NK cell killing resistant cell strain as well as construction method and application thereof

Abstract

The invention discloses an NK cell killing resistant cell strain as well as a construction method and application thereof. The NK cell killing resistant cell strain is named as a human liver cancer cell HepG2-NK-R Homo sapiens, and is preserved in the China Center for Type Culture Collection on November 05, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2025298. The human hepatoma cell line HepG2 is exposed to NK cells for a long time for co-culture to obtain the human hepatoma cell line HepG2. The cell strain has a stable biological phenotype through in-vitro simulation of NK cell mediated long-term immune editing. Compared with parental cells, the cell line shows remarkably enhanced malignant biological behaviors, can be used for researching occurrence, development and metastasis of liver cancer or preparing a tumor cell model or a tumor animal model, is greatly improved in tumor forming ability, in-vivo proliferation speed and metastasis speed, can remarkably shorten an experimental period, and can highly restore invasion characteristics of tumors after evolution.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology and oncology, and particularly relates to a cell strain resistant to NK cell killing and a construction method and application thereof. BACKGROUND

[0002] In recent years, the concept of tumor evolution has provided a new perspective for understanding the occurrence, development, metastasis and drug resistance of cancer. Studies have shown that tumor cells are not static and unchanging, but a dynamic population that continuously evolves under internal and external environmental pressure. Among them, the monitoring and removal of the immune system is considered to be a key internal force driving tumor evolution.

[0003] In the immune microenvironment of the liver, natural killer (NK) cells, as an important natural immune cell, are abundant and play a "first line of defense" role in the clearance of precancerous lesions and early tumor cells. However, recent studies have found that in the early evolution of liver cancer, the sustained immune pressure mediated by NK cells not only can clear sensitive tumor cells, but also can directly drive and select a subpopulation of tumor cells with resistance to NK cell killing and more invasive. This NK cell-driven evolution of liver cancer cells often exhibits stronger stem cell characteristics, metabolic reprogramming, and potential resistance to subsequent treatment. This process truly simulates the trajectory of some liver cancer patients from early lesions to invasive and refractory cancer in the clinic.

[0004] However, at present, there is a serious lack of liver cancer cell strain models that can stably simulate and represent this "evolutionary" phenotype after NK cell immune editing in the fields of scientific research and drug screening. This lack makes it difficult for researchers to accurately study the specific molecular mechanisms of NK cell-driven tumor evolution in vitro and in vivo, and to screen new drugs that can overcome such immune escape and drug resistance.

[0005] Therefore, the establishment of a liver cancer cell strain model that can stably resist NK cell killing and has highly malignant biological behavior such as rapid metastasis not only fills the gap in existing tool models, but also provides a powerful and highly clinically relevant research platform for revealing the immune escape mechanism of liver cancer, studying the relationship between metabolic reprogramming and tumor evolution, and developing precise treatment options for "post-evolution" advanced liver cancer. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application proposes a cell strain resistant to NK cell killing and a construction method and application thereof, which fills the gap in liver cancer cell strain models with highly malignant biological behavior, and provides a powerful and highly clinically relevant research platform for revealing the immune escape mechanism of liver cancer, studying the relationship between metabolic reprogramming and tumor evolution, and developing precise treatment options for advanced liver cancer.

[0007] The application provides a cell strain resistant to NK cell killing, which is named human hepatocarcinoma cell HepG2-NK-R Homo sapiens, and has been preserved in the China Center for Type Culture Collection (CCTCC) on November 5, 2025, with a preservation number of CCTCC NO: C2025298 and a preservation address of China. Wuhan. Wuhan University, postal code: 430072.

[0008] The application also provides a descendant cell of the cell strain resistant to NK cell killing.

[0009] A construction method of a cell strain resistant to NK cell killing, which comprises the following steps: Step 1, co-culturing wild-type hepatocarcinoma cell HepG2 cells with NK92MI cells.

[0010] Step 2, after the co-culturing is completed, the survived tumor cells are collected, and then the tumor cells are recovered and proliferated in fresh complete culture medium until the cell confluence reaches a target.

