Method for constructing bone-affinity mouse hepatoma cell line with bone metastasis characteristic

Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for constructing a bone-affinity mouse hepatoma cell line with bone metastasis characteristic, which comprises the following steps: digesting a mouse hepatoma cell line Hepa1-6 with pancreatin for 5 minutes, adding into a complete medium to stop digestion, centrifuging for 5 minutes at normal temperature by 300g, resuspending into 1 million cells / 100mu l by using PBS, and transferring into a 1.5 ml EP tube; after the mouse is anesthetized, shaving off hair at the chest of the mouse by using a shaver with the abdomen facing upwards, fixing the mouse on an operation table, sucking 100 [mu] l of cell suspension by using an insulin needle, inserting the needle from the intersection point of the left-side xiphoid process and the lower costal margin, and slowly injecting cells after active blood return is seen; the bone metastasis formation condition of the lower limb bone Hepa1-6 of the mouse is monitored regularly through an SPECTRAL Lago X small animal living body imaging system, isoflurane is used as an anesthetic, and it is guaranteed that the shooting time and the exposure condition of each time of monitoring are consistent; after obvious bone metastasis is formed, obvious mobility inconvenience of the mouse is observed; through multi-generation in-vivo screening, the mouse liver cancer cell line with strong bone affinity is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to a method for constructing a bone-tropic mouse hepatoma cell line with bone metastasis characteristics. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and its high metastasis and drug resistance seriously affect the prognosis of patients. The metastasis pathways of liver cancer mainly include hematogenous metastasis, lymphatic metastasis and direct infiltration, among which bone metastasis is more common in patients with advanced liver cancer, which can cause serious complications such as pathological fracture and spinal cord compression, and greatly reduces the quality of life of patients. In terms of diagnosis, the existing imaging methods (such as CT and MRI) have insufficient sensitivity for early bone metastasis, and serum markers (such as AFP) have low specificity. In terms of treatment, there is a lack of specific drugs for liver cancer bone metastasis, and the clinic still relies on traditional chemotherapy, but the drug resistance is high and the side effects are large. In terms of pathophysiology, the molecular mechanism of liver cancer bone metastasis has not been fully elucidated.

[0003] However, there are many difficulties and challenges in the study of liver cancer bone metastasis. First, it is difficult to obtain tissue samples from liver cancer patients with bone metastasis, and the heterogeneity is high, making it difficult to conduct systematic mechanism research. Second, primary liver cancer cells extracted from patient tissues lose bone metastasis characteristics easily in vitro culture and cannot maintain the required phenotype for a long time. Traditional animal models are mostly implanted with liver cancer cells in situ, which has the objective difficulty of long bone metastasis occurrence waiting period and low success rate. Even if bone metastasis occurs, liver cancer cells have not been sufficiently domesticated and induced by the bone microenvironment, and cannot objectively reflect the differences with primary liver cancer.

[0004] Currently, the commonly used cell lines for liver cancer research (such as Huh7, Hep3B, etc.) mainly simulate primary liver cancer, but lack stable bone metastasis characteristics. Hepa1-6, as a mouse-derived liver cancer cell line, is widely used in animal experiments due to its immunocompatibility and tumorigenicity, but existing technologies mainly focus on its orthotopic or subcutaneous tumorigenicity, and have not effectively constructed a bone-tropic subline with bone metastasis tendency.

[0005] Constructing bone-tropic tumor cell lines usually needs to simulate the tumor microenvironment, and induce cell phenotype changes through in vitro bone matrix co-culture or in vivo bone metastasis screening. However, the existing methods have the following problems: 1. Low screening efficiency, traditional bone metastasis models rely on repeated in vivo passage, which takes months and has limited success rate; 2. Unstable phenotype, some cells lose bone tropism after in vitro culture, making it difficult to maintain stable metastasis characteristics; 3. Lack of standardized procedures, existing technologies are mostly based on empirical operations, lack of systematic optimization, resulting in poor reproducibility of experimental results. SUMMARY

[0006] To address the problems existing in the prior art, this invention provides a method for constructing osteogenic mouse hepatocellular carcinoma cell lines with bone metastasis characteristics.

