KR20190059864A - Multicellular tumor spheroid for hepatocellular carcinoma therapy and drug screening method using the same - Google Patents

Abstract

The present invention relates to a multicellular tumor spheroid for hepatocellular carcinoma therapy and a drug screening method using the same. The multicellular tumor spheroid of the present invention makes it possible to construct a three-dimensional tumor microenvironment platform and to screen for a patient-specific hepatocellular carcinoma therapeutic agent capable of alleviating side effects caused by nonspecific drugs.

Description

[0001] The present invention relates to a multicellular tumor spheroid for the treatment of liver cancer, and a drug screening method using the same. [0002]

The present invention relates to a multicellular tumor spheroid for the treatment of liver cancer and a drug screening method using the same. More particularly, the present invention relates to a method of screening a patient-customized therapeutic agent using a multicellular tumor scaffold comprising liver cancer cells and stromal cells at a specific ratio and using the same.

Cancer is still the leading cause of human death, and hepatocellular carcinoma cells (HCC) is one of the most serious of these cancers. A typical primary treatment of hepatocellular carcinoma involves surgical resection of the tumor, and the secondary therapy is performed using sorafenib, a multiple tyrosine protein kinase inhibitor for metastatic or unresectable HCC. However, both therapies are often not effective in treating patients. Chronic infections due to hepatitis B and C and alcohol intoxication are the main causes of hepatocellular carcinoma with metastasis from other areas of the body. The recurrence rate after 3 years of resection is about 80%, and mortality rate is high when recurrence occurs after resection. From a clinical point of view, awareness of clinical aspects of patients during treatment is very important. The researchers sought to identify target genes and drug candidates for the diagnosis and treatment of hepatocellular carcinoma. However, the development of targeted drugs has not yet significantly improved the prognosis.

Because this difficulty is related to the in vivo condition of cancer in vitro, the inventors have developed a new model for drug screening in the relevant biological context. Tumor microenvironment (TME) plays an important physiological role in cell differentiation, tumorigenesis, metastasis and therapeutic efficacy. It is difficult to obtain relevant results regarding the formation of TME without considering clinical tumor conditions. Efforts to discover preclinical cancer drugs are currently being made using a two-dimensional (2D) cell-based assay model. However, 2D cell-based models do not predict in vivo efficacy and the success rate of new drug development for clinical use is low. Therefore, the 2D analysis system may not be useful because the result data can not be used for translation studies. On the other hand, complex 3D (3D) cell culture systems have better established therapeutically relevant parameters of in vivo tumors such as 3D cell context, pH and oxygen gradient, infiltration of growth factors and distribution of proliferating / necrotic cells, With this system, more effective simulation is possible. Especially in 3D culture systems, hepatocytes perform a number of liver functions better than 2D culture systems, including albumin and urea synthesis, bile secretion and cell polarization. The advantage of 3D cell culture compared to 2D systems is that in the 3D system the cells form multiple layers whereas the cells in the 2D system form a thin layer spread over the plastic surface. When testing a drug in a 2D model, the drug only needs to spread a short distance across the cell membrane to reach the target. On the other hand, situations in a 3D system are better able to express a tumor in vivo, and the drug has to spread to several layers of cells to reach the target.

Numerous references are referenced throughout the specification and are cited therein. The disclosure of the cited document is incorporated herein by reference in its entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2011-0077549

Recently, scientists have published a multicellular tumor model (MCTS) model that mimics the in vivo characteristics of tumors. This has become a powerful way to reproduce tumor complexity for anticancer studies and in vivo drug efficacy predictions.

Based on these considerations, the present inventors have made efforts to develop 3D tumor microenvironment for liver cancer-specific drug screening. As a result, the invention was completed by mimicking the in vivo characteristics and microenvironment of the tumor, constructing and verifying a highly reproducible MCTS-based high-throughput screening (HTS) system for multicellular HCC drug screening.

Accordingly, an object of the present invention is to provide a multicellular tumor spherical body comprising liver cancer cells and stromal cells.

It is another object of the present invention to provide a method for screening a therapeutic agent for liver cancer using the multicellular tumor globule.

Yet another object of the present invention is to provide a method for producing a tumor comprising the multicellular tumor spherical body; And a high-speed three-dimensional fluorescence imaging device.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide multicellular tumor spheroids comprising liver cancer cells and stromal cells.

The present inventors have reported interactions between tumors and stromal cells (including fibroblasts, endothelial cells, hepatic stellate cells and immune cells) in a spherical model system. Interaction between tumor cells and stromal cells can alter the expression of extracellular matrix molecules and epithelial mesotransfer (EMT) -related proteins in the MCTS model. Thus, the MCTS model mimics EMT behavior and in vivo propagation of cancer cells.

In this study, we improved the culture conditions of cancer cells and provided proof of concept to determine whether the MCTS model closely replicated TME in vivo. In addition, since 3D spherical body formation is a costly, cumbersome, and complex process for applying the MCTS model to a high-efficiency screening system, we have developed the most efficient method for producing 3D spherical bodies. Despite these difficulties, the present inventors have found that a single spherical body of uniform wall size and the same composition can be used as a highly reproducible MCTS-based high-throughput screening (HTS) for HCC drug screening using multi-wall plates to successfully screen possible drugs for HCC. System was built and verified. Thus, the MCTS-based HTS system provides a new model for anticancer drug screening that greatly improves the efficacy and reliability of drug identification for HCC treatment.

In a preferred embodiment of the present invention, the ratio of the number of liver cancer cells: stromal cells contained in the multicellular tumor globule is 49:51 to 30:70.

Since hepatocellular carcinoma cells are the most important key components in hepatocarcinoma tissues, it has been suggested that hepatocellular carcinomas should be the main constituents of tumor cells. Thus, the ratio of hepatocellular: stromal cells in MCTS was 7: 3 and the maximum ratio was 1: 1.

Furthermore, when cultured MCTS containing both spherical bodies (Huh7 spheres) composed of hepatocarcinoma cells and stromal cells, Huh7 spherical bodies exhibited tumor growth much faster than MCTS (Fig. 11), and more than 50% It has been assumed that it should be composed of tumor cells.

However, the inventors of the present invention have found that when the ratio of the number of epileptic cells exceeds 50%, they show greater resistance to the anticancer agent, which is surprising result.

Therefore, in order to provide a drug screening model with greater resistance to an anticancer drug, it is preferable that the MCTC of the present invention contains less than 50% of liver cancer cells and more than 50% of interstitial cell counts, more preferably, It is preferable that the ratio of cell number of cell: stromal cell is in the range of 49:51 to 30:70, and most preferably the ratio of cell number of liver cancer cell: stromal cell is 40:60.

In the MCTS of the present invention, the stromal cells are characterized by comprising at least one selected from the group consisting of hepatic stellate cells, fibroblasts, and endothelial cells.

Hepatic stellate cells (HSCs) are pericytes found in the perisinusoidal space around the liver and are one of the major cell types associated with hepatic fibrosis, which occurs in response to liver damage and forms scar tissue. The hepatic stellate cells are activated by chronic liver injury and lose the fat stored from the cells that store the steady state fat, and form the cytoskeletal protein α-SMA (smooth muscle α-actin) and the rough endoplasmic reticulum After activation with myofibroblasts, a large amount of extracellular matrix can be generated to promote hepatic fibrosis. Various receptors and intracellular signaling systems work to activate and sustain hepatic stellate cells.

Fibroblasts are a type of cell that synthesizes collagen and extracellular matrix, a stroma for animal tissue, and plays an important role in wound healing. Fibroblasts are known to be the most common cells in animal connective tissues, forming structural frameworks in animal tissues, producing glycoproteins, elastic fibers, collagen and glycosaminoglycans found in extracellular matrix and cytokines have.

Endothelial cells are vascular wall cells that not only produce vasoactive substances but also control the function of substances that are already produced and secreted. It is a cell that plays a crucial role in the interaction with various cells such as smooth muscle cells, fibrous cells and elastin which are distributed in the blood vessel walls. Endothelial cells do not have fixed and unchanging simple functions, and they have dynamic functions that can cope with various situations.

In addition, the multicellular tumor spheroids of the present invention may further include liver cancer stem cells, and may include liver cancer cells, hepatic stellate cells, fibroblasts, endothelial cells, and liver cancer stem cells according to an embodiment.

Stem cells refer to cells that have self-renewal ability and ability to differentiate into various cells to produce unlimited numbers of the same cells as themselves. In addition to the ability of stem cells to self-renew and differentiate, It refers to a cell capable of resistance to initiation, metastasis, recurrence, and anticancer drugs. Cancer stem cells can be generated by the degeneration of cells that have been transformed or differentiated by genetic and eugenic changes in normal stem cells.

Hepatocellular carcinoma originates from liver cancer stem cells. Various cells in the hepatic lineage can be reprogrammed into hepatoma stem cells and may originate from other cells.

In a preferred embodiment, the multicellular tumor spheroids of the present invention may be formed by three-dimensional stereochemistry of hepatocarcinoma cells derived from hepatocarcinoma patients. When such primary hepatocarcinoma-derived hepatocellular carcinoma cells are used, And to provide a guideline for personalized care for each patient, thereby reducing side effects caused by nonspecific drugs.

The culture can be performed using a medium containing stem cell culture medium to form a patient-customized multicellular tumor sphere containing liver cancer stem cells. The stem cell culture medium may be those conventionally used in the art. For example, growth factors derived from embryonic stem cells, fetal brain tissue, or human adult brain tissue may be added to the basic medium, It is not. The stem cell culture medium may also be purchased commercially, for example, B27 supplement from Invitrogen, Cellvento? Medium, a stem cell culture medium from Gibco, and the like, but the present invention is not limited thereto. Another aspect of the present invention is to provide a method for screening a therapeutic agent for liver cancer using the multicellular tumor globule according to the present invention.

Another aspect of the present invention is a method for preparing a tumor cell, comprising: (a) forming a multicellular tumor spheroid comprising liver cancer cells and stromal cells by three-dimensionally culturing liver cancer cells derived from a patient suffering from a liver cancer; (b) treating the candidate substance with the multicellular tumor scaffold; And (c) measuring a change in the size of the multicellular tumor spheroid. When the size of the multicellular tumor spheroid decreases due to the treatment of the tumor, The material is judged to be the patient-customized liver cancer treatment agent.

