Individualized patient-derived tumor organoids

Abstract

The present disclosure provides compositions and methods for preparing and using personalized patient-derived tumor organoids (IPTOs). Human iPSC-derived cerebral organoids can be used as hosts for tumor tissue grafts. Patient material can be obtained directly after surgery and dissected into small pieces. The small tumor tissue is inserted into the cerebral organoid to prepare a hybrid organoid. The hybrid organoid can be grown for weeks to months, and can grow slow-growing tumors such as gliomas and pilocytic astrocytomas with IDH mutations. The tumor pieces preferably contain both tumor cells and adjacent stromal cells, allowing for the testing of immunotherapeutic agents as well as conventional chemotherapeutic agents.

Description

[Technical field]

[0001] Prediction of drug response is one of the major bottlenecks for selecting the best drug candidates for clinical treatment in personalized cancer therapy, which requires access to reliable patient material to test individual drugs before clinical treatment. [Background technology]

[0002] Patient-derived cancer cells (PDCs) and patient-derived xenografts (PDXs) are often used as tumor models, but each has many drawbacks: PDCs lack diversity in terms of cell type, spatial organization, and microenvironment, while PDXs have low engraftment success rates and require long culture periods.

[0003] Recently, organoids have been developed as three-dimensional cell cultures. Organoids are cell masses constructed in vitro and can be generated from embryonic stem cells, induced pluripotent stem cells (iPSCs) or somatic stem cells (SSCs). Organoids have the capacity for self-renewal and proliferation, and can maintain the physiological structure and function of the tissue from which they are derived.

[0004] Several brain tumor organoid models have been deciphered, but these models lack native tissue structure or interactions between tumor and non-tumor cells, which are major limitations of brain tumor organoid models. Summary of the Invention [Problem to be solved by the invention]

[0005] This disclosure describes a new personalized patient-derived tumor organoid (IPTO) system that overcomes the limitations of traditional tumor organoid systems. Human iPSC-derived cerebral organoids can be used as hosts for tumor tissue transplantation. Patient material can be obtained directly after surgery and dissociated into small pieces. The small tumor tissue is inserted into the cerebral organoid to prepare a hybrid organoid. The hybrid organoid can grow for weeks to months, even in slow-growing tumors such as gliomas and pilocytic astrocytomas with IDH mutations. IPTOs preferably contain both tumor cells and adjacent stromal cells, allowing both efficacy and safety of anticancer drugs to be tested.

[0006] In one embodiment, the present disclosure provides a hybrid organoid comprising tumor tissue embedded in a cerebral organoid.

[0007] In some embodiments, cerebral organoid is differentiated from stem cell.In some embodiments, stem cell is induced pluripotent stem cell (iPSC).In some embodiments, cerebral organoid expresses at least one marker selected from the group consisting of neuroepithelial stem cell protein (nestin), doublecortin (DCX), neuron-specific class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin and T-box brain transcription factor 1 (TBR1).

[0008] In some embodiments, the size of the tumor tissue when placed in the cerebral organoid is 0.2mm to 5mm in diameter.In some embodiments, the size of the tumor tissue when placed in the cerebral organoid is 0.5mm to 2mm in diameter.

[0009] In some embodiments, the tumor tissue is a brain tumor tissue, hi some embodiments, the brain tumor is selected from the group consisting of glioblastoma, pilocytic astrocytoma, oligodendroglioma, and a metastatic tumor derived from another tissue.

[0010] In some embodiments, a portion of the brain tumors are characterized by a mutation in isocitrate dehydrogenase (IDH). In some embodiments, the tumor tissue comprises tumor cells and adjacent non-tumor stromal cells.

[0011] In some embodiments, the tumor tissue has expanded in tumor cell number by at least 50% compared to when the tumor tissue was initially placed in the cerebral organoid.

[0012] In some embodiments, tumor tissue is at least twice as proliferated as tumor cell number when tumor tissue is initially placed in cerebral organoid.In some embodiments, tumor tissue is at least twice as large as tumor tissue when tumor tissue is initially placed in cerebral organoid.

[0013] In some embodiments, the hybrid organoids express at least one marker selected from the group consisting of platelet endothelial cell adhesion molecule (PECAM-1, CD31), protein tyrosine phosphatase, receptor type, C (PTPRC, CD45), cluster of differentiation 68 (CD68), doublecortin (DCX), glial fibrillary acidic protein (GFAP), glycerol-3-phosphate dehydrogenase 1 (GPD1), allograft inflammatory factor 1 (Iba1), proliferation marker Ki-67 (Ki67), microtubule-associated protein 2 (MAP2), neuroepithelial stem cell protein (nestin), oligodendrocyte transcription factor (Olig2), S100beta chain, SRY (sex determining region Y)-box 2 (Sox2), and neural specific class III beta-tubulin (TuJ1).

[0014] In one embodiment, a method for evaluating a candidate anticancer drug is provided, comprising contacting a hybrid organoid described in any one of claims 1 to 13 with a candidate anticancer drug and examining a change in cell number within the hybrid organoid.

[0015] In some embodiments, the reduction of viable tumor cells in the hybrid organoid indicates the efficacy of anti-cancer drugs.In some embodiments, the reduction of viable host cells in the hybrid organoid indicates the toxicity of anti-cancer drugs.

[0016] In some embodiments, the hybrid organoid is cultured for 1-16 weeks after tumor tissue is initially placed in the cerebral organoid.In some embodiments, the hybrid organoid is cultured for 1-3 weeks after tumor tissue is initially placed in the cerebral organoid.In some embodiments, the hybrid organoid is cryopreserved and collected.

[0017] Another embodiment provides a method for preparing the hybrid organoids of the present disclosure, comprising placing tumor tissue into an incision of a cerebral organoid.

[0018] In some embodiments, the method further comprises covering the hybrid organoid with a Matrigel® solubilized basement membrane matrix and solidifying the matrix. In some embodiments, the size of the tumor tissue when placed in the cerebral organoid is 0.2 mm to 5 mm in diameter. In some embodiments, the size of the tumor tissue when placed in the cerebral organoid is 0.5 mm to 2 mm in diameter. In some embodiments, the tumor tissue is a brain tumor tissue. In some embodiments, the brain tumor is selected from the group consisting of glioblastoma, pilocytic astrocytoma, oligodendroglioma, and metastatic tumors derived from another tissue.

[0019] In some embodiments, cerebral organoid is prepared from induced pluripotent stem cell (iPSC).In some embodiments, cerebral organoid expresses at least one marker selected from the group consisting of neuroepithelial stem cell protein (nestin), doublecortin (DCX), neural specific class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin and T-box brain transcription factor 1 (TBR1).

