METHOD FOR PREPARING FIBROSIS-ENCAPSULATED TUMOROID, AND USE THEREOF

Abstract

The present invention relates to a method for producing a fibrosis-encapsulated tumoroid (FET), and the use of the fibrosis-encapsulated tumoroid. According to the present invention, an analogue that is close to real solid cancer tissue is produced using induced pluripotent stem cell-derived cell. The analogue has significant improvement over conventional tumoroids, which fail to perfectly reflect the characteristics of human solid cancer and have a very high probability of failure in the clinical validation stage. Thus, the present invention is expected to be widely used in the fields of new anticancer drug development and precision medicine.

Claims

1. A method for producing a fibrosis-encapsulated tumoroid, the method comprising steps of (a) obtaining cancer cells isolated from a subject; and (b) co-culturing the cancer cells with induced pluripotent stem cells (iPSCs) or induced pluripotent stem cell-derived cells to form a spheroid-shaped culture.

2. The method of claim 1, wherein the cancer cells comprise non-spheroid-forming cells (NSFs).

3. The method of claim 2, wherein the non-spheroid-forming cells (NSFs) are formed into the spheroid-shaped culture by the induced pluripotent stem cells or iPSC-derived mesenchymal stem cells (iMSCs).

4. The method of claim 1, wherein the co-culture in step (b) is performed by mixing and culturing the cancer cells and the induced pluripotent stem cells (iPSCs) or induced pluripotent stem cell-derived cells at a cell number ratio of 0.01:1 to 100:1.

5. The method of claim 4, wherein the co-culture in step (b) is performed by mixing and culturing the cancer cells and the induced pluripotent stem cells (iPSCs) or induced pluripotent stem cell-derived cells at a cell number ratio of 1.5: 1 to 2.5:1.

6. The method of claim 1, further comprising step (c) of inducing a fibrotic layer on the culture.

7. The method of claim 6, further comprising a step of compacting the induced fibrotic layer.

8. The method of claim 6, further comprising step (d) of selecting a fibrosis-encapsulated tumoroid (FET) on which the fibrotic layer has been induced.

9. The method of claim 6, wherein step (c) comprises encapsulating the fibrotic layer by at least one cell type selected from the group consisting of fibroblasts, fibroblast precursor cells, cancer-associated fibroblasts, induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), and iPSC-derived cancer-associated fibroblasts.

10. The method of claim 8, further comprising step (e) of inducing a vascular cell layer on the fibrotic layer.

11. The method of claim 10, wherein the vascular cell layer is induced by endothelial cells or vascular endothelial cells.

12. The method of claim 11, wherein the endothelial cells or vascular endothelial cells are induced from induced pluripotent stem cells (iPSCs).

13. The method of claim 1, wherein the cancer is a cancer selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, and combinations thereof.

14. The method of claim 1, wherein the co-culture is performed in at least one culture medium selected from the group consisting of DMEM (Dulbeco’s modified Eagle’s medium), IMDM (Iscove’s modified Dulbecco’s medium), a-MEM (alpha modification of Eagle’s medium), TI-free OGM (TI-free osteoblast growth medium), F12 (nutrient mixture F-12), RPMI 1640 (RPMI 1640 medium), Williams’ s medium E, McCoy’s 5A, essential 8 (E8) medium, SFM medium (StemPro-34 SFM medium), N2B2 medium, OBM medium (OGM osteoblast growth medium), growth medium-5, growth medium-10, and DMEM/F12 (Dulbecco’s modified Eagle medium: nutrient mixture F-12).

15. The method of claim 14, wherein the culture medium further contains B27, N2, G5, or forskolin.

16. The method of claim 14, wherein the culture medium further contains cancer-associated fibroblasts, endothelial cells, mesenchymal stem cells (iMSCs) differentiated from induced pluripotent stem cells (iPSCs), or a growth factor.

17. The method of claim 16, wherein the growth factor is at least one selected from the group consisting of vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGFA), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1).

18. The method of claim 1, wherein the co-culture is performed for 2 days to 14 days.

19. A fibrosis-encapsulated tumoroid produced by the method of any one of claims 1 to 18.

20. (canceled)

21. A method for screening a substance for treatment or alleviation of cancer, the method comprising steps of: (a) preparing the tumoroid of claim 19; (b) treating the tumoroid with an anticancer candidate substance; and (c) determining that the candidate substance is the substance for treatment or alleviation of cancer, when the tumoroid exhibits an anticancer effect.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1 is a schematic view showing an experimental design for producing a solid cancer analogue using induced pluripotent stem cells.