[0011] Step 3, the survived cells after proliferation are subcultured, the subcultured cells are co-cultured with NK92MI cells and returned to step 2 to enter a new round of stimulation and recovery, and the process is repeated for multiple times, so that the cell strain resistant to NK cell killing is obtained.

[0012] Preferably, the process of stimulation and recovery is repeated for more than 20 times.

[0013] The application also provides an application of the cell strain resistant to NK cell killing, which comprises the following steps: a. used for the research on the occurrence, development and metastasis of hepatocarcinoma.

[0014] Preferably, the hepatocarcinoma is hepatocellular carcinoma.

[0015] b. used for preparing a tumor cell model or a tumor animal model.

[0016] Preferably, the tumor animal model comprises an animal hepatocarcinoma subcutaneous ectopic tumor model and a lung metastasis model.

[0017] Preferably, the hepatocarcinoma subcutaneous ectopic tumor model is obtained by inoculating a certain amount of the cell strain resistant to NK cell killing into the subcutis of an immunodeficient mouse.

[0018] Preferably, the hepatocarcinoma metastasis model is obtained by injecting a certain amount of the cell strain resistant to NK cell killing through the tail vein of a mouse, and after 8 weeks.

[0019] The present invention has the following beneficial effects: 1. For the first time, a stable, heritable cell line resistant to NK cell killing was presented, realistically simulating the key evolutionary process of liver cancer under immune pressure. This cell line, through in vitro simulation of long-term NK cell-mediated immune editing, achieved a stable "evolved" biological phenotype, successfully replicating the process of tumor cells malignantly enhancing to evade immune surveillance and providing strong experimental validation for related clinical evolution theories. As a direct manifestation of this evolutionary process, this cell line exhibits significantly enhanced malignant biological behavior compared to its parent cells, with significantly improved tumorigenicity, in vivo proliferation rate, and tumorigenesis and metastasis rates in subcutaneous, orthotopic, and lung metastasis models. This significantly shortened the experimental period and highly replicated the invasive characteristics of the evolved tumor.

[0020] 2. The aforementioned NK cell-resistant cell lines are not only key research tools for in-depth analysis of how liver cancer cells achieve immune escape through metabolic reprogramming and stemness enhancement, but also ideal models for specifically screening and evaluating novel therapies aimed at overcoming immune escape and treatment resistance. They can provide prospective guidance for clinical drug strategies for advanced and refractory liver cancer, and provide a precise platform for developing novel drugs that can reverse immune escape, possessing both outstanding and far-reaching scientific research value and clinical translation potential. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the construction process of NK-resistant cell lines.

[0022] Figure 2 NK cell-mediated cytotoxicity in WT and NK-R cell lines.

[0023] Figure 3 The difference in tumor stemness between WT cell lines and NK-R cell lines.

[0024] Figure 4 The electron microscopy results show the morphological differences between WT and NK-R cell lines in contact with NK cells.

[0025] Figure 5 The difference in growth between WT cell line and NK-R cell line in mice.

[0026] Figure 6 The differences in immunohistochemical analysis between WT cell lines and NK-R cell lines after tumor formation were identified.

[0027] Figure 7 The difference in in vitro transfer ability between WT cell lines and NK-R cell lines was investigated. Detailed Implementation

[0028] The present application is further illustrated by the following examples without limiting the present application. The experimental methods in the following examples, unless otherwise specified, are generally carried out according to the conventional conditions.

[0029] Example 1

[0030] This example provides a method for constructing a cell strain resistant to NK cell killing, as shown in the following specific steps: Figure 1 Step 1, select human hepatoma cell line HepG2 as the parent wild-type cell, denoted as WT hepatoma cell.

[0031] Under standard conditions, the WT hepatoma cells are cultured in DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture environment is maintained at 37°C, 5% CO2 constant humidity environment.

[0032] Step 2, use human NK92MI cell line as effector cell to simulate the killing effect in the immune system. Under standard conditions, the human NK92MI cell line is cultured in a special NK medium, and the culture environment is maintained at 37°C, 5% CO2 constant humidity environment.