The screening method of the present invention can be carried out in various ways, and can be carried out in a high throughput manner according to various binding assays known in the art.

In the screening method of the present invention, the change in the size of the spheroids is detected by labeling hepatocarcinoma cells or other test substances with a detectable label and measuring the fluorescence intensity of a high-speed three-dimensional fluorescence imaging device, preferably a confocal imaging device And analyzing and measuring the spherical image obtained by using the spherical body.

The detectable label may be a chemical label (e.g., biotin), an enzyme label (such as horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase,? -Galactosidase and? - glucosidase), a radiolabel (e. g., C ^14, I ^125, P ³² and S ^35), fluorescent labels [e.g., coumarin, fluorescein, FITC (fluoresein Isothiocyanate), rhodamine 6G (rhodamine 6G), Roda Rhodamine B, TAMRA, Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl And FAM, luminescent markers, chemiluminescent markers, fluorescence resonance energy transfer (FRET) markers or metal markers (e.g., gold and silver), and the like.

The candidate substance analyzed by the screening method of the present invention is a single compound or a mixture of compounds (e.g., a natural extract or a cell or tissue culture). The candidate substance is preferably a compound library. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA).

Candidate materials to be tested can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, 1-compound 1 " library method, and synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl . Acad . Sci . USA . 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci. USA . 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed. Engl. 33,2059,1994; Carell et al., Angew . Chem . Int . Ed. Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994, and the like.

Another aspect of the present invention relates to the above-described multicellular tumor spherical body according to the present invention; And a high-speed three-dimensional fluorescence image device.

The liver cancer-specific therapeutic screening apparatus of the present invention provides a new type of drug screening platform that can overcome discrepancy in drug efficacy in vivo and in vitro, and is expected to be applicable to various types of cancer including liver cancer .

The multicellular tumor spheroids of the present invention can obtain the effect of a one-to-one mutation which not only presents new therapeutic agents but also therapeutic targets in new tumor microenvironment. Such a three-dimensional tumor microenvironment platform for patient-tailored treatment can reduce side effects caused by nonspecific drugs by providing standardized patient-specific care guidelines.

Figure 1: hepatocellular carcinoma (HCC) multicellular tumor spheroids (MCTS) that mimics the tissues of patients with established environmental model
Figure 1A shows drug susceptibility to sorapenib at 10 [mu] M for 96 hours in tumor spheres using HCC cell lines (Huh7, SNU449, PLC / PRF / 5 and Hep3B).
Figure 1B shows drug susceptibility to sorapenib at 10 [mu] M for 96 hours in tumor tissue derived from a patient tissue.
Fig. 1c shows representative histochemical images of CD31 (green), p75 (yellow) and FAP (red) expression after Hoechst 33342 staining of tissue nuclei from liver cancer patients.
Figure 1d shows drug susceptibility to 10 μM sorapenib in the constituent MCTS model in which the ratio of HCC and stromal cells (hepatic stellate cells, fibroblasts and vascular endothelial cells) is different.
Figure 1e shows the different cell viability after treatment with indicated concentrations of sorapenib in MCTS models with different compositions of HCC and stromal cells using resazurin assay.
Figure 1f shows HCC cell migration in MCTC models with different compositions of HCC and stromal cells (green for HUVEC, blue for WI38, red for LX2) after reattachment to the collagen coated plate.
All images were obtained using the Operetta® High Content search system, and the data were expressed as mean and standard deviation (SD) of three experiments (scale bar: 200 μm).
Figure 2: Multicellular tumor spheroid ( MCTS ) model as a model system to mimic the in vivo tumor microenvironment
FIG. 2A shows a gene expression map showing the magnification change ratio of MCTS model versus tumor spheroid.
Figure 2B shows a plot of gene cluster enrichment analysis of the upregulated gene (left panel) or downregulated gene (right panel) of the MCTS model versus normal liver (GSE64041).
Figure 2c shows a horizontal bar graph of molecular functions and biological processes exaggerated in a highly abundant gene of Huh7 (blue) and MCTS (orange) lysates.
Figure 3: Xenotransplantation model from multicellular tumor spheroids ( MCTS ) as a new and effective tool for the growth of human hepatocellular carcinoma in a mouse model
3A shows a xenograft model derived from MCTS prepared by subcutaneous injection of Huh7.5 cells, Huh7.5 spheroids or MCTS into nude mice.
Figure 3b shows the results of two measurements per week of animal body weight and tumor volume until the tumor size reached 2,000 mm < ³ &gt;.
Figure 3c shows a representative image of a trichrome-stained tumor of hematoxylin and eosin- or Masson in Huh7.5, Huh7.5 spheroids, or MCTS-derived xenografts. Inset is the macroscopic image of the tumor sample (scale bar: 100 μm).
Figure 3d shows the expression of CD31 and Ki-67 and immunofluorescence staining of nuclei in tumor sections of xenografts derived from Huh7.5, Huh7.5 spheroids or MCTS models. Inset shows a detailed area containing the blood vessel (scale bar: 100 μm).
Figure 4: HCC development of multicellular tumor spheroids (MCTS) model based phenomic screening for drug discovery
Figure 4a shows a MCTS model homogenously constructed with Huh7, LX2, WI38 and human umbilical vail endothelial cells.
FIG. 4B shows the drug susceptibility to tumor antigens (cisplatin, 5-FU and doxorubicin) of tumor spheroids and MCTS models.
Figure 4c shows the validation of phenotypic screening using the MCTS-based assay. Spherical images were obtained on 96-well and 384-well plates using Operetta® High Content imaging equipment. The obtained images were analyzed by measuring the size and intensity.
FIG. 4d shows the phenotype screening test using the MCTS-based assay. In the case of sorafenib IC ₅₀ as a low control and 0.5% DMSO (v / v) as a high control at 10 days, the brightfield and RFP- (Magnification: 10 times).
Figure 4e shows RFP intensities of Huh7 spheres in 96- and 384-well plates at 10 days. All images were obtained using the Operetta® High Content search system (scale bar: 200 μm, DMSO = dimethyl sulfoxide, 5-FU = 5-fluorouracil, RFP = red fluorescent protein).
5: Heat compound
5A is a bar graph showing the pharmacological action of the multicellular tumor complex blocking compound along the horizontal axis. The vertical axis represents the logarithm of the number of compounds having a specific pharmacological action. The blue bars represent the total number of compounds, including certain pharmacological actions (All). The red bars represent the number of compounds identified by heat from the primary screening (primary) for each pharmacological action. The green bars indicate the number of major heat compounds identified by pharmacological action (identified).
FIG. 5B shows a pilot screen result. Acid scatter analysis showed positive (red; sorafenib IC ₅₀ ) and negative (black; 0.5% dimethylsulfoxide) controls in the assay plate. Points represent each well tested.
FIG. 5C shows a pilot screen result. A total of 4,763 compounds were screened at a concentration of 10e validation plate. Points represent a single test well. Dots are a hit from the sorafenib control. The correlation coefficient (r2 = 0.8) was calculated using the obtained optimum number of components.
Figure 6: multicellular tumor spheroids (MCTS), normal liver, and hepatocellular carcinoma (HCC) heat drug sensitivity caused by the primary screening of a cell line according to the concentration.
Figure 6a shows the sphere size and red fluorescent protein (RFP) signal intensity of the MCTS model after treatment with the indicated concentrations of ponatinib, ouabain, camptothecin, actinomycin D or digitoxigenin.
Figure 6b shows the dose-response curve of the RFP signal intensity of the MCTS model after treatment with ponatinib, ouabain, camptothecin, actinomycin D or digitoxigenin.
Figure 6c shows the toxicity of five heat compounds to normal hepatocytes (Fa2N-4) and Huh7 cells at indicated concentrations in two-dimensional (2D) culture conditions after 48 hours of treatment.
6d shows the cell viability of HCC cell lines (Huh7, Hep3B, Huh6, PLC / PRF / 5, SNU449 and SNU475) under 2D culture conditions after 48 hours treatment with ponatinib, ouabain, camptothecin, actinomycin D or digitoxigenin. Reaction curve. All images were obtained using the Operetta® High Content search system. Data were expressed as mean and standard deviation (SD) of three experiments (scale bar: 200 μm).
Figure 7: Figure 7 shows the biological process that is significantly overexpressed in the secretomes of the Huh7 tumor spheroid (blue) compared to the multicellular tumor spheroids (orange) shown on the bar chart.
Figure 8: Molecular function enhanced or depleted in lysates of Huh7 tumor spheroid compared to multicellular tumor spheres.
Figure 9: Figure 9 shows the biological process enhanced or depleted in lysates of Huh7 tumor spheroid compared to multicellular tumor spheres.
Figure 10: Biological pathways enhanced or depleted in lysates of Huh7 tumor spheroid compared to multicellular tumor spheroids.
Figure 11 shows the drug susceptibility of Huh7 tumor spheroids and multicellular tumor spheroids to typical cytotoxic compounds and anticancer drugs. All images were obtained using the Operetta® High Content search system (scale bar: 200 μm).
Figure 12: Sphere size and RFP signal intensity after compound treatment
Figure 12a shows the sphere size and RFP signal intensity after treatment of the primary heat (57 compounds) selected at the indicated concentrations.
12B is a continuation of Fig.
Fig. 12C is a flowchart
All images were obtained using the Operetta® High Content search system (scale bar: 200 μm; RFP = red fluorescent protein).
Figure 13: Characterization of primary HCC cells
13A shows the morphology of AMC-H1 and AMC-H2 HCC cells under monolayer culture conditions.
FIG. 13B shows the results of examining the cell origin of primary HCC cells by immunostaining AMC-H1 and AMC-H2 cells with AFP, albumin and HepPar-1.
13C shows the AFP and albumin mRNA expression levels in AMC-H1 and AMC-H2.
FIG. 13d shows the results of Western blotting of the expression levels of EGFR, β-carderine, p53, PTEN, ERK and Akt in AMC-H1 and AMC-H2. Activation of the signaling pathway was confirmed by measuring the degree of phosphorylation of ERK and Akt.
FIG. 13E shows the spheroid culture of AMC-H1 and AMC-H2 in a stem cell permissive medium for 5 days. The degree of expression of each molecule was quantified by concentration analysis.
FIG. 13f shows the CD133 immunostaining results of AMC-H1 and AMC-H2 cells.
FIG. 13g shows the dose-response curves of AMC-H1 and AMC-H2 treated with 5-FU, cisplatin and sorapenib at a designated concentration for 48 hours under monolayer culture conditions. Bright field images were acquired using a Zeiss microscope. Fluorescence images were acquired and analyzed using the Operetta® High Content Screening (HCS) system. Data are presented as the mean ± SD of three experiments. Scale bar = 200 μm
Figure 14: Drug sensitivity of tumor spheroids obtained from primary HCC cells and time-dependent spheroid- forming ability
Figure 14a shows the ability to form tumor spheroids of AMC-H1 and AMC-H2 at the indicated time. Cells were cultured on 96-well ultra-low attachment (ULA) plates for 96 hours.
FIG. 14B shows drug sensitivity to tumor spheres of AMC-H1 and AMC-H2. Cells were cultured on 96-well ULA plates for 3 days and then treated with 5-FU, cisplatin and sorafenib for 7 days at the indicated concentrations. Tumor spheroids were then stained with ethidium homodimer-1 (EthD-1; left panel) and analyzed for staining intensity (right panel). All images were acquired and analyzed using the HCS system. Data are presented as the mean ± SD of three experiments. Scale bar = 200 μm. * p < 0.05, ** p < 0.005
Figure 15: Multicellular spheroid formation and drug sensitivity of the third day HCC cells
15A is a schematic diagram of a multicellular spherical body model showing a biological microenvironment.
Fig. 15B shows tumor morphology and multicellular spherical morphology of AMC-H1 and AMC-H2 cells.
Figure 15c shows the drug sensitivity of AMC-H1 and AMC-H2 MCTS-type cells grown with hepatic stellate cells (HSC; LX2), fibroblasts (WI38), and endothelial cells (HUVECs). Cells were cultured in 96-well ULA plates for 3 days and then treated with 5-FU, cisplatin and sorafenib for 7 days at the indicated concentrations. MCTS was then stained with EthD-1 (upper panel) and analyzed for staining intensity (bottom panel).
FIG. 15d shows the EthD-1 intensity of AMC-1 and AMC-H2 composed of the tumor-treated spheroids and multicellular spheroids treated with the indicated drugs. All images were acquired and analyzed using the HCS system. Data were expressed as the mean ± SD of three experiments. Scale bar = 200 μm * p <0.05, ** p <0.005
Figure 16: Analysis of wound healing of primary HCC cells. Wound healing analysis
Figure 16a shows the results of introducing wound after cells reached 80-90% confluence. Cell migration was monitored under the microscope for the indicated times. Scale bar = 500 μm.
FIG. 16B shows the result of quantifying the migration rate expressed as% of the control group by monitoring the cell growth for 7 days.
Figure 17: AMC -H1 and AMC -H2 Karyoviews of.
The blue bar represents the gain, the red bar represents loss, and the purple bar represents LOH.
Figure 18: Characterization of various basic HCC cells.
Figure 18A shows the capacity of various primary HCCs forming tumoral spheroids and MCTS.
FIG. 18B shows the result of examining the cell origin of primary hepatocytes by albumin and HepPar-1 immunostaining.
Figure 18c shows AFP and albumin mRNA expression levels in primary HCC cells (PDF 790 kb).
Figure 19 shows the quantification results of HBV DNA and HBsAg in AMC-H1 and AMC-H2 (DOCX 25 kb).
Figure 20 shows chromosome loss detected by SNP arrays in AMC-H1 and AMC-H2 (DOCX 26 kb).
21: SNP array results
Figure 21a shows the chromosomal gains detected by SNP arrays in AMC-H1 and AMC-H2 (DOCX 26 kb).
Fig. 21B is a continuation of Fig.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention more specifically and that the scope of the present invention is not limited by these embodiments.