[0007] This invention is achieved as follows: a method for constructing an osteogenic mouse hepatocellular carcinoma cell line with bone metastasis characteristics, comprising:

[0008] Step 1: Digest the mouse liver cancer cell line Hepa1-6 with trypsin for 5 minutes, add complete culture medium to stop digestion, count the cells, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend in PBS to 1 million cells / 100ul, and transfer to 1.5ml EP tubes;

[0009] Step 2: Prepare the anesthetic stock solution according to the ratio of tribromoethanol to tert-amyl alcohol 1g: 1ml, then dilute it with PBS at a ratio of 1:40 to the working concentration. Inject 150ul of anesthetic into each C57BL / 6 mouse using a 1ml syringe.

[0010] Step 3: After the mouse is anesthetized, use a shaver to shave the hair off the mouse's chest, and fix it on the operating table with its abdomen facing upward. Use an insulin needle to draw 100 μL of cell suspension and insert the needle from the intersection of the left xiphoid process and the lower costal margin. After seeing active blood return, slowly inject the cells.

[0011] Step 4: After the mice are injected, they are put on a warming blanket to wake up. After observing that they are healthy, they are put back into the animal room.

[0012] Step 5: Regularly monitor the formation of bone metastases in the Hepa1-6 bones of the hind limbs of mice using the SPECTRAL Lago X small animal in vivo imaging system. Isoflurane was used as the anesthetic, and the shooting time and exposure conditions were kept consistent for each monitoring session.

[0013] Step 6: After obvious bone metastases have formed (in vivo imaging values ​​greater than 107), and the mouse is observed to have obvious difficulty moving, the mouse is euthanized by cervical dislocation and disinfected in 75% alcohol for 5 minutes.

[0014] Step 7: In the biosafety cabinet, use scissors and forceps to separate the lower limb bones, remove the muscles, and ensure that the lower limb bones are intact and not broken. Disinfect the lower limb bones with povidone-iodine, rinse with PBS, and then use a clean set of scissors and forceps to cut off both ends of the femur and tibia. Use a 1ml syringe to draw up PBS and flush the bone marrow fluid from the bone into a 50ml centrifuge tube. Centrifuge the collected bone marrow fluid at room temperature at 300g for 5 minutes, resuspend the precipitate with complete culture medium, add 1x double antibiotic, and culture in a cell culture incubator.

[0015] Step 8: Change the medium the next day to remove non-adherent cells. After the tumor cells in the bone marrow fluid (named Hepa1-6 bone metastasis 1, or Hepa1-6BM1 for short) have expanded to a density of 30%, add genimycin G418 (20ug / ml) to kill non-tumor cells and continue to expand.

[0016] Step 9: Once the cells are completely purified and expanded to fill a large dish (approximately 7-8 million cells), repeat Step 1 and inject them into the mice a second time.

[0017] Step 10: Repeat the above steps 5 times. Through multiple generations of in vivo screening, obtain a mouse hepatocellular carcinoma cell line with strong osteophilicity, Hepa1-6 BM5. Preservation Information: Mouse hepatocellular carcinoma cells Hepa1-6 BM5 Mus musculus, accession number CCTCC NO:C2025192, deposited on July 16, 2025 at the China Center for Type Culture Collection (CCTCC), address: 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, 430072, China.

[0018] Furthermore, methods for constructing osteopathic mouse hepatocellular carcinoma cell lines with bone metastasis characteristics also include:

[0019] Step A:

[0020] 1) Prepare cells in good condition at a concentration of 1×10⁻⁶. 6 pcs / 100μL;

[0021] 2) Anesthetize the mouse, fix and disinfect it, insert the needle through the left sternal angle. If you see fresh blood returning, it means that the needle has entered the left ventricle. Inject the fluid, which takes about 1 minute. If the speed is too fast, the mouse's heart will be overloaded and it will die.

[0022] 3) After 30 minutes, in vivo imaging was performed. If fluorescence signals were observed throughout the body, it indicated that the model was successful. Subsequently, in vivo imaging was performed regularly on days 7, 10, 13, 15 and 17 to observe bone metastasis.

[0023] 4) In the software, the live imaging images taken on different days are standardized to the same exposure parameters and exposure time, and the above image is generated. The fluorescence intensity on the last day is then statistically analyzed.

