KR102115674B1 - Patient-derived hepatocellular carcinoma cell lines and the method for screening anticancer agents using thereof - Google Patents

Abstract

The present invention relates to patient-derived hepatocellular carcinoma cell lines (AMC-H1, AMC-H2) and a method for screening an anticancer agent using the same. The patient-derived hepatocellular carcinoma cell lines according to the present invention not only exhibit characteristics of liver cancer cells but also can continuously secrete HBV DNA and thus, can be used as a very useful platform for research on the onset and treatment of liver cancer related to HBV infection. In addition, according to the present invention, the patient-derived hepatocellular carcinoma cell lines can be used to discover the optimal treatment agent and treatment method for liver cancer and can be applied to discover a novel target for liver cancer treatment.

Description

Patient-derived liver cancer cell line and anti-cancer agent screening method using the same{Patient-derived hepatocellular carcinoma cell lines and the method for screening anticancer agents using thereof}

The present invention relates to a patient-derived liver cancer cell line (AMC-H1, AMC-H2) and an anticancer agent screening method using the same.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and is known to have a very poor prognosis. Since liver cancer is composed of cells with very heterogeneous molecular properties, it is difficult to study liver etiology and identify appropriate treatment targets. Liver cancer patients are generally less resistant to systemic chemotherapy due to liver dysfunction, and have resistance to conventional chemotherapeutic agents. Nevertheless, effective chemotherapy and other treatment strategies for liver cancer patients are lacking.

In particular, hepatitis B virus (HBV) is known to be infected with hepatocytes, causing acute hepatitis and chronic hepatitis, and is closely related to the risk of human liver cancer. Currently, "HBV" carriers do not necessarily become "liver cancer", and even "HBV" carriers often pass without suffering from liver cancer. Even hepatitis B virus carriers often pass through life without developing liver cancer, and about 30% of carriers develop liver cancer within 20 years after infection, and how molecular markers develop liver cancer. It is not known exactly whether it is transferred to other tissues. Therefore, there is a lack of research on therapeutic agents or treatment methods suitable for patients with liver cancer due to hepatitis B virus infection.

Therefore, clinical specialists have made efforts to select effective anti-cancer drugs to improve the quality of life of patients with liver cancer. As a result, personalized medicine has been proposed as a tailored treatment method with optimal response and highest safety, which can reduce unnecessary exposure to conventional chemotherapy, which has limited therapeutic efficacy and can cause acute toxic reactions. In addition, it is known as an optimal treatment method that can guarantee an optimal treatment for a patient. However, due to the lack of biopsy of liver cancer, low incidence of genetic mutations present in liver cancer, and tumor heterogeneity, it has been difficult to develop a customized treatment for liver cancer patients. Therefore, in order to overcome these problems and to study the exact mechanism for the development, progression and metastasis of liver cancer, a suitable in vitro model is essential.

Therefore, the present inventors tried to construct a primary liver cancer cell line by separating liver cancer cells from a large number of liver cancer patients infected with hepatitis B virus (HBV), and successfully constructed a liver cancer cell line separated from two patients. The present invention was completed by confirming that hepatocellular carcinoma marker expression and hepatocarcinoma-related signaling pathway in the cell line revealed hepatocarcinoma properties, and confirming hepatitis B virus (HBV) DNA secretion properties.

Korean Registered Patent No. 10-0118123

An object of the present invention is to provide a patient-derived liver cancer cell line and a screening method for liver cancer treatment using the same.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present inventors isolated liver cancer cells derived from a liver cancer patient infected with HBV, which is one of the most common carcinogenic factors of liver cancer, and constructed it as a cell line.

The present invention provides a liver cancer cell line derived from a patient deposited with accession number KCLRF-BP-00461 (AMC-H1) or accession number KCLRF-BP-00462 (AMC-H2).

According to one aspect of the invention, the cell line may be isolated from a liver cancer patient infected with Hepatitis B virus (Hepatitis Virus B, HBV).

According to an aspect of the present invention, the cell line may secrete hepatitis B virus DNA.

According to one aspect of the invention, the cell line may be to express at least one of Hep Par-1 (Hepatocyte Specific Antigen), AFP (alpha fetoprotein) and albumin.

