082] (4) 1.25ml of N2 supplement (N-2 Supplement (100X) Gibco 17502048);

[0083] (5) 62.5 μl of insulin (insulin, Sigma-Aldrich 91077C);

[0084] (6) 2.5ml of GlutaMAX supplement(GlutaMAX™ Supplement (100X) Gibco 35050061);

[0085] (7) 1.25ml of MEM-NEAA (MEM Non-Essential Amino Acids Solution (100X) Gibco 11140050);

[0086] (8) 2.5 ml of penicillin-streptomycin (Gibco 15140122); and

[0087] (9) B27 supplement (B-27 Supplement Gibco 17504044) 2.5ml.

[0088] After preparing each of the above compositions, 2-mercaptoethanol was prepared by diluting 1:100, and 87.5 μl of this was added to the medium to prepare the final culture medium. Therefore, in one embodiment of the present invention, in the step (S2), the brain cancer tissue can be cultured in a culture medium containing amino acids, vitamins, inorganic salts, glucose, lipids, N2 supplements, insulin, L-glutamine, penicillin-streptomycin, antioxidant enzymes, proteins, vitamins, fatty acids, and 2-mercaptoethanol, but is not limited thereto.

[0089] In one embodiment of the present invention, the two-dimensional brain cancer cells and the three-dimensional brain cancer spheroids may be derived from brain cancer tissue of the same individual, but are not limited thereto.

[0090] In the present invention, “simultaneous” may mean culturing from brain cancer tissue collected from the same single individual.

[0091] In one embodiment of the present invention, steps (S1) and (S2) may be performed semi-continuously, but are not limited thereto.

[0092] In the present invention, “performing semi-continuously” may mean performing semi-continuously an operation of separating tissue floating in the brain cancer tissue culture medium while leaving the two-dimensional brain cancer cells that have settled and attached to the bottom of the culture vessel intact from the brain cancer tissue culture medium collected from the same single individual and culturing the tissue, adding a culture medium for producing three-dimensional spheroids, and culturing the separated tissue. In the present invention, when two-dimensional brain cancer cells are formed by culturing brain cancer tissue in step (S1), a three-dimensional brain cancer spheroid is produced by collecting and culturing brain cancer tissue floating in the upper layer of the brain cancer tissue culture medium, wherein steps (S1) and (S2) may not be completely separated in time, and once step (S1) is performed, step (S2) may be repeated semi-continuously one or more times.

[0093] In addition, the present invention provides two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities manufactured by the above-described simultaneous manufacturing method.

[0094] In one embodiment of the present invention, the two-dimensional brain cancer cells may be one or more selected from the group consisting of, but are not limited to:

[0095] a) Contains homogeneous brain cancer cells; and

[0096] b) No drug resistance.

[0097] In one embodiment of the present invention, the brain cancer may be any one selected from the group consisting of meningocele meningioma, glioblastoma, pituitary adenoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor, but is not limited thereto.

[0098] In the present invention, glioblastoma may include primary glioblastoma or secondary glioblastoma.

[0099] In one embodiment of the present invention, the metastatic brain cancer may be any one selected from the group consisting of metastasized lung cancer, metastasized breast cancer, metastasized systemic lymphoma, metastasized melanoma, metastasized sarcoma, and metastasized gastrointestinal cancer, but is not limited thereto.

[0100] The World Health Organization (WHO) classifies primary brain tumors into four categories. WHO grades I and II are low-grade gliomas, while anaplastic astrocytomas and anaplastic oligodendrogliomas (WHO grade III), as well as glioblastomas (GBMs) (WHO grade IV), are collectively referred to as malignant gliomas. The prognosis for most primary and secondary brain tumors is very poor, due to the lack of effective treatments.

[0101] Metastatic brain tumors begin as cancers anywhere in the body and spread to the brain. Breast cancer, lung cancer, melanoma, colon cancer, and kidney cancer are common metastases. Often, these metastatic brain tumors are discovered before the primary tumor. Metastatic brain tumors are the most common brain tumor in adults.

