Mouse colon cancer immunotherapy drug-resistant cell line as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a drug-resistant cell line MC38-IR for immunotherapy of mouse colon cancer as well as a preparation method and application of the drug-resistant cell line MC38-IR. The cell line is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025358. Experimental results show that the MC38-IR cell line has a remarkable PD-1 antibody drug resistance characteristic while maintaining the in-vitro proliferation and migration capabilities of parent cells. An in-vivo experiment shows that the tumor volume of a mouse inoculated with the cell line is not obviously inhibited after PD-1 antibody treatment, the lifetime has no statistical difference compared with that of a control group, and the parent MC38 cell shows obvious treatment response. The invention solves the problem of lack of colon cancer immunotherapy drug resistance models in the prior art, provides an important tool for researching drug resistance mechanisms, screening reverse drug resistance drugs and developing combined treatment strategies, and has higher scientific research and clinical application values.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a mouse colon cancer immunotherapy resistant cell line, its preparation method and application, specifically applicable to the mouse colon cancer cell line MC38 with a C57BL / 6 mouse background. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, with consistently high incidence and mortality rates. According to the World Health Organization, colorectal cancer ranks third in cancer incidence among men and second among women, with over 1.9 million new cases and approximately 930,000 deaths annually. Advanced colorectal cancer patients often experience distant metastasis, such as liver metastasis, and traditional treatments like surgery, chemotherapy, and radiotherapy have limited effectiveness, resulting in a 5-year survival rate of less than 15%. In recent years, the emergence of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, especially antibody drugs targeting programmed death receptor-1 (PD-1) and its ligand PD-L1, which have shown significant efficacy in various solid tumors. For example, pembrolizumab and nivolumab have been approved for patients with microsatellite instability-high (MSI-H) or mismatch repair deficient advanced colorectal cancer, achieving objective response rates of 30-50%. These drugs relieve T-cell immunosuppression by blocking the PD-1 / PD-L1 pathway, thereby activating the body's anti-tumor immune response and bringing long-term survival benefits to some patients.

[0003] The mouse colon cancer cell line MC38 is a commonly used syngeneic model in the C57BL / 6 background. Due to its strong immunogenicity and ability to mimic the human tumor microenvironment, it is widely used to evaluate the efficacy of immunotherapy and drug resistance mechanisms. In basic research, by constructing mouse subcutaneous tumor or liver metastasis models, the process of patients receiving PD-1 antibody therapy can be simulated, thereby exploring the biological basis of drug resistance. Studies have shown that the MC38 model is sensitive to PD-1 monoclonal antibodies, and tumor shrinkage or complete remission can be observed after treatment, providing a platform for understanding immune activation mechanisms. Furthermore, cell line models play an irreplaceable role in drug screening, enabling high-throughput evaluation of the effects of candidate compounds and revealing the roles of signaling pathways (such as IFN-γ and WNT / β-catenin) in drug resistance.

[0004] However, existing technologies have significant limitations and problems. First, there is a severe lack of immunotherapy resistance models for colorectal cancer. Although the MC38 cell line is widely used, publicly available PD-1 resistance-derived cell lines are almost nonexistent. This leads to research relying heavily on patient-derived xenograft models or in vitro induction methods. The former is costly, time-consuming, and suffers from imperfect humanized mouse immune systems; the latter struggles to simulate dynamic interactions within the tumor microenvironment, such as T cell exhaustion or stromal cell influences, resulting in incomplete research on resistance mechanisms. Second, existing resistance models often focus on single mechanisms, such as gene mutations or pathway abnormalities, while colorectal cancer resistance often involves multiple synergistic factors, such as regulatory T cell infiltration or metabolic reprogramming in the microenvironment. Existing models cannot systematically integrate these elements, reducing their clinical predictive value. Furthermore, resource accessibility is poor. Commercial cell banks lack standardized resistance cell lines, requiring researchers to construct their own, but this involves inconsistent methods, low success rates, and a lack of cross-cancer applicability validation. These problems constrain the depth of resistance research and the efficiency of drug development, necessitating a reliable and reproducible tool for identifying colorectal cancer-specific resistance.