The cell line of the present invention is a liver cancer cell line isolated and cultured from 110 liver cancer samples obtained from surgical surgery, and two liver cancer cell lines are successfully constructed. Using the newly constructed cell line in the present invention, cell morphological differences, expression of hepatocyte-specific markers, release of HBV DNA, growth kinetics, invasiveness and number of copies of genes (CN, copy) nimber).

As a result, it was confirmed that the shape of the two cell lines (FIG. 1) and the metastatic ability (FIG. 4) were different, from which it was found that AMC-H2 was more invasive than AMC-H1. In addition, the cell lines of the present invention all express hepatocyte-specific markers Hep Par-1 (Hepatocyte Specific Antigen), AFP (alpha fetoprotein) and albumin (Figs. 2 and 3), stably express HBV DNA It was confirmed to discharge (Table 3). As a result of SNP microarray analysis, instability of chromosomes was observed in both cell lines, and it was confirmed that copy number variation (CNV) was significantly increased (FIG. 6).

The present invention provides a composition for screening for an anticancer agent comprising a liver cancer cell line derived from a patient deposited with accession number KCLRF-BP-00461 (AMC-H1) or accession number KCLRF-BP-00462 (AMC-H2).

The patient-derived liver cancer cell line AMC-H1 or AMC-H2 according to the present invention is a liver cancer cell containing HBV DNA, and thus a liver cancer-specific biomarker is expressed, thereby screening candidate substances that induce or inhibit the expression of a liver cancer-specific marker. Can be useful.

According to an embodiment of the present invention, after culturing a patient-derived liver cancer cell line of the present invention, reactivity to different anticancer agents was measured. Specifically, for the treatment of liver cancer, sorafenib (sorafenib, Nexavar ^TM ), the only targeted therapeutics approved by the FDA, and 5-fluorouracil (5-FU, 5-fluorouracil) and cisplatin, which are known as various solid cancer drugs, Reactivity was measured. As a result, AMC-H1 and AMC-H2 showed different reactivity to each drug, and it was confirmed that both AMC-H1 and AMC-H2 showed resistance to sorafenib.

As described above, not only can a drug that is most suitable for a patient be selected from among various candidate anticancer drugs by using a property in which the reactivity to an anticancer agent is different depending on the type of the liver cancer cell line of the present invention, but also a new liver cancer target anticancer drug can be screened from the candidate drugs have.

The present invention also comprises the steps of: (a) treating a candidate for treatment for liver cancer on a liver cancer cell line derived from a patient deposited with accession number KCLRF-BP-00461 (AMC-H1) or accession number KCLRF-BP-00462 (AMC-H2); And (b) measuring reactivity to the candidate substance.

The candidate drug for treating liver cancer of the present invention may include sorafenib (sorafenuib), which has already been approved as a drug for treating liver cancer, but is not limited thereto. Specifically, the candidate agent for treating liver cancer may be applied to any drug that exhibits a therapeutic effect on various cancers, as well as any anticancer drug candidate that is expected to exhibit anticancer effects.

The step (b) of the present invention is a step of measuring a change in the cell line after treating the candidate material on the liver cancer cell line, the change in viability, growth or metastasis of the liver cancer cell line, the change in the expression of a specific biomarker for liver cancer, It may include changes in the specific signaling pathway of liver cancer, changes in the amount of HBV DNA secretion, and the like, but is not limited to, as long as it can confirm treatment or improvement of liver cancer.

The present invention also comprises the steps of: (a) performing candidate anti-cancer therapy on a liver cancer cell line derived from a patient deposited with accession number KCLRF-BP-00461 (AMC-H1) or accession number KCLRF-BP-00462 (AMC-H2); And (b) measuring the reactivity to the treatment.

According to an aspect of the present invention, the candidate anti-cancer therapy may include one or more selected from the group consisting of chemotherapy, immunotherapy and radiation therapy. The chemotherapy may include treating an anticancer drug candidate, the immunotherapy may include treating an immune enhancer or treating immune cells, and the radiation therapy may irradiate cell lines with radiation. It may include a step, but is not limited to this.

When a specific anti-cancer treatment is performed on a patient-derived liver cancer cell line of the present invention, reactivity can be measured and compared to predict the most effective treatment method for the patient, thereby improving treatment efficacy.