Example

I. Example  One

1. Materials and Methods

Cell lines and culture conditions

Human immortalized hepatocyte stem cell Fa2N-4 cells were purchased from Xenotech (Lenexa, KS, USA) at the China Cell Line Bank, Huh7, Hep3B, PLC / PRF / 5 and SNU449. The Huh7.5-red fluorescent protein- (RFP) -NLS-IPS reporter cell line was provided by Dr. Marc Windisch (Korea Pasteur Institute). Stromal cells such as human lung fibroblast (WI38), human hepatic stellate cell (LX2) and HUVECs (human umbilical vein endothelial cells) were purchased from ATCC (Manassas, Va., USA), Merck Millipore (Darmstadt, Germany) and Lonza Basel, Switzerland). Fa2N-4 (human immortal liver cell line) was purchased from Xenotech. All cells were maintained at 37 ° C in a humidified atmosphere of 5% CO ₂ .

HCC cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Welgene) supplemented with heat-inactivated 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1 페 penicillin-streptomycin , Daegu, Korea). WI38 cells were cultured in a minimal essential medium (Welgene) supplemented with heat-inactivated 10% FBS and 1X penicillin-streptomycin. LX2 cells were cultured in DMEM supplemented with heat-inactivated 2% FBS and 1X penicillin-streptomycin. Endothelial basal medium was purchased from PromoCells (Heidelberg, Germany) for HUVEC culture. Fa2N-4 cells were plated on collagen coated plates. Serum-containing plating medium (XenoTech) was replaced with support culture medium (XenoTech) after cell attachment (about 3 to 6 hours).

Hepatocarcinoma  Primary culture

Immediately after surgery, a portion of the tumor was immersed in Hank's balanced salt solution (HBSS; Gibco) and transferred from the operating room to the laboratory at 0 ° C. Samples were collected under sterile conditions and rinsed 2-3 times with calcium and magnesium-free HBSS to remove blood. After removal of blood, liver samples were resected, cut into small pieces, and gently dispersed in HBSS containing 0.03% pronase, 0.05% IV collagenase, and 0.01% deoxyribonuclease (DNase small pancreas) Respectively. The resulting suspension was filtered through a 100 μm nylon filter (BD Falcon, Franklin Lakes, NJ, USA) and centrifuged at 50 ° C for 2 minutes at 4 ° C to obtain hepatocytes. The pellet was washed twice in HBSS containing 0.005% DNase. The final cell suspension was incubated in a humidified 5 &lt; RTI ID = 0.0 &gt; (5%) &lt; / RTI &gt; medium using collagen-coated T25 flasks (BD Falcon) in F12 / DMEM (Gibco) supplemented with 20% FBS, 1% NEAA (Gibco), 1% glutamine and 1% P / They were cultured in the incubator% CO _2. After 24 hours of incubation, the medium was changed to remove dead cells and debris. When confluency reached 70% to 80%, cells were reconstituted using a 1: 1 mixture of DMEM medium and F12 / DMEM as a supplement. After 5 passages, cells were grown in DMEM medium supplemented with 10% FBS and 1% P / S. The confluent cells were trypsinized, counted, and divided into 1: 3-1: 5 in all passages. The cell lines were maintained over 30 passages, collected and stored in liquid nitrogen.

Ethical approvals and patient agreements

This study was conducted according to the Helsinki Declaration. It was approved by the Human Research Ethics Committee of Asan Medical Center. Asan Hospital Medical Institutions Screening Committee has complied with ICH, KGCP, bioethics and safety regulations. A written agreement on the use of the tissue for research was obtained from the patient at the time of procurement of the tumor specimen.

Immunohistochemistry

Liver cancer tissues of formalin-fixed and paraffin-embedded tissues were analyzed by AccuMax? (Abacam, Cambridge, UK), p75 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and fibroblast activation protein (FAP; Abcam) We used slides arranged for. Deparaffin and rehydration were performed using xylene and ethanol (Sigma-Aldrich, St Louis, MO, USA), and the pretreated slides were incubated in 3% H ₂ O ₂ (Sigma-Aldrich) To remove endogenous peroxidase activity. Tissues were incubated with anti-CD31, anti-p75 and anti-FAP for 16 h at 4 째 C, washed with phosphate buffered saline (PBS) for 10 min and then incubated with goat anti rabbit Alexa Fluor 633 (Invitrogen, Eugene, OR, USA) and goat anti-mouse Alexa Fluor 488 (Invitrogen) secondary antibody. After washing three times with PBS, the coverslip was mounted on a microscope slide using a VECTASHIELD mounting medium containing 4 ', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Slides were analyzed using Operetta® high-content screening system (Perkin Elmer, Waltham, Mass., USA).

tumor Spherical body (spheroids) and the creation of medications

Cells suspended in complete medium to produce spheroids and multicellular tumor squamous epithelium were cultured in 384- or 96-well round bottom ULA microplates (Corning Life Sciences, Amsterdam, The Netherlands) And seeded at a density of 6 × 10 ³ cells / well depending on the ratio of stromal cells. The plate was incubated in humidified atmosphere of 5% CO ₂ at 37 ℃ 3 days. After 3 days, 10 [mu] M of cytotoxic or anticancer agent was added and further cultured for 7 days. A solution of 0.5% dimethyl sulfoxide (DMSO) was used as negative control. All images were obtained 10 days after cell seeding using Operetta® Harmony software (Perkin Elmer).

Drugs (methotrexate, roscovitine, prinixic acid, cycloheximide, aminopterin, etoposide, salinomycin, cisplatin, 5-fluorouracil (5-FU) and doxorubicin) were purchased from Sigma-Aldrich. Other drugs (Scriptaid, vinblastine and gefitinib) were purchased from Tocris (Bristol, UK). Cyclosporin (Japan Wako), Simvastatin (Tokyo, Japan), ALLN (Millipore, Billerica, MA, USA), and Santa Cruz Biotechnology.

Resazurin  analysis

Spheroids were generated as described above and treated with sorafenib at 1, 5 or 10 μM. The spheres were incubated for 5 hours in the presence of 50 μM resazurin (Sigma-Aldrich) and the reduction of Resazurin was measured by colorimetry (570/600 nm) using an EnSpire plate reader (Perkin Elmer).