[0033] Step 3, the logarithmic growth phase of WT hepatoma cells and NK92MI cells are used as target cells and effector cells respectively, and co-cultured in a new culture system. In order to exert effective selection pressure without complete elimination, the effector-target ratio (E:T ratio) is (1~10):1, and the specific effector-target ratio value is determined by pre-experiment of series gradient test to ensure that about 30% of WT hepatoma cells survive after co-culture incubation for 24 hours, that is, the killing rate is 70%.

[0034] Step 4, after co-culturing for 24 hours, the supernatant culture medium is aspirated and gently washed with sterile PBS buffer to selectively remove most of the NK92MI cells, and then the remaining adherent surviving WT hepatoma cells are collected. The surviving WT hepatoma cells are transferred to a culture dish containing fresh complete medium for 48~72 hours of recovery proliferation until the cell confluence reaches about 80%.

[0035] Step 5, the recovered and proliferated cells are subjected to conventional trypsin digestion, passaging, and returned to step 3 for a new round of co-culture induction, forming a stimulation-recovery cycle, which is repeated for more than 20 times. After a series of long-term continuous stimulation and directional screening, tumor cells showing stable and significant resistance to NK92MI-mediated cytotoxicity are obtained, that is, the cell strain resistant to NK cell killing, denoted as NK-R cells.

[0036] Comparative Example 1 ​To ensure that the NK-R cell phenotype obtained in Example 1 is derived from specific immune pressure of NK cells rather than a population bottleneck effect caused simply by a sudden decrease in cell number, a parallel control experiment was performed: human hepatocellular carcinoma cell line HepG2 was cultured alone, and when the cells reached confluence, about 70% of the cell population was randomly removed, and only the remaining 30% of the cells were subcultured to simulate the survival rate of cells after each round of screening in Example 1. The remaining cells were designated as WT cells.

[0037] Test Example 1 To verify the stability of the NK-R cell phenotype, the NK-R cells obtained in Example 1 and the WT cells obtained in Comparative Example 1 were cultured continuously for more than 10 generations in conventional culture medium without any NK cells, and then co-cultured with NK cells at an effector-to-target ratio of 0:1, 2:1, 5:1, 10:1, and standard cell killing experiments were performed, as shown in Figure 2 Without continuous NK cell contact, the NK-R cells of the progeny still efficiently maintained their resistant phenotype against NK cell killing, proving that a stably heritable cell model that simulates tumor immune escape has been successfully constructed. The WT cells of the progeny still stably maintained high killing sensitivity in the in vitro NK cell-mediated killing system.

[0038] Test Example 2 To verify the stemness characteristics and self-renewal ability of the NK-R cells, in vitro sphere formation experiments were performed on the NK-R cells obtained in Example 1 and the WT cells obtained in Comparative Example 1.

[0039] The target cell strain in the logarithmic growth phase was collected after digestion and centrifugation, and serum-free stem cell sphere culture medium was prepared. An ultra-low adsorption surface 96-well round-bottom culture plate was selected, and then cell seeding and culture were performed. The collected target cell pellet was resuspended in the above-mentioned sphere culture medium, filtered through a 200-mesh cell sieve to remove cell clumps, and then counted using a hemocytometer. The cell concentration was adjusted to 5×10 2 μL of cell suspension was added to each well of the ultra-low adsorption 96-well plate, and 3-5 biological replicate wells were set up for each group. The inoculated culture plate was then placed in a constant-temperature incubator at 37°C, 5% CO2, and 95% humidity for static culture. The culture period was 7-14 days, during which the culture plate was avoided from shaking to prevent cell sphere breakage. Cell sphere formation was observed every 2 days through an inverted microscope, and fresh sphere culture medium was supplemented at 50% volume when the culture time exceeded 7 days. At 3 days, 7 days, and 14 days of culture, the morphology, size, and integrity of the cell spheres in each well were observed using an inverted phase contrast microscope (100x or 200x objective lens), and 3 different fields of view in each well were selected for photography. Figure 3As shown, the increase in cell sphere volume directly reflects the enhancement of cell proliferation aggregation ability and self-renewal activity, indicating that the NK-R cell strain is significantly stronger than the wild strain of the same source, i.e., the WT cell, in the level of stemness ability.