[0102] Although the situation is slightly better than with GBMs, the prognosis for metastatic brain cancer is generally poor. Again, a combination of surgery, therapy, and chemotherapy is employed, but the exact combination within these options varies depending on the nature and stage of the metastatic cancer. Surgery and radiotherapy are standard treatments. Chemotherapy is sometimes employed. Unfortunately, these have not been very successful to date. This is partly due to the need for chemotherapeutic agents that exhibit good CNS penetration (and, of course, good anti-neoplastic activity and an acceptable toxicity profile). Many existing chemotherapeutic agents have shown poor blood-brain barrier penetration. There is an urgent need for novel therapeutics that address these issues, as well as the development of research systems for their development, such as markers and methods for screening, organoids (broadly including spheroids) for assessing therapeutic efficacy, and cell cultures.

[0103] In the present invention, "metastasis" refers to cancer cells leaving the primary organ and moving to another organ. The term "cancer" as used herein encompasses "cancer stem cells." The spread of cancer to other parts of the body can be broadly categorized into two types: cancerous tissue growing from the primary tumor and directly infiltrating surrounding organs, and distant metastasis via blood vessels or lymphatic vessels to distant organs. Metastasis can be controlled by suppressing the expression of genes associated with cancer development or by inhibiting the protein activity of said genes.

[0104] In the present invention, “cancer stem cell” refers to a comprehensive cancer cell that has self-renewal or differentiation capabilities, which are unique abilities of stem cells, and may include, for example, a sphere-shaped cancer cell population or cancer tissues with an unclear shape and poor prognosis. Under normal tumor growth conditions of the cancer stem cell (the “normal tumor growth conditions” refer to a state in which nutrients (glucose) necessary for cell growth are sufficient and the growth conditions of the tumor microenvironment are abundant, so that there is no cell stress), the cancer stem cell may proliferate at a slower rate than general cancer cells or maintain a dormant state, thereby having resistance to anticancer drugs. For example, the expression of transcriptional regulatory factors such as PGC-1a may be controlled differently from that of normal tumor cells, and thus the functions of major metabolic regulatory substances may be different compared to general cancer cells. This term encompasses cells that possess different metabolic regulatory capabilities and, through modulation of mechanistically linked cell signaling pathways, acquire resistance to apoptosis in nutrient-deprived conditions and possess invasive and / or metastatic potential. However, it is not limited to cells capable of differentiating into typical cancer cells.

[0105] In the present invention, “cancer stem cell treatment” means including the death of cancer stem cells, inhibition of cancer stem cell maintenance, inhibition of cancer stem cell malignancy, and inhibition of cancer stem cell invasive activity.

[0106] The present invention provides a method for producing an in vitro model of brain cancer, comprising the following steps:

[0107] (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and

[0108] (S2) A step of producing a 3D brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

[0109] In one embodiment of the present invention, the in vitro model of brain cancer may be, but is not limited to, a two-dimensional brain cancer cell and a three-dimensional brain cancer spheroid.

[0110] The present invention provides an in vitro model of brain cancer prepared by the above method.

[0111] The present invention provides a method for screening a substance for preventing or treating brain cancer, comprising the following steps:

[0112] (S1) a step of treating a candidate substance to at least one of the two-dimensional brain cancer cells or three-dimensional brain cancer spheroids manufactured by the above simultaneous manufacturing method; and

[0113] (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one of two-dimensional brain cancer cells or three-dimensional brain cancer spheroids treated with the above candidate substance.

[0114] In one embodiment of the present invention, the screening method comprises:

[0115] (S3) If the effect of preventing or treating brain cancer is confirmed, an additional step of determining the candidate substance as a substance for preventing or treating brain cancer may be included, but is not limited thereto.

[0116] Alternatively, the present invention provides a method for screening a substance for preventing or treating brain cancer, comprising the following steps:

[0117] (S1) a step of treating a candidate substance to at least one selected from the group consisting of two-dimensional brain cancer cells, three-dimensional brain cancer spheroids, and in vitro models of the brain cancer manufactured by the above simultaneous manufacturing method; and

[0118] (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one selected from the group consisting of two-dimensional brain cancer cells treated with the above candidate substance, three-dimensional brain cancer spheroids, and in vitro models of the above brain cancer.

[0119] In one embodiment of the present invention, the screening method comprises:

[0120] (S3) If the effect of preventing or treating brain cancer is confirmed, an additional step of determining the candidate substance as a substance for preventing or treating brain cancer may be included, but is not limited thereto.