Spherical body  Movement analysis

Prior to the generation of spheroids, the cells were cultured in complete medium in a 37 ° C incubator at a ratio of 1: 500 for 20 minutes using DiD (633 nm), DiI (546 nm) or Dio (488 nm) (Invitrogen) LX2, WI38 cells and HUVEC were stained. After washing with DPBS (Welgene), spheres were formed over 3 days and each spherical body was transferred to collagen I-coated 384-well plate (Greiner Bio-one, Monroe, NC, USA) Lt; / RTI &gt; The medium was replaced once every two days after transfer. Four days later, the cells were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and washed twice with DPBS. For nuclear staining, cells were incubated with Hoechst 33342 (Invitrogen) at a ratio of 1: 500 in DBPS for 10 minutes at room temperature. After washing twice with DPBS, fluorescent images were captured according to the optimal excitation and emission wavelength of each probe. To capture the entire image, 25 image fields starting from the center of the well were collected in each well using an HCS system with a 10x objective.

Xenotransplantation studies

(LX2 cells, WI38 cells or HUVEC) at a density of 1 x 10 ⁴ cells / well in 96-well round bottom ULA microplate (Corning) to Huh7.5-RFP cells to generate spheroids of multicellular tumors Respectively. The plate was incubated in humidified atmosphere of 5% CO ₂ at 37 ℃ 3 days. A total of 1 x ¹⁰⁶ cells were subcutaneously injected into a 5-week-old male BALB / c nude mouse in a 2D or 3D culture system containing 0.1 mL of Matrigel (BD Biosciences with reduced matrix growth factor, San Jose, CA, USA). Animals were weighed and tumor size was measured twice a week with calipers. Tumor volume (mm 3) was calculated using the following formula: Volume = (width) 2 Х length / 2. Seven mice in each group were studied.

Histological analysis

Tumor tissues were fixed in 4% PFA, cut into 4 μm paraffin embedded sections and stained with hematoxylin and eosin (H & E) and Mars Trichrome. For immunohistochemistry, tumor tissue was fixed overnight in 4% PFA, embedded in paraffin, and sectioned. After paraffin removal and dehydration, antigen was searched by heating in 10 mM citrate buffer (pH 6.0) for 15 minutes. Antibodies used in immunohistochemistry include antibodies against anti-CD31 (ab28364; Abcam) and Ki67 (ab15580; Abcam). Secondary antibodies were anti-IgG conjugated with Alexa Fluor 546 (Invitrogen) and observed under a microscope (Nikon, Tokyo, Japan).

Microarray  analysis

Global gene expression analysis was performed using an Affymetrix GeneChip® Human Gene 2.0 ST array. Sample preparation was carried out in accordance with the manufacturer's instructions and recommendations. The total RNA of Huh7.5 globular and multicellular tumor spheres was isolated using an RNeasy Mini Kit column (Qiagen, Hilden, Germany). RNA quality was assessed using an Agilent 2100 bioanalyzer using RNA 6000 Nano Chip (Agilent Technologies), and the amount was determined using a Nanodrop-1000 spectrophotometer (Thermo Scientific). For RNA analysis, 300 ng was used as input using the Affymetrix procedure recommended in the protocol (http://www.affymetrix.com). Briefly, 300 ng of total RNA from each sample was converted to double-stranded cDNA using a random hexamer containing the T7 promoter and the amplified RNA was generated from double-stranded cDNA templates using in vitro transcription reactions Next, purified using the Affymetrix sample cleanup module. cDNA was regenerated by randomly primed reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented by UDG and APE1 restriction endonuclease and end-labeled using a terminal transferase reaction with biotinylated dideoxynucleotide introduced. The fragmented, final fragmented cDNA was hybridized to a GeneChip (R) Human Gene 2.0 ST array at 45 DEG C, 60 rpm for 17 hours as described in the Gene Chip Entire Sense Sensitive Target Labeling Analysis Manual (Affymetrix). After hybridization, the chips were stained, washed in Genechip Fluidics Station 450 (Affymetrix) and scanned using a Genechip Array 3000 7G scanner (Affymetrix). Expression intensity data was extracted from scanned images using Affymetrix Command Console software version 1.1 and saved as a CEL file.

Data Analysis

The robust Multi-array Average algorithm implemented in the Affymetrix Expression Console software, version 1.3.1, was used to normalize the intensity values of the CEL file data to eliminate bias between the arrays (http://www.affymetrix.com). By importing the entire standardized data into the programming environment R, version 3.0.2, and using the Bioconductor Project (http://www.bioconductor.org) (M2) to verify proper normalization, Signal distributions were compared. After confirming whether the data were properly standardized, a differentially expressed gene (DEG) was manually selected which showed a difference of more than 2 times between the mean signal value of the control and the treatment group. Also, the normalized data of the selected DEGs were brought to the programming environment R for the t-test, and the genes with values of p <0.05 were extracted with DEG for later study. Hierarchical clustering analysis was performed using Multi Experiment Viewer software version 4.4 (http://www.tm4.org) to classify co-expressing gene groups with similar expression patterns. Finally, DEG is functionally annotated for web-based tools Annotation, Visualization, and Integrated Discovery, and used with http://david.abcc.ncifcrf.gov and includes genes such as OMIM disease, Gene Ontology, KEGG pathway and BioCarta database Based on functional information and identified the regulatory networks they are associated with.

Analysis of biological and chemical informatics

Functional abundance analysis of genes with> 2-fold change (absolute value) was performed using Funrich software. We evaluated microarray analysis of secretions of Huh7 tumor globules and MCTS models to determine which genes were enriched or depleted of molecular functions, biological processes and biological pathways. DEG was functionally classified to account for overexpressed molecular functions, biological processes, and biological pathways. (Pathan M, Keerthikumar S, Ang CS, Gangoda L, Quek CY, Williamson NA, Mouradov D, Sieber OM, Simpson RJ, Salima, Bacica, Hill AF, Stroudde, Ryan MT, Agnina JI, Mariadason JM, Burgess AW, Mathivanan S. FunRich: Open Access Standalone Enhancement and Interaction Network Analysis Tool Proteomics, 2015 Aug; 15 (15): 2597-601.)

Information on the pharmacokinetics of each compound was combined with information provided by the supplier, as well as the ChemIDPlus, MESH and SciFinder databases. All pharmacological actions have been described and not limited to the main action. Once relevant information was collected, the pharmacological action was manually reevaluated to classify all compounds into 69 pharmacological behaviors.

High Throughput Screening

Libraries of 4,763 compounds were assembled in various supplies (Tocris, Selleck, LOPAC and Prestwick) and screened in 0.5% DMSO (v / v) in an anti-HCC MTCS model at a final concentration of 10 μM.

Four cell lines were inoculated into low attachment U-bottom 96-well plates (Corning Life Sciences, Corning, NY, USA) at a density of 6 x 10 ³ cells / well to produce unattached spheroids. To facilitate cell aggregation, the plate with the cell suspension was centrifuged at 1,000 rpm for 1 minute before incubation. After that the plate at 37 ℃ in a humidified incubator of 5% CO ₂ and incubated for 3 days.

For compound testing, 2 μL of each compound was transferred to an intermediate 384-well polypropylene plate (Greiner) using a liquid processor (Apricot Personal Pipettor; Apricot Design, Covina, CA, USA). The compound was mixed with 78 [mu] l / well of complete medium. Subsequently, 20 [mu] l of compound was dispensed into each well of a 96-well assay plate for 3 days. The plates at 37 ℃ in a humidified incubator of 5% CO ₂ and incubated for 7 days.

Control groups were added to each assay plate with low IC ₅₀ control and sorafenib high control with 0.5% DMSO (v / v). Seven days later, spheroid images were acquired using a high-content imaging device (Operetta®, PerkinElmer).

The size and intensity of the spheres were measured using self - developed algorithms. The heat compound was selected using a threshold based on 3 s (standard deviation) from the IC ₅₀ of sorapenib.

Statistical analysis

All experiments were performed at least three times. Results were expressed as mean ± SD. Statistical analysis was performed using Student's t-test.

2. Experimental results

The interactions between hepatocytes and stromal cells MCTS  In the model Sorapanib  Resistance and HCC movement Promote .

In a previous study, we constructed a simple MCTS model with simple forms of epileptic cells including HCC cell line and LX2 [human hepatocyte (HSCs)], WI38 (human fibroblast) and HUVEC to determine tumor complexity and heterogeneity Respectively. Interaction between each type of stromal cells and hepatocellular carcinoma promoted the simplicity of tumor mass and increased chemotherapy and hepatocellular carcinoma migration. However, the previous MCTS model was not derived from a mixture of HCC cells with both hepatocellular carcinoma, fibroblasts, and vascular endothelial cells.

In this study, a more complex MCTS model derived from a mixture of HSC, fibroblast and vascular endothelial cells and HCC cells was developed to reproduce the in vivo tumor microenvironment of hepatocytes because hepatocellular carcinoma is accompanied by extensive inflammation and fibrosis . Before developing the MCTS model, we compared the drug sensitivity between tumor spheroids and tumor - derived ellipsoids of HCC patients after treatment with 10 μM sorafenib.

The size of spheroids derived from HCC cell lines was significantly reduced by treatment with sorapenib (Fig. 1a). However, the size of tumor cells derived from HCC patient tissue was not changed by sorapenib treatment compared to spheres derived from HCC cell line (Fig. 1b). Since different sensitivities to sorafenib have been observed from both spheres, immunohistochemical probes for CD31 (vascular endothelial cell marker), p75 (HSC marker) and FAP (fibroblast marker) Respectively. As a result, the tissues of patients with liver cancer were composed of specific ratios of interstitial cells such as HSC, fibroblasts, vascular endothelial cells and HCC (Fig. 1c). These results suggest that cross-talk between TME (tumor microenvironment) and HCC cell components may induce chemotherapy in HCC tumor-derived tumor ellipsoid. In order to confirm this possibility, the inventors of the present invention examined the effects of intercellular interactions between hepatocytes and hepatocytes on chemical resistance and cell migration, in order to determine the effect of various MCTSs (HCCs, fibroblasts, vascular endothelial cells) The model was constructed.

The present inventors selected the MCTS model best suited for HCC drug screening by characterizing susceptibility to sorapenib through spheroid size measurements and resazurin analysis. The MCTS model, which grew into epidermal and hepatocellular carcinomas, was found to be more resistant to sorapenib according to the percentage of stromal cells in the MCTS model (Fig. 1d). On the other hand, the size of HCC spheroids decreased dramatically after treatment with sorapenib. Consistent with spheroid size, cell viability after treatment with sorapenib was substantially increased in proportion to the proportion of stromal cells in the MCTS model (Fig. 1e).