[0076] FIG. 2 shows a process of inducing spheroid formation from non-spheroid-forming cells (NSCs).

[0077] FIG. 3 shows results indicating that spheroid formation was induced from various cancer cells.

[0078] FIG. 4 shows results indicating the natural death of linker cells.

[0079] FIG. 5 is a schematic view sequentially showing processes of inducing of FET and eFET, which are fibrosis-encapsulated tumoroids.

[0080] FIG. 6 shows a series of processes of screening an anticancer agent using the fibrosis-encapsulated tumoroid.

[0081] FIG. 7 shows the results of observing the morphology of specific fibroblasts shown in various breast cancer cell lines.

[0082] FIGS. 8A and 8B show images after fluorescence staining of a fibrosis-encapsulated tumoroid (FET or eFET).

[0083] FIG. 9 shows the results of confirming the possibility of controlling the thickness of a fibrotic layer using fibroblasts.

[0084] FIGS. 10A and 10B show the results of comparing anticancer agent resistance between a simple tumoroid and a fibrosis-encapsulated tumoroid.

BEST MODE

[0085] One embodiment of the present invention is directed to a method for producing a fibrosis-encapsulated tumoroid, the method comprising steps of: (a) obtaining cancer cells isolated from a subject; and (b) co-culturing the cancer cells with induced pluripotent stem cells (iPSCs) or induced pluripotent stem cell-derived cells to form a spheroid-shaped culture.

[0086] Another embodiment of the present invention is directed to a fibrosis-encapsulated tumoroid produced by the method of the present invention.

[0087] Still another embodiment of the present invention is directed to a non-human animal model transplanted with the fibrosis-encapsulated tumoroid produced by the method of the present invention.

[0088] Yet another embodiment of the present invention is directed to a method of screening an anticancer agent using the fibrosis-encapsulated tumoroid produced by the method of the present invention.

MODE FOR INVENTION

[0089] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

Example 1: Culture of Cancer Cell Lines and Preparation of Linker Cells

1.1 Culture of Cancer Cell Lines

[0090] The present inventors conducted all experiments with the approval of the Institutional Review Board (IRB) of the Yonsei University College of Medicine, and used the previously established induced pluripotent stem cell pool in the experiments. In addition, human breast cancer cell lines (BT-474, MCF-7, SK-BR-3, BT-20, MDA-MB-231 and Hs578T) and pancreatic cancer cell lines (PANC-1, MIA Paca-2, AsPC-1 and BxPC-3) were obtained from the American Type Culture Collection (ATCC), cultured in 10% FBS-containing media, and used in the experiment. All media used in the Examples below to culture other cells in addition to the culture of cancer cells have known compositions or are commercially available products. In the present invention, for optimization of culture, a mixture of two or more culture media was used or VEGFA or the like was added to the medium.

1.2 Preparation of Linker Cells

[0091] Using an embryonic body (EB) formation medium (EBFM, AggreWell, STEMCELL Technologies), an embryonic body (EB) was prepared with 1x10.sup.4 induced pluripotent stem cells (iPSCs). The embryo body was cultured for 5 days in Essential 8 (E8) medium containing the components shown in Table 1 below. On day 5, the embryo body was attached to a 0.1% gelatin coated dish and additionally cultured for 2 weeks in a mesenchymal stem cell (MSC) culture medium composed of the components shown in Table 2 below and allowed to differentiate into iPSC-derived mesenchymal cells (iMSCs), thus preparing linker cells.

TABLE-US-00001 Essential 8 (E8) medium component Final concentration DMEM/F12 with glutamine and HEPES 1 x NaHCO.sub.3 543 .Math.g/ml Sodium selenite 14 ng/ml Transferrin 10.7 .Math.g/ml Insulin 19.4 .Math.g/ml L-ascorbic acid-2-phosphate magnesium 64 .Math.g/ml FGF2 100 ng/ml TGF-b1 100 ng/ml

TABLE-US-00002 MSC culture medium component Final concentration Minimum Essential Medium Eagle - alpha modified (α-MEM) 1 x Fetal calf serum (FCS) 10% penicillin/streptomycin 50 U.Math.ml.sup.-1 / 50 mg.Math.ml.sup.-1 Sodium pyruvate 1 mM 1-ascorbate-2-phosphate 100 .Math.M 1-Glutamine 2 mM non-essential amino acids 1 x HEPES 10 mM

[0092] The present invention utilized induced pluripotent cells in a manner differentiated from conventional inventions, and FIG. 1 is a schematic view showing an experimental design for producing a solid cancer analogue using the induced pluripotent cells. Hereinafter, a process of producing a fibrosis-encapsulated tumoroid according to the design will be described in detail.