[0020] In some embodiments, the method further comprises cryopreserving the hybrid organoid. In some embodiments, cryopreservation is performed in a cryopreservation medium that comprises a ROCK inhibitor. [Brief description of the drawings]

[0021] [Figure 1] Figure 1a is a schematic diagram showing the procedure for establishing IPTO, and Figure 1b shows the immunostaining results for various neuronal markers in the host organoids. [Diagram 2] Figure 2a shows IDHmut glioma cultured on IPTO. The cell density of IDHmut glioma-derived IPTO was comparable to that of the parental tumor (H&E staining, left), and 2-HG secretion from IDHmut glioma-derived IPTO was detectable. Figure 2b shows IDHmut glioma cultured on IPTO. Representative IHC (IDHR132H) images showed the spatial distribution of IDHmut glioma cells in the corresponding IPTO. Figure 2c shows pilocytic astrocytoma cultured on IPTO. Tumor cell density was high in pilocytic astrocytoma-derived IPTO, and proliferating cells (EdU+) were identified in the tumor area (GFP-). Figure 2d shows pilocytic astrocytoma cultured on IPTO. Tumor cells were shown to have GFAP expression and a lack of GFP expression. Figure 2e shows tumor proliferation by luciferase activity. [Diagram 3]Figure 3a shows the preservation of stromal cells. Representative immunofluorescence images showing proliferating macrophages / microglia (yellow arrows) after 2 and 4 weeks of culture. Figure 3b shows the quantification of CD68+ labeled macrophages / microglia. Figure 3c shows that various amounts of T cells were identified in IPTO based on FACS analysis. Figure 3d shows that immunohistochemical staining detected CD4+ T cells in brain metastasis-derived IPTO. Figure 3e shows that immunohistochemical staining showed CD8+ T cells (red arrows) in brain metastasis-derived IPTO. Figure 3f shows that H&E staining revealed the preservation of vascular structures (red circles) in IPTO. Figure 3g shows that CD31 / CD34 labeled endothelial cells were detected in both parental tumor tissue and parental tumor-derived IPTO. [Figure 4] Fig. 4a shows the therapeutic response in IPTO. It shows the drug efficacy evaluation by quantifying the ratio of BrdU+ cells to GFP- cells. Fig. 4b shows the therapeutic response in IPTO. It shows the drug toxicity evaluation by quantifying the ratio of BrdU+ cells to GFP+ cells. [Diagram 5] Genome-wide DNA methylation arrays used for molecular classification of human brain tumors. The t-SNE plot shows that IPTO ("Liu_IPTO") clusters very closely with the parent tumor ("Liu_T"). [Figure 6] FIG. 1 shows that single-cell RNA sequencing of IPTO and parental tumors demonstrated that IPTO maintained the entire cellular diversity of the parental tumors and their molecular identity. [Figure 7] Figure 1 shows that IPTO chemosensitivity predicted the PFS (progression-free survival) of patients. After testing the TMZ sensitivity of each IPTO, the PFS of the patients (all of whom received TMZ for treatment) was followed up. The results showed that IPTO predicted the TMZ sensitivity and the response of the patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] [Definition] The following description sets forth exemplary embodiments of the present technology, however, it should be recognized that such description is not intended to limit the scope of the disclosure, but is instead provided as a description of exemplary embodiments.

[0023] [Definition] As used herein, the following words and symbols are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0024] As used herein, certain terms may have the following defined meanings. As used in this specification and claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly indicates otherwise. For example, the term "a cell" includes not only a single cell, but also a plurality of cells, including mixtures thereof.

[0025] [Individualized Patient-Derived Tumor Organoids (IPTO)] Organoids are three-dimensional cultures of cells that mimic the original tissue. Tumor organoids are promising tools for disease modeling and drug screening. However, existing tumor organoids either lack the original tissue structure (da Silva et al., 2018; Hubert et al., 2016; Linkous et al., 2019) or lack the interactions between tumor and non-tumor cells (Jacob et al., 2019).

[0026] Furthermore, growing brain tumor organoids is challenging, especially for slow-growing brain tumors such as gliomas and pilocytic astrocytomas, especially those with IDH (isocitrate dehydrogenase) mutations.

[0027] The present inventors have developed a new individualized patient-derived tumor organoid (IPTO) technology that overcomes various limitations of conventional tumor organoid technology. In an exemplary embodiment, a small piece of tumor tissue (e.g., 1 mm in diameter) is inserted into a host cerebral organoid. The host cerebral organoid is preferably developed from stem cells, such as iPSC-derived cerebral organoids. The resulting hybrid organoid can be grown for several months. If the tumor piece contains both tumor cells and adjacent stromal cells, both types of cells can be well maintained within the hybrid organoid.

[0028] IPTO has many advantages over existing systems. First, with IPTO technology, the inventors achieved a success rate of nearly 100% in culturing brain tumor organoids. Approximately 120 patient tumors were cultured in IPTO, including both pediatric and adult glioblastomas, IDH mutant gliomas, low-grade brain tumors such as pilocytic astrocytoma and oligodendroglioma, as well as a wide variety of brain metastatic tumors (e.g., melanoma, brain lymphoma, lung cancer, pancreatic cancer, colorectal cancer). Among these, IDH mutant gliomas and low-grade brain tumors are known to be particularly difficult to culture.

[0029] IDH mutations are found in 70-80% of WHO grade 2 or 3 astrocytomas and oligodendrogliomas. Therefore, the ability to culture slow-growing brain tumor cells was highly valuable and unexpected. IPTO could benefit from the presence of host cerebral organoids without disrupting the architecture of the tumor tissue.

[0030] Second, IPTO can and preferably does include stromal cells, such as vascular cells, microglial cells, and T cells, which can be expanded or maintained within the organoid. In a recently published study culturing glioblastoma tissue (Jacob et al., 2019), the macrophage / microglial cell population gradually decreased. In contrast, in the IPTO system, the macrophage / microglial cell population remained stable or even enriched in the majority of samples tested (Figure 3). This is another unexpected finding and an important advantage of the present disclosure. The inclusion of such stromal cells allows for the testing of therapeutics such as immunotherapy.

[0031] Third, IPTO can be expanded, passaged, cryopreserved, and harvested, as shown in the experimental examples. Moreover, preparation, expansion, passaging, etc. can be performed very efficiently and quickly. For example, the entire procedure from surgery to establishing organoids can be completed within 2 weeks.

[0032] Fourth, as shown in the figure, the cellular and molecular pathological features are maintained from patient to organoid, making it ideal for testing drug sensitivity in patients. Finally, the culture system harbors healthy host cells, allowing the safety of candidate drugs to be evaluated by these healthy host cells.

[0033] These and other advantages of IPTO technology are summarized in Table 1 below.

[0034] [Table 1]

[0035] Thus, according to one embodiment of the present disclosure, a hybrid organoid is provided comprising tumor tissue embedded in a cerebral organoid.

[0036] Cerebral organoids can be prepared by methods known in the art (Lancaster and Knoblich, 2014; Lancaster et al., 2013). An example of a protocol is shown in Example 1. In some embodiments, cerebral organoids are differentiated from stem cells, preferably pluripotent stem cells such as induced pluripotent stem cells (iPSCs).

[0037] As used herein, "stem cells" are defined as cells that have the ability to divide indefinitely in culture and give rise to specialized cells. Non-limiting examples of stem cell types include somatic (adult) stem cells, embryonic stem cells, parthenogenetic stem cells (Cibelli et al., 2002; Janus, 2008; Kim, 2010) and / or induced pluripotent stem cells (iPS cells or iPSCs).

