NK cell-killing-resistant cell strain, construction method and application thereof

Abstract

This invention discloses a cell line resistant to NK cell killing, its construction method, and its applications. The NK cell line, named HepG2-NK-R Homo sapiens (human hepatocellular carcinoma cells), was deposited at the China Center for Type Culture Collection (CCTCC) on November 5, 2025, with accession number CCTCC NO: C2025298. It was obtained by co-culturing the HepG2 human hepatocellular carcinoma cell line with NK cells for an extended period. This cell line exhibits a stable biological phenotype by mimicking long-term NK cell-mediated immune editing in vitro. Compared to parental cells, it displays significantly enhanced malignant biological behavior and can be used to study the occurrence, development, and metastasis of liver cancer, or to prepare tumor cell models or tumor animal models. Its tumorigenicity, in vivo proliferation rate, and metastasis rate are all significantly improved, which can significantly shorten the experimental cycle and highly replicate the invasive characteristics of evolved tumors.

Description

Technical Field

[0001] This invention belongs to the fields of biology and oncology, specifically relating to a cell line resistant to NK cell killing, its construction method, and its application. Background Technology

[0002] In recent years, the concept of tumor evolution has provided a novel perspective for understanding the occurrence, development, metastasis, and drug resistance of cancer. Research indicates that tumor cells are not static but rather dynamic populations that constantly evolve under internal and external environmental pressures. The surveillance and clearance functions of the immune system are considered a key intrinsic force driving tumor evolution.

[0003] In the liver's immune microenvironment, natural killer (NK) cells, as important innate immune cells, are abundant and play a "first line of defense" role in clearing precancerous lesions and early-stage tumor cells. However, recent research has found that in the early evolution of liver cancer, the persistent immune pressure mediated by NK cells not only eliminates sensitive tumor cells but also acts as a selective pressure, directly driving and screening out a more aggressive subset of tumor cells with NK cell-resistant killing abilities. These NK cell-driven liver cancer cells typically exhibit stronger stem cell characteristics, metabolic reprogramming, and potential resistance to subsequent treatments. This process realistically mimics the trajectory of some liver cancer patients in clinical practice as they progress from early-stage lesions to aggressive, refractory cancer.

[0004] However, in the fields of scientific research and drug screening, there is a severe lack of liver cancer cell line models that can stably mimic and represent this "evolutionary" phenotype after NK cell immune editing. This deficiency makes it difficult for researchers to accurately study the specific molecular mechanisms by which NK cells drive tumor evolution in vitro and in vivo, and also makes it impossible to specifically screen for novel drugs that can overcome such immune evasion and drug resistance.

[0005] Therefore, establishing a liver cancer cell line model that can stably resist NK cell killing and exhibit highly malignant biological behaviors such as rapid metastasis can not only fill the gap in existing tools and models, but also provide 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 precision treatment options for "evolved" advanced liver cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a cell line resistant to NK cell killing, its construction method, and its applications. This fills the gap in liver cancer cell line models with highly malignant biological behavior, providing a powerful and clinically relevant research platform for revealing the immune escape mechanism of liver cancer, studying the relationship between metabolic reprogramming and tumor evolution, and developing precision treatment strategies for advanced liver cancer.

[0007] This invention provides a cell line resistant to NK cell killing, named HepG2-NK-RHomo sapiens, a human hepatocellular carcinoma cell line. It was deposited on November 5, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C2025298, at Wuhan University, Wuhan, China, 430072, China.

[0008] The present invention also provides progeny cells of the NK cell-resistant cell line described above.

[0009] A method for constructing a cell line resistant to NK cell killing involves co-culturing a human hepatocellular carcinoma cell line, HepG2, with NK cells for an extended period to obtain the NK cell line resistant to NK cell killing. The specific steps are as follows:

[0010] Step 1: Co-culture wild-type liver cancer cells HepG2 cells with NK92MI cells.

