Method for constructing in-vivo lentinib acquired drug resistance model of hepatocellular carcinoma

Abstract

The invention relates to the field of animal models, and discloses a hepatocellular carcinoma in-vivo lentinib acquired drug resistance model construction method. The method comprises the following steps: step 1, inoculating mouse hepatoma carcinoma cells under the skin of a mouse to form a transplanted tumor; step 2, when the transplanted tumor grows to a predetermined size, carrying out Lenvatinib intragastric administration on the mouse; 3, under the condition of lavage administration of the lenvatinib, carrying out continuous passage on the surviving tumor tissues in the bodies of similar mice; and 4, repeating the steps 2 and 3 for multiple times until no statistical difference exists between the tumor volume of the subcultured tumor and the tumor volume of the non-dosed control group under the condition of dosing the lenvatinib. The construction method has the advantages of being easy and convenient to operate, short in construction period, stable in drug resistance and the like, the acquired drug resistance mechanism of the lentinib in the human body can be effectively simulated, and a reliable and repeatable experimental model basis is provided for hepatocellular carcinoma drug screening, drug resistance mechanism research and individualized treatment scheme development.

Description

Technical Field

[0001] This application relates to the technical field of animal models, and more specifically, it relates to a method for constructing an in vivo lentinib acquired resistance model of hepatocellular carcinoma. Background Technology

[0002] Primary liver cancer is a common malignant tumor of the digestive system, with more than 850,000 new cases and 700,000 deaths worldwide each year. Half of these cases occur in China, and the incidence rate is increasing annually, making it the second leading cause of cancer death in the country. Among all primary liver cancers, hepatocellular carcinoma (HCC) is the most common type, accounting for approximately 90%, characterized by high recurrence, metastasis, high mortality, and poor prognosis. Clinically, most liver cancer patients are diagnosed at an intermediate or advanced stage, thus missing the opportunity for surgical treatment. Therefore, medication becomes the primary treatment option for unresectable liver cancer and for patients after liver cancer resection.

[0003] Lenvatinib, as a first-line drug for the clinical treatment of advanced hepatocellular carcinoma (HCC), exerts its effects by inhibiting tumor angiogenesis, suppressing tumor growth, and promoting tumor cell apoptosis. Although it is more suitable for use in East Asian populations with a history of hepatitis compared to sorafenib, some patients fail to benefit from it due to primary resistance, and acquired resistance that develops during use further limits its clinical application. Therefore, constructing a lenvatinib-related HCC resistance model is fundamental to studying the mechanisms of HCC resistance and is of great significance for preventing postoperative recurrence and metastasis, improving clinical treatment efficacy, and increasing patient survival rates.

[0004] Traditional drug resistance models are typically constructed based on in vitro cultured cell lines, gradually inducing and maintaining drug resistance through escalating drug concentrations. However, these in vitro models only simulate the changes in the patient's tumor cells themselves after drug resistance develops, failing to mimic the tumor cell microenvironment and neglecting the influence of both the overall tumor tissue and the tumor microenvironment on treatment resistance. Consequently, they often fail to reflect the true in vivo situation. Therefore, constructing an in vivo lenvatinib resistance model can elucidate the dynamic changes in the microenvironment of hepatocellular carcinoma under lenvatinib treatment, explore novel mechanisms mediating acquired lenvatinib resistance, and propose rational treatment strategies to truly serve clinical patients. Summary of the Invention

[0005] The purpose of this invention is to address the technical shortcomings of traditional drug resistance models, which are usually based on in vitro cultured cell lines and cannot simulate the microenvironment of tumor cells. This invention provides a lenvatinib resistance model for hepatocellular carcinoma that can simulate the real in vivo microenvironment, so as to study its resistance mechanism more accurately.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution: In a first aspect, this application discloses a method for constructing an in vivo lentinib acquired resistance model in hepatocellular carcinoma, comprising the following steps: Step 1: Inoculate mouse-derived liver cancer cells subcutaneously into mice to form xenografts; Step 2: When the transplanted tumor grows to the predetermined size, the mice are administered lenvatinib by gavage; Step 3: Under the condition of lenvatinib gavage administration, the surviving tumor tissue was continuously passaged in mice of the same species; Step 4: Repeat steps 2 and 3 multiple times until the tumor volume of the passaged tumors under lenvatinib administration conditions is not statistically different from that of the unadministered control group, thus obtaining a stable in vivo lenvatinib acquired resistance model of hepatocellular carcinoma.

[0007] Furthermore, in step 1, the mouse-derived liver cancer cells are Hep1-6 cells.

[0008] Furthermore, in step 1, the subcutaneous inoculation of mice refers to preparing a cell suspension at a concentration of 5 x 10⁻⁶. 6 / 100μL / was injected subcutaneously in the hypochondrial region of the abdomen of 4-week-old male C57BL / 6 mice.

[0009] Furthermore, in step 2, the predetermined size refers to an average tumor diameter of 5 mm.

[0010] Furthermore, in step 2, the administration of lenvatinib by gavage refers to treatment by gavage of 500 μL of lenvatinib mesylate at a concentration of 10 mg / kg.