[0038] As used herein, the term "pluripotent stem cells" refers to cells that: (i) are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype even after extended periods of culture; and (iii) maintain the potential to differentiate into derivatives of all three embryonic germ layers (endoderm, mesoderm, ectoderm) even after extended periods of culture. Non-limiting examples of currently available pluripotent stem cells include embryonic stem cells and iPSCs.

[0039] Cerebral organoids can be examined and confirmed with known markers such as neuroepithelial stem cell protein (nestin), doublecortin (DCX), neural specific class III beta tubulin (TuJ1), microtubule associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin, T-box brain transcription factor 1 (TBR1). Nestin is a marker for neural stem cells. DCX is a marker for neuroblasts. TuJ1 is a marker for immature neurons. KI67 indicates proliferating neurons. PAX6 indicates forebrain neural progenitor cells. Phosphorylated vimentin indicates radial glia. TBR1 indicates deep layer neurons. MAP2 protein is a neural specific cytoskeletal protein enriched in dendrites and perikarya, suggesting a role in determining and stabilizing neuronal morphology during neuronal development. In some embodiments, at least two of these markers are detectable in cerebral organoids. In some embodiments, at least three, four, five, or six of these markers are detectable in cerebral organoids.

[0040] In some embodiments, cerebral organoid is grown to a size suitable for embedding tumor tissue.In some embodiments, suitable size is, but not limited to, 0.5-20 mm in diameter, for example, 1-15 mm in diameter, 1-10 mm in diameter, 1-8 mm in diameter, 2-6 mm in diameter, or 2-4 mm in diameter.

[0041] In some embodiments, incision is made in cerebral organoid to allow tumor fragment placement.In some embodiments, incision has a depth of about half to 1 / 3 of the diameter of cerebral organoid.In some embodiments, incision is in the approximate center of cerebral organoid.

[0042] In some embodiments, the tumor tissue embedded in cerebral organoid is brain tumor tissue.In some embodiments, the brain tumor is high grade brain tumor.In some embodiments, the brain tumor is low grade brain tumor.In some embodiments, the brain tumor is slow growing brain tumor.

[0043] Brain tumors can be classified into four grades. Grades 1 and 2 are also called low-grade tumors. Grades 3 and 4 are also called high-grade tumors. The criteria for defining brain tumors are known in the art. In general, grade 1 brain tumors grow slowly and are unlikely to metastasize. They can often be treated with surgery; grade 2 brain tumors are unlikely to grow and metastasize, but are likely to recur after treatment; grade 3 brain tumors are likely to have rapidly dividing cells but no dead cells; and grade 4 brain tumor cells are actively dividing.

[0044] In some embodiments, the brain tumor is a glioblastoma, a pilocytic astrocytoma, or an oligodendroglioma. In some embodiments, the brain tumor, such as a glioma, is characterized by one or more isocitrate dehydrogenase (IDH) mutations. A high percentage of low-grade gliomas have mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes. IDH mutations may be a driver of oncogenesis. Somatic mosaicism of IDH1 or IDH2 at R132 causes enchondromatosis syndrome, Ollier disease, and Maffucci syndrome, which are characterized by hemangiomas and cartilage tumors, and increase the risk of glioma. Also, introduction of mutated IDH into normal cells results in increased proliferation, increased colony formation, and failure to differentiate.

[0045] In some embodiments, the brain tumor is a metastatic tumor originating from a different tissue. The tumor originating from a different tissue may be, but is not limited to, melanoma, brain lymphoma, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, prostate cancer, and thyroid cancer.

[0046] In some embodiments, the tumor embedded in the cerebral organoid has a suitable size. For example, the tumor tissue has a diameter of 0.2mm to 5mm. In some embodiments, the tumor tissue has a diameter of, but not limited to, 0.2 to 4mm, or 0.4 to 3mm, 0.5 to 2mm, or 0.8 to 1.5mm.

[0047] In some embodiments, the diameter of the tumor tissue is about 10% to 90% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 20% to 80% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 25% to about 75% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 30% to about 70% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 40% to 60% of the diameter of the cerebral organoid.

[0048] In some embodiments, the tumor tissue embedded in cerebral organoid also comprises adjacent stromal cells.In some embodiments, stromal cells comprise vascular cells, microglial cells and / or T cells.

[0049] The hybrid organoid of the present disclosure can be in different growth stages.In some embodiments, after a period of culture, the number of tumor cells in hybrid organoid increases at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or 20-fold since being embedded in host cerebral organoid.

[0050] In some embodiments, after a period of culture, the size of the tumor tissue within the hybrid organoid increases by at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or 20-fold in diameter since being embedded in the host cerebral organoid.

[0051] In some embodiments, hybrid organoid can be cultured for at least 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 4 months or longer.Cultivation can be carried out in medium such as modified DMEM medium of Example 2.In some embodiments, hybrid organoid can be cryopreserved or frozen and then restored for further use.

[0052] In some embodiments, the hybrid organoids express one or more of the following markers: platelet endothelial cell adhesion molecule (PECAM-1, CD31), protein tyrosine phosphatase, receptor type, C (PTPRC, CD45), differentiation cluster 68 (CD68), doublecortin (DCX), glial fibrillary acidic protein (GFAP), glycerol-3-phosphate dehydrogenase 1 (GPD1), graft inflammatory factor 1 (Iba1), proliferation marker Ki-67 (Ki67), microtubule-associated protein 2 (MAP2), neuroepithelial stem cell protein (nestin), oligodendrocyte transcription factor (Olig2), S100beta chain, SRY (sex determining region Y)-box 2 (Sox2), and neural specific class III beta-tubulin (TuJ1). In some embodiments, at least two of these markers are detectable in the cerebral organoids. In some embodiments, at least three, four, five or six of these markers are detectable in cerebral organoids.

[0053] In one embodiment, the hybrid organoid expresses CD31. In one embodiment, the hybrid organoid expresses CD45. In one embodiment, the hybrid organoid expresses CD68. In one embodiment, the hybrid organoid expresses DCX. In one embodiment, the hybrid organoid expresses GFAP. In one embodiment, the hybrid organoid expresses GPD1. In one embodiment, the hybrid organoid expresses Iba1.

[0054] In one embodiment, the hybrid organoid expresses Ki67. In one embodiment, the hybrid organoid expresses MAP2. In one embodiment, the hybrid organoid expresses nestin. In one embodiment, the hybrid organoid expresses Olig2. In one embodiment, the hybrid organoid expresses S100beta chain. In one embodiment, the hybrid organoid expresses Sox2. In one embodiment, the hybrid organoid expresses TuJ1.

[0055] [How to use IPTO] The individualized patient-derived tumor organoids (IPTOs) prepared according to certain embodiments of the present technology can be used as a model system for various purposes. In some embodiments, the model system can be used to clarify the biological mechanism of tumorigenesis. Importantly, the model system can also be used to evaluate candidate drugs that may be useful in the treatment of tumors.