Since the interaction between HCC cells and their tumor microenvironment is considered to be important for promoting cell migration characteristic of cancer cells, the MCTS model with a ratio of HCC cells to stroma cells was tested using tumor spheroid-based migration assay , And the distance from the center of the MCTS model to the leading edge of moving HCC cells was measured. This analysis showed that the interaction between stromal cells and HCC cells in the MTCS model is an important determinant of hepatocyte transplantation capacity (Fig. 1f).

MCTS  A model system that mimics the in vivo tumor microenvironment .

The MCTS model is a powerful tool for anticancer studies because it mimics the complexity and heterogeneity of tumor tissue, includes 3D cellular structures, and includes the pathophysiological gradient of in vivo tumors. However, it is difficult to obtain valuable results using the MCTS model without considering clinical tumor status. The inventors thus determined whether the model closely replicates the in vivo tumor microenvironment.

Gene expression profiling was performed to normalize the profile of tumor spheroids in the MCTS model system. We identified 1,596 DEGs between the MCTS and tumor spheroids and found a> 2-fold difference and a P <0.01 value (Fig. 2a). Gene set enrichment analysis (GSEA) was performed using the Gene Expression Omnibus (GEO) data set of two independent HCC cohorts (GSE64041) to demonstrate the MCTS model specific signature associated with the phenotype of patient-derived HCC tissue. As a result, the MCTS-specific signature was enriched in HCC tumor tissues when compared to non-tumor tissues suggesting that the MCTS model is an effective tool for mimicking the HCC microenvironment in vivo [Fig. 2b].

Functional enrichment analysis comparing the number of molecular functions between monolayer cultured Huh7 and MCTS models showed that the constitutive extracellular matrix structural function, structural components of the cytoskeleton, cytokine activity, transcription factor activity and genes involved in DNA binding Was overexpressed much more in MCTS models than Huh7 cells (Fig. 2c). Genes involved in the regulation of nucleobases, nucleosides, nucleotides, nucleic acid metabolism, cell growth, immune responses and anti-apoptotic responses in the biological process were enriched in the MCTS model (Fig. 2d). In addition, integrin-related genes involved in cell surface interactions became more abundant in the MCTS model (Fig. 7; Table 1].

[Table 1] Summary of remarkably enriched molecular functions, biologic processes and biological pathways from microarray analysis of MCTS models and Huh7 cells

In displacement-based analysis, biological processes and biological pathways are more detailed and quantity-based. In the MCTS model, genes involved in cofactor binding, cytokine activity, nucleic acid binding and extracellular matrix structural components became enriched. Notably, the genes involved in glucosidase activity are largely depleted (-15-fold changes) and further work will be required [8]. Regarding biological processes, genes involved in peptide metabolism, endocytosis regulation, anti-apoptosis, muscle contraction, cell-cell adhesion and specific signaling pathways were more than doubled [Figure 9]. Functions such as the biological pathways involved in hepatic pathology, and the synthesis and secretion and deacylation of the Gli protein, Ghrelin, and Hedgehog signaling mediated by vitamin A uptake in intestinal cells, were depleted in the MCTS model (Fig. 10).

MTCS  Derived xenograft mouse model is an effective tool for characterizing the reproduction of human hepatocytes .

To demonstrate that the MCTS model mimics the microenvironment of the tumor, the inventors made subcutaneous xenografts in monolayer cultures of Huh7 cells, Huh7 spheroids or MCTS model systems in nude mice. The general xenotransplantation model, a cell harvested from a monolayer culture, produced relatively white and graphene-like aggregated tumors. In contrast, the spheroid-derived xenotransplantation model formed a deep red, smoothly condensed glob-type tumor (Fig. 3a). When we performed comparative analysis of tumor growth between Huh7 cells, Huh7 spheroids and MCTS system, all of the xenotransplantation models did not change the body weight of the Huh7 cells> Huh7 spheroids> Thereby increasing the volume (Fig. 11). Tumor formation was faster in Huh7 cells than in Huh7 spheroids and MCTS models, but tumor growth was faster in spheroid culture systems after tumor formation (Fig. 3b).

We performed H & E staining following dissection of three tumor tissues from monolayer cultured Huh7 cells, Huh7 spheroids or MCTS-xenografted mice. Three different tissues showed different morphology, structure and vascular distribution as a result of staining (Fig. 3c, left). There was also a difference in the degree of fibrosis between the three types of tumor tissue in monolayer cultured Huh7 cells, Huh7 spheroids, or MCTS-xenografted mice using Masson's trichrome staining method. Late late fibrosis was observed in the tissues of MCTS-xenografted mice (Fig. 3c, right).

Antibodies to CD31 were used primarily to confirm the presence of vascular endothelial cells in tissue sections. Tumor tissue of Huh7 spheroids or MCTS xenografted mice showed that CD31 was expressed in a compartmentalized form, while CD31 was irregularly distributed in tumor tissue of monolayer cultured Huh7 xenografted mice in a relatively large area , left]. Immunohistochemical analysis of Ki67 was performed in tumor tissues to measure tumor cell proliferation around the blood vessels. In particular, tumor tissue of MCTS xenografted mice showed the highest expression among the three tissues (Fig. 4c, right). Taken together, the morphology and composition of tumor tissues differed according to cell culture methods for transplantation, especially the xenograft model derived from MCTS, which is very similar to in vivo HCC.

HCC  For the discovery of therapeutic agents MCTS  base phenomic  Screening

As described above, the present inventors have established the most appropriate MCTS model for anticancer screening consisting of Huh7-RFP cells, WI38 cells, LX2 cells, and HUVECs in order to mimic the HCC tumor microenvironment in accordance with HCC histological analysis. Since Huh7-RFP cells were labeled with fluorescence, a uniformly configured MCTS and a specific effect on HCC were observed in the screening process (Fig. 4a). The present inventors confirmed the clinical significance of the effects of anticancer drugs such as cisplatin, 5-FU and doxorubicin on the growth of spherical bodies or MCTS models composed of Huh7 alone. The results showed that the chemotherapeutic agent was less effective in the MCTS model compared to the Huh7 spheroid suggesting that the interaction between the HCC and the tumor microenvironment contributes to the chemical resistance of the MCTS model (Fig. 4b). The present inventors also showed similar results by characterizing the effects of various cytotoxic compounds and anticancer agents on the growth of spheres composed of Huh7 alone or MCTS model (Fig. 12).

Next, the present inventors focused on developing an MCTS-based screening platform for HCC drug discovery. To construct an MCTS-based phenomic screening platform for HTS / HCS, the inventors have developed an efficient method for generating 3D spheres. We have successfully generated homogeneous size, composition, and single spheroids in 96-well or 384-well plates. The spherical image is Operetta? It was obtained in only 30 minutes using the HTS system. The inventors therefore established a simple method for establishing highly reproducible (< 10% size change) spheres with respect to high speed image acquisition [Fig. 4c]. We also developed appropriate image analysis software to detect the intensity of Huh7-RFP to determine the size of the globe and the cytotoxic effect on HCC (Fig. 4d). RFP signal intensity of Huh7-RFP was measured to mimic the in vivo growth of cancer cells and tumor growth was characterized using multiple well plates. Depending on the intensity of the RFP signal, the HCC population grew steadily as MCT cells grew on multiwell plates (Fig. 4e).

MTCS  base phenomic  Identification of therapeutic agents for liver cancer using screening

We collected 4,763 compounds consisting of FDA-approved drugs with known molecular targets and identified new drugs for HCC treatment. The pharmacological action of each compound is provided by the ChemIDPlus database (https://chem.nlm.nih.gov/chemidplus/), the MESH database (https://meshb.nlm.nih.gov/search) and SciFinder, version 2017 I refer to the information provided by the vendor. Since it is difficult to define the main pharmacological actions of known drugs, all pharmacological actions have been described and are not confined to the main pharmacology (Fig. 5a).

Screening was then performed to identify compounds that specifically altered the intensity of the Huh7-RFP cell signal in the MTCS model and the size of these models was determined. Compounds were screened at an initial concentration of 10 [mu] M to determine the reduced intensity of the RFP signal or to determine the magnitude of the MTCS. Positive and negative controls were 10 μM sorapenib and 0.5% DMSO, respectively [FIG. 5 b]. We screened all compounds twice to confirm the reproducibility of the observed effects. The Z 'factor 0.46 and the correlation coefficient 0.89 for duplicate images indicated that the test was reliable [Figure 5-C]. 87 compounds significantly inhibited the size of MTCS and the intensity of RFP signal. The primary hit included 49 categories, and the hit contained 39 categories. Most of these 39 categories were associated with anticancer therapies such as tyrosine kinase inhibitors (Fig. 5A).

To verify the heat of the primary screening, a dose response study was performed to quantify the efficacy of the selected heat using the MCTS model. Five compounds (ponatinib, ouabain, camptothecin, actinomycin D and digitoxigenin) sufficiently inhibited the RFP signal by more than 60% and reduced the MCTS size at 1 μM compared to 10 μM sorapenib (Fig. Table 2].

[Table 2] IC ₅₀ values for RFP intensity of HCC-multicellular tumor sphere

The dose-response curve was also used to determine the effective concentration required to reduce the growth of _50% (EC 50) in Huh7 cells and normal liver cells (Fa2N-4 cells) [Fig. 6c]. To determine the effect of heat compounds on human HCC cells, the IC ₅₀ values of the heat compounds for growth of various HCC cell lines were determined. The dose-response curve showed that the impinging compound fully induces apoptosis in HCC cells (Fig. 6d; Table 3].

Table 3: IC ₅₀ values of HIT compounds for growth of various HCC cell lines

3. Discussion

HCC is the sixth most common malignancy and the second leading cause of cancer-related deaths in the world. The highest incidence occurs in eastern Asia and sub-Saharan Africa. In addition, the incidence and mortality rate of liver cancer in Korea is the 18th highest in the world.