[0121] In the present invention, “screening” may mean selecting a substance having a specific desired property from a candidate group consisting of various substances through a specific manipulation or evaluation method.

[0122] For the purpose of the present invention, the screening method of the present invention may mean a series of processes including, but not limited to, a step of treating a candidate substance to two-dimensional brain cancer cells and three-dimensional brain cancer spheroids of the present invention and confirming the therapeutic response and effect thereof in order to identify a brain cancer treatment agent that produces the best therapeutic effect in brain cancer patients, and then a step of determining the brain cancer treatment agent that produces the best therapeutic effect as the brain cancer treatment agent.

[0123] In the present invention, the step of confirming the treatment response and effect may be repeated several times depending on the therapeutic candidate substance, and may additionally include steps used in the art as a general screening method, such as adding additional substances or steps to confirm the treatment response and effect, but is not limited thereto. In addition, the step for confirming the treatment response and effect is not limited to a specific method. Therefore, in order to confirm the treatment response and effect, methods generally performed in the art, such as increasing or decreasing the expression of a specific cancer marker at the mRNA or protein level, and whether or not the symptoms of brain cancer in the subject are improved, may be applied. The screening method of the present invention is characterized by using the two-dimensional brain cancer cells and / or three-dimensional brain cancer spheroids produced in the present invention, but is not limited thereto.

[0124] In the present invention, the term "verification" may correspond to, but is not limited to, the meaning of "analysis." In the present invention, "analysis" may preferably mean "measurement," and in this case, the analysis may include qualitative analysis and quantitative analysis. Qualitative analysis may mean measuring and confirming the presence or absence of a target substance to confirm a therapeutic response. In addition, quantitative analysis may mean measuring and confirming changes in the presence level (expression level) or amount of a target substance. In the present invention, the analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and may be performed as quantitative measurement.

[0125] In the present invention, the brain cancer treatment candidate includes all formulations and methods that can be used to treat various brain cancers. For example, it may be an anticancer agent used to treat brain cancer, but is not limited thereto.

[0126] The term "anticancer agent" refers to a general term for chemotherapeutic agents used to treat malignant tumors. Most anticancer agents interfere with various metabolic pathways in cancer cells, primarily inhibiting nucleic acid synthesis or exhibiting anticancer activity. Anticancer agents currently used in cancer treatment are classified into six categories based on their biochemical mechanisms of action.

[0127] (1) Alkylating agents: These are highly reactive substances capable of introducing an alkyl group R-CH2 into a compound. When they are applied to cells, most of them react with the N7 of guanine in DNA, modifying the DNA structure and causing chain breaks, resulting in anticancer and cytotoxic effects. Drugs belonging to this group include: ① Nitrogen mustards: nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, etc. ② Ethyleneimine: thiotepa ③ Alkyl sulfonates: busulfan ④ Triazines, hydrazines: DTIC (dacarbazine), procarbazine ⑤ Nitrosoureas: BCNU, CCNU, methyl-CCNU, etc.

[0128] (2) Antimetabolites: These have the effect of inhibiting the metabolic process necessary for the proliferation of cancer cells, and include ① folic acid derivatives: methotrexate (MTX) ② purine derivatives: 6-mercaptopurine (6-MP), 6-thioguanine ③ pyrimidine derivatives: 5-fluorouracil, cytarabine, etc.

[0129] (3) Antibiotics: Antibiotics produced by bacteria that exhibit anticancer effects, such as adriamycin, daunorubicin, bleomycin, mitomycin-C, and actinomycin-D.

[0130] (4) Mitotic inhibitors (vinca alkaloids): These are mitotic phase-specific drugs that stop cell division in the metaphase of mitosis. Examples include vincristine, vinblastine, VP-16-213, and VM-26.

[0131] (5) Hormonal agents: Certain types of cancer can be treated effectively by administering hormones. Male hormones are effective for breast cancer, female hormones for prostate cancer, and progesterone for endometrial cancer. Adrenal cortex hormones are used to treat acute lymphoblastic leukemia and lymphoma, and tamoxifen, an anti-female hormone, is used for breast cancer. The anticancer agent as the therapeutic substance of the present invention may include a hormonal anticancer agent that can be applied to brain cancer.