[0056] Thus, in one embodiment, the present disclosure provides a method for evaluating a candidate drug, which comprises contacting the hybrid organoid of the present disclosure with a candidate anti-cancer drug, and examining the effect of the candidate drug on the hybrid organoid.In some embodiments, the tumor tissue embedded in the cerebral organoid also comprises adjacent stromal cells.In some embodiments, the stromal cells comprise vascular cells, microglial cells and / or T cells.In general, the higher the death rate of tumor cells, the higher the anti-cancer effect, and the lower the death rate of normal cells (e.g., the cells of the host organoid), the higher the safety of the drug.

[0057] As shown in the examples, tumor cells in IPTO can grow at a high rate. After about 2 weeks of embedding the original tumor pieces into the cerebral organoids, IPTO reaches high tumor cell density, providing a suitable model for drug testing. Thus, in some embodiments, the hybrid organoids are cultured for about 2 weeks after embedding the tumor tissue into the host cerebral organoids. Alternatively, in some embodiments, the hybrid organoids are cultured for at least 1 week, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks after embedding the tumor tissue into the host cerebral organoids. In some embodiments, the hybrid organoids are cultured for 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks or less after embedding the tumor tissue into the host cerebral organoids.

[0058] The hybrid organoid of the present disclosure can be in different growth stages.In some embodiments, after a period of culture, the number of tumor cells in hybrid organoid increases at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or 20-fold since being embedded in host cerebral organoid.

[0059] In certain embodiments, after a period of culture, the size of the tumor tissue within the hybrid organoid has increased in diameter by at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 20-fold since implantation into the host cerebral organoid.

[0060] The test can also be customized for a specific tumor or a specific patient. In some embodiments, the tumor tissue embedded in the cerebral organoid is a brain tumor tissue. In some embodiments, the brain tumor is a high-grade brain tumor. In some embodiments, the brain tumor is a low-grade brain tumor. In some embodiments, the brain tumor is a slow-growing brain tumor. In some embodiments, the brain tumor is a glioblastoma, a pilocytic astrocytoma or an oligodendroglioma. In some embodiments, the brain tumor, such as a glioma, is characterized by one or more isocitrate dehydrogenase (IDH) mutations. In some embodiments, the brain tumor is a metastatic tumor originating from different tissues. The tumor originating from different tissues can be, but is not limited to, melanoma, brain lymphoma, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, prostate cancer and thyroid cancer.

[0061] Thus, the efficacy of the candidate agents tested herein is specific to the tumor tissue used or to the tumor tissue of a particular patient, thus providing rapid treatment to the patient.

[0062] The IPTO system disclosed herein is useful not only for testing traditional chemotherapeutic drugs, but also for immunotherapies such as PD-L1 inhibitors and CD47 inhibitors, due to the presence of stromal cells, including immune cells, in the hybrid organoids.

[0063] The term immunotherapy refers to treating tumors by activating or suppressing the immune system, resulting in tumor cell death, rather than directly killing tumor cells. In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In some embodiments, the immunotherapy is an antibody. In some embodiments, the immunotherapy includes a cytokine. In some embodiments, the immunotherapy is a CAR-T (chimeric antigen receptor T cell) therapy or a TCR (T cell receptor T cell) therapy.

[0064] [Preparation method of IPTO] Another embodiment provides the method for preparing the hybrid organoid of the present disclosure.In some embodiments, the method comprises placing tumor tissue in the incision of cerebral organoid.

[0065] Cerebral organoids can be prepared by methods known in the art (Lancaster and Knoblich, 2014; Lancaster et al., 2013). An example of a protocol is shown in Example 1. In some embodiments, cerebral organoids are differentiated from stem cells, preferably pluripotent stem cells such as induced pluripotent stem cells (iPSCs).

[0066] Cerebral organoids can be examined and confirmed with known markers such as neuroepithelial stem cell protein (nestin), doublecortin (DCX), neural specific class III beta tubulin (TuJ1), microtubule associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin and T-box brain transcription factor 1 (TBR1). Nestin is a marker for neural stem cells. DCX is a marker for neuroblasts. TuJ1 is a marker for immature neurons. KI67 indicates proliferating neurons. PAX6 indicates forebrain neural progenitor cells. Phosphorylated vimentin indicates radial glia. TBR1 indicates deep layer neurons. MAP2 protein is a neural specific cytoskeletal protein enriched in dendrites and perikarya, suggesting a role in determining and stabilizing neuronal morphology during neuronal development. In some embodiments, at least two of these markers are detectable in cerebral organoids. In some embodiments, at least three, four, five, or six of these markers are detectable in cerebral organoids.

[0067] In some embodiments, cerebral organoid is grown to a size suitable for embedding tumor tissue.In some embodiments, suitable size is, but not limited to, 0.5-20mm in diameter, for example, 1-15mm in diameter, 1-10mm in diameter, 1-8mm in diameter, 2-6mm in diameter, or 2-4mm in diameter.

[0068] In some embodiments, incision is made in cerebral organoid to allow tumor fragment placement.In some embodiments, incision has a depth of about half to 1 / 3 of the diameter of cerebral organoid.In some embodiments, incision is in the approximate center of cerebral organoid.

[0069] In some embodiments, the tumor tissue embedded in cerebral organoid is brain tumor tissue.In some embodiments, the brain tumor is high grade brain tumor.In some embodiments, the brain tumor is low grade brain tumor.In some embodiments, the brain tumor is slow growing brain tumor.

[0070] Brain tumors are classified into four grades. Grades 1 and 2 are also called low-grade tumors. Grades 3 and 4 are also called high-grade tumors. The criteria for defining brain tumors are known in the art. In general, grade 1 brain tumors grow slowly and are unlikely to metastasize. They can often be treated with surgery; grade 2 brain tumors are unlikely to grow and metastasize, but are likely to recur after treatment; grade 3 brain tumors are likely to have rapidly dividing cells but no dead cells; and grade 4 brain tumor cells are actively dividing.

[0071] In some embodiments, the brain tumor is a glioblastoma, a pilocytic astrocytoma, or an oligodendroglioma. In some embodiments, the brain tumor, such as a glioma, is characterized by one or more isocitrate dehydrogenase (IDH) mutations. A high percentage of low-grade gliomas have mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes. IDH mutations may be a driver of oncogenesis. Somatic mosaicism of IDH1 or IDH2 at R132 causes enchondromatosis syndrome, Ollier disease, and Maffucci syndrome, which are characterized by hemangiomas and cartilage tumors, and increase the risk of glioma. Also, introduction of mutated IDH into normal cells results in increased proliferation, increased colony formation, and failure to differentiate.

[0072] In some embodiments, the brain tumor is a metastatic tumor originating from a different tissue. The tumor originating from a different tissue may be, but is not limited to, melanoma, brain lymphoma, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, prostate cancer, and thyroid cancer.

[0073] In some embodiments, the tumor embedded in the cerebral organoid has a suitable size. For example, the tumor tissue has a diameter of 0.2mm to 5mm. In some embodiments, the tumor tissue has a diameter of, but not limited to, 0.2 to 4mm, or 0.4 to 3mm, 0.5 to 2mm, or 0.8 to 1.5mm.