The first regimen of HCC is surgical resection of the tumor, and the second regimen is the treatment with sorafenib, a kinase inhibitor. However, both therapies are effective only for patients with liver cancer. In addition, HCC is rapidly resistant to sorafenib. To improve treatment options, additional drugs have been developed via the sorafenib method. However, these efforts still did not lead to better efficacy. Based on clinical considerations, the inventors have developed an MCTS model for HCC drug screening, a new and effective method of mimicking tumor complexity and heterogeneity.

Differences in susceptibility to sorapenib between single-cell tumor ellipsoid and hepatocellular carcinoma tissue-derived tumors emphasized the importance of the interaction between TME and HCC for the determination of chemotherapy chemotherapy in tissue-derived tissues from hepatocellular carcinoma patients -1c]. The present inventors have also characterized the effect of sorapenib on the growth of MCT spheres composed of Huh7 monocyte spheroids or HCC cells, fibroblasts, HSCs, cancer stem cells and vascular endothelial cells. This system mimics the microenvironment of HCC tumors based on analysis of HCC histology and shows that sorapenib is less effective in MCTS when compared to the effect on Huh7 spheroids (Fig. 1d, 1e). The present inventors also confirmed the efficacy of various anti-cancer drugs and cytotoxic drugs using HCC spheroids and MCTS models. As expected, MCTS showed a stronger tolerance to cytotoxic drugs compared to HCC spheroids (Fig. 12). Taken together, these results suggest that interaction between HCC and TME in 3D culture systems is an important consideration in the development of an effective drug screening process.

Various types of MCTS models have been used to characterize interactions between tumors and stromal cells. The co-culture of HUVEC and hepatocytes in the heterocellular 3D system was used to monitor cancer angiogenesis. Co - culture of prostate cancer epithelial cells and stromal cells in 3D conditions affected the secretion of E - cadherin 23. 3D co-culture system Cloned cancer cells and interstitial fibroblasts showed stronger invasive phenotype than 3D clone cancer cells alone. Dynamic analysis of HCC MCTS formation showed a fundamental role of E-cadherin and β1-integrin in cell aggregation and multicellular tumor compaction. According to the results, co-culture of hepatocellular carcinoma and stromal cells reported an increase in cancer progression and a change in cytokine expression profile through activation of specific signal pathways.

In order to identify the cause of TME-related drug resistance, it is necessary to develop a sophisticated methodology to reflect the TME. In this study, the inventors have shown that the MTCS model closely mimics the in vivo tumor microenvironment. First, gene expression measurements and GSEA showed that MCTS-specific signals were enriched in HCC tumor tissue when compared to non-tumor tissues. Second, the present inventors have established a xenotransplantation model derived from MCTS. Macroscopically, liver cancer appears as a nodular or invasive tumor. The nodular tumor type may be solitary (large) or multiple (when developing into complications of cirrhosis). Tumor nodules are circular oval and have an external angle. Diffuse nodular type penetrates portal or hepatic vein because of lack of boundary. The MCTS-derived xenograft model is more like nodular hepatocellular carcinoma in vivo as compared to the single cell-derived xenograft model (Fig. 3).

However, it is not known whether cells removed in patients with liver cancer and proliferated in vitro in terms of tumor type, signal and cellular status have the same characteristics as in vivo. Thus, extensive collaboration with basic researchers and clinicopathologists is essential, as well as ongoing research into the dynamic changes in TME in patient tissues.

II. Example  2

1. Experimental Method

Cell lines and culture conditions

Cells of WI38 (human fibroblast), LX2 (human hepatic stellate cells) and HUVECs (human umbilical vein endothelial cells), which are stromal cells, were obtained from ATCC (Manassas, VA, USA), Merck Millipore (Darmstadt, Germany) and PromoCells ). All cells were incubated in a 5% CO ₂ incubator at 37 ° C.

WI38 cells were treated with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1x penicillin-streptomycin (P / S; Gibco) in a minimum essential nutrient medium (MEM; Welgene, Daegu, Korea) And cultured. LX2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Welgene) supplemented with 2% heat-inactivated FBS and P / S. HUVECs were cultured in endothelial basal medium (EBM) purchased from PromoCell.

Patient origin  Liver cancer cell culture

Immediately after surgery, a portion of the resected tumor was transferred to the laboratory from the operating room at 0 ° C after soaking in Hanks balanced salt solution (HBSS; Gibco). Specimens were collected under aseptic conditions and washed 2-3 times with calcium and magnesium free HBSS to remove blood. After removal of blood, the liver samples were finely sectioned and gently dispersed and the samples were incubated with 0.03% pronase (Gibco), 0.05% type IV collagenase (Gibco), 0.01% deoxyribonuclease (Dnase, from bovine pancreas , Gibco) at 37 &lt; 0 &gt; C for 20 minutes. The resulting suspension was filtered through a 100-μm-nylon filter (BD Falcon, Franklin Lakes, NJ, USA) and centrifuged at 50 xg for 2 minutes at 4 ° C to obtain hepatocytes. The pellet was washed twice with HBSS containing 0.005% DNase. The final cell suspension was suspended in hepatocellular basal medium supplemented with 10% heat-inactivated FBS, 1 ng / ml hepatocyte growth factor (HGF, Prospec, Rehovot, Israel) and 1x antibiotic (antimycotic (HBc) , Basal, Switzerland) in a T25 flask (BD Falcon) coated with collagen at 37 ° C in a 5% CO ₂ incubator. After the incubation, the medium was changed for 24 hours to remove dead cells and debris. When the cells reached 70-80% confluence, the cells were transferred to supplemented HBM medium. Confluent cells were trypsinized, counted, diluted 1: 3-1: 5 for each passage and cultured. When the cell line was cultured 30 times or more, the cells were cryopreserved.

Ethical Victory and Consent to participation

This study was conducted in accordance with the principle of the Helsinki Declaration. The study was approved by the Asan Medical Center Human Research Ethics Committee (license number: 2007-0332). The Asan Hospital's Institutional Review Board abided by all the guidelines including the ICH, KGCP, Bioethics and Safety Act. Consent for the tissue to be used for the study was obtained when procuring a tumor sample from the patient. AMC-H1 was obtained from a 55-year-old female patient, and AMC-H2 was from a 51-year-old male patient. All of the causes of hepatocellular carcinoma were HBV infection.

Immune cell chemistry

Cells were fixed with 4% paraformaldehyde (PFA; Sigma, St. Louis, Mo., USA) for 10 min at room temperature and incubated for 30 min at room temperature with Triton X-100 (Sigma) washed with phosphate-buffered saline (DPBS; Welgene) and then washed three times with DPBS. The following antibodies were used: mouse monoclonal anti-human serum albumin (ALB; 15C7, ab10241, 1: 500; Abcam, Cambridge, UK), mouse monoclonal anti-human Hep par-1 (Clone OCH1E5, M7158, 1: 100; Agilent Technologies, Santa Clara, Calif., USA) and mouse monoclonal anti-human CD133 / 1 (AC133, 130-090-422, 1: 100; Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were treated with antibodies at 4 ° C for 16 hours and then with DPBS for 10 minutes three times. A secondary antibody used for staining is Alexa Fluor ^® 488 is combined with (Invitrogen, Eugene, OR, USA ) goat anti-mouse IgG and goat anti bond, Alexa Fluor ^® 488-rabbit IgG were used. The sample was then treated with the secondary antibody for 1 hour at room temperature and then washed five times with DPBS for 10 minutes each. For nuclear staining, Hoechst 33342 (Invitrogen) was treated at room temperature for 10 minutes and washed rapidly twice with PBS. All fluorescence images were obtained using the Operetta ^® High Content Screening (HCS) System (Perkin Elmer, Waltham, Mass., USA).

AMC -H1 and AMC Growth characteristics of -H2

1x10 ³ AMC-H1 and AMC-H2 cell lines were seeded in 96-well plate wells and grown for 1-7 days. Cell viability was measured via the MTS assay (Promega, Madison, WI, USA) at indicated times.

Stem cell of liver cancer ( LCSC ) spheroid formation

AMC-H1 and AMC-H2 cells were seeded in 6-well ultra-low attachment (ULA) plates (Corning Life Sciences, Amsterdam, The Netherlands) and cultured in liver cancer stem cell medium (1x B27 (Invitrogen), 20 ng / ml basic fibroblast The cells were incubated with DMEM / F12 (Gibco) supplemented with growth factor (bFGF; Invitrogen), 20 ng / ml epidermal growth factor (EGF; Invitrogen) and 25 ug / ml insulin (Sigma- Aldrich) Formation.

Wound healing assay

A wound healing assay was performed to confirm the possibility of metastasis. Cells were transferred to 6-well plates and cultured until they showed 80-90% confluence. Once the cells reached the appropriate area, they scraped the cells using a pipette tip. Cell migration was observed at the edge of the cell. Time-lapse images were captured using a microscope equipped with a Nikon 3 camera.

Western blot

Molecular profiles were confirmed by Western blot analysis. The cells were harvested and centrifuged at 1000 rpm for 3 minutes. The pellet was collected and dissolved vigorously vortexing for 30 min at 4 &lt; 0 &gt; C using RIPA buffer (InTron, Junwon-gu, South Korea). The resulting lysate was centrifuged at 12,500 rpm for 20 minutes at 4 ° C, and the supernatant was collected. Protein concentration was measured by BCA assay (Promega). After SDS-PAGE and transfer, the membranes were incubated with Epithelial Growth Factor Receptor (EGFR, Cell Signaling, Danvers, MA, USA), beta -catenin, N-cardinal, ERK (Cell Signaling), and Akt (Cell Signaling). Appropriate secondary antibodies were attached and detected using ECL reagent (GE, Fairfield, CT, USA).

Single nucleotide polymorphism (SNP) Microarray

SNP array analysis was performed using Affymetrix CytoScan ™ 750K (Affymetrix, Santa Clara, Calif., USA) to investigate gene mutations in AMC-H1 and AMC-H2 cells. DNA was isolated from AMC-H1 and AMC-H2 using the AxyPrep Multisource genomic DNA kit (Axygen, Union City, CA, USA). SNP microarrays were performed according to standardized protocols supplied by the manufacturer. Raw data was analyzed using Chromosome Analysis Suite 2.0 software (Affymetrix) to detect changes in copy number (CN) and loss of heterozygotes (LOH).