[0040] Test Example 3 The NK-R cells and WT cells were inoculated on glass coverslips at a density of 5 x 10 4 cells / well, respectively. Then, the NK92MI cells were added to the wells at an effector-to-target ratio of 2:1, and co-incubated at 37°C for 1 hour. Then, the samples were washed with 0.1 M phosphate buffer, pH 7.4, and fixed in 2.5% glutaraldehyde solution at 4°C overnight. The next day, the samples were fixed again in 1% osmium tetroxide solution for 1.5 hours, and washed with PBS for 3 times. Subsequently, the samples were dehydrated using 50%, 70%, 90% and 100% ethanol, respectively, and dried using a CPD 300 critical point dryer. Finally, the dried samples were sputter-coated with platinum-palladium alloy at an 8 mA current for 60 seconds using a Quorum Q150T ES plus high-vacuum ion sputter coater, and observed using a Nova Nano450 scanning electron microscope at an accelerating voltage of 5 kilovolts, as shown in Figure 4 As shown, the morphology of the NK-R cells and WT cells changed, such as an increase in cell membrane blebbing and an epithelial-mesenchymal transition morphology, and the number of immunological synapses formed between the NK-R cells and NK cells significantly decreased.

[0041] Test Example 4 NCG mice aged 4 to 6 weeks and weighing about 18-20 grams were selected, and were adaptively fed in a SPF level barrier environment for at least one week, during which a standard light cycle was maintained and the mice were freely provided with feed and drinking water. A mouse subcutaneous xenograft model was constructed, and the specific steps were as follows: Step 1, logarithmically growing NK-R cells and WT cells were collected after being trypsinized. The cell pellets were washed twice with serum-free medium, and the cells were counted using a hemocytometer, and the cell concentration was adjusted to 4 x 10 5 cells / mL.

[0042] Step 2, the cells were resuspended in PBS to prepare a single-cell suspension without visible aggregation, and the concentration was accurately adjusted to 2 x 10 7 cells / mL.

[0043] Step 3, the mice were properly fixed using a specially designed fixer, and 100 microliters of cell suspension containing 2 x 10 6 cells was slowly and uniformly injected into the lower limbs of the NCG mice, and the mice were closely observed for liquid extravasation to ensure that all the cells entered.

[0044] Step 4, after injection, the mice were put back to the original cage, and the general conditions such as body weight, behavior, food intake and respiratory status were observed daily. From the first week after injection, the subcutaneous tumors were observed and detected every week.

[0045] Step 5, after 8 weeks or when the mice showed signs meeting the humane endpoint criteria, the mice were euthanized by carbon dioxide inhalation and quickly dissected. The results are shown in Figure 5 As shown in the results, the tumor volume of NK-R cells was much larger than that of WT cells.

[0046] Test Example 5 To evaluate the angiogenesis of tumor tissue, CD31 (PECAM-1) was used as a specific marker for vascular endothelial cells to perform immunohistochemical analysis on mouse transplanted tumor tissue. After routine fixation, embedding and sectioning, antigen detection and signal color development were performed according to the standard immunohistochemical process. Specific primary antibodies against CD31 were used for labeling, and negative controls were set to verify the specificity.

[0047] Step 1, after the subcutaneous tumors formed by NK-R cells and WT cells in NCG mice were separated, they were immediately fixed with 4% paraformaldehyde solution for 24 hours, then deparaffinized in xylene, and then dehydrated, paraffin-embedded and sectioned with gradient alcohol (100%, 95%, 90%, 85%).

[0048] Step 2, use citric acid buffer with pH 6.0, water bath 100℃ for 15 minutes for antigen repair.

[0049] Step 3, use 3% hydrogen peroxide to treat the section, incubate at room temperature for 10 minutes to inactivate endogenous enzyme activity.

[0050] Step 4, use 10% goat serum to block at room temperature for 30 minutes.

[0051] Step 5, add CD31 primary antibody dropwise, incubate overnight at 4℃, then wash the section with PBS buffer.

