Anticancer drug-resistant and sensitive liver cancer cell line, and method for screening liver cancer therapeutic agents by using same

Abstract

The present invention relates to anticancer drug-resistant and sensitive liver cancer cell lines, and a method for screening liver cancer therapeutic agents by using same. Specifically, provided are a liver cancer cell line PLLC15 (Accession No. 00000) that does not have sorafenib resistance and / or a liver cancer cell line PLLC15_Sora (Accession No. 00000) that has sorafenib resistance, or a composition for screening anticancer agents including one or more thereof, and a method for screening anticancer agents, and thus an anticancer immunotherapeutic agent or method using immune function of an experimental animal can be screened and time and cost can be reduced by reviewing in advance a sorafenib resistance problem in the development stage of liver cancer therapeutic agents.

Description

Anticancer drug-resistant and non-resistant liver cancer cell lines and liver cancer treatment drug screening method using the same

[0001] The present invention relates to anticancer drug-resistant and non-anticancer drug-resistant liver cancer cell lines and a method for screening liver cancer therapeutic drugs using the same. More specifically, the invention relates to the sorafenib-resistant liver cancer cell line PLLC15_Sora (Accession No.: KCTC15968BP) and the sorafenib-non-resistant liver cancer cell line PLLC15 (Accession No.: KCTC15967BP), and a method for screening liver cancer therapeutic drugs using the same.

[0002] Liver cancer is a fatal type of cancer with high incidence and mortality rates worldwide. Currently, various anticancer drugs are being used to treat liver cancer. Among them, sorafenib is widely used as a first-line anticancer drug for liver cancer, with proven efficacy in treating hepatocellular carcinoma (HCC).

[0003] Sorafenib is a protein kinase inhibitor that has activity against protein kinases. It is known as an oral targeted anticancer drug (Oral Multikinase Inhibitor) that treats cancer cells and vascular endothelial cells that supply nutrients to cancer cells by simultaneously inhibiting Raf-kinase, a serine / threonine kinase in the signaling pathway, along with VEGFR-2, platelet-derived growth factor receptor (PDGFR)-β, and c-kit, which are overexpressed in tumor cells or tumor blood vessels, leaving normal cells untouched.

[0004] In addition, sorafenib is known to inhibit tumor growth by inducing autophagy, and as a potent soluble epoxide hydrolase inhibitor, it is used as a treatment for unresectable hepatocellular carcinoma (HCC) by reducing side effects.

[0005] However, drug resistance develops in a significant number of patients treated with sorafenib, leading to a decline in therapeutic efficacy. In particular, because sorafenib targets multiple therapeutic targets, its mechanism of action is ambiguous, and the underlying causes of drug resistance are also not well understood. The occurrence of such sorafenib resistance acts as a major obstacle to the treatment of liver cancer, necessitating patients to receive alternative or cross-drug therapy. Accordingly, there is a need for cell line-based models to identify drugs specifically designed for sorafenib resistance and to propose new treatment strategies.

[0006] Meanwhile, immunotherapy has recently emerged as a new treatment method for cancer patients. However, the development of new anticancer immunotherapies and therapeutic drugs is facing difficulties due to a lack of appropriate models that link immunotherapy with clinical diagnosis and treatment strategies. Specifically, most established xenograft tumor models are created using immunodeficient mice, and most of these models neglect the effects of T cells. Since xenograft tumor models in this environment provide a tumor microenvironment that excludes immune function, they are unsuitable as tumor models for immunotherapy that utilizes the patient's own immune system to fight cancer. Therefore, there is a need to establish tumor models that are rich in immune cells within the tumor microenvironment.

[0007] The objective of the present invention is to provide anticancer drug-resistant and non-anticancer drug-resistant liver cancer cell lines and a method for screening liver cancer therapeutic drugs using the same.

[0008] To solve the above problem, the present invention provides a sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP) that does not have sorafenib resistance.

[0009] In addition, the present invention provides a sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP) having sorafenib resistance.