Example 2: Induction of Tumoroid Formation From Non-Spheroid-Forming Cancer Cells

[0093] Cancer cells and iMSCs were detached from culture plates and washed with PBS, and then a pellet form was prepared. Cancer cells (8 x 10.sup.4 cells/ml) and iMSCs (4 x 10.sup.4 cells/ ml) were prepared using a mixed medium of organoid basal medium (OBM) and EB formation medium (EBFM). Here, the mixed medium was maintained at an OBM: EBFM ratio of 1:1. The composition of the OBM medium is shown in Table 3 below.

TABLE-US-00003 OBM medium components Final concentration A83-01 500 nM Y-27632 5 mM SB202190 500 nM B27 1 x N-Acetylcysteine 1.25 mM Nicotinamide 5 mM GlutaMax 100x 1 x HEPES 10 mM Penicillin/streptomycin 100 U.Math.ml.sup.-1 / 100 mg.Math.ml.sup.-1 Advanced DMEM / F12 1 x

[0094] On a plate (ultra-low attachment, U-shape plate), 12.5 .Math.l of the above-prepared cancer cells and 12.5 .Math.l of iMSCs were mixed together. Spheroid formation was induced for 1 day in a cell culture incubator at 37° C. under 5% CO.sub.2. After the spheroid formation was induced, compaction of the spheroid was induced for additional one day. In this case, when the number of the cells was increased while maintaining the ratio of the cancer cells to the iMSCs at 2:1 (e.g., 1x10.sup.3 cells: 5×10.sup.2 cells), the size of the tumoroid could be increased. However, it was confirmed that the above-mentioned number of cells was most suitable to optimize the additional construction of a fibrotic layer and a vascular cell layer. In addition, the same volume (25 .Math.l) of growth medium-10 was added to induce spheroid compaction. In this case, the maintenance of a serum-free state for more than 24 hours could lead to changes in transcript expression in the cells, and thus a medium containing serum was additionally added. Changes in transcript expression could lead to changes in cell properties, and the final serum concentration was set to 5%. The composition of the growth medium-10 is shown in Table 4 below.

TABLE-US-00004 Growth medium components Final concentration Advanced DMEM / F12 1 x FBS 10% Penicillin / streptomycin 100 U.Math.ml.sup.-1 / 100 mg.Math.ml.sup.-1

[0095] FIG. 3 shows depicts photographs showing the results of inducing spheroid formation from the non-spheroid-forming cells, prepared according to Example 2, in different types of cancer cells. In FIG. 3, NSF#1 represents the result obtained using the breast cancer cell line MDA-MB-231, NSF#2 represents the result obtained using the breast cancer cell line SK-BR-3, and NSF#3 represents the results obtained using the pancreatic cancer cell line AsPC-1. Referring to FIG. 3, it could be confirmed that non-spheroid-forming cells of various carcinomas were formed into spheroids within 1 day by linker cells (iMSCs). As a result of staining the linker cells (which are iMSCs differentiated from iPSCs) with a green fluorescent substance, it was confirmed that 90% or more of the linker cells were dead within 5 days after being used for spheroid formation (see FIG. 4). Due to the death of these linker cells, the characteristics of the tumoroid could be maintained. Another difference from the conventional inventions is that formation of the non-spheroid-forming cells into spheroids was induced using the linker cells.

Example 3: Induction of Fibrotic Layer Formation (Fibrosis-Encapsulated Tumoroid (FET)) Using Induced Pluripotent Stem Cells