The patient-derived liver cancer cell lines AMC-H1 and AMC-H2 of the present invention not only exhibit the characteristics of liver cancer cells, but also can continuously secrete HBV DNA, and are very useful for research on the process and treatment of liver cancer associated with HBV infection. It is expected to be a platform. In addition, it is expected that the optimal treatment and treatment method for liver cancer using the patient-derived liver cancer cell line may be applied to discover new targets for treatment of liver cancer.

1 shows the morphology of the AMC-H1 and AMC-H2 cell lines of the present invention.
2 is a result of immunostaining the AMC-H1 and AMC-H2 cell lines of the present invention with AFP, albumin (ALB) and HepPar-1.
3 is a result of confirming the mRNA expression levels of AFP and albumin (ALB) in the AMC-H1 and AMC-H2 cell lines of the present invention.
Figure 4 shows the results of wound healing analysis of the AMC-H1 and AMC-H2 cell lines of the present invention.
5 is a result of Western blot expression of EGFR, b-catenin, N-cadherin, p53, PTEN, ERK and Akt in AMC-H1 and AMC-H2 cells. The activity of the signaling pathway was confirmed by measuring phosphorylation of ERK and Akt.
Figure 6 It is the result of karyotype analysis of AMC-H1 and AMC-H2. Blue bars indicate chromosomal increase, red bars indicate chromosome reduction, and purple bars indicate loss of heterozygosity (LOH).
7 is a result of spheroid culture of AMC-H1 and AMC-H2 cells.
8 is a result of CD133 immunostaining of AMC-H1 and AMC-H2 cells.
9 is a result of confirming the reactivity of AMC-H1 and AMC-H2 cells to 5-FU, cisplatin and sorafenib in a single-layer culture condition.
10 is a result of photographing changes over time of tumor spheroids made from patient-derived liver cancer cells AMC-H1 and AMC-H2.
11 is a result confirming the susceptibility to 5-fluorouracil (5-FU) in tumor spheroids of AMC-H1 and AMC-H2 HCC cells.
12 is a result confirming the susceptibility to cisplatin (cisplatin) in tumor spheroids of AMC-H1 and AMC-H2 HCC cells.
13 is a result confirming the susceptibility to sorafenib (sorafenib) in tumor spheroids of AMC-H1 and AMC-H2 HCC cells.

Below, The present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

[Example]

1. Patient-derived liver cancer cell line production

In the present invention, primary liver cancer cells were isolated from liver samples cut from hepatocellular carcinoma (HCC) patients to provide more data for clinical drug application and to develop a platform for selecting the optimal anticancer agent.

Specifically, immediately after liver cancer surgery of liver cancer patients due to hepatitis B virus (HBV) infection, the incised tumor portion was immersed in HBSS (Hanks balanced salt solution) and transported to the laboratory at 0°C. After collecting samples in a sterile state, blood was removed by washing with HBSS-free calcium and magnesium several times. The tumor sample is then cut into small pieces, 0.03% pronase, 0.05% type IV collagenase, and 0.01% deoxyribonuclease (DNase, from bovine pancreas) It was soaked in the included HBSS at 37°C for 20 minutes. The resulting suspension was filtered through a 100-μm nylon filter and centrifuged at 50 xg and 4°C for 20 minutes to obtain hepatocytes. The liver cell pellet was washed with HBSS containing 0.005% DNase. The final cell suspension was hepatocyte basal medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated FBS, 1 ng/ml hepatocyte growth factor (HGF), and 1x antibiotic-antifungal agent. The collagen-coated T25 flask (BD Falcon) was placed and cultured in a 37°C incubator containing 5% CO ₂ . The medium was changed after 24 hours to remove dead cells and cellular debris. When the cell density reached 70-80%, the cells were transferred to the HBM medium. Dense cells were counted after trypsin treatment and diluted 1:3 to 1:5 step by step. When the cell line was maintained over 30 passages, the cells were collected and stored in liquid nitrogen.

2. Immunocytochemical analysis

Immunocytochemical analysis was performed to confirm whether the primary liver cancer (HCC) cell lines of the two species isolated from the patient retain the characteristics of the original liver cancer.