[0010] Accordingly, the present invention provides a composition for anticancer drug screening comprising one or more of the sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP) which does not have sorafenib resistance and the sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP) which has sorafenib resistance.

[0011] In addition, the present invention comprises the steps of: (1) treating one or more liver cancer cell lines among PLLC15 (accession number: KCTC15967BP), a sorafenib-non-resistant liver cancer cell line that does not have sorafenib resistance, and PLLC15_Sora (accession number: KCTC15968BP), a sorafenib-resistant liver cancer cell line that has sorafenib resistance, with a liver cancer therapeutic agent; and

[0012] (2) A method for screening anticancer drugs including the step of measuring the responsiveness to the above-mentioned liver cancer treatment candidate substance.

[0013] The present invention comprises the steps of: (1) performing a candidate anticancer therapy on one or more liver cancer cell lines among PLLC15 (accession number: KCTC15967BP), a sorafenib-non-resistant liver cancer cell line that does not have sorafenib resistance, and PLLC15_Sora (accession number: KCTC15968BP), a sorafenib-resistant liver cancer cell line that has sorafenib resistance; and

[0014] (2) A method for evaluating the efficacy of anticancer therapy including a step of measuring responsiveness to the above candidate anticancer therapy.

[0015] The above candidate anticancer therapies may be one or more selected from the group consisting of chemotherapy, immunotherapy, and radiation therapy, but are not limited thereto.

[0016] The sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP), which does not possess sorafenib resistance, and the sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP), which possesses sorafenib resistance, are cell lines established from liver cancer cells formed by inducing hepatocellular carcinoma in immunocompromised mice with a liver cancer-inducing substance. By establishing mouse liver cancer cell lines from immunocompromised mice without using immunodeficient experimental animals, they can be used to evaluate the efficacy of anticancer drugs or anticancer therapies targeting the immune function of liver cancer animal models transplanted allogeneically or xenogeneically from the liver cancer cell lines.

[0017] Accordingly, the present invention comprises the step of (1) injecting one or more liver cancer cell lines among PLLC15 (accession number: KCTC15967BP), a sorafenib-non-resistant liver cancer cell line that does not have sorafenib resistance, and PLLC15_Sora (accession number: KCTC15968BP), a sorafenib-resistant liver cancer cell line that has sorafenib resistance, into an animal having immunity to form a tumor;

[0018] (2) A step of treating an animal injected with the above liver cancer cell line with a liver cancer treatment candidate substance; and

[0019] (3) A method for screening anticancer drugs including the step of measuring the responsiveness to the above-mentioned liver cancer treatment candidate substance.

[0020] The above-mentioned liver cancer treatment candidate substance may be an immunotherapy drug.

[0021] The present invention comprises the step of (1) injecting one or more liver cancer cell lines selected from PLLC15 (accession number: KCTC15967BP), a sorafenib-non-resistant liver cancer cell line that does not have sorafenib resistance, and PLLC15_Sora (accession number: KCTC15968BP), a sorafenib-resistant liver cancer cell line that has sorafenib resistance, into an animal with immunity to form a tumor;

[0022] (2) A step of performing a candidate anticancer therapy on an animal injected with the above liver cancer cell line; and

[0023] (3) A method for evaluating the efficacy of anticancer therapy including a step of measuring responsiveness to the above candidate anticancer therapy.

[0024] The above candidate anticancer therapy may be one or more selected from the group consisting of chemotherapy, immunotherapy, and radiation therapy.

[0025] The present invention relates to anticancer drug-resistant and non-resistant liver cancer cell lines and a method for screening liver cancer therapeutic drugs using the same. Specifically, by providing a sorafenib-non-resistant liver cancer cell line PLLC15 (Deposit No.: KCTC15967BP) that does not possess sorafenib resistance and / or a sorafenib-resistant liver cancer cell line PLLC15_Sora (Deposit No.: KCTC15968BP) that possesses sorafenib resistance, or a composition for screening anticancer drugs and a method for screening anticancer drugs comprising one or more of these, it is possible to screen anticancer immunotherapies or methods using the immune function of experimental animals, and time and costs can be saved by reviewing the issue of sorafenib resistance in advance during the liver cancer therapeutic drug development stage.

