A method for constructing a liver cancer drug-resistant animal model simulating a clinical drug resistance process

Abstract

This disclosure provides a method for constructing a drug-resistant animal model of liver cancer and the resulting drug-resistant animal model of liver cancer. The method is simple and convenient, and can realistically simulate the occurrence and development of drug resistance in liver cancer as an in situ tumor model.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and more specifically, to a method for constructing a drug-resistant animal model of liver cancer, and the drug-resistant animal model of liver cancer constructed by this method. Background Technology

[0002] Compared to other tumor types, basic and clinical research on drug resistance in liver cancer is severely lacking, particularly in suitable animal models. Previous studies, aimed at quantitatively monitoring the dynamic changes in drug-resistant liver cancer in animals, have used nude mice subcutaneously inoculated with drug-resistant tumor cells or tissue. Clinical liver cancer involves the invasion of liver tissue by liver cancer cells, while subcutaneously inoculated tumor cells are deprived of the liver's nutritional and metabolic environment. More importantly, drug-resistant tumor cells or tissues develop resistance through in vitro cell culture techniques, rather than through chemotherapy treatment of liver tumors. Therefore, both tumor growth and the process of developing resistance to chemotherapy drugs differ significantly from clinical liver cancer drug resistance.

[0003] Existing models include human tumor tissue xenograft models and human tumor cell xenograft subcutaneous transplantation models. However, all three models have certain drawbacks. For example, human tumor tissue xenograft requires strict ethical review of specimens, is complex, and has poor availability. Existing human tumor cell xenograft models require tumor-bearing mice to be severely immunodeficient nude mice, which have demanding feeding conditions and are expensive. Furthermore, the transplantation site is not the primary site of liver cancer, thus failing to accurately simulate the development and progression of liver cancer. Therefore, while previously reported patient-derived tumor xenograft models and cell-derived xenografts have played an important role in liver cancer research, they suffer from drawbacks such as difficulty in obtaining specimens and complex procedures.

[0004] Therefore, there is an urgent need for an in situ tumor model that is simple and convenient to construct and can realistically simulate the occurrence and development of drug resistance in liver cancer. Summary of the Invention

[0005] The purpose of this disclosure is to provide an in situ tumorigenesis model that is simple and convenient to construct and can realistically simulate the occurrence and development of drug resistance in liver cancer.

[0006] To achieve the above objectives, this disclosure provides a method for constructing a drug-resistant animal model of liver cancer, the method comprising the following steps:

[0007] S1. Culture of fluorescently labeled liver cancer cells;

[0008] S2. The fluorescently labeled liver cancer cells are transplanted into the liver of mice to obtain transplanted mice;

[0009] S3. After culturing the transplanted mice for 1-3 weeks, a fluorescent imaging agent was injected into the tail vein, and the initial fluorescence intensity of liver cancer cells in the mice before drug intervention was monitored using animal in vivo imaging.

[0010] S4. Treat mice with anti-hepatocellular carcinoma chemotherapy drugs. After 3-8 weeks of administration, monitor the fluorescence intensity of hepatocellular carcinoma cells in mice using in vivo animal imaging. If the fluorescence intensity of hepatocellular carcinoma cells in mice after drug intervention is not less than the initial fluorescence intensity of hepatocellular carcinoma cells in mice before drug intervention, then the drug-resistant animal model of hepatocellular carcinoma is obtained.

[0011] According to this disclosure, the mice are 4-6 week old BALB / c nude mice.

[0012] According to this disclosure, the anti-hepatocellular carcinoma drug is sorafenib, and the dosage is 15-60 mg / kg every 2 days, preferably 30 mg / kg every 2 days.

[0013] According to this disclosure, in step S2, the amount of liver cancer cells transplanted is (2-5)×10⁻⁶. 6 One HepG2 / Luc cell (adjust the volume of cells to 20-50 μL with PBS depending on the cell count).

[0014] According to this disclosure, the fluorescent imaging agent comprises D-fluorescein potassium salt and / or 18 F-FDG.

[0015] According to this disclosure, the liver cancer cells are prepared by the following method: PGL4.17, an expression plasmid carrying the neo resistance gene and the luciferase reporter gene, is encapsulated in liposomes and transfected in vitro into human liver cancer cells HepG2.