Specifically, AMC-H1 and AMC-H2 cell lines were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature, and treated with 0.1% Triton X-100 for 30 minutes at room temperature to form permeability, followed by DPBS ( Dulbecco's Phosphate-Buffered Saline, Dulbecco's Phosphate Buffered Saline). Next, each cell sample was incubated with a primary antibody at 4°C for 16 hours, and washed several times with DPBS. Mouse monoclonal anti-human serum albumin (ALB; 15C7, ab10241, 1:500; Abcam, Cambridge, UK) as primary antibody, mouse monoclonal anti-human hepatocytes (mouse monoclonal anti-human human Hep Par-1, Clone OCH1E5, M7158, 1:100; Agilent Technologies, Santa Clara, CA, USA), and mouse monoclonal anti-human CD133/1, AC133, 130- 090-422, 1:100; Miltenyi Biotec, Bergisch Gladbach, Germany). Next, for cell staining, the sample was incubated for 1 hour at room temperature in the secondary antibody and darkroom, and then washed several times with DPBS. As a secondary antibody for the dye Alexa ^® Fluor 488 is coupled goat anti-mouse IgG (goat anti-mouse IgG) and Alexa ^® Fluor the 488 is coupled goat anti-rabbit IgG (goat anti-rabbit IgG) was used . Next, in order to stain the nucleus, the cells were incubated with Hoechst 33342 (Invitrogen) for 10 minutes at room temperature in the dark and then washed with PBS. All fluorescence images were taken using the Operetta ^® High Content Screening (HCS) System (Perkin Elmer, Waltham, MA, USA).

3. Wound healing assay

In order to confirm the metastatic ability of the cell line, a wound healing analysis was performed. Specifically, the cell lines were placed in 6-well plates, respectively, and the cell density was grown to 80-90%. Cells grown to an appropriate level were scratched using a pipette tip. Cell movement was observed from the boundary of the wound, and a time-lapse image was taken with a microscope equipped with a camera (Nikon 3).

4. Western blot analysis

Western blot analysis was performed to confirm the molecular properties of the cell lines of the present invention. Specifically, the obtained cells were centrifuged at 1000 rpm for 3 minutes, and then pellets were obtained and eluted using RIPA (radioimmunoprecipitation assay) buffer (InTron, South Korea). The eluted cells were centrifuged at 4°C and 12,500 rpm for 20 minutes, and the supernatant was separated.

Protein concentration was measured by BCA (bicinchonic acid) assay (Promega), epidermal growth factor receptor (EGFR), β-catenin, N-cadherin, p53, Western blot was performed using antibodies against PTEN, pERK, ERK, pAkt, and Akt. Protein signals were detected using electrochemiluminescence (ECL) reagents (GE, Fairfield, CT, USA).

5. SNP microarray

To confirm the genetic specificity of the AMC-H1 and AMC-H2 cells of the present invention, a single-base polymorphic array analysis was performed using Affymetrix CytoScan ^™ 750K (Affymetrix, Santa Clara, CA, USA). Specifically, DNA was separated from AMC-H1 and AMC-H2 using AxyPrep Multisource Genomic DNA Kit (Axygen, Union City, CA, USA), and SNP microarray was performed according to standard usage. Experimental data were analyzed using Chromosome Analysis Suite 2.0 software (Affymetrix) to detect copy number variations and loss of heterozygosity (LOH).

6. Liver cancer stem cells (LCSC) spheroid formation

Primary cells of AMC-H1 and AMC-H2 were supplemented with 1Х B27, 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epithelial growth factor (EGF) and DMEM/F12 supplemented with 25 μg/ml insulin ( Gibco) was embedded in a 6-well ultra-low attachment (ULA) plate (Corning Life Sciences, Amsterdam, The Netherlands). Cells were cultured for 5-7 days without replacing the culture medium.

7. Tumor spheroid formation and drug treatment

To generate tumor spheroids, cells cultured suspended in HBM were planted in 96-well round bottom ULA microplates at a density of 6Х10 ³ cells/well. Plates with cells were incubated at 5% CO ₂ at 37° C. for 3 days. After 3 days, 5-fluorouracil (5-FU; Sigma-Aldrich), cisplatin (cisplatin; Sigma-Aldrich), and sorafenib (sorafenib; Santa Cruz Biotechnology) were added and further cultured for 7 days. As a negative control, a 0.5% DMSO (dimethylsulfoxide, dimethyl sulfoxide; Sigma-Aldrich) solution was used.