[0011] Step 2: After co-culture, collect the surviving tumor cells and allow them to recover and proliferate in fresh complete culture medium until the cell confluence reaches the target.

[0012] 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.

[0013] Preferably, the stimulation and recovery process is repeated more than 20 times.

[0014] This invention also provides applications of the NK cell-resistant cell lines described above, including:

[0015] a. Used for research on the occurrence, development, and metastasis of liver cancer.

[0016] Preferably, the liver cancer is hepatocellular carcinoma.

[0017] b. Prepare tumor cell models or prepare tumor animal models.

[0018] Preferably, the tumor animal model includes an animal liver cancer subcutaneous ectopic xenograft model and a lung metastasis model.

[0019] Preferably, a subcutaneous heterotopic liver cancer xenograft model is obtained by subcutaneously inoculating a certain number of the NK cell-resistant cell lines into immunodeficient mice.

[0020] Preferably, a liver cancer metastasis model is obtained by injecting a certain number of the NK cell-resistant cell lines into the tail vein of a mouse after 8 weeks.

[0021] The present invention has the following beneficial effects:

[0022] 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.

[0023] 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

[0024] Figure 1 This is a flowchart illustrating the construction process of NK-resistant cell lines.

[0025] Figure 2 NK cell-mediated cytotoxicity in WT and NK-R cell lines.

[0026] Figure 3 The difference in tumor stemness between WT cell lines and NK-R cell lines.

[0027] Figure 4 The electron microscopy results show the morphological differences between WT and NK-R cell lines in contact with NK cells.

[0028] Figure 5 The difference in growth between WT cell line and NK-R cell line in mice.

[0029] Figure 6 The differences in immunohistochemical analysis between WT cell lines and NK-R cell lines after tumor formation were identified.

[0030] Figure 7The difference in in vitro transfer ability between WT cell lines and NK-R cell lines was investigated. Detailed Implementation

[0031] The present invention will be further illustrated by the following examples, but the invention is not limited thereto. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions.

[0032] Example 1

[0033] This embodiment provides a method for constructing a cell line resistant to NK cell killing, such as... Figure 1 As shown, the specific steps are as follows:

[0034] Step 1: Select the human hepatocellular carcinoma cell line HepG2 as the parent wild-type cell, denoted as WT hepatocellular carcinoma cell.

[0035] WT hepatocellular carcinoma cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin under standard conditions. The culture environment was maintained at a constant humidity of 37°C and 5% CO2.

[0036] Step 2: Human NK92MI cell line was used as effector cells to simulate the killing effect in the immune system. Under standard conditions, the human NK92MI cell line was cultured in a medium supplemented with specific NK cells, and the culture environment was maintained at a constant humidity of 37°C and 5% CO2.

[0037] Step 3: Logarithmic growth phase WT hepatocellular carcinoma cells and NK92MI cells were co-cultured in a new culture system as target cells and effector cells, respectively. To exert effective selection pressure rather than complete elimination, the effector-to-target ratio (E:T ratio) was (1~10):1. The specific effector-to-target ratio was determined based on a series of gradient tests in preliminary experiments to ensure that approximately 30% of WT hepatocellular carcinoma cells survived after 24 hours of co-culture incubation, i.e., a killing rate of 70%.

[0038] Step 4: After co-culturing for 24 hours, the supernatant culture medium was aspirated and the cells were gently washed with sterile PBS buffer to selectively remove most of the NK92MI cells. The remaining adherent surviving WT hepatocellular carcinoma cells were then collected. The surviving WT hepatocellular carcinoma cells were transferred to culture dishes containing fresh complete culture medium and allowed to undergo restorative proliferation for 48–72 hours until the cell confluence reached approximately 80%.

[0039] Step 5: After recovery and proliferation, the cells are routinely digested with trypsin and passaged, and then returned to step 3 for a new round of co-culture induction, forming a stimulation-recovery cycle, which is repeated more than 20 times. After this series of long-term continuous stimulation and targeted screening, tumor cells that show stable and significant resistance to NK92MI-mediated cytotoxicity are obtained, namely the NK cell-resistant cell line, denoted as NK-R cells.