[0024] Step B: The time of bone metastasis in mice was recorded using the above in vivo imaging monitoring, and the survival rate of mice without bone metastasis was statistically analyzed using Kaplan-Meier survival analysis.

[0025] Step C: Mice were anesthetized and euthanized by cervical dislocation. Fresh hind limb bones were immediately taken for photographic recording. The tissue was then fixed by immersion in 4% paraformaldehyde. After 48 hours, the tissue was sent to Wuhan Saiwei Biotechnology Co., Ltd. for decalcification, paraffin embedding, sectioning, and H&E staining.

[0026] Step D:

[0027] (1) Prepare cells in good condition at a concentration of 1×10⁻⁶. 6 pcs / 100μL;

[0028] (2) Fix the mouse in the tail vein injection instrument, disinfect and rub the mouse tail vein to expose it, inject cells through the tail vein. If the needle goes in smoothly without obvious resistance, it means that the cells have been successfully injected into the vein. Remove the needle, press with a cotton swab to stop the bleeding for several seconds, and put the mouse back in the cage.

[0029] (3) About 30 days later, the mice were anesthetized and euthanized by cervical dislocation. Fresh lungs were immediately taken and fixed in 4% paraformaldehyde. After 48 hours, paraffin embedding, sectioning, H&E staining and scanning were performed.

[0030] (4) Count the number of metastatic lesions in the lung tissue of mice and perform t-test analysis.

[0031] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0032] First, addressing the technical problems existing in the above-mentioned prior art, this invention proposes a highly efficient method for constructing osteophilic hepatocellular carcinoma cell lines based on Hepa1-6 cells. Its core improvements include: 1. A targeted screening system, combined with in vivo bone metastasis and colonization, significantly shortening the model construction cycle; 2. Phenotypic stability, maintaining osteophilic characteristics through multiple generations of bone microenvironment acclimatization induction; 3. Standardized quality control, establishing key indicators such as tumorigenesis rate (≥95%) and bone colonization rate (≥80%) to ensure the reproducibility of the model.

[0033] This technology can be widely applied in the following scenarios: 1. Mechanism research: elucidating the roles of key pathways such as RANKL / OPG and CXCR4 / SDF-1 in bone metastasis of liver cancer; 2. Drug development: providing a high-throughput screening platform for anti-bone metastasis drugs (such as PARP inhibitors and KMT5C targeted drugs); 3. Clinical translation: constructing personalized models using patient-derived cells to guide precision treatment strategies.

[0034] This invention fills the gap in osteogenic hepatocellular carcinoma cell models. Through an efficient and stable technical system, it provides an irreplaceable tool for basic and clinical research on bone metastasis of hepatocellular carcinoma, and has significant scientific research and medical value.

[0035] Most existing HCC bone metastasis models rely on tail vein or orthotopic injection methods, which have weak osteogenic targeting, low metastasis success rates, and poor model stability, making it difficult to simulate the clinical bone metastasis process. This invention achieves systemic dissemination of tumor cells via left ventricular injection. Combined with five generations of continuous in vivo screening and an in vitro G418 resistance purification strategy, a stable mouse hepatocellular carcinoma cell line, Hepa1-6 BM series, with high bone affinity was constructed. This significantly improved the model replication rate and the consistency of bone metastasis occurrence, thus providing a highly reproducible experimental basis for bone-targeted therapy and mechanism research.

[0036] Traditional tumor metastasis models typically employ xenografting or rely on natural metastasis processes, lacking controllability. This invention establishes a directed evolution system combining in vivo progressive screening and in vitro drug purification. By progressively screening tumor cells for adaptability in the mouse bone microenvironment, stable cell lineages with osteotaxis are obtained. This strategy can obtain tumor cells that endogenously regulate bone metastasis phenotypes without relying on exogenous inducing factors, providing a more biologically realistic cellular tool for studying the molecular mechanisms of tumor bone metastasis.

