Mouse liver cancer cell line with high lung metastasis characteristic as well as construction method and application of mouse liver cancer cell line

Abstract

The invention discloses a mouse liver cancer cell line with a high lung metastasis characteristic as well as a construction method and application thereof, and belongs to the technical field of biology and oncology. The cell is preserved in China Center for Type Culture Collection on February 4, 2026, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C202621. The PNV-LM3 cell line provided by the invention is constructed by combining a PTEN KO and NRAS G12V mutated hydrodynamic tail vein injection model with tail vein injection in-vivo domestication, a mouse liver cancer model is successfully induced to generate lung metastasis, and the lung metastasis tendency of the cell line is greatly enhanced. Compared with a traditional Hepa1-6 cell line, the PNV-LM3 cell line has the advantages that the lung metastasis process of the liver cancer is more accurately and efficiently reproduced, and a more real experimental platform is provided for research of a liver cancer metastasis mechanism.

Description

Technical Field

[0001] This invention relates to the fields of biology and oncology, and in particular to a mouse hepatocellular carcinoma cell line with high lung metastasis characteristics, its construction method, and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and mortality rates worldwide. Its high metastatic potential, especially lung metastasis, severely impacts patient survival. Existing mouse HCC cell lines (such as Hepa1-6) are significantly inadequate in simulating HCC metastasis, particularly lung metastasis. They cannot effectively reproduce the HCC microenvironment or realistically simulate the pathophysiological processes of malignant progression and treatment resistance, thus limiting their application in research on HCC metastasis mechanisms and treatment. Summary of the Invention

[0003] The purpose of this invention is to provide a mouse hepatocellular carcinoma cell line with high lung metastasis characteristics, its construction method and application, so as to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a mouse hepatocellular carcinoma cell line PNV-LM3 Musmusculus with lung metastasis characteristics. This cell line was deposited at the China Center for Type Culture Collection on February 4, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202621.

[0005] The second technical solution of the present invention is a method for constructing the mouse hepatocellular carcinoma cell line PNV-LM3, comprising the following steps: (1) The combination of PTEN KO and NRAS G12V mutant plasmids was introduced into the liver of C57BL / 6 mice by hydrodynamic tail vein injection to induce liver cancer formation; (2) Primary cells PNV-HTVi were extracted from liver cancer tissue and cultured in vitro; (3) PNV-HTVi cells were injected into new-generation C57BL / 6 mice via tail vein to induce lung metastasis; (4) Extract cells from lung metastases, culture them in vitro, and repeat step (3) at least twice to obtain the PNV-LM3 cell line with high lung metastasis characteristics.

[0006] The third technical solution of the present invention is a method for establishing an animal model of liver cancer lung metastasis, which uses the PNV-LM3 cell line to inoculate immune healthy mice via tail vein injection or intrahepatic injection.

[0007] The fourth technical solution of the present invention is a method for screening or evaluating drugs against liver cancer metastasis, comprising the following steps: (1) An animal model of liver cancer lung metastasis was established using the PNV-LM3 cell line described above; (2) Administer the drug to be tested; (3) Observe the formation, growth or regression of lung metastases and evaluate the drug effect.

[0008] The fifth technical solution of the present invention is a method for studying the immune microenvironment or immune escape mechanism of liver cancer, which uses an animal model established by the PNV-LM3 cell line to analyze the infiltration of immune cells and the composition of stromal cells in tumor tissue.

[0009] The sixth technical solution of the present invention is the application of the mouse hepatocellular carcinoma cell line PNV-LM3 in the preparation of experimental models for the study of hepatocellular carcinoma metastasis mechanisms, drug screening, or immunotherapy evaluation.

[0010] Based on the above technical solution, the present invention has the following technical effects: 1. More efficiently simulates the process of liver cancer metastasis to the lungs. The PNV-LM3 cell line provided by this invention was constructed through a hydrodynamic tail vein injection model using PTEN KO and NRAS G12V mutants, combined with in vivo domestication via tail vein injection. This successfully induced lung metastasis in a mouse hepatocellular carcinoma model, significantly enhancing the cell line's tendency to metastasize to the lungs. Compared to the traditional Hepa1-6 cell line, the PNV-LM3 cell line more accurately and efficiently reproduces the lung metastasis process of hepatocellular carcinoma, providing a more realistic experimental platform for studying the mechanisms of hepatocellular carcinoma metastasis.