8. Anti-cancer drug susceptibility under single-layer culture conditions

Each liver cancer cell line was planted in a 384-well plate (Greiner Bio-One, Monroe, NC, USA) at a concentration of 2.5 x 10 ³ cells/well and cultured for 16 hours. After 16 hours, 5-fluorouracil (5-FU), cisplatin and sorafenib were added to each well and cultured for 48 hours. After 48 hours, cells were fixed with 4% PFA for 10 minutes at room temperature and washed twice with DPBS. Hoechst 33342 was used for nuclear staining. In order to secure enough cells (> 1,000) for analysis, 5 pictures were obtained from each well, and all pictures were analyzed using HCS system and Harmony software. Cells were counted and normalized to control (0.5% DMSO).

9. Confirm cell death in spheroid

Spheroid cell death was detected with ethidium homodimer-1 (EthD-1; Invitrogen). EthD-1 is a substance with high affinity to nucleic acids, which exhibits weak fluorescence until it binds to DNA, but has the property of emitting bright red fluorescence after binding to nucleic acids of dead cells (excitation/emission maxima ~528/617 ).

After spheroids were incubated for 30 minutes at 37°C with 4 μM EthD-1 in HBM medium, a picture of EthD-1 was taken using an HCS system, and the degree of luminescence was measured.

10. Real-time PCR

Total RNA was isolated from the cell line using TRIzol ^® (Invitrogen). A RT buffer, dNTP solution, RNasin ^® inhibitor, oligo (dT) ¹⁵ primer, total RNA, and M-MLV reverse transcriptase reaction mixture by mixing (Moloney Murine Leukemia Virus) were prepared. The reaction mixture was reacted at 37° C. for 1 hour, and the transfer reaction was terminated by heat treatment at 95° C. for 5 minutes, followed by rapid cooling on ice.

PCR reaction was performed using a StepOnePlus real-time PCR system (Applied Biosystems) in a 96-well plate with a mixture of cDNA, SYBR Green master mix, primer, and DEP (diethyl pyrocarbonate, diethyl pyrocarbonate). .

After reacting at 95°C for 10 minutes, 15 seconds at 95°C and 1 minute at 60°C were repeated 40 times. The CT (threshold cycle) value is defined as the number of cycles of the fluorescence signal passing through a fixed threshold. CT values were calculated by the following equation.

R = 2-△△CT

(The above △CT = CT _{target gene} -CT _GAPDH , △△CT = △CT _test- △CT _control )

CT values were normalized to GAPDH expression.

All real-time PCR showed the results of three repeated experiments as the mean ± standard deviation (SD).

Primers used in this example were purchased or designed from Bioneer and are shown in Table 1 below.

name Kinds Base sequence (5'->3') Sequence number ALB Forward CGC CTG AGC CAG AGA TTT CC One Reverse ATC TCC ATG GCA GCA TTC CG 2 AFP Foward GGA CCC GAA CTT TCC AAG CC 3 Reverse TGG GCC ACA TCC AGG ACT AG 4

[Experiment result]

1. Confirmation of liver cancer (HCC) characteristics of patient-derived cell lines AMC-H1 and AMC-H2

1-1) Check the morphological characteristics

As a result of culturing the cells isolated from patients with liver cancer (HCC) according to the above embodiment, two new liver cancer cell lines, AMC-H1 and AMC-H2, were selected, and deposit numbers KCLRF-BP-00461 (AMC in each depository institution) -H1) or deposit number KCLRF-BP-00462 (AMC-H2).

The characteristics of the two patients at the time of liver cancer resection were shown in Table 2 below.

Characteristic AMC-H1 AMC-H2 Age/Gender 55 years old / female 51 years old / man HBsAg positivity positivity Serum HBV DNA 1.6 x 10 ⁶ copies/mL 7.2 x 10 ⁶ copies/mL Anti-HCV antibodies voice voice Serum-αfetoprotein 128,000 ng/mL 4.4 ng/mL Liver cancer biopsy diagnosis
(HCC Pathologic diagnosis) Multinodular confluent type Expanding nodular type Vascular invasion No vascular invasion Edmondson-Steiner grade 3/2* Edmondson-Steiner grade 3/2* Cirrhosis No cirrhosis AJCC TNM stage T3bN0M0 stage IIIB T1N0M0 stage I

*Edmondson-Steiner grade worst/major.