[0040] Comparative Example 1

[0041] To ensure that the NK-R cell phenotype obtained in Example 1 stemmed from specific immune pressure from NK cells, rather than a population bottleneck effect caused solely by a sharp decrease in cell numbers, a parallel control group experiment was conducted in this comparative example: the human hepatocellular carcinoma cell line HepG2 was cultured separately, and after the cells were pooled, approximately 70% of the cell population was randomly removed, with only the remaining 30% being passaged to simulate the cell survival rate after each round of selection in Example 1. This was repeated more than 20 times, and the remaining cells were designated as WT cells.

[0042] Test Example 1

[0043] 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. Then, they were co-cultured with NK cells at effector-target ratios of 0:1, 2:1, 5:1, and 10:1, and standard cell killing experiments were performed. The results are as follows: Figure 2 As shown, even without continuous NK cell contact, the progeny NK-R cells still efficiently maintained their resistance phenotype to NK cell killing, demonstrating the successful construction of a stably heritable cell model that mimics tumor immune escape. Furthermore, the progeny WT cells maintained a high level of killing sensitivity in an in vitro NK cell-mediated killing system.

[0044] Test Example 2

[0045] To verify the stem cell characteristics and self-renewal capacity of NK-R cells, in vitro spheroidization experiments were conducted on NK-R cells obtained in Example 1 and WT cells obtained in Comparative Example 1.

[0046] Target cell lines in the logarithmic growth phase were collected, digested, centrifuged, and the cell pellet was collected. Serum-free stem cell spheroidizing medium was prepared, and a 96-well round-bottom plate with ultra-low adsorption surface was used for cell seeding and culture. The collected target cell pellet was resuspended in the aforementioned spheroidizing medium, filtered through a 200-mesh cell sieve to remove cell clumps, and then counted using a hemocytometer to adjust the cell concentration to 5 × 10⁻⁶. 2Cells / mL were added to each well of a 96-well ultra-low adsorption plate, with 100 μL of cell suspension. Each group had 3-5 biological replicate wells. The inoculated plates were then statically cultured in a 37°C, 5% CO2, 95% humidity incubator for 7-14 days. During this period, the plates were kept still to prevent cell breakage. Cell formation was observed every 2 days using an inverted microscope. If culture exceeded 7 days, 50% of the fresh cell-forming medium could be added. On days 3, 7, and 14, the morphology, size, and integrity of the cell spheroids in each well were observed using an inverted phase-contrast microscope (100× or 200× objective lens). Three different fields of view were photographed from each well for recording. Figure 3 As shown, the increase in cell spheroid volume directly reflects the enhanced cell proliferation and aggregation capacity and self-renewal activity, indicating that the NK-R cell line is significantly stronger than the wild-type WT cell line of the same origin in terms of stemness.

[0047] Test Example 3

[0048] NK-R cells and WT cells were respectively loaded with 5×10 4 Cells were seeded at a density of 100 cells / well on glass coverslips. Then, 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. The samples were then washed with 0.1 M, pH 7.4 phosphate-buffered saline and fixed overnight in 2.5% glutaraldehyde solution at 4°C. The next day, the samples were refixed with 1% osmium tetroxide solution for 1.5 hours and washed three times with PBS. Subsequently, the samples were dehydrated sequentially with 50%, 70%, 90%, and 100% ethanol, and then dried using a CPD 300 critical point desiccator. Finally, the dried samples were sputtered with a platinum-palladium alloy for 60 seconds at 8 mA using a Quorum Q150T ES plus high-vacuum ion sputtering system, and observed using a Nova Nano450 scanning electron microscope at an accelerating voltage of 5 kV. Figure 4 As shown, the morphology of NK-R cells and WT cells changed, such as increased cell membrane vesicle exudation, exhibiting an epithelial-mesenchymal transition morphology, while the number of immune synapses formed between NK-R cells and NK cells was significantly reduced.