[0011] 2. More realistically simulates the microenvironment of liver cancer lung metastasis. The liver cancer lung metastasis microenvironment established by the PNV-LM3 cell line, particularly in terms of immune cell infiltration characteristics, more closely resembles the pathological features of clinical liver cancer. This cell line exhibits a low proportion of CD8+ T cells and displays a higher proportion of endothelial cells and fibroblasts, better mimicking the dynamic changes in immune escape and the tumor microenvironment during liver cancer metastasis. This provides strong support for research on the liver cancer immune microenvironment and the development of immunotherapy strategies.

[0012] 3. Enhances drug resistance properties The PNV-LM3 cell line of this invention exhibits significant drug resistance characteristics, showing poor response to PD-1 inhibitors and targeted drugs such as lenvatinib, demonstrating strong drug resistance. This characteristic makes the PNV-LM3 cell line an ideal platform for studying the immune escape mechanisms of liver cancer, drug resistance, and optimizing immunotherapy and targeted therapy regimens, and it has important clinical application value.

[0013] 4. Provides an ideal model for liver cancer treatment research. Due to its unique advantages in liver cancer metastasis, immune escape, and drug resistance, the PNV-LM3 cell line not only helps to deepen our understanding of the biological characteristics of liver cancer, but also provides more precise experimental evidence for the early diagnosis, targeted therapy, and immunotherapy development of liver cancer. Its high clinical relevance makes it an important tool in liver cancer treatment research, providing a more reliable experimental platform for evaluating clinical efficacy and developing new therapies. Attached Figure Description

[0014] Figure 1 The basic characteristics of PNV-LM3 mouse hepatocellular carcinoma cells are shown. (A) A schematic diagram of PNV-LM3 cell extraction and in vivo lung metastasis acclimation. The general process involves first inducing hepatocellular carcinoma through hydrodynamic tail vein injection using a combination of PTEN KO and NRAS G12V mutant plasmids. Primary parental cells, PNV-HTVi, are extracted, stabilized in vitro, and passaged. Then, lung metastasis-inducing hepatocellular carcinoma cells are induced via tail vein injection. PNV-LM1 cells are extracted, stabilized in vitro, and passaged again, followed by another tail vein injection. This process, involving three rounds of in vivo lung metastasis induction, ultimately yields PNV-LM3 cells with a strong lung metastasis tendency. (B) A gross image (left) of the in situ tumor formed after intrahepatic injection of PNV-LM3 in C57BL / 6 mice, and the corresponding HE staining results of the liver tissue (right), showing the typical hepatocellular carcinoma tissue structure (scale bar: 50 µm). (C) Hepatocellular carcinoma lung metastases formed after tail vein injection of PNV-LM3 in C57BL / 6 mice, showing typical lung metastatic hepatocellular carcinoma tissue structure (scale bar: 200 µm, 50 µm).

[0015] Figure 2 To validate the lung metastasis tendency of PNV-LM3. (A) Gross image (left) of lung tumors formed after tail vein injection of PNV-LM3 cells in C57BL / 6 mice, and corresponding HE staining results (right), showing typical hepatocellular carcinoma tumor characteristics and stronger lung metastasis ability (scale bar: 1 cm). (B) After tail vein injection of PNV-LM3, lung metastasis formation was periodically detected by in vivo imaging, and the lung metastasis-free survival rate was calculated, suggesting that PNV-LM3 cells have a stronger lung metastasis ability. (C) Gross image (left) of lung tumors formed after tail vein injection of PNV-LM3, and corresponding HE staining results (right), indicating that PNV-LM3 has a stronger lung metastasis ability compared to Hepa1-6 (scale bar: 1 cm). (D) Lung metastasis-free survival rate suggests that PNV-LM3 has a stronger lung metastasis ability compared to Hepa1-6.