[0026] Figure 1 illustrates the process of liver cancer cell line generation in chronological order.

[0027] Figure 2 illustrates the process of securing sorafenib-resistant vesicles in chronological order.

[0028] The inventors confirmed that by establishing anticancer drug-resistant and non-resistant mouse-based liver cancer cell lines possessing immunogenicity, it is possible to screen liver cancer therapeutic agents containing the activation of the animal's own immune function in an allogeneic tumor mouse model, and thus completed the present invention.

[0029] Preferred embodiments of the present invention are described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms, and the contents introduced herein are provided to sufficiently convey the concept of the present invention.

[0030] <Example 1. Establishment of Liver Cancer Cell Line>

[0031] Production of sorafenib-non-resistant cell lines

[0032] Figure 1 schematically illustrates the process of establishing a liver cancer cell line.

[0033] Seven-week-old male C57BL / 6 mice were housed in a pathogen-free animal facility with free access to water and food while maintaining a standard 12-hour light / 12-hour dark cycle. Upon acclimatization, mice were intraperitoneally injected with 100 mg of diethylnitrosamine (DEN, Sigma-Aldrich, Munich, Germany) per kg of body weight along with a high-fat diet. The DEN solution was prepared by mixing it with a 0.9% sodium chloride solution (Sigma-Aldrich). Treatment was administered at 100 mg / kg twice a week (Monday and Friday). At 23 weeks post-injection, the mice were sacrificed and their livers were removed.

[0034] Cells from the obtained liver tissue were isolated using a tumor dissociation kit (Miltenyi Biotec, Korea). Briefly, the liver tissue was cut with sterile animal surgical scissors, immersed in HBSS, and subsequently washed with HBSS to remove non-tumor tissue and necrotic tumor tissue. Then, the tumor was 1–2 mm 3The cells were cut into pieces of a certain size, the pellet was suspended in digestion buffer, and then cultured at 37°C for 2-4 hours. Afterward, single cells were collected using a 70 μm cell strainer and centrifuged at 1,000 rpm for 3 minutes. The cells obtained using the cell strainer were isolated using a tumor cell isolation kit and cultured. After culture, cells were detached from the colonized cells using a colony cylinder in a 0.05% Trypsin-EDTA solution (Gibco), cultured in a new 10 cm culture dish, and obtained as a liver cancer cell line (PLLC15).

[0035] The sorafenib-non-resistant liver cancer cell line PLLC15 obtained above was deposited on July 18, 2024 (Deposit No.: KCTC15967BP).

[0036] Production of sorafenib-resistant cell lines

[0037] Figure 2 schematically illustrates the process of obtaining a sorafenib-resistant cell line in chronological order. Specifically, the sorafenib-non-resistant cell line prepared above was first seeded into a cell culture dish and cultured at 80% density. Subsequently, 20 μM of sorafenib was added to the culture medium and cultured for 72 hours. After culture, dead cells were removed, and the surviving cells were seeded into a new culture dish and cultured for 3 to 5 days until the density reached 80%. Subsequently, the above process including sorafenib treatment was repeated three times to obtain the surviving cells as a sorafenib-resistant liver cancer cell line (PLLC15_Sora).

[0038] The above liver cancer cell line PLLC15_Sora is a sorafenib-resistant liver cancer cell line that was deposited on July 18, 2024 (KCTC15968BP).