[0049] Test Example 4

[0050] NCG mice aged 4 to 6 weeks and weighing approximately 18 to 20 grams were selected and acclimatized in an SPF-grade barrier environment for at least one week, maintaining a standard light cycle and providing free access to food and water. A subcutaneous xenograft model was established in mice, and the specific steps are as follows:

[0051] Step 1: Collect NK-R cells and WT cells in logarithmic growth phase after trypsin digestion. 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 per mL.

[0052] Step 2: Resuspend the cells in PBS to prepare a single-cell suspension without visible aggregation, and precisely adjust its concentration to 2 × 10⁻⁶. 7 per milliliter.

[0053] Step 3: Secure the mouse properly using a special fixator, and draw 100 μL of solution containing 2 × 10⁻⁶ mg / L. 6 A cell suspension containing 100 cells was slowly and evenly injected into one side of the lower limb of an NCG mouse, and the patient was closely observed for any extravasation of fluid to ensure that all cells were injected.

[0054] Step 4: After the injection, return the mice to their original cages and closely observe their weight, behavior, feeding and breathing status daily. Starting from the first week after the injection, observe and test the subcutaneous tumors weekly.

[0055] Step 5, 8 weeks later, or when the mice exhibited signs meeting the humanitarian endpoint criteria, euthanized them using carbon dioxide inhalation and promptly dissected them. Results are as follows: Figure 5 As shown, the tumor volume of NK-R cells is much larger than that of WT cells.

[0056] Test Example 5

[0057] To assess angiogenesis in tumor tissues, CD31 (PECAM-1) was used as a specific marker for vascular endothelial cells for immunohistochemical analysis of mouse xenograft tissues. After routine fixation, embedding, and sectioning, antigen detection and signal development were performed according to standard immunohistochemical procedures. A specific primary antibody against CD31 was used for labeling, and a negative control was included to verify specificity.

[0058] Step 1: After separating NK-R cells and WT cells from subcutaneous tumors formed in NCG mice, the tissues were immediately fixed with 4% paraformaldehyde solution for 24 hours. Subsequently, the tissues were dewaxed in xylene, then dehydrated with a gradient of alcohols (100%, 95%, 90%, 85%), embedded in paraffin, and sectioned.

[0059] Step 2: Use citrate buffer solution with pH 6.0 and heat in a water bath at 100°C for 15 minutes to perform antigen retrieval.

[0060] Step 3: Treat the slices with 3% hydrogen peroxide and incubate at room temperature for 10 minutes to inactivate endogenous enzyme activity.

[0061] Step 4: Block with 10% goat serum at room temperature for 30 minutes.

[0062] Step 5: Add CD31 primary antibody, incubate overnight at 4°C, and then wash the slides with PBS buffer.

[0063] Step 6: Add HRP-labeled secondary antibody, incubate at room temperature for 30 minutes, and then wash the slide.

[0064] Step 7: Develop the color using DAB developer. The results are as follows: Figure 6 As shown, the expression level of CD31 in tumor tissue sections of the NK-R group was significantly higher than that of the WT cell group, indicating a significant difference in the angiogenesis capacity of the two cell types.

[0065] Test Example 6

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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. The application of the NK cell-resistant cell line as described in claim 1, characterized in that: Used to prepare liver cancer tumor cell models or liver cancer animal models.

4. The application of the NK cell-resistant cell line as described in claim 3, characterized in that: The liver cancer mentioned is hepatocellular carcinoma.

5. The application of the NK cell-resistant cell line as described in claim 3, characterized in that: The tumor animal models include animal liver cancer subcutaneous heterotopic xenograft tumor models and lung metastasis models.

6. The application of the NK cell-resistant cell line as described in claim 5, characterized in that: A subcutaneous heterotopic liver cancer xenograft model was obtained by subcutaneously inoculating the NK cell-resistant cell line into immunodeficient mice.

7. The application of the NK cell-resistant cell line as described in claim 5, 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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