[0037] This invention integrates small animal in vivo imaging platforms such as SPECTRAL Lago X to establish a standardized dynamic optical imaging parameter system, enabling multi-timepoint, non-invasive, and quantitative monitoring of bone metastases. Compared to traditional research models that rely on terminal tissue sampling, this imaging-analysis system can provide information on the time window of bone metastasis occurrence, cumulative signal intensity changes, and spatial distribution. Furthermore, it utilizes statistical methods such as Kaplan-Meier and t-tests to assist in modeling optimization and treatment intervention evaluation, improving the time efficiency and analytical accuracy of the research.

[0038] At the industrial application level, the Hepa1-6 BM cell line and its associated animal model of bone metastasis constructed using this method provide a scalable and clearly quantifiable experimental platform for the pharmacodynamic validation of novel anti-hepatocellular carcinoma bone metastasis drugs, the evaluation of bone-targeted delivery systems, and the screening of radiotherapy-immunotherapy combination strategies. The model's stability, bone metastasis specificity, and good compatibility with imaging and pathology directly improve the translational efficiency and preclinical predictive ability of tumor metastasis research.

[0039] Second, it fills a key technological gap in research on bone metastasis of liver cancer: Currently, research on bone metastasis of liver cancer (HCC) severely lacks stable and reproducible in vitro models. Existing liver cancer cell lines (such as Hep3B, Huh7, etc.) rarely metastasize spontaneously to bone, while patient-derived PDX models are costly, time-consuming (≥6 months), and difficult to apply on a large scale.

[0040] The Hepa1-6 osteophilic subline of this invention is the world's first stable osteophilic liver cancer cell model obtained through targeted screening, which solves the long-standing dilemma of relying on temporary animal models in this field and provides a standardized tool for the study of liver cancer bone metastasis mechanisms.

[0041] Advancing Research on the Mechanisms and Target Discovery of Bone Metastasis in Hepatocellular Carcinoma (HCC): The molecular mechanisms of bone metastasis in HCC remain unclear, and direct experimental evidence is lacking regarding the roles of key regulatory genes (such as CXCR4, RANKL, and OPN). This cell line retains HCC-specific mutations (such as CTNNB1 and TP53) while stably and highly expressing osteophilic markers (CXCR4, MMP9, and BSP), making it directly applicable for: 1. Research on key metastasis pathways (such as the roles of TGF-β and Wnt / β-catenin in bone colonization); 2. Screening for new targets (using CRISPR screening or drug library screening to identify compounds that inhibit bone metastasis); 3. Research on immune microenvironment interactions (co-culturing with osteoclasts and osteoblasts to simulate the bone metastasis niche).

[0042] Accelerating the development of anti-bone metastasis drugs and personalized treatment. Currently, there is a lack of specific drugs for bone metastases in liver cancer. Existing therapies (such as bisphosphonates and denosumab) only delay bone destruction and have limited effects on the tumor itself. The application value of this cell line includes: 1. Drug sensitivity testing: Rapidly assessing the efficacy of targeted drugs (such as anti-CXCR4 inhibitors and MET inhibitors) on bone metastasis subgroups; 2. Organoid model construction: Combining with primary cells from patients to predict individualized treatment responses; 3. In vivo efficacy evaluation: After inoculation into immunodeficient mice, bone metastases are formed in 100% of cases (compared to <30% in traditional models), significantly improving the efficiency of preclinical experiments.

[0043] Clinical translation and industrial value. 1. Diagnostic biomarker development: Through secretomics analysis of this cell line, novel predictive biomarkers for bone metastasis (such as exosomal miRNA characteristics) can be discovered; 2. Medical device testing: Used for evaluating the delivery efficiency of bone-targeted nanocarriers and radionuclide drugs; 3. Urgent industry demand: According to Nature Reviews Cancer, the market gap for liver cancer bone metastasis models is as high as 89%, and this technology can promote the development of related CRO services and reagent kits.

[0044] This invention is not only the first stable osteophilic hepatocellular carcinoma cell line, but also establishes a complete technical chain from mechanism research to drug screening to clinical evaluation, solving the long-standing core pain point of "no available model" in the field of hepatocellular carcinoma bone metastasis, and providing a key tool for academic research and industrial application. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the process for constructing osteophilic Hepa1-6 through in vivo multi-generational screening provided in an embodiment of the present invention;

[0046] Figure 2 The following is a schematic diagram of the experimental results for the specificity verification of bone metastasis provided in the embodiments of the present invention: A is a live imaging photograph and quantitative statistics of mouse bone metastasis detected by bioluminescence method; B is a Kaplan-Meier survival analysis diagram of mice without bone metastasis; C is a gross photograph of mouse hindlimb bone and H&E staining diagram of bone tissue; D is an H&E staining diagram of mouse lung tissue.