[0011] Furthermore, the frequency of the gavage treatment is once a day, 6 days a week.

[0012] Furthermore, in step 3, the number of consecutive generations is at least 3.

[0013] Secondly, this application discloses an in vivo lentinib acquired resistance model for hepatocellular carcinoma, which is constructed by any of the construction methods described in the first aspect.

[0014] Thirdly, the application of the lenvatinib acquired resistance model in hepatocellular carcinoma as disclosed in the second aspect of this application in screening anti-hepatocellular carcinoma drugs, studying the lenvatinib resistance mechanism in hepatocellular carcinoma, or identifying tumor drug resistance biomarkers.

[0015] The key to this invention lies in: 1. In vivo construction: Unlike traditional in vitro cell models, this invention establishes a drug resistance model in living animals through in vivo induction. This ensures the involvement of the tumor microenvironment (including blood vessels, immune cells, fibroblasts, etc.).

[0016] 2. Induction and maintenance methods: The key is to establish cell line-derived xenografts (CDX) in animals and then administer lenvatinib in vivo for a long period of time. By applying treatment selection pressure, the process of acquired drug resistance in clinical patients is simulated, thereby screening out in vivo tumor models with drug resistance.

[0017] 3. Systematicity of the model: This model not only includes drug-resistant tumor cells themselves, but also the tumor microenvironment remodeled under drug stress, which can be used more comprehensively to study drug resistance mechanisms and screen combination therapy regimens to reverse drug resistance.

[0018] In summary, this application has the following beneficial effects: This invention is the first to construct an in vivo lenvatinib resistance model for hepatocellular carcinoma, which can be used to analyze changes in the immune microenvironment after lenvatinib resistance in hepatocellular carcinoma, study the mechanism of tumor resistance to lenvatinib and methods to reverse tumor resistance, discover tumor resistance biomarkers, and screen and evaluate novel antitumor drugs. It has high scientific research and production application value.

[0019] The stable drug resistance model obtained by the construction method described in this invention can not only be used to study drug resistance-related molecular events, molecular tags and biomarkers through high-throughput sequencing, microarrays, omics and other means, but also serve as a powerful tool for functional studies and targeted drug screening, and has important application prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the induction and establishment of the Hep1-6 LR drug resistance model for liver cancer. Figure 2 This is a diagram validating the induction of the Hep1-6 LR drug resistance model for liver cancer. Figure 3 These are growth curves of Hep1-6 and Hep1-6 LR; Figure 4 These are Hep1-6 and Hep1-6 LR tumor quality maps. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0022] The mouse liver cancer cells Hep1-6 used in the examples were purchased from the Cell Bank of the Chinese Academy of Sciences; the lenvatinib mesylate used in the examples was the commercially available Lenvatinib (E7080) Mesylate 50mg product from Selleck Chemicals.

[0023] Example: Construction and identification of an in vivo lenvatinib acquired resistance model in C57BL / 6 mouse hepatocellular carcinoma. Hep1-6 cells in the logarithmic growth phase were digested, centrifuged, and resuspended in sterile PBS. An equal volume of Matrigel was added and mixed well. The mixed cell suspension and the remaining Matrigel were then placed back on ice.

[0024] Use a 1mL syringe to dispense the cell suspension at a concentration of (5 x 10). 6 (100 μL / mouse) was injected subcutaneously into the hypochondrial region of the abdomen of 4-week-old male C57BL / 6 black mice. A round wheal was observed to form under the skin. When withdrawing the needle, a cotton swab was used to press the injection site to prevent the fluid from flowing out.

[0025] Mice were divided into two groups (PBS group and drug treatment group), with five mice in each group. After one week, when the average tumor diameter reached 5 mm, mice in the drug treatment group were treated by gavage with 500 μL of lenvatinib mesylate at a concentration of 30 mg / kg. Mice in the PBS group were treated by gavage with 500 μL of PBS once a day for six days a week.

[0026] The weight and tumor size of the nude mice were measured every weekend. After 4 weeks, the tumor was removed and weighed. Tumor volume = 0.5 × longest diameter of tumor × shortest diameter of tumor. 2 .

[0027] Four weeks later, the largest tumors in the PBS group and the drug-treated group were selected. The more viable portion of the tumor near the periphery was selected and cut into 1mm sections under aseptic conditions. 3 spare.

[0028] The mice to be inoculated with the tumor were anesthetized, the hair on the mice's abdomen was removed, a 5mm skin incision was made in the groin area of ​​the mice using surgical scissors, and a tunnel was bluntly dissected into the subcutaneous area of ​​the ipsilateral costal region of the mice using tissue forceps. The cut tumor was implanted at the end of the subcutaneous tunnel, and the skin incision was sutured with mousse sutures.

[0029] Place the mouse on its side in the cage to prevent the tongue from falling back and causing suffocation. Cover the mouse with sterile gauze to keep it warm and observe it until it recovers.

[0030] One week later, when the average tumor diameter reached 5 mm, the mice in the treatment group were treated by gavage with 500 μL of lenvatinib mesylate at a concentration of 30 mg / kg. The mice in the PBS group were treated by gavage with 500 μL of PBS once a day for 6 days a week.