Tumor spheroid , Multicellular Tumor spheroid ( MCTS ) Formation and medication

To construct tumor spheroids, the cells suspended in HBM medium were seeded in 96-well U bottom ULA microplate (Corning Life Science) at a density of 6 x 10 ³ cells / well. The plate was incubated at 37 [deg.] C for 3 days in an incubator maintained at 5% CO2. To make the MCTS, 6 x the cells suspended in HBM medium in 96-well U bottom ULA microplates 10 were planted in ³ cell / well density. This plate was also incubated at 37 [deg.] C for 3 days in an incubator maintained at 5% CO2. After 3 days, 5-fluorouracil (5-FU; Sigma-Aldrich), cisplatin (Sigma-Aldrich), and sorafenib (Santa Cruz Biotechnology) were added and incubated for an additional 7 days. 0.5% dimethylsulfoxide (DMSO; Sigma-Aldrich) solution was used as a negative control.

In single layer culture conditions, drug sensitivity

A patient of liver cancer cells in 384-well plates (Greiner Bio-One, Monroe, NC, USA) 2.5 x 10 3 were seeding the cells / well density. After 16 hours of incubation, 5-FU, cisplatin and sorapenib were treated for 48 hours. After 48 hours, the cells were fixed with 4% PFA for 10 minutes at room temperature and washed twice with DPBS. Hoechst 33,342 was used for nuclear staining. Five image fields were collected for each well starting from the center of the wells in order to analyze enough cells (> 1000) for analysis. All image analysis was performed using HCS system and Harmony software. Cell numbers were calculated and normalized to control (0.5% DMSO).

In the sphere Cell death  detection

Cells of spheroids were detected using ethidium homodimer-1 (EthD-1; Invitrogen), a cell-impermeable viability indicator. EthD-1 is a nucleic acid-affinity dye that produces weak fluorescence and emits red fluorescence until it binds to DNA. Spheroid was cultured in HBM medium containing 4 μM of EthD-1 for 30 minutes at 37 ° C, and the image of EthD-1 was analyzed using HCS system.

Realtime PCR

The total RNA was prepared according to the manufacturer &apos; s instructions, 　 (Invitrogen). &Lt; / RTI &gt; The reaction mixture contained RT buffer (Bio Basic, Amherst, NY, USA), dNTP solution (Bio Basic), RNasin ^® inhibitor (Promega), oligo (dT) ¹⁵ primer (Bioneer, Daejeon, Korea) MLV reverse transcriptase (Invitrogen) is included. The reaction mixture was incubated at 37 [deg.] C for 1 hour and the mixture was heated at 95 [deg.] C for 5 minutes and then rapidly cooled on ice to terminate the transcription reaction. PCR reactions were carried out in 96-well plates containing cDNA, SYBR Green master mix (Applied Biosystems, Waltham, Mass., USA), primers and DEPC using a StepOnePlus real-time PCR system (Applied Biosystems). The reaction conditions were 95 ° C for 10 minutes and 40 cycles at 95 ° C for 15 seconds and 60 ° C for 1 minute. The critical period (CT) is defined as the number of fractional cycles through which fluorescence passes through a fixed threshold. The CT value was calculated as the mathematical model R = 2 - ΔΔCT, and the values are (ΔCT = CT _{target gene -} CT _GAPDH , ΔCT = ΔCT _test - ΔCT _control ). CT values were normalized according to GAPDH expression. All real-time PCR was performed 3 times and data were expressed as mean ± SD. All primers were designed and purchased from Bioneer.

Statistical analysis

All experiments were performed at least three times. Results are expressed as means ± SD. Statistical analysis was performed using Student's t-test.

2. Experimental results

Characterization of Primary Hepatocarcinoma Cells Maintaining the Nature of Liver Cancer Cells

We have isolated primary HCC tumors from hepatocarcinoma liver resection samples to develop a platform for selecting the best anticancer drug by providing useful information for clinical drug applications. Based on this, we selected two HCC cell lines (AMC-H1, AMC-H2) from HBV-infected Korean HCC patients. The characteristics of the two patients are summarized in Table 4 below.

[table 4] HCC  Donor characteristics at the time of ablation

At the beginning of primary culture of all tumors after resection, hepatocytes were observed to spread out from other cell groups. After subculturing 2-3 times, the cells consisted of almost all hepatic cells. Cells were maintained for more than 50 generations in DMEM supplemented with 10% FBS. To determine if the cell line was hepatocytes, we observed the morphology of AMC-H1 and AMC-H2. AMC-H1 cells exhibited homogeneous cell populations in the form of EPITHELIA-LIKE or polygons and showed clear nuclei with multiple phosphorus. The cells grew into an array of roads with distinct interfaces such as faults. AMC-H2 cells, in contrast, showed a fusiform shape rather than a polygonal shape. Immunostaining was performed using hepatocyte-specific markers for hepatocyte-specific antigens (Hep Par-1), alpha-fetoprotein (AFP) and albumin in order to confirm whether the two primary HCC cell lines retained their original HCC characteristics Respectively. HEP Par-1 was expressed in both cell lines from patients and AFP expression was higher in AMC-H2 than in AMC-H1. Albumin was expressed in both cell lines but higher in AMC-H1 (Figure 13b). These results suggest that both AMC-H1 and AMC-H2 have hepatocyte characteristics, but the degree of expression of AFP and albumin is different. To assess the ability of liver function, the expression level of AFP and albumin mRNA was analyzed by real time PCR. There was a difference in mRNA expression of AFP and albumin between AMC-H1 and AMC-H2. AFP mRNA was weakly expressed in AMC-H1 while strongly expressed in AMC-H2. The mRNA expression patterns of both AFP and albumin were consistent with those of immunostaining (Figure 13c)

Both patients in the cell line were able to detect HBV DNA in their blood at the time of resection. HBV DNA and hepatitis B surface antigen (HBsAg) were measured in culture supernatants to determine if two cell lines were maintaining stable HBV infection. As shown in Fig. 19, although the number of copies (CN) decreased, HBV DNA was still detected in both cell lines after a long time in the subculture. We attempted to confirm the presence of whole genomic HBV DNA in the cell lysate of the cell line but were not successful. Nevertheless, our data suggest that AMC-H1 and AMC-H2 secrete at least a portion of the HBV DNA S gene region, which stably integrates the genome and continuously secretes HDV DNA despite long-term subculture. In contrast, HBsAg could not be detected in the culture supernatants from both cell lines (Fig. 19). These results suggest that AMC-H1 and AMC-H2 do not produce a complete virus with infectivity, at least in spite of the fact that some HBV DNA is integrated into the genome, at least in the genome. Cell migration from the wound edge in the Wound healing assay was faster and larger in AMC-H2 than in AMC-H1 (AMC-H1 vs. AMC-H2; 100 vs 250 μm in 24 h; In summary, these results indicate that the two cell lines have similar proliferative capacity, HBV DNA and HBsAg secretion, and AFP and albumin expression but differ in morphology, metastatic capacity, and population of liver cancer stem cells (LCSCs) Suggesting that -H2 is more invasive.

AMC - H1 and AMC Tumor-related characteristic profiles in -H2

It is well known that EGFR, beta -catenin, p53 and PTEN do not function in liver cancer cells. We have identified the expression levels of the aforementioned factors in a number of different cancer cell lines (Huh7, SNU398, SNU449 and SNU475) as well as new cell lines. EGFR was expressed at similar levels in both AMC-H1 and AMC-H2. β- catenin was not overexpressed in the two new cell lines compared to other hepatoma cell lines. The tumor suppressor p53 and PTEN were expressed at low levels in both cell lines suggesting a potentially high survival rate. In addition, N-carderine expression associated with epithelial-mesenchymal transition and metastasis was also observed, which showed higher N-carderine expression in AMC-H2 than in AMC-H1. This finding was consistent with the wound healing assay, which showed higher migration potential in AMC-H2 than AMC-H1. In addition, we identified which signaling pathway was predominantly activated as well as related gene expression. In particular, we examined the two major receptor mediated pathways, RAS / RAF / MEK / ERK and PI3K / Akt pathways. Both RAS / RAF / MEK / ERK pathways did not show activity in both new cell lines and only the AMC-H2 was active in the PI3K / Akt pathway. Thus, AMC-H1 and AMC-H2 suggest that tumor genes and tumor suppressors are similar in expression but different in activated signaling pathways and invasive capacities (Fig. 13d). Genetic modification and instability in liver cancer can play an important role. To measure gene imbalance, SNP arrays were run using Affymetrix CytoScan ™ 750K and microsomal chromosome aberrations were analyzed for CN changes (CNVs) on each chromosome. CNV contains a relatively large portion of the genome that is deleted or replicated in a particular gene and consequently increases or decreases. As shown in the karyotypic analysis (FIG. 17), numerous genetic modifications were observed in most chromosomes of AMC-H1 and AMC-H2 (FIGS. 20 and 21). Previous studies have identified CD133 as a CSC surface marker through the characterization of CSCs in the primary HCC and found that CD133 + primary HCC cells have greater tumoroid-forming ability and are more chemically resistant than CD133-primary HCC cells. For characterization of new primary HCC cell lines, spheroid-forming ability was assessed in suspension cultured oocyte cells since CSCs were enriched in non-adherent spheroids of breast, colon and liver cancer cells. AMC-H2 cells formed spheroids well, whereas AMC-H1 did not form spheroids (Fig. 13e). Next, we detected the expression of CD133 in AMC-H1 and AMC-H2 HCC cells. Immunohistochemistry revealed that CD133 expression was predominant in the membrane of AMC-H2 HCC cells whereas CD133 was expressed only in the cytoplasm of AMC-H1 HCC cell spheroids (FIG. 13f).

Monolayer AMC - H1 and AMC - In H2 cells  Comparison of Sensitivity to Anticancer Drugs

We assessed the sensitivity of AMC-H1 and AMC-H2 HCC cell lines to chemotherapy in a monolayer culture system, as we observed differences in CD133 expression, genetic modification and activated signal transduction pathways between the two cell lines. Sorapenib, 5-FU and cisplatin, which are standard treatments for advanced HCC patients in Korea, were cultured in two cell lines. AMC-H1 and AMC-H2 HCC cells exhibited similar half-maximal inhibitory concentrations (IC ₅₀ ) for 5-FU, cisplatin, and taxol, with slightly different IC ₅₀ values for sorapenib (FIG. Reference).