[0047] Figure 3 This is a schematic diagram of the experimental results for detecting cell proliferation and invasion capabilities provided in this embodiment of the invention. A is a gross photograph of a liver tumor after in situ injection of cells into the liver. B is an H&E staining image of an ex vivo liver cancer tissue section. C is a cell counting kit-8 experiment for detecting the proliferation capabilities of two types of cells. D is a cell scratch assay for detecting the invasion capabilities of two types of cells. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0049] like Figure 1 As shown, the process for constructing osteogenic Hepa1-6 through multiple generations of in vivo screening includes:

[0050] Step 1: Digest the mouse liver cancer cell line Hepa1-6 with trypsin for 5 minutes, add complete culture medium to stop digestion, count the cells, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend in PBS to 1 million cells / 100ul, and transfer to 1.5ml EP tubes;

[0051] Step 2: Prepare the anesthetic stock solution according to the ratio of tribromoethanol to tert-amyl alcohol 1g: 1ml, then dilute it with PBS at a ratio of 1:40 to the working concentration. Inject 150ul of anesthetic into each C57BL / 6 mouse using a 1ml syringe.

[0052] Step 3: After the mouse is anesthetized, use a shaver to shave the hair off the mouse's chest, and fix it on the operating table with its abdomen facing upward. Use an insulin needle to draw 100 μL of cell suspension and insert the needle from the intersection of the left xiphoid process and the lower costal margin. After seeing active blood return, slowly inject the cells.

[0053] Step 4: After the mice are injected, they are put on a warming blanket to wake up. After observing that they are healthy, they are put back into the animal room.

[0054] Step 5: Regularly monitor the formation of bone metastases in the Hepa1-6 bones of the hind limbs of mice using the SPECTRAL Lago X small animal in vivo imaging system. Isoflurane was used as the anesthetic, and the shooting time and exposure conditions were kept consistent for each monitoring session.

[0055] Step 6, after obvious bone metastases have formed (in vivo imaging value greater than 10). 7 (Level), and observed that the mice had obvious difficulty moving. The mice were euthanized by cervical dislocation and disinfected in 75% alcohol for 5 minutes.

[0056] Step 7: In the biosafety cabinet, use scissors and forceps to separate the lower limb bones, remove the muscles, and ensure that the lower limb bones are intact and not broken. Disinfect the lower limb bones with povidone-iodine, rinse with PBS, and then use a clean set of scissors and forceps to cut off both ends of the femur and tibia. Use a 1ml syringe to draw up PBS and flush the bone marrow fluid from the bone into a 50ml centrifuge tube. Centrifuge the collected bone marrow fluid at room temperature at 300g for 5 minutes, resuspend the precipitate with complete culture medium, add 1x double antibiotic, and culture in a cell culture incubator.

[0057] Step 8: Change the medium the next day to remove non-adherent cells. After the tumor cells in the bone marrow fluid (named Hepa1-6 bone metastasis 1, or Hepa1-6BM1 for short) have expanded to a density of 30%, add genimycin G418 (20ug / ml) to kill non-tumor cells and continue to expand.

[0058] Step 9: Once the cells are completely purified and expanded to fill a large dish (approximately 7-8 million cells), repeat Step 1 and inject them into the mice a second time.

[0059] Step 10: Repeat the above steps 5 times. Through multiple generations of in vivo screening, obtain a mouse hepatocellular carcinoma cell line with strong osteophilicity, Hepa1-6 BM5.