[0016] Figure 3Analysis of the microenvironment characteristics of PNV-LM3 in hepatocellular carcinoma lung metastases. Among them, (A) flow cytometry analysis of lung tissue tumors formed after tail vein injection of PNV-LM3 showed that, compared to Hepa1-6, PNV-LM3 tumors had significantly higher CD8+ levels. + The proportion of T cell infiltration was significantly reduced, while the proportions of endothelial cells and fibroblasts were significantly increased. (B) Multiplex immunofluorescence staining of lung tissue tumors formed after PNV-LM3 tail vein injection: green: CD8 (cytotoxic T cells), white: PDCD1 (programmed cell death protein 1), red: GZMB (granzyme B), yellow: CD31 (endothelial cell marker), and DAPI staining of the nucleus. The results showed that CD8 in PNV-LM3 tumors... + The proportion of T cell infiltration was significantly reduced, and depleted CD8 cells were also present. + There are more T cells and activated CD8 cells. + There were fewer T cells and a significantly increased proportion of endothelial cells (scale bar: 50 µm).

[0017] Figure 4 This section describes the response of the PNV-LM3 orthotopic tumor model to targeted immunotherapy. (A) C57BL / 6 mice were inoculated with PNV-LM3 and Hepa1-6 cells to form orthotopic tumors, and then treated with either PD-1 monoclonal antibody and lenvatinib combined with targeted immunotherapy, or as a control. The figure shows gross liver images at the end of treatment for both groups of mice, indicating a significant reduction in tumor volume after Hepa1-6 tumor treatment, but no significant change in tumor volume after PNV-LM3 tumor treatment. (B) Survival curve analysis showed that Hepa1-6 tumor treatment significantly prolonged survival time in mice, but there was no significant difference in survival time before and after PNV-LM3 tumor treatment (log-rank test). Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides a mouse hepatocellular carcinoma cell line PNV-LM3 with lung metastasis characteristics. This cell line was deposited on February 4, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202621.

[0020] In some specific implementations, the cell line is obtained by inducing mouse liver cancer through hydrodynamic tail vein injection of a combination of PTEN KO and NRAS G12V mutant plasmids, extracting primary cells PNV-HTVi, and then subjecting them to at least three rounds of in vivo tail vein injection for lung metastasis training.

[0021] In some specific implementations, the cell line was able to form lung metastases after tail vein injection in C57BL / 6 mice, and the lung metastasis rate was significantly higher than that of the Hepa1-6 cell line.

[0022] In some specific implementations, CD8 is present in the tumor microenvironment formed by the cell line. + The proportion of T cell infiltration was lower than that of the Hepa1-6 cell line, and the proportion of endothelial cells and fibroblasts was increased.

[0023] In some specific implementations, the cell lines are resistant to PD-1 inhibitors and lenvatinib.

[0024] This invention also provides a method for constructing the mouse hepatocellular carcinoma cell line PNV-LM3, comprising the following steps: (1) The combination of PTEN KO and NRAS G12V mutant plasmids was introduced into the liver of C57BL / 6 mice by hydrodynamic tail vein injection to induce liver cancer formation; (2) Primary cells PNV-HTVi were extracted from liver cancer tissue and cultured in vitro; (3) PNV-HTVi cells were injected into new-generation C57BL / 6 mice via tail vein to induce lung metastasis; (4) Extract cells from lung metastases, culture them in vitro, and repeat step (3) at least twice to obtain the PNV-LM3 cell line with high lung metastasis characteristics.

[0025] This invention also provides a method for establishing an animal model of liver cancer lung metastasis, using the PNV-LM3 cell line, which is injected into immunocompetent mice via tail vein injection or intrahepatic injection.

[0026] This invention also provides a method for screening or evaluating drugs against liver cancer metastasis, comprising the following steps: (1) An animal model of liver cancer lung metastasis was established using the PNV-LM3 cell line described above; (2) Administer the drug to be tested; (3) Observe the formation, growth or regression of lung metastases and evaluate the drug effect.

[0027] This invention also provides a method for studying the immune microenvironment or immune escape mechanism of liver cancer, using an animal model established by the PNV-LM3 cell line to analyze the infiltration of immune cells and the composition of stromal cells in tumor tissue.

[0028] This invention also provides the application of the mouse hepatocellular carcinoma cell line PNV-LM3 in the preparation of experimental models for studying the mechanism of liver cancer metastasis, drug screening, or evaluation of immunotherapy.