[Table 5] Half maximal inhibitory concentration (IC ₅₀ ) of anticancer drugs in AMC-H1 and AMC-H2 primary HCC

AMC -H1 and AMC -H2 cells In the tumor sphere  Comparison of Sensitivity to Anticancer Drugs

3D tumor spheroids may better reflect tumor features in vivo than conventional monolayer cultured cells, and it is expected that tumor somatosensitivity will contribute to the development of optimized therapies for patients with hepatocellular carcinoma. The AMC-H1 cells showed limited ability to form spheroids, compared to AMC-H2 cells having strong cohesive power in a short period of time. In drug treatment studies on spheroids, homogenous size generation of single spheres of patients in multi-well plates is essential for the comparison of reactions at various compound and concentration conditions. To determine the feasibility of these constraints, we successfully created a single spherical body of uniform size in a multi-well plate. The Operetta HTS system was able to get images in just 10 minutes. In this way, we have established a simple method for obtaining high-speed images with high reproducibility (<10% size change) spheres. EthD-1 was used to bind dead DNA and detect fluorescence to detect dead cells in the globes. The intensity of EthD-1 staining was analyzed to quantify the degree of cell damage. Interestingly, tumor spheres of AMC-H1 and AMC-H2 cells exhibited different drug sensitivities under monolayer culture conditions (Fig. 14B). The spheres of AMC-H2 cells showed a strong resistance to sorapenib, 5-FU and cisplatin whereas the spheres of AMC-H1 cells significantly decreased cell survival rates for the aforementioned drugs. Differences in drug reactivity of cisplatin and 5-FU between AMC-H1 and AMC-H2, which were not apparent under monolayer culture conditions, were observed under 3D culture conditions. In general, when experimenting with monolayer culture conditions, the cell membrane must be diffused across the distance to reach the target only, whereas under 3D culture conditions the drug must spread across several layers of the cell to reach the target It is more realistic. Therefore, it is considered that the chemical resistance is larger in the 3D culture condition than in the single layer culture. However, unlike the idea, drug reactivity was more sensitive in 3D culturing conditions than monolayer culture condition for HCC cell line. These results indicate that sorapenib has an effective anticancer effect on AMC-H1 cells and that no drug is an effective treatment for AMC-H2 cells.

Multicellular Tumor spheroid And  Primary HCC  Sensitivity to anticancer drugs in cells

3D tumor spheroids are similar to in vivo tissues and represent the microenvironment of the tumor in relation to the distribution of oxygen, metabolites and nutrients as well as drug penetration. ; However, this structure lacks other key elements of tumor tissue. In particular, the tumor microenvironment includes epithelial cells, fibroblasts, activated hepatic stellate cells, invasive immune cells, endothelial cells, and ECM molecules such as collagen and integrins. In order to reduce this gap, we used HCC cell lines to mimic the previously observed tumor heterogeneity in vivo with LX2 (human hepatic stellate cells), WI38 (human fibroblasts) or HUVEC (vascular endothelial cell) 1 ratio to create a simple multicellular tumor spheroid model. This system is limited because it is mixed with one type of HCC cells and epileptic cells. In this study, we aimed to construct multicellular tumor cells using patient cells as a model for clinical application to investigate drug sensitivity. For this, AMC-H1 cells and AMC-H2 cells were incubated with three types of stromal cells (LX2, WI38, HUVEC) under 3D culture conditions. AMC-H1 and AMC-H2 MCTS showed a firmer and more sharp shape than tumor spheroids. In addition, the intensity of EthD-1 staining after treatment with 5-FU, sipflatin and sorafenib was analyzed at the indicated concentrations. Interestingly, similar results were obtained for the reactivity of the tumor spheroids and multicellular spheres with the three chemotherapeutic agents. In particular, sorapenib was effective against AMC-H1 cells and no drugs tested against AMC-H2 cells were effective. Regarding drug sensitivity, AMC-H1 multicellular globules and AMC-H2 multicellular globules were more sensitive than simple tumor spheres. In particular, the sensitivity to sorapenib, which was not observed in the AMC-H2 spheres, was observed in AMC-H2 multicellular spheres, and it was confirmed that sorapenib was the best chemotherapeutic method for AMC-H2 cells (FIG. These results indicate that despite the drug sensitivity observed in primary HCC cells, the sensitivities of drug therapy may change depending on the culture method. Furthermore, this finding suggests that 3D structure tumor microenvironment and the interaction between tumor and epileptic cells affect sensitivity to chemotherapy.

Primary HCC  Monolayer cultured cells, tumor spheroids and To MCTS  Comparison of Drug Sensitivity

AMC-H1 and AMC-H2 HCC cells were obtained from liver patient specimens and stabilized with cancer cell lines. Therefore, we investigated drug sensitivity to 8 primary HCC cells of monolayer culture cells, tumor spheroids and multicellular spheroids obtained from liver patients only 3-6 times subculture. When primary HCC was cultured, several cell types were cultured together, but hepatocytes survived only in stromal cell-free cultures after subculture 3-6 times in hepatocyte-specific culture conditions. Prior to investigating drug sensitivity of these cell conditions, tumor cells and multicellular globular bodies were determined for their ability to form (Figs. 18A). Six of the eight primary HCC cells tested formed tightly packed tumor spheroids; However, 8513B and 51532B showed spherical forms of loose agglomerates. All primary HCC cells formed solid and multicolored multicellular tumor morphology compared to tumor spheroids, with intercellular interactions helped by mixed cultures of stromal cells. In addition, the expression of liver or liver cancer markers showed albumin and HepPar-1 expression in the cytoplasm (Fig. 18B) as much as the AFP and albumin mRNA expression (Fig. 18C), even though the degree of difference between primary HCC cells was seen. We investigated drug sensitivity using a monolayer culture system, tumor spheroids and multicellular spheroids, and compared various primary HCC cells (see Table 6, below).

[Table 6] Drug sensitivity in monolayer culture system, tumor globule antibody and multicellular spheroids (MCTSs) obtained from patients

The method of analysis varied depending on the 2D and 3D culture systems. Nuclei were stained in the monolayer culture system, the number of cells was determined, and the survival rate was shown for the control (0.1% DMSO). On the other hand, tumor spheroids and multicellular spheroids were stained with EthD-1 to detect dead cells and cell death rates were compared to the control (0.1% DMSO). Because of this difference, the sensitivity to anticancer drugs was not compared to specific inhibitory concentrations such as IC50. Instead, drug sensitivity was classified as: " - " means a cell survival rate of 90% or more in monolayer cultures and less than 10% of cell death in tumor spheres and multicellular spheres, " + " Of tumor cells, and less than 30% and more than 10% of cell deaths in tumor cells and multicellular spheroids, while " ++ " means cell survival rates of less than 50% and 30% (X &gt; 90% and Y &lt; 10%, &quot; + &quot; 70% &lt; X &lt; 90% and 10% < Y < 30 %, "++" 30% <X <50% and 30% <Y <50%, X = survival rate in monolayer culture Y = cell death in spheroids and multicellular spheroids). According to Table 6, all primary HCCs respond sensitively to 5-FU in monolayer cultures, whereas only half of the primary HCCs respond to 5-FU in tumor spheres and multicellular globular systems. The selective response of 5-FU was most prominent in the multicellular spheroid culture system. The response to cisplatin was completely different between 2D and 3D culture systems, and no primary HCCs multicellular congeners were sensitive to cisplatin. Thus, primary HCCs MCTS showed the strongest cisplatin resistance among the three culture systems. In contrast, primary HCCs MCTS appeared to be the most sensitive to sorafenib. In the monolayer culture system, the primary HCC did not respond to sorafenib treatment, but the primary HCC tumor supernatant showed a mild response in sorapenib. Based on the following data, we concluded that primary HCC cell cultures differ in drug sensitivity depending on the culture method. Simplifying the assay system for monolayer cultures will not be useful as an optimized treatment for HCC patients with HCC because the results data can not be used for translation studies. Taken together, the tumor cell population reflects the 3D cellular environment, pathological gradients and in vivo heterogeneity, suggesting that the multicellular tumor sphere culture system is the best way to select an optimal treatment for patients with liver cancer.

Claims (13)

A multicellular tumor spheroid (MCTS) containing hepatocellular carcinoma cells and stromal cells. The multicellular tumor spherical body according to claim 1, wherein the ratio of the number of cells of the liver cancer cell: stromal cell is from 49:51 to 30:70. The multicellular tumor complex according to claim 1, wherein the stromal cell comprises at least one selected from the group consisting of hepatic stellate cells, fibroblasts and endothelial cells. The multicellular tumor spherical body according to claim 1, wherein the multicellular tumor spherical body further comprises liver cancer stem cells. [Claim 3] The multicellular tumor spherical body according to claim 1, wherein the multicellular tumor spherical body comprises liver cancer cells, hepatic stellate cells, fibroblasts, endothelial cells and liver cancer stem cells. [Claim 2] The multicellular tumor spherical body according to claim 1, wherein the multicellular tumor spherical body is formed by three-dimensional stereochemistry of liver cancer cells derived from a patient suffering from liver cancer. 7. The multicellular tumor spherical body according to claim 6, wherein the culture is carried out using a culture medium containing stem cell culture medium A method for screening a therapeutic agent for liver cancer using the multicellular tumor spherical body according to any one of claims 1 to 7. 9. The method of claim 8, wherein the liver cancer therapeutic agent is a patient-customized therapeutic agent. A method for screening a patient-customized liver cancer therapeutic comprising the steps of:
(a) culturing hepatocarcinoma cells derived from liver cancer patients in a three-dimensional stereochemistry to form multicellular tumor spheroids including liver cancer cells and stromal cells;
(b) treating the candidate substance with the multicellular tumor scaffold; And
(c) measuring a change in size of the multicellular tumor spheroid,
Here, when the size of the multicellular tumor spheroid decreases due to the treatment of the substance, the substance is determined to be the patient-customized liver cancer treatment agent. 11. The method of claim 10, wherein the change in size of the spherical body is measured by analyzing a spherical image obtained using a high-speed three-dimensional fluorescence imaging apparatus. 12. The method of claim 11, wherein the fast three-dimensional fluorescent imaging device is a confocal imaging device. A multicellular tumor spherical body according to any one of claims 1 to 7; And a high-speed three-dimensional fluorescence imaging device.

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