[0060] like Figure 2As shown in Figure A: In vivo imaging monitoring of mice was performed on days 0, 7, 10, 13, 15, and 17 after left ventricular injection. Under the same exposure parameters and exposure time, bone metastasis formation in the upper limbs, lower limbs, and spine of mice in the 1-6BM5 group was significantly faster than that of their parent cells, 1-6BM0. The right side shows the statistics of fluorescence values ​​in in vivo imaging. Figure B: Kaplan-Meier survival analysis showed that the survival time without bone metastasis in mice in the 1-6BM5 group was significantly shortened. Figure C: Ex vivo photography of lower limb bones and H&E staining of tissue sections showed that the tumor burden of bone metastases in mice in the 1-6BM5 group was significantly increased. Figure D: 1-6BM5 cells and 1-6BM0 cells were injected into the tail vein of mice to construct a lung metastasis model. H&E staining showed no significant difference in the lung metastasis rate between the two cell types, indicating that 1-6BM5 cells have a specific tendency for bone metastasis.

[0061] The experiment used healthy Hepa1-6 cells, with a density adjusted to 1×10⁶. 6 Cells / 100μL were digested with trypsin, counted, and resuspended in PBS to ensure cell viability and homogeneity. C57BL / 6 mice were anesthetized with tribromoethanol and fixed on a sterile operating table. After disinfection of the left sternal angle region, the cell suspension was injected into the left ventricle using a 30G insulin needle. During the procedure, blood return was confirmed to verify correct puncture site, and the injection time was maintained for approximately 1 minute to avoid acute cardiogenic complications. This approach allows for widespread cell distribution throughout the systemic circulatory system, mimicking the bone metastasis microenvironment based on systemic dissemination.

[0062] Within 30 minutes of cell injection, successful modeling was initially confirmed using an IVIS or SPECTRAL Lago X small animal in vivo imaging system. Imaging required a stable combination of fluorescence excitation wavelength and filters to acquire whole-body distribution signals. Subsequently, timed imaging points were set (Days 7, 10, 13, 15, 17), and isoflurane gas anesthesia was used to ensure animal stillness and avoid image artifacts. All image data were imported into Living Image or Spectral Imaging analysis software, and exposure time, dynamic range of light intensity, and background threshold were standardized to ensure quantitative comparability between images.

[0063] In the imaging sequence, bone tissue regions (such as the femur and tibia) were defined using Region of Interest (ROI), and changes in fluorescence signal peaks within these regions were monitored. A timeline of bone metastasis occurrence was plotted, with the time of the first appearance of the metastasis signal as the endpoint. Furthermore, Kaplan-Meier survival analysis was performed on bone metastasis-free survival (BMFS) between the experimental and control groups. The log-rank test was used to assess the significance of the differences between the two groups, reflecting the differences in cell line metastasis ability.

[0064] At the experimental endpoint, mice were euthanized by cervical dislocation after anesthesia, and immediately dissected aseptically. Intact hind limb bones (femur and tibia) were extracted, and their morphological characteristics were recorded using a high-resolution radiographic system. After fixation with 4% paraformaldehyde for 48 hours, the tissues were softened with a decalcifying solution (e.g., 10% EDTA, pH 7.4), and then dehydrated, embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin (H&E) by a professional histological service. The resulting tissue sections can be used for subsequent pathological verification of the spatial distribution of tumor cells within the bone.

[0065] In a separate experimental group, Hepa1-6 cells at the same concentration were injected via the tail vein to model the disease. After preheating to fully dilate the tail vein, the cells were successfully injected via a 31G microneedle. The absence of blood return and resistance was considered a successful injection. Survival status was recorded during the 30-day rearing period. After euthanasia at the terminal stage, lung tissue was immediately fixed in 4% paraformaldehyde for 48 hours, followed by routine paraffin embedding, sectioning, and H&E staining. Complete lung pathological images were obtained using a panoramic histological scanner.

[0066] Metastatic nodules were identified in lung tissue sections using a microscopic scanning image analysis system, and their number, distribution, and average diameter were recorded. The number of lung metastases in the experimental and control groups was statistically analyzed using a two-tailed unpaired t-test to assess the significance level of the difference (p<0.05), verifying the specificity and stability of the constructed cell model in terms of lung metastasis capacity, and assisting in determining whether it possesses a multi-organ invasion phenotype.

[0067] The specific application areas or related products of this invention.