[0096] FIG. 5 schematically shows a process of inducing precursor cells (iMSCs) of activated fibroblasts (major cells constituting fibrotic tissue) from induced pluripotent stem cells (iPSCs), producing an FET by inducing the formation of a fibrotic layer on the outside of the tumoroid, and producing an eFET (endothelial cell-layered FET) by inducing the formation of a vascular cell layer. The fibroblast precursor cells derived or originating from induced pluripotent stem cells are called iMSCs, which are differentiated into activated fibroblasts by TGF-β (transforming growth factor-beta) secreted by cancer cells. In this experiment, iMSCs overexpressing a fluorescent protein was used to confirm the formation of the fibrotic layer or the vascular cell layer. First, using encapsulation medium 1, on a plate (ultra-low attachment, U-shape plate), the formation of a fibrotic layer on the outside of the tumoroid was induced using 3x10.sup.3 iMSCs that are triple the number of cells used for tumoroid formation. Here, the iMSCs were prepared at a concentration of 1.2×10.sup.5 cells/ml and used in an amount of 25 .Math.l, and as encapsulation medium 1 used, a mixed medium of equal amounts of TGF-b inhibitor (TI)-free organoid growth medium (OGM) and embryonic body (EB) formation medium (EBFM/AggreWell - STEMCELL Technologies) was used. The composition of the TI-free OGM medium is shown in Table 5 below.

TABLE-US-00005 TI-free OGM medium components Final concentration R-Spondin 1 conditioned medium 10% or R-Spondin 3 250 ng.Math.ml.sup.-1 Neuregulin 1 5 nM FGF 7 5 ng.Math.ml.sup.-1 FGF 10 20 ng.Math.ml.sup.-1 EGF 5 ng.Math.ml.sup.-1 Noggin 100 ng.Math.ml.sup.-1 Y-27632 5 mM SB202190 500 nM B27 supplement 1x N-acetylcysteine 1.25 mM Nicotinamide 5 mM GlutaMax 100x 1x Hepes 10 mM Penicillin/streptomycin 100 U.Math.ml.sup.-1 / 100 mg.Math.ml.sup.-1 Primocin 50 mg.Math.ml.sup.-1 Advanced DMEM/F12 1x

[0097] Thereafter, the cells were subjected to spheroid formation and compaction processes in a cell culture incubator at 37° C. under 5% CO.sub.2 for additional 2 days. Changes in the morphology of fibroblasts activated by culture media for various human breast cancer cell lines (BT-474, MCF-7, SK-BR-3, BT-20, MDA-MB-231, and Hs578T) that have undergone the above-described processes were observed with a fluorescence microscope, and the results are shown in FIG. 7. In light of the above results, it can be confirmed that various types of fibroblasts were observed depending on the cancer cell line. In addition, unlike a simple tumoroid (Sci Rep. 2016 Jan 11; 6:19103.) consisting of central dead cells, intermediate quiescent cells and outer proliferating cells, a fibrotic layer was formed on the outer layer of a conventional tumoroid, and the resulting tumoroid was fluorescently stained and the fluorescence image thereof was acquired. As a result, as shown in FIG. 8A, it could be confirmed that a fibrotic layer was produced on the outside of the cancer tissue.

Example 4: Induction of Vascular Cell Layer (Endothelial Cell-Layered FET, eFET) Formation Using Induced Pluripotent Stem Cells

[0098] Using encapsulation medium 2, on a plate (ultra-low attachment, U-shape plate), formation of an EC layer on the outside of the FET subjected to the compaction process was induced using 9 x 10.sup.3 endothelial cells (ECs) which are triple the number of cells used to form the fibrotic layer. Here, the ECs were prepared at a concentration of 3.6 x 10.sup.5 cells/ml and used in an amount of 25 .Math.l, and encapsulation medium 2 was prepared by adding 50 ng/ml of VEGFA to encapsulation medium 1. Next, spheroid formation was induced in a cell culture incubator at 37° C. under 5% CO.sub.2 for 1 day, and maintenance medium was prepared by adding 20 ng/ml of VEGFA to growth medium-5 shown in Table 6 below. Final spheroid formation was induced by replacing the medium with the maintenance medium.

TABLE-US-00006 Growth medium 5 components Final concentration Advanced DMEM / F12 1 x FBS 5% Penicillin / streptomycin 100 U.Math.ml.sup.-1 / 100 mg.Math.ml.sup.-1

[0099] FIG. 8B shows an image of the eFET produced by the above-described method. The vascular endothelial cells were not fluorescently stained, and thus the vascular endothelial cell layer on the fluorescence image was indicated by a white dotted line. It was confirmed that, in the case of the fibrosis-encapsulated tumoroid (FET or eFET) produced by the method of Examples 3 and 4, the thickness of the fibrotic layer varied according to the degree of progression of carcinoma or cancer (see FIG. 9). The thickness of the fibrotic layer can be controlled by controlling the number of fibroblasts, and a fibrosis-encapsulated tumoroid with each fibrotic layer thickness can be produced.