[0029] This invention provides a novel hepatocellular carcinoma cell line, PNV-LM3, derived from primary PNV-HTVi cells extracted from a hepatocellular carcinoma model constructed using a hydrodynamic tail vein injection (HTVi) model combined with PTEN KO and NRAS G12V mutant plasmids. After three generations of in vivo acclimatization via tail vein injection, this invention successfully established a stable lung metastasis cell line capable of effectively simulating the occurrence, development, and lung metastasis of hepatocellular carcinoma. Compared to the traditional Hepa1-6 cell line, this cell line exhibits significant advantages in simulating lung metastasis tendency and microenvironment, more realistically reflecting the pathophysiological processes and molecular mechanisms of hepatocellular carcinoma metastasis. This cell line also demonstrates resistance to targeted and immunotherapies, further enhancing its value as a clinical research model.

[0030] The cell line of this invention provides a more precise and reliable experimental platform for the study of liver cancer metastasis mechanisms, the exploration of immune escape mechanisms, and the evaluation of targeted immunotherapy, and has important research and clinical application value.

[0031] The technical solution of the present invention includes the following aspects: 1. Primary cell extraction using a combination of PTEN KO and NRAS G12V hydrodynamic plasmids This invention successfully induced tumor formation in mouse livers using a combination of PTEN gene knockout (KO) and NRAS G12V mutant plasmid in a hydrodynamic tail vein injection (HTVi) model. Primary hepatocellular carcinoma cells, PNV-HTVi, were extracted from the liver cancer tumors generated by this model and used as the parental cell line for this invention.

[0032] 2. Induction and domestication of in vivo third-generation lung metastasis tendency after tail vein injection To enhance the lung metastasis capacity of cells, this invention involves three generations of in vivo domestication of primary PNV-HTVi cells via tail vein injection. After each generation of injection, the cells form lung metastatic tumors in mice, allowing for further screening and culture of cell populations with a strong tendency for lung metastasis, ultimately resulting in a stable PNV-LM3 cell line.

[0033] 3. Verification of transfer tendency This invention, by comparing the PNV-LM3 cell line with the parental cell line (PNV-HTVi) and the commonly used mouse hepatocellular carcinoma cell line Hepa1-6, verifies the advantage of the PNV-LM3 cell line in lung metastasis tendency. In vivo animal experiments showed that the PNV-LM3 cell line exhibited significantly stronger lung metastasis ability, demonstrating that it can more realistically simulate the lung metastasis process of hepatocellular carcinoma compared to the Hepa1-6 cell line.

[0034] 4. Detection and comparison of the tumor microenvironment This invention further analyzed the tumor microenvironment characteristics formed by the PNV-LM3 cell line. By comparing it with the tumor microenvironment formed by the Hepa1-6 cell line, the results showed that the tumor microenvironment of the PNV-LM3 cell line more closely resembled the pathological characteristics of clinical liver cancer patients. Particularly regarding immune cell infiltration, the PNV-LM3 cell line showed increased CD8+ in tumor cells. + The proportion of T cells was low; it also showed a significant advantage in the proportion of tumor-associated endothelial cells and fibroblasts, simulating immunosuppression and microenvironmental changes during the lung metastasis of liver cancer.

[0035] 5. Comparison of responses to targeted therapy and immunotherapy This invention compares the responses of the PNV-LM3 cell line and the Hepa1-6 cell line to targeted therapy and immunotherapy. The results show that the PNV-LM3 cell line responded significantly worse to lenvatinib and PD-1 inhibitors, exhibiting significant drug resistance. This characteristic is highly consistent with the immune escape and drug resistance observed in clinical liver cancer patients during treatment, indicating that the PNV-LM3 cell line can better mimic the treatment response of liver cancer, and is particularly valuable in the evaluation of targeted therapy and immunotherapy.