[0132] (6) Others: Cisplatin, L-asparaginase, o,p-DDD, etc. Despite ongoing efforts to develop effective anticancer agents, current leading treatments mainly consist of surgery, radiation, and chemotherapy. Chemotherapeutic approaches can be primarily used to treat metastatic or particularly aggressive cancers.

[0133] The therapeutic candidate substances to which the screening method according to the present invention is applied may include not only specific drug preparations such as the anticancer agent, but also all anticancer therapies such as radiation therapy, electricity, electromagnetic waves, etc., or chemotherapy.

[0134] In one embodiment of the present invention, the brain cancer may be any one selected from the group consisting of glioblastoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor, but is not limited thereto.

[0135] In addition, the present invention provides a method for treating brain cancer, which further comprises, in the screening method, a step of administering the therapeutic agent to an individual in need thereof.

[0136] The present invention provides a method for preventing or treating brain cancer comprising the following steps:

[0137] (S1) a step of treating a candidate substance to at least one of the two-dimensional brain cancer cells or three-dimensional brain cancer spheroids manufactured by the above simultaneous manufacturing method; and

[0138] (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one of two-dimensional brain cancer cells or three-dimensional brain cancer spheroids treated with the above candidate substance.

[0139] (S3) If the effect of preventing or treating brain cancer is confirmed, a step of determining the candidate substance as a substance for preventing or treating brain cancer; and

[0140] (S4) A step of administering the above therapeutic substance to an individual in need thereof.

[0141] In addition, the present invention provides a kit for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer individuals, comprising a composition for culturing brain cancer cells and a composition for culturing brain cancer spheroids.

[0142] In one embodiment of the present invention, the kit may additionally include instructions. Furthermore, the instructions may include, but are not limited to, instructions for the manufacturing method.

[0143] In the present invention, a “kit” may mean, but is not limited to, a tool including a preparation capable of producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids, including a composition for each medium for producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids from brain cancer tissue of a brain cancer subject, and an instruction manual describing a method for producing the same.

[0144] In addition, the present invention provides a kit for screening a brain cancer prevention or treatment agent comprising two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from a brain cancer entity.

[0145] At this time, in the present invention, “kit” may mean a tool including a preparation capable of screening a preparation for preventing or treating brain cancer, including two-dimensional brain cancer cells and three-dimensional brain cancer spheroids produced by the method of the present invention, and an instruction manual describing a method for producing the preparation thereof, but is not limited thereto.

[0146] The kit of the present invention may include, in addition to the composition and instructions according to the present invention, other components, compositions, solutions, devices, etc. that are typically required for the manufacturing method of the present invention, and there is no limitation on the order in which the above materials are applied, and the application of each material may be carried out simultaneously or microscopically.

[0147] In the present invention, the kit may further include, but is not limited to, a container. The container may serve to package the material, and may also serve to store and secure the material. The material of the container may be, but is not limited to, plastic, glass, or the like.

[0148] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0149]

[0150] [Example]

[0151]

[0152] Example 1. Acquisition of brain cancer tissue

[0153] Cancer tissue was obtained from a patient with glioblastoma brain cancer. Specifically, cancer tissue samples isolated from brain cancer surgery were placed in a tissue transfer medium (DMEM, 10% FBS media) within 24 hours. The obtained tissue was finely chopped into pieces of approximately 1–2 mm in size using sterilized scissors and washed at least twice with PBS to remove blood and dead tissue. The tissue was then cultured in an incubator at 37°C and 5% CO2 for 1 day (Figures 1a and 1b).

[0154]

[0155] Example 2. Simultaneous production of two-dimensional brain cancer cells and three-dimensional brain cancer spheroids using brain cancer tissue.

[0156] The brain cancer tissue obtained in Example 1 was transferred to a dish. At this time, for brain cancer cell culture, DMEM medium (Gibco 11965092) supplemented with 10% FBS and 5% penicillin-streptomycin was cultured for approximately 10 days. As a result, it was confirmed that 2D brain cancer cells derived from brain cancer patients, which exhibit anchorage-dependent characteristics of attaching to the bottom surface of the dish and proliferating, were formed in a layer (Fig. 2a). These were named “2D brain cancer cells” in the present invention.