[0074] In some embodiments, the diameter of the tumor tissue is about 10% to 90% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 20% to 80% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 25% to about 75% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 30% to about 70% of the diameter of the cerebral organoid. In some embodiments, the diameter of the tumor tissue is about 40% to 60% of the diameter of the cerebral organoid.

[0075] In some embodiments, the tumor tissue embedded in cerebral organoid also comprises adjacent stromal cells.In some embodiments, stromal cells comprise vascular cells, microglial cells and / or T cells.

[0076] The hybrid organoid of the present disclosure can be in different growth stages.In some embodiments, after a period of culture, since being embedded in host cerebral organoid, the number of tumor cells in hybrid organoid increases at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or 20-fold.

[0077] In some embodiments, after a period of culture, the size of the tumor tissue within the hybrid organoid increases by at least 20%, 40%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or 20-fold in diameter since being embedded in the host cerebral organoid.

[0078] In some embodiments, hybrid organoid can be cultured for at least 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 4 months or longer.Cultivation can be carried out in medium such as modified DMEM medium of Example 2.In some embodiments, hybrid organoid can be cryopreserved or frozen and then restored for further use.

[0079] In some embodiments, the hybrid organoids express one or more of the following markers: platelet endothelial cell adhesion molecule (PECAM-1, CD31), protein tyrosine phosphatase, receptor type, C (PTPRC, CD45), differentiation cluster 68 (CD68), doublecortin (DCX), glial fibrillary acidic protein (GFAP), glycerol-3-phosphate dehydrogenase 1 (GPD1), graft inflammatory factor 1 (Iba1), proliferation marker Ki-67 (Ki67), microtubule-associated protein 2 (MAP2), neuroepithelial stem cell protein (nestin), oligodendrocyte transcription factor (Olig2), S100beta chain, SRY (sex determining region Y)-box 2 (Sox2), and neural specific class III beta-tubulin (TuJ1). In some embodiments, at least two of these markers are detectable in the cerebral organoids. In some embodiments, at least three, four, five or six of these markers are detectable in cerebral organoids.

[0080] In one embodiment, the hybrid organoids express CD31. In one embodiment, the hybrid organoids express CD45. In one embodiment, the hybrid organoids express CD68. In one embodiment, the hybrid organoids express DCX. In one embodiment, the hybrid organoids express GFAP. In one embodiment, the hybrid organoids express GPD1. In one embodiment, the hybrid organoids express Iba1. In one embodiment, the hybrid organoids express Ki67. In one embodiment, the hybrid organoids express MAP2. In one embodiment, the hybrid organoids express nestin. In one embodiment, the hybrid organoids express Olig2. In one embodiment, the hybrid organoids express S100beta chain. In one embodiment, the hybrid organoids express Sox2. In one embodiment, the hybrid organoids express TuJ1.

[0081] In some embodiments, the method further comprises cryopreserving the hybrid organoid.In some embodiments, cryopreservation is carried out in the cryopreservation medium containing ROCK inhibitor.In some embodiments, the medium comprises 10% DMSO containing retinoic acid (RA) and 10 μM ROCK inhibitor (for example, Y-27632). EXAMPLES

[0082] Example 1. Preparation of cerebral organoids (CO) derived from human induced pluripotent stem cells (hiPSCs) This example describes the generation of cerebral organoids (COs) derived from human induced pluripotent stem cells (hiPSCs). The procedure was adapted from previously published methods (Lancaster and Knoblich, 2014; Lancaster et al., 2013).

[0083] Briefly, on day 0, hiPSCs were dissociated into single cells and seeded at a concentration of 12,000 cells per well in ultra-low attachment 96-well plates in stem cell medium supplemented with 4ng / mL bFGF and 50μM Rho-associated protein kinase (ROCK) inhibitor. On day 3, the medium was replaced with fresh stem cell medium. From day 5, organoids were transferred to ultra-low attachment 24-well plates in neural induction medium: DMEM-F12 supplemented with 1x N2 supplement, 1μg / mL heparin solution, 1x GlutaMAX and 1x MEM-NEAA, and the medium was refreshed every other day. On day 11, organoids were embedded in droplets of Matrigel and transferred to 6-well plates containing NeuroDMEM (50% DMEM-F12, 50% Neurobasal medium, 1x N2, 1x B27), Vitamin A, 2.5mg / mL insulin, 0.05mM BME, 1x GlutaMAX, 1x MEM-NEAA, 1x penicillin / streptomycin. On day 15, the medium was replaced with differentiation medium consisting of 50% DMEM-F12, 50% Neurobasal medium, 1x N2, 1x B27, 2.5mg / mL insulin, 0.05mM BME, 1x GlutaMAX, 1x MEM-NEAA, 1x penicillin / streptomycin, and culture was continued on an orbital shaker. Medium was replaced every 2-3 days.

[0084] To confirm successful differentiation of hiPSCs into the nervous system, organoids can be fixed and serial sections taken using a cryostat. Sections are stained with markers to confirm cortical and forebrain identity. This includes immunostaining for KI67 (proliferating cells), PAX6 (forebrain neuronal progenitors), phosphorylated vimentin (radial glia) and TBR1 (deep layer neurons). The presence of KI67, PAX6, phosphorylated vimentin and TBR1 antibody markers can confirm successful differentiation into early cortical forebrain identity.

[0085] Furthermore, the distinct spatial localization of each marker allows us to reveal the self-organized structural nature of 3D organoids: for example, the progenitor marker PAX6 and the postmitotic neuronal marker TBR1 occupy distinct spatial locations and can be seen to represent the in vitro equivalents of the ventricular zone and cortical plate, respectively.

[0086] Example 2. Preparation of patient-derived hybrid organoids In this example, we describe a procedure for co-culturing fresh patient-derived tumor pieces with cerebral organoids and then passaging, cryopreserving, and recovering individualized patient-derived tumor organoids (IPTOs).

[0087] [Preparation of tumor pieces] A variety of brain tumors can be cultured in this system, including primary / recurrent GBM, metastatic lymphoma, and metastatic melanoma. Suitable tumor tissues are those close to the tumor border without significant necrosis.

[0088] Once the tumor tissue was excised, it was placed in a 15 mL Falcon tube and immersed in PBS. The tumor was then washed with DPBS to remove debris and blood cells. To perform the dissection, the tumor was transferred to a 10 cm dish on ice and RA+ (retinoic acid+) medium was added. Next, the tumor bulk was cut into small pieces with a diameter of 1-2 mm with a scalpel, and then washed three times with RA+ medium. The tumor pieces were stored in modified DMEM medium supplemented with retinoic acid (RA) and containing the ROCK inhibitor (RI) Y-27632 (10 μM).

[0089] [Preparation of host cerebral organoids (CO)] Host COs can be prepared as described in Example 1. Host COs from 4 weeks to 16 weeks of age are generally acceptable. The appropriate size of the CO is 2-3 mm in diameter. A gradual expansion was observed in the cerebral tissue, and the resulting organoids shrank after 6 months of culture. Staining showed that immature and mature neurons were observed in the cerebral organoids cultured for 1 month, as indicated by TUJ1 (neuron-specific class III β-tubulin) and MAP2 (microtubule-associated protein 2), respectively. Similarly, in the cerebral organoids aged 1 to 5 months, astrocytes were abundant and expressed GFAP and S100beta. Thus, these organoids retain diverse neuronal populations and are suitable for co-culture after 1 to 5 months of culture.