[0036] Example 1 1. Establishment of a mouse model and hydrodynamic tail vein injection (HTVi) Eight- to ten-week-old male C57BL / 6 mice, weighing approximately 20-25 grams, were used as experimental animals. Mice were injected with hydrodynamic plasmids via tail vein injection (HTVi). The plasmid combination for hydrodynamic injection included a CRISPR / Cas9 system plasmid for PTEN gene knockout (KO) (a PTEN knockout sequence inserted into the pSpCas9(BB)-2A-GFP, #48138, Addgene plasmid) and an NRAS G12V mutant plasmid (pT / Caggs-NRASV12, #20205, Addgene), as well as an SB transposase plasmid (pCMV(CAT)T7-SB100, #34879, Addgene). Each mouse was injected with 2 mL of a plasmid mixture containing 10 μg of PTENKO plasmid, 10 μg of NRAS G12V mutant plasmid, and 1 μg of SB transposase plasmid, administered over 4-7 seconds to ensure sufficient hydrodynamic pressure for effective plasmid entry into the mouse liver. After injection, the mice were allowed to rest for a period to observe for the development of liver tumors. After approximately 4-6 weeks, tumors began to form in localized areas of the liver in the plasmid-injected mice, and the tumors gradually increased in size.

[0037] 2. Extraction and culture of primary hepatocellular carcinoma cells After tumor formation, the mouse liver tumors significantly enlarged approximately 6-8 weeks later. At this point, the tumor tissue was excised and subjected to enzymatic digestion. First, the tumor tissue was cut into small pieces and digested with 0.25% trypsin and 1 mg / mL collagenase solution at 37°C for 1-2 hours until the tissue was broken down into a single-cell suspension. Subsequently, the suspension was filtered through a 70 μm cell filter to remove undigested tissue fragments. Next, the single-cell suspension was collected, the supernatant was removed by centrifugation, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. The primary hepatocellular carcinoma cells formed a stable cell population after culture, named PNV-HTVi.

[0038] 3. The process of lung metastasis acclimatization after in vivo tail vein injection Primary hepatocellular carcinoma cells, PNV-HTVi, were extracted and injected into new-generation C57BL / 6 mice via tail vein injection. Each mouse received 1 × 10⁻⁶ cells. 6PNV-HTVi cells were injected. Tumor formation and lung metastasis were observed in mice after injection. The dynamic process of tumor metastasis was monitored using in vivo imaging techniques (such as IVIS imaging system). To enhance the lung metastasis tendency of PNV-HTVi cells, these mice were injected via tail vein three times. Two to three weeks after each injection, in vivo imaging and anatomical examination were used to confirm whether lung metastasis had occurred. If lung metastasis was confirmed, the mouse lung tumor was isolated, and primary cells were extracted from the lung following the aforementioned steps. After a period of culture, a stable cell population was formed. Each experimental cycle lasted approximately 4-5 weeks, allowing the cells to complete in vivo adaptation and acclimatization, ultimately successfully establishing a stable PNV-LM3 cell line.

[0039] 4. Verification of transfer tendency The metastatic tendency of the PNV-LM3 cell line was verified through in vivo xenotransplantation experiments. In this experiment, PNV-HTVi, Hepa1-6, and PNV-LM3 cells were injected into the tail vein or liver of C57BL / 6 mice, respectively, and tumor metastasis was monitored weekly using an IVIS imaging system. After 4-6 weeks, metastasis to the lungs and other organs was analyzed by dissection and H&E staining. The results showed that the lung metastasis rate of the PNV-LM3 cell line was significantly higher than that of the PNV-HTVi and Hepa1-6 cell lines, demonstrating that the PNV-LM3 cell line has a significant tendency for lung metastasis.

[0040] 5. Detection and comparison of tumor microenvironment characteristics After tumor formation in the PNV-LM3 cell line, lung tumor tissue was collected for immunofluorescence staining and flow cytometry analysis to assess the immune cell components in the tumor microenvironment, with particular attention to the proportions of macrophages, CD8+ T cells, and other stromal cells such as endothelial cells and fibroblasts. Compared with tumors formed by the Hepa1-6 cell line, the proportion of CD8+ T cell infiltration in the PNV-LM3 cell line was significantly lower than that in the Hepa1-6 cell line, while the proportions of endothelial cells and fibroblasts were also significantly increased. These results indicate that the tumor microenvironment characteristics of the PNV-LM3 cell line are more similar to the immune microenvironment of clinically metastatic liver cancer and have high clinical relevance.