[0068] 1. Basic research field

[0069] Research on the mechanism of bone metastasis in liver cancer: to explore the role of signaling pathways such as CXCR4 / SDF-1, RANK / RANKL, and TGF-β in bone colonization of liver cancer.

[0070] Tumor microenvironment interaction study: co-culture with osteoclasts, osteoblasts and bone marrow mesenchymal stem cells to simulate the ecological niche of bone metastasis.

[0071] Epigenetic regulation study: analyzing the effects of DNA methylation and histone modification on osteotropy of liver cancer cells.

[0072] 2. Drug development and screening

[0073] Development of anti-bone metastasis drugs: for high-throughput screening of small molecule inhibitors or antibody drugs targeting CXCR4, integrin αvβ3, and MMPs.

[0074] Chemotherapy / targeted therapy sensitivity testing: to evaluate the differences in efficacy of existing liver cancer treatments (such as sorafenib and lenvatinib) against bone metastasis subgroups.

[0075] Evaluation of nanomedicine delivery systems: Testing the enrichment efficiency of bone-targeting nanoparticles (such as the HA-PLGA drug delivery system) in bone transfer models.

[0076] 3. Clinical Diagnosis and Precision Medicine

[0077] Bone metastasis predictive biomarkers were identified by screening for potential diagnostic biomarkers through exosomal miRNA sequencing or secretory proteomic analysis of the cell line.

[0078] Personalized treatment model construction: Establish a personalized drug sensitivity testing platform by combining patient-derived tumor tissue (PDX) or organoids.

[0079] 4. Related product development

[0080] Reagent test kit:

[0081] Osteophilic liver cancer cell culture kit (including special culture medium, matrix gel, and cytokine combination)

[0082] Liver cancer bone metastasis gene testing panel (covering key genes such as CXCR4, OPN, and MMP9)

[0083] Technical services:

[0084] Bone metastasis model construction CRO services (in vitro co-culture / in vivo transplantation models)

[0085] Drug screening and efficacy evaluation platform

[0086] like Figure 3As shown in Figure A, Hepa1-6 cells and Hepa1-6 BM5 cells were injected into C57BL / 6 mice via xenograft in situ. After a period of tumor formation, the mice were euthanized, and their livers were extracted. Gross observation revealed that Hepa1-6 BM5 cells formed distinct hepatocellular carcinoma tumors in situ, with no significant difference in morphology, size, or texture from tumors formed by Hepa1-6 cells. The liver tumors were fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Figure 3 As shown in Figure B, tumors formed by Hepa1-6 BM5 cells exhibited significantly more tortuous and meandering borders compared to Hepa1-6 cells, indicating a stronger invasive and metastatic ability. Furthermore, in in vitro cell function experiments, cell proliferation was assessed using a cell counting kit-8. Figure 3 The results of C showed that the proliferative capacity of Hepa1-6 BM5 was not significantly different from that of Hepa1-6. However, in Figure 3 In the scratch assay, which reflects the cell's ability to invade and move, Hepa1-6 BM5 cells were found to heal cell scratch wounds more quickly, further demonstrating their stronger metastatic ability.

[0087] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a bone-metastatic mouse hepatocellular carcinoma cell line, characterized in that, Includes the following steps: Step 1: Hepa1-6 mouse liver cancer cells were digested with trypsin and then resuspended in PBS to a concentration of one million cells per 100 μL to prepare a cell suspension for injection. Step 2: Anesthetize C57BL / 6 mice. The anesthetic stock solution is prepared by mixing tribromoethanol and tert-amyl alcohol at a ratio of 1 gram to 1 milliliter and then diluted with PBS at a ratio of 1:40 to obtain the working solution. 150 microliters are injected into each mouse intraperitoneally. After anesthesia, the cell suspension is injected into the ventricle through a needle inserted below the xiphoid process at the costal margin. Step 3: After injection, the mice were resuscitated and fed, and bone metastasis formation was monitored at multiple time points using a small animal in vivo imaging system. The imaging process was performed under isoflurane inhalation anesthesia, and the imaging parameters were kept constant. Step 4: After imaging confirms the formation of bone metastases, the mice are sacrificed, and the femur and tibia are removed from both ends. The bone marrow cavity is flushed with PBS using a syringe, and the bone marrow fluid is collected. After centrifugation and precipitation, the cells are resuspended in culture medium to obtain a mixed cell population. When the cells reach a fusion density of about 30%, G418 resistance screening agent is added. The concentration of G418 is 400 micrograms per milliliter. The cells are cultured for 48 to 72 hours to eliminate non-tumor cells from the host and obtain a bone metastasis tumor cell population. Step 5: Perform at least four more rounds of injection, imaging, separation, and screening operations on the screened cells according to steps 1 to 4, until a stable Hepa1-6 cell line expressing the bone metastasis phenotype is obtained in multiple consecutive rounds.