[0090] Cut the center of the organoid with a scalpel. The depth of the incision is generally half to one-third of the organoid's diameter. Transfer the organoid into a 15 mL Falcon tube, wash it three times with DPBS, and discard it after the organoid sinks to the bottom of the tube. Then, transfer the organoid into improved DMEM medium supplemented with RA and RI.

[0091] [Co-culture of tumor tissue and CO] Organoid embedding sheets were used for co-culture. An example of a commercially available product is catalog number 08579 (Stem Cell Technologies, Vancouver, BC, Canada). The CO was transferred to the embedding sheet and excess medium was removed. The tumor pieces were placed into the host CO along the incision using an autoclaved metal toothpick.

[0092] 30 μL of pre-chilled Matrigel was then added to cover the tumor-organoid hybrids, preferably centered. The tumor-organoid hybrids were incubated at 37° C. for 20 minutes to allow the Matrigel to solidify. The droplets formed were washed with modified DMEM and incubated statically overnight.

[0093] Tumor-organoid hybrids, IPTOs, were transferred to an orbital shaker at 75 rpm. Media was changed after 2 days and then every 2-3 days.

[0094] IPTO prepared in this manner stained positive with antibodies against the following markers: CD31 (platelet endothelial cell adhesion molecule (PECAM-1)), CD45 (protein tyrosine phosphatase, receptor type, C (PTPRC)), CD68 (cluster of differentiation 68), DCX (doublecortin), GFAP (glial fibrillary acidic protein), GPD1 (glycerol-3-phosphate dehydrogenase 1), Iba1 (graft inflammatory factor 1), Ki67 (proliferation marker Ki-67), MAP2 (microtubule-associated protein 2), (nestin), Olig2 (oligodendrocyte transcription factor), S100beta chain, Sox2 (SRY (sex determining region Y)-box 2), and TuJ1 (neuron-specific class III beta-tubulin).

[0095] [Passaging of hybrid organoids] IPTO can be passaged to avoid necrosis. "Mature IPTO" here refers to organoids with acceptable size and tumor cell ratio. For example, the size (diameter) of IPTO should be larger than 3-4 mm or the ratio of tumor cells in IPTO should be higher than 50%.

[0096] The duration of subculture varies from 4 to 16 weeks, depending on the rate of tumor cell proliferation. The IPTO are cut into small pieces (eg, about 1 mm in diameter) and cultured in the same manner as the original IPTO.

[0097] After 2–3 months of culture, tumor cells began to dominate the clear periphery of the organoids and Matrigel. This necessitated subculture to ensure sufficient oxygen and gradient. IPTOs were capable of long-term culture for up to 68 weeks, and a significantly higher proportion of Ki67+ was observed. Staining showed that tumor markers (GFAP for GBM) were abundant and stable in the first 3 months of culture. Interestingly, expression of nestin or SOX2 was fairly stable or enriched over time.

[0098] Mature IPTO can be stored frozen and harvested prior to use.

[0099] Cryopreservation and recovery of tumor samples or tumors in IPTO Samples from surgical tumors or IPTO can be cryopreserved. For both sample sources, samples must first be cut into appropriate sizes on ice. After washing with DPBS / RA+ medium, the sample pieces were placed in RA+ medium supplemented with 10 μM Rock inhibitor and placed on an orbital shaker (75 rpm, 37 °C, 5% CO2) for 1 h. Cryopreservation medium was added and equilibrated for another 10 min. Cryopreservation medium contained RA+ medium, 10% DMSO and 20 μM Rock inhibitor. Finally, the sample pieces were transferred to cryotubes in CoolCell freezing containers at -80 °C overnight. For long-term storage, samples were further transferred and kept in liquid nitrogen.

[0100] To retrieve samples, cryovials were first thawed in a 37°C water bath until a few ice chunks were visible. 1mL of RA+ containing 10μM RI was added to a 6-well plate, and samples were gently transferred to the plate using a P1000 pipette with a cut tip. The DMSO-containing medium was then discarded and replaced with fresh RA+ containing 10μM RI. These retrieved samples were then ready to be co-cultured with fresh cerebral organoids as shown in the generation of IPTOs.

[0101] To explore the optimal working regimen, four cryopreservation media were tested: Group 1 (RA+ plus 10% DMSO), Group 2 (RA+ plus 10% DMSO plus 10 μM RI), Group 3 (RA+ plus 10% DMSO plus 10 μM RI plus 0.5 M trehalose), and Group 4 (FBS plus 10% DMSO plus 10 μM RI). Cerebral organoids were derived from iPS cell clones stably expressing firefly luciferase. As a result, the BL results showed that the signal in Group 1 tended to be low on day 3, but then increased and was comparable to Group 2, Group 3, and Group 4. However, the Ki67 ratio in Group 1 tended to be lower than the other three groups. This suggested the need to add 10 μM RI to the cryopreservation medium. The Ki67 ratio in Group 2 was higher than that in Group 3, suggesting that 0.5 M trehalose did not provide any additional benefit. This is consistent with previous findings that >0.1M trehalose significantly increases osmolality, and the addition of this sugar may counteract the effect of reducing ice crystals. Therefore, RA+ plus 10% DMSO plus 10 μM RI was used for cryopreservation of parental or organoid tissues. Histological characteristics and Ki67 staining were comparable. Furthermore, microglial cells also survived in IPLTs retrieved from cryopreserved tumor tissues.

[0102] [Tumor infection and bioluminescence imaging in IPTO] To ensure that only cells in the tumor samples express firefly luciferase, tumor fragments were infected with lentivirus prior to co-culture. Thus, treated tumor fragments were treated overnight with an optimized concentration of lentivirus. Polybrene (Merck Millipore, #TR-1003-50UL) was used to increase the infection efficiency. The co-culture procedure was performed the following day as described above. For bioluminescence imaging, IPTOs were refreshed with RA+ medium containing 150 μg / mL D-luciferin (BioVision, #7903) and then incubated for 10 min on an orbital shaker at 37 °C. Bioluminescence images (BLI) were taken with an IVIS Lumina II (Perkin Elmer) in five consecutive segments with an exposure time of 60 s. The maximum sum pixels of the five segments were selected for analysis.

[0103] Example 3. Preparation of individualized patient-derived tumor organoids (IPTOs) and their use for drug evaluation. In this example, tumor hybrid organoids were prepared using the processes described in Examples 1 and 2, which were then used to test the efficiency of anticancer drugs.

[0104] hiPSC-derived cerebral organoids (COs) were prepared as described in Example 1. In addition, they were genetically engineered to express green fluorescent protein (GFP) for visualization.