[0157]

[0158] Additionally, after a 10-day culture period, small floaters in the brain cancer tissue culture medium were aspirated with a pipette and transferred to a 15 ml tube. After allowing the tissue to settle to the bottom by gravity for approximately 30 seconds, the culture medium was washed once or twice with the prepared DPBS, and then the culture medium (media) was added. The composition of the culture medium for producing brain cancer spheroids was as follows:

[0159] (1) ∼250ml of medium;

[0160] (2) the medium combines 125ml of DMEM-F12 (Dulbecco's Modified Eagle Medium (DMEM) Gibco 11320033);

[0161] (3) 125ml of basal medium (Neurobasal™ Medium (Gibco 21103049);

[0162] (4) 1.25ml of N2 supplement (N-2 Supplement (100X) Gibco 17502048);

[0163] (5) 62.5 μl of insulin (insulin, Sigma-Aldrich 91077C);

[0164] (6) 2.5ml of GlutaMAX supplement(GlutaMAX™ Supplement (100X) Gibco 35050061);

[0165] (7) 1.25ml of MEM-NEAA (MEM Non-Essential Amino Acids Solution (100X) Gibco 11140050);

[0166] (8) 2.5 ml of penicillin-streptomycin (Gibco 15140122); and

[0167] (9) B27 supplement (B-27 Supplement Gibco 17504044) 2.5ml.

[0168] After preparing each of the above compositions, 2-mercaptoethanol was prepared by diluting it 1:100, and 87.5 μl of this was added to the medium to prepare the final culture solution.

[0169]

[0170] After growing in a shaker for approximately 15 days, spheroids were confirmed to have formed (Fig. 2b). These were named “3D brain cancer spheroids” in the present invention.

[0171]

[0172] According to the experimental method of the present invention, both 2D brain cancer cells derived from two brain cancer patients and 3D brain cancer spheroids can be produced from a single sample. In the case of 2D brain cancer cells, since they contain uniform cancer cells, a homogeneous effect can be confirmed, and the disadvantage of 3D brain cancer spheroids, which are very resistant to drugs, can be offset. In addition, 3D brain cancer spheroids have the effect of confirming complex experimental results that cannot be confirmed in 2D brain tissue cultures. Therefore, through the examples of the present invention, a screening tool that reflects the characteristics of patients was produced that has excellent economic effect while simultaneously utilizing the advantages of both 2D brain tissue cultures and 3D brain cancer spheroids for drug screening, thereby minimizing the disadvantages of each method and increasing the accuracy of drug screening.

[0173]

[0174] Example 3. Confirmation of the excellent reflective ability of 3D brain cancer spheroids using brain cancer tissue to the individual brain cancer pathology tissue.

[0175] It was analyzed whether the 3D brain cancer spheroids produced in Example 2 reflected the characteristics of the pathological tissue of the brain cancer entity from which the spheroids were derived. Meningothelial meningioma, glioblastoma, gonadotroph anterior pituitary neuroendocrine tumor (PitNET), and metastatic carcinoma entities were used as brain cancer entities (Table 1). Brain cancer tissues were isolated from each brain cancer entity according to the method of Example 1, and 3D brain cancer spheroids were produced according to the method of Example 2.

[0176]

[0177] Individual No. Brain Cancer Type Characteristics 1 Meningocortical Meningioma Grade 1 by WHO 2 Glioblastoma IDH-wildtype, CNS WHO Grade 4, 3 Pituitary Adenoma-4 Metastatic Brain Cancer-5 Glioblastoma IDH-wildtype, CNS WHO Grade 4,

[0178]

[0179] Afterwards, H&E staining and Ki67 analysis were performed on the manufactured 3D brain cancer spheroids and the tissues of each derived brain cancer entity according to general methods, and GFAP analysis was additionally performed in cases 1 and 5 of Table 1.

[0180] Since tissues stained with H&E staining can be evaluated for cell structure (nuclear shape, cytoplasmic staining intensity, etc.) through a microscope, the cell structure, distribution of the tissue, and characteristics of inflammation or tumors were observed. GFAP-positive cells are astrocytes or reactive glial cells, and mainly appear in gliomas. Therefore, GFAP staining was used to confirm the distribution of glial cells and whether tumors such as reactive gliosis and glioblastoma are of glial origin. Finally, Ki-67-positive cells are cells that proliferate in the cell cycle, and the cell proliferation index can be obtained by calculating their ratio as a percentage of the total number of cells. Therefore, Ki-67 staining was used to evaluate the proliferation rate of tumors and the degree of inflammatory response.