[0016] According to this disclosure, the nucleotide sequence of the neo resistance gene is shown in SEQ ID NO.1, and the nucleotide sequence of the luciferase reporter gene is shown in SEQ ID NO.2.

[0017] On the other hand, this disclosure provides a liver cancer drug resistance model constructed using the above method.

[0018] Through the above technical solution, this disclosure provides a method and an animal model for constructing a drug-resistant liver cancer model. The animal model constructed using this method can be used to quantitatively monitor the dynamic changes of tumors in drug-resistant liver cancer animals and accurately simulate the occurrence and development of drug resistance in liver cancer, which is of great significance for clinical research on liver cancer.

[0019] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0021] Figure 1 This is a schematic diagram of the timeline of the sorafenib-resistant animal model provided in this disclosure.

[0022] Figure 2 Kaplan-Meier survival curves for each group of animals.

[0023] Figure 3 This is an in vivo imaging image of a mouse.

[0024] Figure 4 for 18 F-FDG MicroPET / CT tumor metabolism assay image.

[0025] Figure 5 This is a quantitative biofluorescence map for in vivo imaging.

[0026] Figure 6 for 18 F-FDG MicroPET / CT tumor metabolism biofluorescence quantitative map.

[0027] Figure 7 This is a graph showing the weight ratio of mouse liver to brain.

[0028] Figure 8 This is a representative image of a liver tumor.

[0029] Figure 9 Images of H&E staining of liver tissue from each group of animals.

[0030] Figure 10 This is a schematic diagram of the pGL4.17[Luc2 / Neo] luciferase expression vector. Detailed Implementation

[0031] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0032] On the one hand, this disclosure provides a method for constructing a drug-resistant animal model of liver cancer, the method comprising the following steps:

[0033] S1. Culture of fluorescently labeled liver cancer cells;

[0034] S2. The fluorescently labeled liver cancer cells are transplanted into the liver of mice to obtain transplanted mice;

[0035] S3. After culturing the transplanted mice for 1-3 weeks, a fluorescent imaging agent was injected into the tail vein, and the initial fluorescence intensity of liver cancer cells in the mice before drug intervention was monitored using animal in vivo imaging.

[0036] S4. Mice were treated with anti-hepatocellular carcinoma chemotherapy drugs. After 3-8 weeks of administration, the fluorescence intensity of hepatocellular carcinoma cells in mice was continuously monitored multiple times using in vivo animal imaging. If the fluorescence intensity of hepatocellular carcinoma cells in mice after drug intervention was not less than the initial fluorescence intensity of hepatocellular carcinoma cells in mice before drug intervention, then the drug-resistant animal model of hepatocellular carcinoma was obtained.

[0037] Optionally, the mice are 4-6 week old BALB / c nude mice.

[0038] Optionally, the anti-liver cancer drug is sorafenib, and the dosage is 15-60 mg / kg every 2 days, preferably 30 mg / kg every 2 days.

[0039] Optionally, in step S2, the amount of liver cancer cells transplanted is (2-5)×10⁻⁶. 6 One HepG2 / Luc cell (adjust the volume of cells to 20-50 μL with PBS depending on the cell count).

[0040] Optionally, the fluorescent imaging agent comprises D-fluorescein potassium salt and / or 18 F-FDG.

[0041] Optionally, the liver cancer cells are prepared by encapsulating the expression plasmid PGL4.17 carrying the neo resistance gene and the luciferase reporter gene in liposomes and transfecting it into human liver cancer cells HepG2 in vitro.

[0042] Optionally, the nucleotide sequence of the neo resistance gene is shown in SEQ ID NO.1, and the nucleotide sequence of the luciferase reporter gene is shown in SEQ ID NO.2.

[0043] On the other hand, this disclosure provides a drug-resistant animal model of liver cancer constructed using the above method.

[0044] The present invention will be further described in detail below through examples.

[0045] Example 1

[0046] This embodiment is used to construct an animal model of sorafenib resistance in hepatocellular carcinoma in situ.