[0039] <Example 2. Characterization of Sorafenib-Resistant Liver Cancer Cell Lines>

[0040] Cell proliferation analysis

[0041] The sorafenib-non-resistant hepatocellular carcinoma cell lines PLLC15 and PLLC15_Sora, and the mouse hepatocellular carcinoma cell line Hepa 1-6 (ATCC) established above were cultured in DMEM (pH 7.2–7.4) containing 1% sodium pyruvate, 3.7 mg / ml sodium bicarbonate, 10% FBS, and 1% antibiotics (including 10,000 unit / ml sodium penicillin G, 10,000 µg / ml streptomycin sulfate, and 25 µg / ml amphotericin B) at 37°C in a high-humidity environment containing 50 mL / L CO2 in the air. Each of the above cells was placed in a 12-well plate at a density of 5 × 10⁶ 5 The cells were seeded into a cell / well. Subsequently, each well was harvested at 24, 48, and 72 hours of culture, and the cell count was measured and converted into a cell proliferation %) relative to 0 hours of culture, as shown in Figure 3. Each result was calculated as the mean ± standard deviation after three repeated measurements. As shown in Figure 3, the number of cells in the mouse liver cancer cell line Hepa 1-6 increased by 309.45% after 72 hours of culture, while the sorafenib-non-resistant liver cancer cell line PLLC15 according to the present invention showed a cell proliferation rate of 376.99%, and the sorafenib-resistant liver cancer cell line PLLC15_Sora showed a cell proliferation rate of 444.33%. That is, the cell proliferation rate of the sorafenib-non-resistant liver cancer cell line PLLC15 according to the present invention increased by 1.22 times compared to the Hepa 1-6 cell line, and the cell proliferation rate of the sorafenib-resistant liver cancer cell line PLLC15_Sora increased by 1.44 times compared to the Hepa 1-6 cell line.

[0042] Analysis of cancer cell transplantation and tumor formation

[0043] The sorafenib-non-resistant hepatocellular carcinoma cell lines PLLC15 and PLLC15_Sora, and the rat hepatocellular carcinoma cell lines Hepa 1-6 established above were cultured in 10 cm culture dishes of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a 37°C incubator. 5-6 week old C57BL / 6 mice were reared under a 12-hour light / 12-hour dark cycle with free access to water and food. After one week of acclimatization, 2×10⁶ phosphate groups suspended in 100 μL phosphate-buffered saline (PBS) were placed on the flanks of the mice. 6 Canine sorafenib-inhibited liver cancer cell line PLLC15, sorafenib-resistant liver cancer cell line PLLC15_Sora, and rat liver cancer cell line Hepa 1-6 were injected subcutaneously. As a control, the same volume of PBS was injected subcutaneously into the same site in mice. Subsequently, the mice were raised for 2 weeks while being allowed to freely consume water and feed. Two weeks after injection, prior to sacrificing the mice, a photograph of the tumors grown in the mice is shown in Fig. 4A. After sacrificing the mice from each group, the tumors grown at the liver cancer cell transplant site were excised and are shown in Fig. 4B. As seen in Figs. 4A and 4B, mice injected with PBS and Hepa 1-6 did not form tumors and did not develop sufficiently, whereas the sorafenib-inhibited liver cancer cell line PLLC15 and the sorafenib-resistant liver cancer cell line PLLC15_Sora according to the present invention formed tumors with maximum diameters of 17.35 mm and 14.5 mm, respectively.

[0044] As shown in the results above, the sorafenib-non-resistant liver cancer cell line PLLC15 and the sorafenib-resistant liver cancer cell line PLLC15_Sora according to the present invention exhibit a very fast growth rate compared to existing mouse cancer cell lines, and form tumors very well in allograft or xenograft models, thus accurately reflecting the tumor growth characteristics of actual patients.

[0045] In addition, by simultaneously applying the efficacy of various anticancer drugs to sorafenib-resistant and non-resistant cell lines or to allogeneic or xenograft models utilizing them, the issue of anticancer drug resistance can be reviewed in advance, thereby saving time and costs in the development process of liver cancer treatments.

[0046] [Consignment Number]

[0047] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0048] Trustee Number: KCTC15967BP

[0049] Date of Trust: 20240718

[0050]

[0051] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0052] Trustee Number: KCTC15968BP

[0053] Date of Trust: 20240718

[0054]

Claims

1. PLLC15, a sorafenib-non-resistant liver cancer cell line that does not possess sorafenib resistance (Accession No.: KCTC15967BP).