[0181]

[0182] As a result, analysis results similar to those in Figures 3a to 3e were confirmed for Nos. 1 to 5, respectively. According to these results, the three-dimensional brain cancer spheroids manufactured according to the method of the present invention were found to reflect the pathological characteristics of each brain cancer entity from which they were derived.

[0183]

[0184] First, the results of the H&E staining analysis are as follows.

[0185] Specifically, the staining results of the 3D organoids derived from individual 1 showed that the tumor cells were arranged in a spiral shape, which is one of the representative characteristics of meningiomas, and this was reflected in the 3D brain cancer spheroids as well. In addition, the nuclei of the tumor cells were found to be round or oval and of a certain size, which confirmed that the diagnosis and classification of meningiomas are possible by observing the histological characteristics of meningiomas through H&E staining.

[0186] 3D organoids derived from individual #2 and individual #5 exhibited a wide range of cell sizes and shapes, with severe dysplasia observed. Nuclei were irregular and polymorphic, and some cells were extremely large. Furthermore, cells with abnormally large and diverse nuclei were frequently observed, and active cell division was observed, reflecting rapid tumor growth.

[0187] The 3D organoids derived from individual 3 were homogeneous in cell size and shape, similar to tumor cells. The cells primarily formed lobular structures or were arranged in a sheet-like pattern. Furthermore, the nuclei were round or oval, uniform in size, and mitotic figures were rarely observed, reflecting the characteristics of pituitary adenomas.

[0188] Finally, 3D organoids derived from individual 4 demonstrated that metastatic tumor cells reflected the characteristics of the primary cancer and displayed a heterogeneous cell arrangement compared to brain tissue. Metastatic tumors had relatively clear boundaries with surrounding normal brain tissue, and some metastases exhibited invasive growth patterns.

[0189]

[0190] These H&E staining analysis results demonstrate that 3D brain cancer spheroids can be used as cell models that accurately reflect the pathological characteristics of each brain cancer entity from which they were derived.

[0191]

[0192] Second, the results of Ki67 staining analysis are as follows.

[0193] Ki-67 is a cell proliferation marker that can provide important information for evaluating the cell division and growth rate of tumors and comparing the degree of cell proliferation and malignancy of tumors.

[0194] Specifically, 3D organoids derived from individual 1 showed staining primarily in the nuclei of tumor cells, with a low percentage of sporadically positive cells.

[0195] In the 3D organoids derived from individual 3, the Ki-67 index was found to be low at less than 3%. The Ki-67 index is an important indicator for distinguishing between non-invasive and invasive, and it was confirmed that it directly reflected the non-invasive characteristics.

[0196] In the 3D organoids derived from the 4th individual, a wide range of Ki-67 indices were observed, and it was confirmed that metastatic cells were proliferating rapidly as they were mainly characterized by a high Ki-67 indices.

[0197] The Ki-67 index of the 3D organoids derived from individuals 2 and 5 was very high, averaging 20-40% or more, which was confirmed to reflect rapid cell proliferation and tumor aggressiveness, and thus may reflect rapid cell proliferation and high malignancy.

[0198]

[0199] Finally, the results of the GFAP analysis performed on subjects 1 and 5 are as follows.

[0200] Specifically, the 3D brain cancer spheroid derived from individual 1 was found to be composed of GFAP-negative cells, which is the same characteristic of individual 1, which has meningocele meningioma that is not of glial origin. In addition, GFAP analysis was performed on the 3D brain cancer spheroid derived from individual 5, and GFAP was found to be positive, and since individual 5 has glioblastoma, it was confirmed that the brain cancer characteristics of the individual from which it was derived were the same as those of the 3D brain cancer spheroid from individual 1. According to the GFAP analysis, it was confirmed that the 3D brain cancer spheroid manufactured by the method of the present invention can directly reflect the characteristics of the brain cancer of the individual.