[0005] Objectively speaking, existing technologies have failed to address shortcomings such as insufficient standardization of colorectal cancer immune resistance models, poor in vivo realism, and narrow mechanism coverage. This invention aims to solve these problems by establishing an MC38-derived PD-1 resistant cell line, providing a reliable tool for elucidating the mechanisms of colorectal cancer immune resistance, screening drug targets to reverse resistance, and developing personalized treatment strategies. Summary of the Invention

[0006] The purpose of this invention is to provide a colorectal cancer immunotherapy-resistant cell line, its preparation method, and its application, to address the problem of the lack of colorectal cancer immunotherapy-resistant models in the prior art. This invention simulates in vivo immune stress to screen and obtain a stable drug-resistant cell line, MC38-IR, whose in vitro proliferation and migration abilities remain unchanged, but which exhibits significant tolerance to PD-1 antibody therapy in vivo.

[0007] The technical solution of this invention is as follows: The first objective of this invention is to provide a mouse colon cancer immunotherapy resistant cell line MC38-IR, which is deposited at the China Center for Type Culture Collection on December 2, 2025, with accession number CCTCC NO: C2025358.

[0008] A second objective of this invention is to provide a method for preparing the aforementioned mouse colon cancer immunotherapy resistant cell line MC38-IR, comprising the following steps: (1) The puromycin resistance gene was stably integrated through lentiviral transduction; (2) A C57BL / 6 mouse subcutaneous tumor model was constructed using the colon cancer cell line MC38, and the mice were treated with immune checkpoint PD-1 antibody. The largest tumors after treatment were selected. (3) The tumor tissue obtained in step (1) was separated under aseptic conditions, cut into pieces, cultured and passaged in DMEM medium containing 10% fetal bovine serum to obtain primary tumor cells. (4) Use the primary cell replica in vivo model obtained in step (2) to screen for PD-1 antibody therapy until the tumor volume of the PD-1 antibody treatment group is not significantly different from that of the control group; (5) The tumor tissue obtained in step (3) was isolated under aseptic conditions, minced and cultured in DMEM medium and stably passaged to obtain the colorectal cancer immunotherapy resistant cell line MC38-IR. Furthermore, in step (1), the colon cancer cell line is a murine colon cancer cell line.

[0009] Furthermore, in step (4), the culture conditions are 37°C and CO2 volume concentration is 5%.

[0010] A third objective of this invention is to provide the application of the aforementioned colorectal cancer immunotherapy resistant cell line MC38-IR in screening anti-colorectal cancer drug targets.

[0011] A fourth objective of this invention is to provide the use of the aforementioned colorectal cancer immunotherapy resistant cell line MC38-IR in screening and / or preparing anti-colorectal cancer drugs.

[0012] A fifth objective of this invention is to provide the application of the aforementioned colorectal cancer immunotherapy resistant cell line MC38-IR in screening and / or preparing diagnostic reagents for evaluating anti-colorectal cancer efficacy.

[0013] Compared with the prior art, the present invention has the following outstanding advantages: (1) Standardized model construction: By simulating the clinical drug resistance evolution process through in vivo screening and introducing screening markers by combining lentivirus transduction technology, a reproducible and standardized colorectal cancer immune drug resistance cell line was established, which solved the problem of lack of consistency in existing models.

[0014] (2) Stable drug resistance characteristics: The MC38-IR cell line maintains a stable PD-1 antibody resistance phenotype in vitro and in vivo, without changing its basic proliferation and cell cycle characteristics, providing a reliable tool for mechanism research.

[0015] (3) Efficient screening process: The use of multiple rounds of in vivo screening and puromycin resistance gene enrichment significantly improves the screening efficiency and purity of drug-resistant cells, overcoming the limitations of traditional in vitro induction methods.

[0016] (4) Wide range of applications: This cell line can be used for high-throughput drug screening, identification of drug resistance-related biomarkers, evaluation of combination therapy strategies and research on tumor microenvironment, promoting innovative development in the field of colorectal cancer immunotherapy.

[0017] This invention not only provides the MC38-IR cell line itself, but also elaborates on its preparation method and verification process, ensuring the reproducibility and practicality of the technical solution, and providing complete technical support for research on immunotherapy resistance in colorectal cancer. Attached Figure Description

[0018] Figure 1 The construction process and validation results of the MC38-IR drug-resistant cell line, including: Figure 1 A shows the flowchart for constructing the MC38-IR drug-resistant cell line.