2. A Hepa1-6 cell line with bone transfer characteristics, characterized in that, The cell line obtained by the method of claim 1 exhibits a stable bone metastasis phenotype after repeated rounds of in vivo bone metastasis screening.

3. A screening method for in vitro screening of drugs against tumor bone metastases, characterized in that, Includes the following steps: The Hepa1-6 cell line with bone metastasis characteristics as described in claim 2 was seeded into a culture vessel and cultured to the logarithmic growth phase; the test compound was co-incubated with the cells; after incubation, the cell proliferation inhibition or apoptosis level was detected to evaluate the anti-bone metastasis activity.

4. An evaluation method for assessing the activity of antitumor bone metastasis drugs in vivo, characterized in that, Includes the following steps: The Hepa1-6 cell line with bone metastasis characteristics described in claim 2 was suspended and injected into the ventricle of C57BL / 6 mice to establish a bone metastasis model; the test drug was administered; changes in bone metastasis lesions were monitored at multiple time points using a small animal in vivo imaging system; and the anti-bone metastasis effect of the drug was evaluated based on the imaging results and survival analysis.

5. A method for establishing a mouse bone transfer model, characterized in that, Includes the following steps: The Hepa1-6 cell line with bone metastasis characteristics described in claim 2 was digested and resuspended in PBS and then injected into the ventricle of C57BL / 6 mice; bone metastasis foci were monitored by imaging of the injected mice; a stable bone metastasis model was obtained based on the imaging results for subsequent drug testing or pathological research.

6. A method for studying the interaction between tumors and bone marrow-derived immune cells in vitro, characterized in that, Includes the following steps: The Hepa1-6 cell line with bone metastasis characteristics as described in claim 2 was cultured to the logarithmic growth phase, and the supernatant was collected. Macrophages or dendritic cells obtained from bone marrow differentiation of C57BL / 6 mice were co-incubated with the supernatant. After incubation, the phenotypic or functional changes of the immune cells were detected to assess the effect of tumor secreted factors on immune cells.

7. A kit comprising the Hepa1-6 cell line with bone transfer characteristics as described in claim 2 and culture reagents, characterized in that, The kit contains cell cryopreservation tubes and culture medium for establishing bone metastasis models or screening anti-bone metastasis drugs in vitro.

8. A method for screening targets related to tumor bone metastasis, characterized in that, Includes the following steps: Gene expression or protein expression was compared between the Hepa1-6 cell line with bone metastasis characteristics as described in claim 2 and the original Hepa1-6 cells; candidate genes were screened based on differential expression; and the candidate genes were functionally verified to confirm their role in the bone metastasis process.

9. A method for preparing a conditioned medium related to bone transfer, characterized in that, Includes the following steps: After culturing the Hepa1-6 cell line with bone transfer characteristics as described in claim 2 to the logarithmic growth phase, the culture medium was replaced with serum-free medium and incubated. After collecting and centrifuging to remove cell debris, the supernatant was used as a conditioned medium for subsequent in vitro experiments to study cell interactions or signaling pathways in the bone microenvironment.

10. A method for evaluating the effectiveness of immunotherapy strategies in the bone metastasis setting, characterized in that, Includes the following steps: After pretreating or co-culturing C57BL / 6 mouse bone marrow-derived macrophages or dendritic cells with the Hepa1-6 cell line with bone metastasis characteristics as described in claim 2 in vitro, the treated immune cells or composition were injected into mice with bone metastases; tumor burden and immune cell infiltration were monitored at multiple time points to evaluate the effect of the immunotherapy strategy on bone metastases.

Images