Example 5: Application of Optimal Medium Composition Ratio for Efficient EC Differentiation

[0100] iPSCs were dispensed on a matrigel-coated dish at a density of 4 x 10.sup.4 cells/cm.sup.2, and the medium was replaced with pre-warmed N2B27 medium supplemented with CHIR-99021 (8 .Math.M) and hBMP4 (25 ng/ml) in order to induce lateral mesoderm, and the cells were maintained for 3 days without additional medium replacement. The composition of the medium is shown in Table 7 below, and the ratio of DMEM/F12: Meurobasal medium was set to 1:1.

TABLE-US-00007 N2B27 medium components Final concentration DMEM/F12 1 x Meurobasal medium 1 x B27 1.94% N2 0.97% P-mercaptoethanol 0.097%

[0101] On days 4 and 5, the medium was replaced with StemPro-34 SFM medium supplemented with vascular endothelial growth factor (VEGF, 200 ng/ml) and forskolin (2 .Math.M) in order to induce endothelial cells, and at this time, the medium was replaced daily, unlike during the above three days. On day 6, endothelial cells were sorted and only CD144-positive endothelial cells were selected and plated again. At this time, StemPRo-34 supplemented with VEGF (50 ng/ml) was used as the medium, and the cells were dispensed on fibronectin-coated plates. The composition of the StemPro-34 SFM medium purchased from ThermoFisher Scinentific is shown in Table 8 below.

TABLE-US-00008 StemPro-34 SFM medium components Final concentration StemPro-34 SFM medium 1 x Penicillin / Streptomycin 100 U.Math.ml.sup.-1 / 100 mg.Math.ml.sup.-1 GlutaMax 100x 1 x

[0102] The StemPro-34 medium contains StemPro-34 SFM medium and StemPro-34 supplement. From day 7, the cells were cultured and maintained in Vasculife complete medium.

Example 6: Examination of Anticancer Drug Resistance of Fibrosis-Encapsulated Tumoroid

[0103] The present inventors performed a drug resistance test in order to evaluate whether it was possible to select drugs, which can be effective in the clinical stage, using the fibrosis-encapsulated tumoroid which is a solid cancer tissue analogue produced by the methods of Examples 1 to 4 (see FIGS. 10A and 10B). In addition, FIG. 6 shows the time flow from the above-described tumoroid formation process to the production of the fibrosis-encapsulated tumoroid and the drug test process. The drug test was conducted through cell viability measurement. The cell viability of each of the group containing the anticancer drug and the control group not containing the anticancer drug was measured after replacing the medium with maintenance medium. At this time, the fluorescent signal was measured using a confocal microscope, and the living cancer cells on the tumeroid that did not display fluorescence were confirmed by staining with Calsein-AM-blue, and the dead cancer cells were confirmed by staining with propidium iodide (PI). The cell viability of the solid cancer tissue analogue was measured using Promega’s CellTiter-Glo luminescent cell viability assay as another measurement method, and the death of cancer cells by the anticancer drug could be indirectly confirmed by comparison with the control group. The experimental results are shown in FIGS. 10A and 10B. It was confirmed that the fibrosis-encapsulated tumoroid (FET) showed high anticancer drug resistance, that is, high cell viability, and a stronger Calsein-AM signal, compared to a simple tumoroid (tumor only) (see FIG. 10A). Referring to FIG. 10B, it can be confirmed that the fibrosis-encapsulated tumoroid (FET) shows statistically significant resistance to the anticancer drug (cisplatin, 100 .Math.M), compared to the simple tumoroid. The fibrosis-encapsulated tumoroid produced as described above mimicked a series of processes in which the anticancer agent passes sequentially through the blood vessel wall and fibrotic tissue and reaches cancer tissue, in the same way as in the human body. Thus, it is preferable to finally produce the eFET and conduct an anticancer drug test using the eFET, but only the FET may be selectively used in order to reduce cost or time. When the FET is used, the experimental period can be shortened by 2 days, enabling rapid preemptive selection of anticancer drugs for cancer patients having time and physical limitations. Thus, the FET is expected to be widely used in the precision medical field.

[0104] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0105] The present invention relates to a method for producing a fibrosis-encapsulated tumoroid (FET) and the use of the fibrosis-encapsulated tumoroid (FET). When the method of the present invention is used, it may dramatically increases the success probability of a candidate substance in the clinical verification stage by perfectly reflecting the characteristics of human solid cancer. Thus, the method may be widely used in the fields of new anticancer drug development and precision medicine.