*Acronym: HBV, hepatitis B virus; HCV, hepatitis C virus; AJCC, American Joint Committee on Cancer.

When the primary culture of the entire tumor was started after resection, hepatocytes were observed to extend from other types of cells. After the 2nd to 3rd generation culture, almost all cultured tissues were composed of hepatocytes. Hepatocyte-rich culture tissues were maintained over 50 generations in DMEM with 10% FBS.

In order to confirm whether the constructed cell line was a liver cell, the morphology of AMC-H1 and AMC-H2 was observed under a microscope. During the proliferation phase, AMC-H1 cells had a homogeneous cell configuration of epithelial-like or polygonal shape and showed a transparent nucleus with multiple nucleoli. Cells grew in a monolayer with clear boundaries. On the other hand, AMC-H2 appeared in a thread shape (Fig. 1).

1-2) Liver cancer (HCC) cell marker identification

Hepatocyte-specific markers Hepocyte-1 (Hepatocyte Specific Antigen), AFP (alpha fetoprotein) and albumin are used to confirm whether the primary liver cancer cell lines of the two species isolated from the patient maintain the original liver cancer characteristics. To perform immunocytochemistry analysis and real-time PCR (real-time PCR) analysis.

As a result, Hep Par-1 was expressed in both patient-derived cell lines, and AFP expression was significantly higher in AMC-H2 than in AMC-H1. Albumin was also expressed in both cell lines and higher in AMC-H1 (FIG. 2 ). From these results, it was found that the levels of AFP and albumin are different in AMC-H1 and AMC-H2, but both cell lines have characteristics of hepatocytes.

In addition, as a result of evaluating the mRNA expression levels of AFP and albumin (ALB) through real-time PCR analysis, AFP and albumin mRNA expression was different in AMC-H1 and AMC-H2. Specifically, AFP mRNA was stronger in ANC-H2, while ALB mRNA expression was higher in AMC-H1. It was found that the expression pattern of AFP and ALB mRNA was consistent with the result of the immunostaining (FIG. 3).

1-3) Hepatitis B virus characterization

The two patients who provided the cell line were liver cancer patients diagnosed with HBV infection, and both were detected with HBV DNA in the blood at the time of resection. We have identified HBV DNA and hepatitis B surface antigen (HBsAg) in cell culture to ensure that HBV infection is stably maintained in both cell lines.

As a result, as shown in [Table 3], the viral copy number (CN) decreased, but HBV DNA was detected in both cell lines. Although the whole genome of HBV DNA in the cell eluate of the cell line could not be confirmed, from the above results, the AMC-H1 and AMC-H2 cell lines of the present invention stably retain at least a portion of the HBV DNA gene in the genome, and It has been found that despite time passage culture, HBV DNA can be continuously secreted.

However, HBsAg could not be detected from the culture of each cell line. This means that although a portion of HBV DNA is inserted into the genomes of AMC-H1 and AMC-H2, each cell line does not produce an intact form of virion with infectivity.

AMC-H1 AMC-H2 Experiment date 2008. 12. 01 2012.06.13 2008. 12. 01 2012.06.13 HBV DNA
(copies/mL) 8,670 1,550 2,685 700 HBsAg (IU/mL) NA voice NA voice

*Acronym: HBV, hepatitis B virus; HBsAg, hepatitis B virus surface antigen; NA, not applicable

1-4) Cell mobility check

Wound healing assay was performed to confirm the cell migration properties of the patient-derived liver cancer cell line of the present invention. As a result, the cell migration distance from the wound boundary for 24 hours was confirmed to be 100 μm in AMC-H1 and 250 μm in AMC-H2, and the cell migration was larger and faster in AMC-H2 (FIG. 4 ).

From these results, the proliferation capacity of two cell lines, HBV DNA and HBsAg secretion capacity, AFP and albumin expression show similar characteristics, but the shape, metastasis capacity, and composition of liver cancer stem cells (LCSCs) of the two cell lines are similar. It was confirmed that it was different, from which it was found that AMC-H2 was more invasive than AMC-H1.