[0052] Step 6, add HRP-labeled secondary antibody dropwise, incubate at room temperature for 30 minutes, and wash the section.

[0053] Step 7, use DAB color developing solution for color development. The results are shown in Figure 6 As shown in the results, the expression of CD31 in the tumor tissue section of the NK-R group was significantly higher than that of the WT cell group, indicating that there was a significant difference in the angiogenesis ability of the two cells.

[0054] Test Example 6 Step 1: Observe the growth status of NK-R cells and WT cells in the logarithmic growth phase. Take cells with good growth status, remove them from the culture medium, and add serum-free culture medium to starve them for 24 hours.

[0055] Step 2: Add 500µl of complete culture medium containing 10% FBS to the lower chamber of a 24-well plate. Use tweezers to place the Transwell chamber into the 24-well plate, ensuring that the connection between the wells is tight and no air bubbles are generated.

[0056] Step 3: Collect the cells after digestion with trypsin. Wash the cell pellet twice with serum-free culture medium, count the cells using a hemocytometer, and adjust the cell concentration to 4 x 10⁻⁶. 5 The cells / mL were evenly distributed on the filter membrane. The 24-well plate was then incubated at 37°C, 5% CO2, and 90% humidity for 24–48 hours.

[0057] Step 4: After migration is complete, remove the Transwell plate, wipe the cells in the upper wells with a cotton swab, and then wash the cells in the lower wells with PBS or cell culture medium.

[0058] Step 5: Remove the cells from the lower well and centrifuge to collect the cell pellet. After removing the supernatant, wash the cell pellet with PBS to remove residual culture medium. Then fix the cells with 4% paraformaldehyde to allow them to adhere to the slide.

[0059] Step 6: The fixed cells can be stained with crystal violet for observation and counting. Results are as follows... Figure 7 As shown, the migration ability of NK-R group cells was significantly stronger than that of WT group cells.

Claims

1. A cell line resistant to NK cell killing, named HepG2-NK-R Homosapiens, was deposited at the China Center for Type Culture Collection on November 5, 2025, with accession number CCTCC NO:C2025298.

2. A progeny cell of the NK cell-resistant cell line of claim 1.

3. A method for constructing a cell line resistant to NK cell killing, characterized in that: By co-culturing the human hepatocellular carcinoma cell line HepG2 with NK cells for an extended period, a cell line resistant to NK cell killing as described in claim 1 or 2 was obtained.

4. The method for constructing a cell line resistant to NK cell killing as described in claim 3, characterized in that: The specific steps are as follows: Step 1: Co-culture wild-type liver cancer cells HepG2 cells with NK92MI cells; Step 2: After co-culture, collect the surviving tumor cells and then allow them to recover and proliferate in fresh complete culture medium until the cell confluence reaches the target. Step 3: Passage the proliferated surviving cells, co-culture the passaged cells with NK92MI cells and return to step 2 for a new round of stimulation and recovery. Repeat this process multiple times to obtain the cell line resistant to NK cell killing.

5. The method for constructing a cell line resistant to NK cell killing as described in claim 4, characterized in that: The stimulation and recovery process is repeated more than 20 times.

6. The application of the NK cell-resistant cell line as described in claim 1 or 2, characterized in that: Used for research on the occurrence, development and metastasis of liver cancer, or for the preparation of tumor cell models or tumor animal models.

7. The application of the NK cell-resistant cell line as described in claim 6, characterized in that: The liver cancer mentioned is hepatocellular carcinoma.

8. The application of the NK cell-resistant cell line as described in claim 6, characterized in that: The tumor animal models include animal liver cancer subcutaneous heterotopic xenograft tumor models and lung metastasis models.

9. The application of the NK cell-resistant cell line as described in claim 6, characterized in that: A subcutaneous heterotopic liver cancer xenograft model was obtained by subcutaneously inoculating the NK cell-resistant cell line into immunodeficient mice.

10. The application of the NK cell-resistant cell line as described in claim 6, characterized in that: A liver cancer metastasis model was obtained by injecting the NK cell-resistant cell line into the tail vein of a mouse after a period of time.

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