2. In Paragraph 1, The sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP), which does not possess sorafenib resistance, is characterized by having a cell proliferation rate of at least 1.22 times compared to the Hepa 1-6 cell line after 72 hours of culture.

3. Sorafenib-resistant liver cancer cell line PLLC15_Sora (Accession No.: KCTC15968BP).

4. In Paragraph 2, The sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP) having sorafenib resistance is characterized by having a cell proliferation rate of 1.44 times or more compared to the Hepa 1-6 cell line after 72 hours of culture.

5. A composition for anticancer drug screening comprising one or more of the sorafenib-non-resistant liver cancer cell line PLLC15 (Accession No.: KCTC15967BP) which does not possess sorafenib resistance, and the sorafenib-resistant liver cancer cell line PLLC15_Sora (Accession No.: KCTC15968BP) which possesses sorafenib resistance.

6. (1) A step of treating one or more liver cancer cell lines among sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP) and sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP) with a liver cancer therapeutic candidate substance; and (2) A method for screening anticancer drugs comprising the step of measuring the responsiveness to the liver cancer treatment candidate substance in one or more of the sorafenib-non-resistant liver cancer cell lines PLLC15 (accession number: KCTC15967BP) and sorafenib-resistant liver cancer cell lines PLLC15_Sora (accession number: KCTC15968BP) that do not have sorafenib resistance.

7. (1) A step of performing a candidate anticancer therapy on one or more of the sorafenib-non-resistant liver cancer cell lines PLLC15 (accession number: KCTC15967BP) that do not have sorafenib resistance and sorafenib-resistant liver cancer cell lines PLLC15_Sora (accession number: KCTC15968BP) that have sorafenib resistance; and (2) A method for evaluating the efficacy of an anticancer therapy comprising the step of measuring the responsiveness to the candidate anticancer therapy in one or more of the sorafenib-non-resistant liver cancer cell lines PLLC15 (accession number: KCTC15967BP) and sorafenib-resistant liver cancer cell lines PLLC15_Sora (accession number: KCTC15968BP).

8. In Paragraph 7, A method for evaluating the efficacy of anticancer therapy, characterized in that the above-mentioned candidate anticancer therapy is one or more selected from a group consisting of chemotherapy, immunotherapy, and radiation therapy.

9. (1) A step of injecting one or more liver cancer cell lines among PLLC15 (accession number: KCTC15967BP), a sorafenib-non-resistant liver cancer cell line that does not have sorafenib resistance, and PLLC15_Sora (accession number: KCTC15968BP), a sorafenib-resistant liver cancer cell line that has sorafenib resistance, into an immunized animal to form a tumor; (2) A step of treating an animal injected with the above liver cancer cell line with a liver cancer treatment candidate substance; and (3) A step of measuring the responsiveness to the liver cancer treatment candidate substance in an animal injected with the above liver cancer cell line; an anticancer drug screening method comprising: a step of measuring the responsiveness to the above liver cancer treatment candidate substance in an animal injected with the above liver cancer cell line.

10. (1) A step of injecting one or more liver cancer cell lines, selected from sorafenib-non-resistant liver cancer cell line PLLC15 (accession number: KCTC15967BP) which does not possess sorafenib resistance and sorafenib-resistant liver cancer cell line PLLC15_Sora (accession number: KCTC15968BP) which possesses sorafenib resistance, into an immunized animal to induce tumor formation; (2) A step of performing a candidate anticancer therapy on an animal injected with the above liver cancer cell line; and (3) A method for evaluating the efficacy of an anticancer therapy comprising the step of measuring the responsiveness to the candidate anticancer therapy in an animal injected with the above liver cancer cell line.

11. In Paragraph 10, A method for evaluating the efficacy of anticancer therapy, characterized in that the above-mentioned candidate anticancer therapy is one or more selected from a group consisting of chemotherapy, immunotherapy, and radiation therapy.

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