2. Confirmation of tumor-related characteristics of patient-derived cell lines AMC-H1 and AMC-H2

2-1) Confirmation of gene marker expression related to liver cancer

EGFR, β-catenin, p53, and PTEN are genes well known as liver cancer (HCC) markers. Expression of the liver cancer markers was confirmed by Western blot in two types of patient-derived liver cancer (HCC) cell lines and different liver cancer cell lines (Huh7, SNU398, SNU449, and SNU475). As a result, EGFR was expressed at similar levels in AMC-H1 and AMC-H2, and β-catenin was not overexpressed in AMC-H1 and AMC-H2 compared to other liver cancer cell lines. Tumor suppressors p53 and PTEN were expressed at very low levels in both cells (Fig. 5).

In addition, as a result of confirming the expression of N-cadherin associated with epithelial-mesenchymal transition and cancer metastasis, the expression of N-cadherin in AMC-H2 was higher than that of AMC-H1. Appeared higher. This is consistent with the results of wound healing analysis, which showed higher cell migration capacity in AMC-H2 than in AMC-H1 (Fig. 5).

In addition, it was confirmed whether the specific signaling pathway is activated as well as the expression of genes related to cancer. In particular, with respect to the major receptor-mediated active pathways RAS/RAF/MEK/ERK and PI3K/Akt, the RAS/RAF/MEK/ERK pathway was not activated in both cell lines, and the PI3K/Akt pathway was AMC-H2 Was activated only in FIG. 5 (FIG. 5 ).

From this, it was found that AMC-H1 and AMC-H2 have similar expressions of carcinogenic genes and cancer inhibitory genes, but different invasive and active signaling pathways.

2-2) Chromosome mutation confirmation

It is known that chromosomal variation and instability play an important role in the development of liver cancer. SNP array using Affymetrix CytoScanTM 750K was performed to confirm that such genetic imbalance appeared in the patient-derived cell line of the present invention, and it was analyzed by gene copy number variations (CNVs) of each chromosome. CNVs contain relatively large regions of the genome that are deleted or overlapped on a particular chromosome, resulting in increased or decreased chromosomes.

As a result of karyotype analysis, as shown in FIG. 6, genetic variation was confirmed in most chromosomes of the AMC-H1 and AMC-H2 cell lines.

2-3) Confirmation of tumor spheroid formation ability

CD133 has been reported to be a surface marker of CSC (cancer stem cells), and CD133+ liver cancer cells are known to have greater tumor spheroid forming ability and chemical resistance compared to CD133-.

As a result of evaluating whether the new liver cancer cell lines AMC-H1 and AMC-H2 of the present invention have the ability to form tumor spheroids in culture, AMC-H2 cells aggressively formed spheroids, while AMC-H1 cells were spheroids It was found that does not form (Fig. 7).

Next, as a result of analyzing CD133 expression through immunohistochemical analysis, CD133 expression was predominantly expressed in the cell membrane of AMC-H2 liver cancer cells, whereas in the case of AMC-H1 liver cancer cells, expression of CD133 was confirmed only in the cytoplasm (FIG. 8).

3. Confirmation of anti-cancer drug susceptibility under single-layer culture conditions

Through the above examples, it was confirmed that the AMC-H1 and AMC-H2 cell lines of the present invention differ in signaling pathway activation, genetic variation, and CD133 status, and whether the two cell lines show differences in sensitivity to anticancer agents. I wanted to confirm.

As a result, AMC-H1 and AMC-H2 liver cancer cells showed similar half maximal inhibitory concentrations (IC ₅₀ ) for 5-FU, cisplatin and taxol, and slightly different for sorafenib ( Fig. 9).

IC ₅₀ (μM) IC ₅₀ ER AMC-H1 AMC-H2 5-FU 2.05 1.71 1.198830409 Cisplatin 4.91 3.71 1.323450135 Taxol 0.125 0.127 0.984251969 Sorafenib 7.92 21.08 0.375711575

*IC ₅₀ ER: IC ₅₀ with enhancement ratio (AMC-H1/AMC-H2)

4. Confirmation of anticancer drug susceptibility in tumor spheroids

Since the three-dimensional tumor spheroid can exhibit the in vivo characteristics of the tumor better than the conventional single-layer cultured cells, the tumor spheroid is expected to greatly contribute to the development of an anticancer agent that is most suitable for patients. do.