[0105] Brain tumor tissue was obtained from patients with IDH (isocitrate dehydrogenase) mutant glioma and pilocytic astrocytoma. The tissue was dissected into seeds and co-cultured with hiPSC-derived COs as described in Example 2 (Fig. 1a). At 4 weeks of age, host organoids were used for co-culture with tumor tissue. At this time point, both normal and B2M (beta2-microglobulin) knockout organoids expressed markers of neural stem cells (nestin), neuroblasts (DCX) and immature neurons (TUJ1) (Fig. 1b).

[0106] As shown in Figure 2a, the cell density of IDHmut glioma-derived tumor organoids (IPTO) was comparable to that of the parent tumor (H&E staining, left), and 2-HG secretion was detected. Representative IHC (IDHR132H) images showed the spatial distribution of IDHmut glioma cells in the hybrid tumor organoids (Figure 2b). As shown in Figure 2d, the expression of glial fibrillary acidic protein (GFAP), a marker for glioblastoma, was very high in the tumor area. Meanwhile, the tumor area was also indicated by the lack of GFP expression (Figure 2d). Similarly, the tumor cell density in pilocytic astrocytoma-derived organoids was also high, and proliferating cells in the tumor area (GFP-) were identified (Figure 2c).

[0107] Most IPTOs reached high cell populations (as indicated by bioluminescence signal intensity) around 2 weeks after co-culture, and therefore IPTOs at approximately 2 weeks were considered suitable for drug testing.

[0108] Culturing brain tumors has proven challenging. Furthermore, there are no models of brain tumors that harbor mutations in IDH, which are found in 70-80% of WHO grade 2 or 3 astrocytomas and oligodendrogliomas. Therefore, the observation that IDHmut gliomas could be cultured in hybrid tumor organoids, as evidenced by high tumor cell density and 2-hydroxyglutarate (HG) secretion (Figure 2b), was unexpected.

[0109] Also, compared to high-grade gliomas, few preclinical models of pilocytic astrocytoma have been validated. Encouragingly, in this instance, the pilocytic astrocytoma survived in the IPTO derived from it (Fig. 2c), thus demonstrating that IPTO is an excellent medium for culturing brain tumors, including slow-growing ones.

[0110] DNA methylation profiles have been shown to lead to diagnostic accuracy compared to standard methods. To investigate whether IPTO recapitulates the molecular pathology of the original tumor, DNA methylation analysis was performed, and findings revealed that the epigenetic signature of glioblastoma-derived IPTO is similar to the corresponding parent tumor. Whole-genome DNA methylation arrays have been used for molecular classification of human brain tumors, which has become the new standard for WHO2021 brain tumor classification. Figure 5 shows a t-SNE plot showing that similar tumors cluster together. Each spot represents a sample (parent tumor or IPTO). As shown in the figure, IPTO clusters very closely with the parent tumor, especially in the inflated ones.

[0111] Furthermore, copy number variations (CNVs) in IPTO derived from brain metastases were largely identical to those in the original tumors.Similarly, single-cell RNA sequencing (Figure 6) showed that IPTO samples co-clustered with the parental tumors, indicating that IPTO maintains the tumor microenvironment of the parental tumor.

[0112] In a recent study of direct culture of glioblastoma tissue alone (Jacob et al., 2019), various stromal cells were maintained in the patient-derived organoids (PDO); however, macrophage / microglial cell populations were gradually diminished. In contrast, IPTO benefited from support from the host organoid, and macrophage / microglial cell populations from the majority of samples were stable or even enriched (Figure 3a-b). Of note, tumor-residual T cells were detectable when tumors were cultured in IPTO (Figure 3c-e). Furthermore, microvascular structures (Figure 3f) and endothelial cells (Figure 3g) were preserved in the derived IPTO.

[0113] Maintaining the three-dimensional tissue structure and crosstalk between non-tumor and tumor cells is believed to better recapitulate the brain tumor microenvironment and better support the proliferation of tumor and stromal cells. The excellent reproducibility of tumor organoids in the tumor ecosystem should lead to precise therapeutic responses. The IPTOs prepared here were then used to test the efficacy and safety of candidate drugs.

[0114] Four compounds, ComP11, ComP31, ComP12, and ComP60, were added to IPTO and their effects on cell viability were assessed by BrdU staining. Drug efficacy was quantified as the ratio of BrdU+ cells to GFP- cells (tumor cells, Figure 4a), and drug safety was quantified as the ratio of BrdU+ cells to GFP+ cells (normal cells, Figure 4b). ComP11 and ComP60 appeared to be both effective and safe, as assessed by these IPTOs.

[0115] Example 4. Drug susceptibility testing using IPTO and clinical validation In this example, the IPTO model and drug screening procedures illustrated in Example 3 were used to select drugs and validate them in human patients.

[0116] Before drug testing, IPTO were transferred to a 24-well plate, one IPTO per well, and 3–6 IPTO were randomly selected as groups for drug testing. The viability of IPTO was monitored by bioluminescence imaging using a Quick View 3000 Imaging System (Bio-Real Sciences). Luciferase expressed in IPTO converts the substrate D-luciferin to oxyluciferin in an oxygen- and ATP-dependent process, leading to the emission of photons. D-luciferin was added to the medium of IPTO at a concentration of 150 μg / mL. After 15 min, photons emitted from IPTO were monitored with a Quick View 3000 Imaging System, and the total signal was calculated in photons / second (p / s).

[0117] After signal monitoring, the average signal of IPTO in the drug test group was normalized to the average signal of the control group, and the fold change of the signal at the end of the drug test relative to the signal at the beginning was calculated. If the signal continuously decreased and the fold change was less than 1, the IPTO was considered to be sensitive to the drug; if the signal increased or did not change and the fold change was greater than 1, the IPTO was considered to be insensitive to the drug.

[0118] In this study, IPTO prepared from some glioblastoma patients was determined to be sensitive to temozolomide (TMZ) and others were determined to be insensitive. All patients were treated with TMZ. The follow-up period was 15 months. As shown in Figure 7, patients determined to be sensitive to TMZ (TMZ-sensitive) had a significantly longer progression-free survival (PFS) than patients determined to be insensitive to TMZ (TMZ-resistant). Thus, these results confirmed the accuracy of IPTO for testing the sensitivity of patients to potential treatments.