[0201]

[0202] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0203] The present invention relates to a method for simultaneously producing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from a brain cancer individual, and it has been confirmed that two-dimensional brain cancer cells can be obtained by extracting cancer tissue from a brain cancer individual, and three-dimensional brain cancer spheroids can be produced from brain tissue discarded in the process. That is, according to the present invention, since two screening agents, two-dimensional brain cancer cells and three-dimensional brain cancer spheroids, can both be produced from a single sample, the economic effect is excellent, and since the advantages of each drug screening can all be utilized, it is expected that the disadvantages of each method can be minimized to create an effect of increasing the accuracy of drug screening, and thus the industrial applicability is recognized.

Claims

1. A method for simultaneously manufacturing two-dimensional brain cancer cells and three-dimensional brain cancer spheroids derived from brain cancer entities, comprising the following steps: (S1) A step of producing a brain cancer tissue culture medium by culturing brain cancer tissue isolated from a brain cancer subject, and obtaining two-dimensional brain cancer cells therefrom; and (S2) A step of producing a three-dimensional brain cancer spheroid by collecting and culturing brain cancer tissue from the brain cancer tissue culture medium.

2. In paragraph 1, A simultaneous manufacturing method wherein the above two-dimensional brain cancer cells are cells that have settled and attached in the brain cancer tissue culture medium at step (S1).

3. In paragraph 1, A simultaneous manufacturing method, wherein, in the above step (S1), brain cancer tissue is cultured in a culture medium containing amino acids, vitamins, inorganic salts, glucose, and lipids.

4. In paragraph 1, A simultaneous manufacturing method wherein the brain cancer tissue collected and cultured in the above step (S2) is brain cancer tissue floating in the brain cancer tissue culture medium in the step (S1).

5. In paragraph 1, A simultaneous manufacturing method, wherein in the above (S2) step, brain cancer tissue is cultured in a culture medium containing amino acids, vitamins, inorganic salts, glucose, lipids, N2 supplements, insulin, L-glutamine, penicillin-streptomycin, antioxidant enzymes, proteins, vitamins, fatty acids, and 2-mercaptoethanol.

6. In paragraph 1, A simultaneous manufacturing method wherein the above step (S1) is performed for 5 to 15 days.

7. In paragraph 1, A simultaneous manufacturing method wherein the above (S2) step is performed for 10 to 30 days.

8. In paragraph 1, A simultaneous manufacturing method wherein the two-dimensional brain cancer cells and the three-dimensional brain cancer spheroids are derived from brain cancer tissue of the same individual.

9. In paragraph 1, A simultaneous manufacturing method wherein the above steps (S1) and (S2) are performed semi-continuously.

10. In paragraph 1, The above two-dimensional brain cancer cells are characterized by at least one selected from the group consisting of: a) Contains homogeneous brain cancer cells; and b) No drug resistance.

11. In paragraph 1, A simultaneous manufacturing method, wherein the brain cancer is any one selected from the group consisting of meningiomatosis, glioblastoma, pituitary adenoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor.

12. In paragraph 11, A simultaneous manufacturing method, wherein the metastatic brain cancer is any one selected from the group consisting of metastasized lung cancer, metastasized breast cancer, metastasized systemic lymphoma, metastasized melanoma, metastasized sarcoma, and metastasized gastrointestinal cancer.

13. A method for screening a substance for preventing or treating brain cancer, comprising the following steps: (S1) A step of treating a candidate substance to at least one of the two-dimensional brain cancer cells or three-dimensional brain cancer spheroids manufactured by the simultaneous manufacturing method of paragraph 1; and (S2) A step of confirming the progress of brain cancer prevention or treatment in at least one of two-dimensional brain cancer cells or three-dimensional brain cancer spheroids treated with the above candidate substance.

14. In the 13th paragraph, the screening method, (S3) A screening method, which further includes a step of determining the candidate substance as a substance for preventing or treating brain cancer, if the effect of preventing or treating brain cancer is confirmed.

15. In any one of paragraphs 13 to 14, A screening method, wherein the brain cancer is any one selected from the group consisting of meningiomatosis, glioblastoma, pituitary adenoma, metastatic brain cancer, pediatric brain cancer, malignant glioma, lymphadenoma, and germ cell tumor.

16. In paragraph 15, A screening method, wherein the metastatic brain cancer is any one selected from the group consisting of metastasized lung cancer, metastasized breast cancer, metastasized systemic lymphoma, metastasized melanoma, metastasized sarcoma, and metastasized gastrointestinal cancer.

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