[0031] Four weeks later, the largest tumor mass from both groups was selected again, and the passage was repeated three times. It was confirmed that there was no statistically significant difference in tumor volume between the drug-treated group and the PBS group and that the phenotype was stable. Thus, the drug-resistant mouse hepatocellular carcinoma strain Hep1-6 LR was obtained through in vivo screening.

[0032] Twenty C57BL / 6 mice were randomly divided into four groups (Hep1-6 PBS group, Hep1-6 treatment group, Hep1-6 LR PBS group, and Hep1-6 LR treatment group), with five mice in each group. After one week, when the average tumor diameter reached 5 mm, the mice in the treatment group were treated by gavage with 500 μL of lenvatinib mesylate at a concentration of 10 mg / kg. The mice in the PBS group were treated by gavage with 500 μL of PBS once a day for six days a week.

[0033] The weight and tumor size of the nude mice were measured every weekend. After 4 weeks, the tumor was removed and weighed. Tumor volume = 0.5 × longest diameter of tumor × shortest diameter of tumor. 2 .

[0034] By comparing the tumor volume and weight of each group, the success of establishing an in vivo lenvatinib acquired resistance model for hepatocellular carcinoma was assessed.

[0035] The experimental results showed that the tumor growth rate in mice treated with Hep1-6 LR was significantly slower than that in mice treated with Hep1-6 LR, and there was no significant difference compared with the Hep1-6 LR PBS group. This indicates that the Hep1-6 LR cell line developed significant acquired resistance to lenvatinib in vivo, and the lenvatinib acquired resistance model in hepatocellular carcinoma was successfully established.

[0036] The beneficial effects of this invention are as follows: Using immune-healthy mouse C57BL / 6 as a vector and the mouse hepatocellular carcinoma cell line Hep1-6 as the research object, this invention constructs an in vivo acquired resistance model for hepatocellular carcinoma (HCC) to lenvatinib by gavage at a concentration of 10 mg / kg (500 μL). Through three consecutive in vivo screenings, this model is stable and passivable, and more closely resembles the in vivo resistance process of lenvatinib in HCC. The construction method described in this invention is simple and easy to implement, and can maximize the simulation of the influence of the tumor tissue microenvironment on tumor drug resistance. It has significant application value for research on the immune microenvironment-related resistance mechanisms of lenvatinib in HCC, clinical medication guidance, and the development of new drugs and targets.

[0037] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for constructing an in vivo lentinib acquired resistance model in hepatocellular carcinoma, characterized in that, Includes the following steps: Step 1: Inoculate mouse-derived liver cancer cells subcutaneously into mice to form xenografts; Step 2: When the transplanted tumor grows to the predetermined size, the mice are administered lenvatinib by gavage; Step 3: Under the condition of lenvatinib gavage administration, the surviving tumor tissue was continuously passaged in mice of the same species; Step 4: Repeat steps 2 and 3 multiple times until the tumor volume of the passaged tumors under lenvatinib administration conditions is not statistically different from that of the unadministered control group, thus obtaining a stable in vivo lenvatinib acquired resistance model of hepatocellular carcinoma.

2. The method for constructing an in vivo lentinib acquired resistance model for hepatocellular carcinoma according to claim 1, characterized in that, In step 1, the murine hepatocellular carcinoma cells are Hep1-6 cells.

3. The method for constructing an in vivo lentinib acquired resistance model for hepatocellular carcinoma according to claim 1, characterized in that, In step 1, the subcutaneous inoculation of mice refers to the administration of cell suspension at a concentration of 5 x 10⁻⁶. 6 / 100μL / was injected subcutaneously in the hypochondrial region of the abdomen of 4-week-old male C57BL / 6 mice.

4. The method for constructing an in vivo lentinib acquired resistance model for hepatocellular carcinoma according to claim 1, characterized in that, In step 2, the predetermined size refers to an average tumor diameter of 5 mm.

5. The method for constructing an in vivo lentinib acquired resistance model for hepatocellular carcinoma according to claim 1, characterized in that, In step 2, the administration of lenvatinib by gavage refers to the administration of 500 μL of lenvatinib mesylate at a concentration of 10 mg / kg by gavage.

6. The method for constructing an in vivo lentinib acquired resistance model for hepatocellular carcinoma according to claim 5, characterized in that, The frequency of the gavage treatment is once a day, 6 days a week.

7. The method for constructing an in vivo lentinib acquired resistance model in hepatocellular carcinoma according to claim 1, characterized in that, In step 3, the number of consecutive generations is at least 3.

8. An in vivo model of acquired resistance to lentinib in hepatocellular carcinoma, characterized in that, It is constructed by the construction method described in any one of claims 1-7.

9. The application of the in vivo lenvatinib acquired resistance model for hepatocellular carcinoma as described in claim 8 in screening anti-hepatocellular carcinoma drugs, studying the lenvatinib resistance mechanism in hepatocellular carcinoma, or identifying tumor drug resistance biomarkers.

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