[0041] 6. Detection of response to targeted therapy and immunotherapy In evaluating the response to targeted therapy and immunotherapy, tumor-bearing mice with PNV-LM3 and Hepa1-6 cell lines were treated with lenvatinib and a PD-1 inhibitor. The drugs were administered orally with lenvatinib (10 mg / kg / day) and injected with the PD-1 inhibitor every three days (10 mg / kg). Tumor size was measured weekly during treatment, and tumor monitoring was performed using an IVIS imaging system. The results showed that the PNV-LM3 cell line responded poorly to lenvatinib and the PD-1 inhibitor, exhibiting significant drug resistance and slower tumor growth. This indicates that the PNV-LM3 cell line can effectively mimic the immune escape and drug resistance mechanisms of hepatocellular carcinoma, which is of significant importance for research on the treatment of hepatocellular carcinoma.

[0042] 7. Long-term culture and stability of cell lines The PNV-LM3 cell line maintained a strong tendency for lung metastasis and immune escape characteristics even after multiple passages. The cell line was maintained in standard medium (DMEM, 10% FBS, 1% penicillin and streptomycin) and cultured at 37°C and 5% CO2. At each passage, the cells were diluted 1:3 to maintain cell growth stability and metastatic properties.

[0043] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A mouse hepatocellular carcinoma cell line PNV-LM3 with lung metastasis characteristics, characterized in that, The cells were deposited on February 4, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: C202621.

2. The mouse hepatocellular carcinoma cell line PNV-LM3 according to claim 1, characterized in that, The cell line was obtained by inducing mouse liver cancer through hydrodynamic tail vein injection of a combination of PTEN KO and NRAS G12V mutant plasmids, extracting primary cells PNV-HTVi, and then subjecting them to at least three rounds of in vivo tail vein injection for lung metastasis.

3. The mouse hepatocellular carcinoma cell line PNV-LM3 according to claim 2, characterized in that, The cell line was able to form lung metastases after tail vein injection in C57BL / 6 mice, and the lung metastasis rate was significantly higher than that of the Hepa1-6 cell line.

4. The mouse hepatocellular carcinoma cell line PNV-LM3 according to claim 3, characterized in that, The proportion of CD8⁺ T cell infiltration in the tumor microenvironment formed by the cell line was lower than that of the Hepa1-6 cell line, and the proportion of endothelial cells and fibroblasts was increased.

5. The mouse hepatocellular carcinoma cell line PNV-LM3 according to any one of claims 1-4, characterized in that, The cell line was resistant to PD-1 inhibitors and lenvatinib.

6. A method for constructing the mouse hepatocellular carcinoma cell line PNV-LM3 according to any one of claims 1-5, characterized in that, Includes the following steps: (1) The combination of PTEN KO and NRAS G12V mutant plasmids was introduced into the liver of C57BL / 6 mice by hydrodynamic tail vein injection to induce liver cancer formation; (2) Primary cells PNV-HTVi were extracted from liver cancer tissue and cultured in vitro; (3) PNV-HTVi cells were injected into new-generation C57BL / 6 mice via tail vein to induce lung metastasis; (4) Extract cells from lung metastases, culture them in vitro, and repeat step (3) at least twice to obtain the PNV-LM3 cell line with high lung metastasis characteristics.

7. A method for establishing an animal model of liver cancer lung metastasis, characterized in that, The PNV-LM3 cell line according to any one of claims 1-5 was used to inoculate immunocompetent mice via tail vein injection or intrahepatic injection.

8. A method for screening or evaluating drugs against liver cancer metastasis, characterized in that, Includes the following steps: (1) Establish an animal model of liver cancer lung metastasis using the PNV-LM3 cell line according to any one of claims 1-5; (2) Administer the drug to be tested; (3) Observe the formation, growth or regression of lung metastases and evaluate the drug effect.

9. A method for studying the immune microenvironment or immune escape mechanism of liver cancer, characterized in that, An animal model was established using the PNV-LM3 cell line as described in any one of claims 1-5 to analyze the infiltration of immune cells and the composition of stromal cells in tumor tissue.

10. The use of the mouse hepatocellular carcinoma cell line PNV-LM3 according to any one of claims 1-5 in the preparation of experimental models for studying the mechanism of liver cancer metastasis, drug screening or evaluation of immunotherapy.

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