[0047] like Figure 1As shown, 100 4-6 week old BALB / c nude mice were used, 5 mice per cage, and placed in a specific pathogen-free room. They were housed at 25℃±1℃ with a 12-hour day-night cycle and were given free access to food and water. Ten mice served as a normal control group, and the remaining animals were inoculated with hepatocellular carcinoma cells. First, a hepatocellular carcinoma model based on HepG2 cell orthotopic xenograft transplantation was established. Specifically, the expression plasmid PGL4.17 (as shown in SEQ ID NO.1) carrying the neo resistance gene (as shown in SEQ ID NO.2) was encapsulated in liposomes. Figure 10 (As shown), HepG2 human liver cancer cells were transfected in vitro to obtain HepG2 / Luc cells; nude mice were anesthetized with tribromoethanol, and the liver was exposed through a midline abdominal incision. 2×10⁶ cells were then inserted into the liver. 6 HepG2 / Luc cells were implanted into the largest lobe of the liver, the wound was sealed with medical OB glue, and the abdominal incision was sutured with 5-0 silk sutures to obtain transplanted mice. To monitor tumor growth, two new technologies, small animal in vivo imaging and MicroPET / CT, were used for cross-validation to improve accuracy. One week after surgery, the transplanted mice were injected intravenously with D-Luciferin (15 mg / mL, 200 mL). The Lumina III small animal in vivo imaging system (PerkinElmer, Waltham, MA, USA) was used to monitor the bioluminescence of liver tumors in mice (fluorescence in the liver indicates the presence of a tumor, and stronger fluorescence indicates a larger tumor). After an interval of 3-5 days (both detection methods require anesthesia of the mice, hence the necessary interval), the transplanted mice were injected via the tail vein with 11.1 MBq (300 mCi). 18F-FDG was used to detect the bioluminescence of liver tumors in mice using Inveon MicroPET / CT (Siemens Medical Solution, GER). (If a tumor is present in the liver, the fluorescence intensity at the tumor site is significantly stronger than the background fluorescence intensity at the normal liver site, and the fluorescence intensity is proportional to the tumor size.) Nude mice carrying tumors were designated as the hepatocellular carcinoma (HCC) group and treated with different doses of sorafenib (15 / 30 / 60 mg / kg / 2 days) starting in week 2. Three weeks after administration (i.e., 4 weeks post-surgery), the inhibitory effect of sorafenib on the tumor was monitored using the same two tumor imaging techniques. Compared with the first imaging after surgery, animals with continuously shrinking tumors (i.e., decreased fluorescence intensity in IVIS small animal in vivo imaging or decreased glucose metabolism in Inveon MicroPET / CT) were classified as the sorafenib-sensitive group (SEN), and animals with continuously growing tumors (i.e., increased fluorescence intensity in IVIS small animal in vivo imaging or increased glucose metabolism in Inveon MicroPET / CT) were classified as the sorafenib-resistant group (RES). Seven and nine weeks after surgery (i.e., six and eight weeks after drug administration), the same two techniques were used to conduct the third and fourth tumor imaging experiments to continue monitoring whether the tumor growth in the animals conformed to the corresponding groups. If the tumors of some mice in the drug-sensitive group first decreased and then increased, they were reclassified to the drug-resistant group; or if the tumors of some mice in the drug-resistant group first increased and then decreased, they were reclassified to the drug-sensitive group. Finally, the animals were anesthetized and sacrificed, and samples were collected.

[0048] One week after surgery, tumor imaging showed that all 90 nude mice had tumors. Fifteen mice served as a liver cancer control group, while the remaining 75 were divided into three groups (25 mice per group), receiving different doses of sorafenib (15 / 30 / 60 mg / kg / 2 days) starting from the second week post-surgery. Three weeks after administration (four weeks post-surgery), tumor imaging showed that compared to the first tumor imaging, some mice in the three sorafenib treatment groups exhibited decreased fluorescence intensity in their liver tumors; these mice were considered the drug-sensitive group (SEN). The remaining mice showed increased fluorescence intensity in their liver tumors; these mice were considered the drug-resistant group (RES). The third (six-week) and fourth (eight-week) tumor imaging tests showed that the tumor growth trends in the vast majority of mice conformed to their respective groups, with only a small number of mice showing inconsistent tumor growth. For individual mice in the drug-sensitive group whose tumor fluorescence intensity first decreased and then increased, these mice were classified into the drug-resistant group; conversely, for individual mice in the drug-resistant group whose tumor fluorescence intensity first increased and then decreased, these mice were classified into the drug-sensitive group. In short, from the second week post-surgery, the liver tumor fluorescence intensity and glucose metabolism in the hepatocellular carcinoma (HCC) group mice continued to increase (i.e., tumor continued to grow). Subsequently, treatment with different doses of sorafenib resulted in a gradual decrease in liver tumor fluorescence intensity and glucose metabolism in the sorafenib-sensitive group (SEN) mice starting from the fourth week post-surgery (3 weeks of treatment). Even after sorafenib treatment, the liver tumor fluorescence intensity and glucose metabolism in the sorafenib-resistant group (RES) continued to increase. These results demonstrate the successful establishment of a sorafenib-resistant animal model. Figure 3-6 Furthermore, the proportion of liver cancer mice that were eventually classified as resistant after treatment with sorafenib at a dose of 30 mg / kg / 2 days (approximately 70%) was higher than that of mice treated with doses of 15 mg / kg / 2 days (approximately 60%) and 60 mg / kg / 2 days (approximately 55%), indicating the highest success rate in inducing drug resistance.