[0119] [References] Cibelli, JB, Grant, KA, Chapman, KB, Cunniff, K., Worst, T., Green, HL, Walker, SJ, Gutin, PH, Vilner, L., Tabar, V., et al. (2002). Parthenogenetic stem cells in nonhuman primates. Science (New York, NY) 295, 819. 10.1126 / science.1065637. da Silva, B., Mathew, R.K., Polson, E.S., Williams, J., and Wurdak, H. (2018). Spontaneous Glioblastoma Spheroid Infiltration of Early-Stage Cerebral Organoids Models Brain Tumor Invasion. SLAS discovery : advancing life sciences R & D 23, 862-868. 10.1177 / 2472555218764623. Hubert, C.G., Rivera, M., Spangler, L.C., Wu, Q., Mack, S.C., Prager, B.C., Couce, M., McLendon, R.E., Sloan, A.E., and Rich, J.N. (2016). A Three-Dimensional Organoid Culture System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo. Cancer research 76, 2465-2477. 10.1158 / 0008-5472.can-15-2402. Jacob, F., Salinas, R.D., Zhang, D.Y., Nguyen, P.T.T., Schnoll, J.G., Wong, S.Z.H., Thokala, R., Sheikh, S., Saxena, D., Prokop, S., et al. (2019). A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity. Cell. 10.1016 / j.cell.2019.11.036. Janus, E.S.R.L.N.K.N.A.T.J.D. (2008). Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Kim, K.D., G (2010). Methods for producing embryonic stem cells from parthenogenetic embryos. patent application 12 / 440,964 Lancaster, M.A., and Knoblich, J.A. (2014). Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc 9, 2329-2340. 10.1038 / nprot.2014.158. Lancaster, M.A., Renner, M., Martin, C.A., Wenzel, D., Bicknell, L.S., Hurles, M.E., Homfray, T., Penninger, J.M., Jackson, A.P., and Knoblich, J.A. (2013). Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379. 10.1038 / nature12517. Linkous, A., Balamatsias, D., Snuderl, M., Edwards, L., Miyaguchi, K., Milner, T., Reich, B., Cohen-Gould, L., Storaska, A., Nakayama, Y., et al. (2019). Modeling Patient-Derived Glioblastoma with Cerebral Organoids. Cell reports 26, 3203-3211 e3205. 10.1016 / j.celrep.2019.02.063.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0121] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like, are intended to be interpreted broadly and without limitation. Furthermore, the terms and expressions employed herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention as claimed.

[0122] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications, improvements and variations of the invention disclosed and embodied therein may be adopted by those skilled in the art, and such modifications, improvements and variations are deemed to be within the scope of the present invention. The materials, methods and examples provided herein are representative of preferred embodiments and are illustrative, and are not intended to limit the scope of the invention.

[0123] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a provisos or negative limitation removing any subject matter from the genus, whether or not the excised material herein is specifically described.

[0124] Furthermore, where features or aspects of the invention are described in terms of the Markush group, those skilled in the art will recognize that the invention is thereby also described in terms of individual members or subgroups of members of the Markush group.

[0125] All publications, patent applications, patents, and other documents mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0126] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative, but not limiting, of the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which this disclosure pertains.

Claims

1. A hybrid organoid containing tumor tissue embedded in a cerebral organoid.

2. The hybrid organoid according to claim 1, wherein the cerebral organoid is differentiated from stem cells.

3. The hybrid organoid according to claim 1, wherein the stem cells are induced pluripotent stem cells (iPSCs).

4. The hybrid organoid according to claim 1, wherein the cerebral organoid expresses at least one marker selected from the group consisting of neuroepithelial stem cell protein (nestin), doublecortin (DCX), neuron-specific class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin, and T-box brain transcription factor 1 (TBR1).

5. The hybrid organoid according to claim 1, wherein the size of the tumor tissue when placed within the cerebral organoid is 0.2 mm to 5 mm in diameter.

6. The hybrid organoid according to claim 1, wherein the size of the tumor tissue when placed within the cerebral organoid is 0.5 mm to 2 mm in diameter.

7. The hybrid organoid according to claim 1, wherein the tumor tissue is brain tumor tissue.

8. The hybrid organoid according to claim 7, wherein the brain tumor is selected from the group consisting of glioblastoma, pilocytic astrocytoma, oligodendroglioma, and metastatic tumors originating from another tissue.

9. The hybrid organoid according to claim 8, characterized in that a portion of the brain tumor has a mutation in isocitrate dehydrogenase (IDH).

10. The hybrid organoid according to claim 1, wherein the tumor tissue comprises tumor cells and adjacent non-tumor stromal cells.

11. The hybrid organoid according to claim 1, wherein the number of tumor cells in the tumor tissue has increased by at least 50% compared to when the tumor tissue was first placed in the cerebral organoid.

12. The hybrid organoid according to claim 1, wherein the number of tumor cells in the tumor tissue has increased at least twice compared to when the tumor tissue was first placed in the cerebral organoid.

13. The hybrid organoid according to claim 1, wherein the tumor tissue is at least twice as large as when it was first placed in the cerebral organoid.

14. The hybrid organoid according to claim 1, expressing at least one marker selected from the group consisting of platelet endothelial cell adhesion molecule (PECAM-1, CD31), protein tyrosine phosphatase, receptor type, C (PTPRC, CD45)), differentiation cluster 68 (CD68), double cortin (DCX), glial fibrillary acidic protein (GFAP), glycerol-3-phosphate dehydrogenase 1 (GPD1), allograft inflammation factor 1 (Iba1), proliferation marker Ki-67 (Ki67), microtubule-associated protein 2 (MAP2), neuroepithelial stem cell protein (nestin), oligodendrocyte transcription factor (Olig2), S100beta chain, SRY (sex-determining region Y)-box 2 (Sox2), and neuron-specific class III β-tubulin (TuJ1).

15. A method for evaluating a candidate anticancer drug, comprising contacting a hybrid organoid described in any one of claims 1 to 14 with the candidate anticancer drug and examining the change in the number of cells in the hybrid organoid.

16. The method according to claim 15, wherein a reduction in the number of living tumor cells within the hybrid organoid indicates the effectiveness of the anticancer agent.

17. The method according to claim 15, wherein a decrease in the number of living host cells within the hybrid organoid indicates toxicity of the anticancer drug.

18. The method according to claim 15, wherein the hybrid organoid has been cultured for 1 to 16 weeks after the tumor tissue was first placed in the cerebral organoid.

19. The method according to claim 15, wherein the hybrid organoid has been cultured for 1 to 3 weeks after the tumor tissue was first placed in the cerebral organoid.

20. The method according to claim 18 or 19, wherein the hybrid organoid is cryopreserved and recovered.

21. A method for preparing a hybrid organoid according to any one of claims 1 to 14, comprising placing tumor tissue in the incision site of the cerebral organoid.

22. The method according to claim 21, further comprising covering the hybrid organoid with a Matrigel® solubilized basement membrane matrix and solidifying the matrix.

23. The method according to claim 21, wherein the size of the tumor tissue when placed within the cerebral organoid is 0.2 mm to 5 mm in diameter.

24. The method according to claim 23, wherein the size of the tumor tissue when placed within the cerebral organoid is 0.5 mm to 2 mm in diameter.

25. The method according to claim 21, wherein the tumor tissue is brain tumor tissue.

26. The method according to claim 25, wherein the brain tumor is selected from the group consisting of glioblastoma, pilocytic astrocytoma, oligodendroglioma, and metastatic tumors originating from another tissue.

27. The method according to claim 21, wherein the cerebral organoid is prepared from induced pluripotent stem cells (iPSCs).

28. The method according to claim 27, wherein the cerebral organoid expresses at least one marker selected from the group consisting of neuroepithelial stem cell protein (nestin), doublecortin (DCX), neuron-specific class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), proliferation marker Ki-67 (KI67), paired box 6 (PAX6), vimentin, and T-box brain transcription factor 1 (TBR1).

29. The method according to claim 21, further comprising cryopreserving the hybrid organoid.

30. The method according to claim 29, wherein the cryopreservation is carried out in a cryopreservation medium containing a ROCK inhibitor.

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