While spheroids were formed with the patient-derived cell line of the present invention and observed, AMC-H2 cells showed strong cohesion for a short time, while AMC-H1 cells confirmed that their ability to improve spheroid strength was limited (FIG. 10). ).

In the drug efficacy test for spheroids, the patient's cells are first derived from the patient of the present invention because it is essential to compare the drug responsiveness under various drug and concentration conditions in a multi-well plate in which the patient's cells are produced as a homogeneous size Cell lines were generated as single spheroids of uniform size in multi-well plates. Next, a bright photograph was taken with the Operetta HTS system for 10 minutes. EthD-1 was used to confirm cell death in the spheroid, and EthD-1 staining intensity was analyzed by the amount of cell damage concentration (FIGS. 11 to 13 ).

As a result, as shown in FIGS. 11 to 13, tumor spheroids of AMC-H1 and AMC-H2 cells showed different anticancer drug susceptibility than in monolayer culture. AMC-H2 spheroids have strong resistance to sorafenib, 5-FU, and cisplatin, while cell survival was significantly reduced by this drug in AMC-H1 spheroids.

The difference in drug responsiveness to 5-FU and cisplatin in AMC-H1 and AMC-H2 was not observed in single-layer culture conditions, but was clearly observed in three-dimensional culture conditions. In general, when testing drug reactivity, in a single-layer culture, the drug spreads only along a membrane of a target cell for a short distance, whereas in a three-dimensional culture condition, it spreads along multiple layers of cells, making it a more realistic model. Therefore, in general, resistance to chemotherapy is thought to be greater in 3D culture than in monolayer culture. However, unexpectedly, in the liver cancer cell line, drug reactivity was more sensitive in 3D culture than in monolayer culture. From these results, it was found that sorafenib is the most effective anticancer agent for AMC-H1 cells and no effective anticancer agent for AMC-H2 cells.

Korea Cell Line Bank KCLBKCLRF-BP-00461 20181010 Korea Cell Line Bank KCLBKCLRF-BP-00462 20181010

<110> THE ASAN FOUNDATION UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION <120> Patient-derived hepatocellular carcinoma cell lines and the method for screening anticancer agents using thereof <130> 1063969 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ALB <400> 1 cgcctgagcc agagatttcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ALB <400> 2 atctccatgg cagcattccg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for AFP <400> 3 ggacccgaac tttccaagcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for AFP <400> 4 tgggccacat ccaggactag 20

Claims (8)

Sorafenib-resistant liver cancer cell line derived from a patient deposited with accession number KCLRF-BP-00462 (AMC-H2).
According to claim 1,
The cell line is a patient-derived sorafenib-resistant liver cancer cell line isolated from a liver cancer patient infected with Hepatitis Virus B (HBV).
According to claim 1,
The cell line is a patient-derived sorafenib-resistant liver cancer cell line characterized by secreting hepatitis B virus DNA.
According to claim 1,
The cell line is a patient-derived sorafenib-resistant liver cancer cell line characterized by expressing Hep Par-1 (Hepatocyte Specific Antigen), AFP (alpha fetoprotein) and albumin.
A composition for screening an anticancer agent comprising a patient-derived sorafenib-resistant liver cancer cell line deposited with accession number KCLRF-BP-00462 (AMC-H2).
(a) treating a candidate for liver cancer treatment with a patient-derived sorafenib-resistant liver cancer cell line deposited with accession number KCLRF-BP-00462 (AMC-H2); And
(B) measuring the reactivity to the candidate substance; liver cancer treatment screening method comprising a.
(a) performing candidate anticancer therapy on a patient-derived sorafenib-resistant liver cancer cell line deposited with accession number KCLRF-BP-00462 (AMC-H2); And
(B) measuring the reactivity to the treatment; anti-cancer treatment efficacy evaluation method comprising a.
The method of claim 7,
The candidate anti-cancer therapy is a method for evaluating the efficacy of an anti-cancer therapy that is one or more selected from the group consisting of chemotherapy, immunotherapy and radiation therapy.

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