[0049] The overall survival time of nude mice in the hepatocellular carcinoma (HCC) group and the drug-resistant group (RES) was significantly shorter than that in the drug-sensitive group, and the tumor size and weight were significantly greater in the latter group. Figure 2 7-8). H&E staining results showed that the HCC and RES groups also exhibited similar pathological changes, including numerous necrotic areas in the tumor lesions, vacuolar changes in a few normal cells, and high infiltration of inflammatory cells. However, the SEN group showed significantly better tumor lesions and inflammatory cell infiltration than the HCC and RES groups. Figure 9 Black tailless arrows, short-tailed black arrows, and long-tailed black arrows represent necrotic areas, vacuolar changes, and inflammatory infiltration, respectively. Scale bar: 50 or 100 μm. Data are expressed as mean ± standard deviation; *p<0.05, **p<0.01, ***p<0.001).

[0050] In summary, the method disclosed herein successfully constructed an animal model of drug-resistant liver cancer, with an overall success rate of approximately 70% in inducing resistance, which is relatively high. This animal model can realistically simulate the microenvironment of liver cancer resistance to chemotherapeutic drugs, and is of great significance for clinical research on liver cancer.

[0051] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0052] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0053] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for constructing a drug-resistant animal model of liver cancer, characterized in that, The method includes the following steps: S1. Culture of fluorescently labeled liver cancer cells: The expression plasmid PGL4.17 carrying the neo resistance gene and the luciferase reporter gene was encapsulated in liposomes and transfected into human liver cancer cells HepG2 in vitro; the nucleotide sequence of the neo resistance gene is shown in SEQ ID NO.1, and the nucleotide sequence of the luciferase reporter gene is shown in SEQ ID NO.

2. S2, the fluorescently labeled liver cancer cells are then subjected to (2-5) × 10⁻⁶ 6 One transplant volume was transplanted into the liver of 4-6 week old BALB / c nude mice to obtain transplanted mice; S3. After culturing the transplanted mice for 1-3 weeks, a fluorescent imaging agent is injected via the tail vein. The initial fluorescence intensity and / or initial metabolic level of liver cancer cells in the mice before drug intervention are monitored using in vivo animal imaging and / or MicroPET / CT methods. The fluorescent imaging agent includes D-fluorescein potassium salt and / or... 18 F-FDG; S4. Mice were treated with an anti-hepatocellular carcinoma drug. After 3-8 weeks of treatment, the fluorescence intensity and / or metabolic level of hepatocellular carcinoma cells in mice were monitored using in vivo animal imaging and / or MicroPET / CT. The anti-hepatocellular carcinoma drug was sorafenib, and the dosage was 15-60 mg / kg every 2 days. The drug-resistant animal models of liver cancer were classified based on the trends in changes in tumor fluorescence intensity and / or metabolic levels before and after drug intervention. If the fluorescence intensity and metabolic level of liver cancer cells in mice after drug intervention are not less than the initial fluorescence intensity and initial metabolic level of liver cancer cells in mice before drug intervention, then the liver cancer drug-resistant animal model is determined to be obtained. If the fluorescence intensity and metabolic level of liver cancer cells in mice after drug intervention are consistently lower than the initial fluorescence intensity and initial metabolic level of liver cancer cells in mice before drug intervention, then a liver cancer drug sensitivity model is determined.

2. The method according to claim 1, wherein, The dosage of the anti-liver cancer drug is 30 mg / kg every 2 days.

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