[0019] Figure 1 B represents the tumor growth curve of mice in the PBS control group.

[0020] Figure 1 C represents the tumor growth curve of mice in the anti-PD-1 treatment group.

[0021] Figure 1 D represents the morphological characteristics of the parental MC38 cells after 24 hours of in vitro culture (200×).

[0022] Figure 1 E represents the morphological characteristics of MC38-IR cells after 24 hours of in vitro culture (200×).

[0023] Figure 2 In vitro functional comparison of MC38-IR cells and parental MC38 cells, including: Figure 2 Image A is a representative image from a cloning experiment.

[0024] Figure 2 B is a bar chart showing the number of clones formed.

[0025] Figure 2 C is a cell cycle distribution map detected by flow cytometry.

[0026] Figure 2 D is a statistical graph showing the proportion of cells in the G2 / M phase.

[0027] Figure 3 . Validation of the in vivo proliferation capacity of MC38-IR cells, including: Figure 3 Image A shows a subcutaneous xenograft tumor in C57BL / 6 mice. The tumor at the top is from the MC38-IR group, and the tumor at the bottom is from the parental MC38 group.

[0028] Figure 3 B represents the dynamic curve of tumor growth.

[0029] Figure 3 C is the endpoint, a statistical bar chart of tumor volume.

[0030] Figure 4 In vivo validation of PD-1 antibody resistance in MC38-IR cells, including: Figure 4 A is the experimental design flowchart.

[0031] Figure 4 B represents the dynamic curves of tumor growth in different treatment groups.

[0032] Figure 4 C is a bar chart showing the tumor volume at the end of day 25.

[0033] Figure 4 D. Bar chart of tumor volume between different treatment groups.

[0034] Figure 4 E represents the survival analysis curve.

[0035] Biological Preservation Instructions: The mouse colon cancer cell line MC38-IR, a drug-resistant cell line, has been deposited at the China Center for Type Culture Collection (CCTCC), classified and named as: Mouse colon cancer cell line MC38-IR Mus musculus, deposited on December 2, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: C2025358. Detailed Implementation

[0036] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms "contains," "includes," "has," "contains," etc., used in this article are all open-ended terms, meaning they include but are not limited to.

[0042] This invention uses murine colon cancer cell lines MC38 and C57BL / 6 mice to construct subcutaneous tumor models. Primary tumor cells are isolated from PD-1 antibody-resistant mouse tumors, cultured, and passaged to obtain the PD-1 antibody-resistant colon cancer cell line MC38-IR, as detailed below: ① Culture colon cancer MC38 cells under sterile conditions, collect tumor cells in the logarithmic growth phase according to cell passage methods, resuspend the cells in sterile 1×PBS and count them, and adjust the cell concentration to 1×10⁻⁶. 7 / mL.

[0043] ②Use a 1mL syringe to inoculate 100μL of cell suspension into the subcutaneous tissue on the back of C57BL / 6 mice to construct a subcutaneous tumor model.

[0044] ③ Seven days after tumor implantation, use vernier calipers to measure the tumor's major diameter a and minor diameter b. The volume V is calculated using the formula: V = major diameter × minor diameter. 2 / 2 Calculate tumor volume. Select a volume of 200±20 mm. 3 Mice within the range were randomly divided into two groups, and were given anti-PD-1 monoclonal antibody or control PBS via intraperitoneal injection, 200 μg / mouse each time, once every 3 days, for a total of 4 times.

[0045] ④ The tumor volume of mice was measured and calculated every 3 days. After 20 days of tumor bearing, the tumor proliferation curve of mice was plotted. The largest mouse tumor in the anti-PD-1 monoclonal antibody treatment group was considered to have PD-1 resistance potential.

[0046] ⑤ Isolate the mouse tumor tissue in a sterile operating table and transfer it to a cell culture dish. Wash the tissue three times with sterile 1×PBS, discard the waste liquid, add 1 ml of DMEM complete medium containing 10% fetal bovine serum, and then cut the tumor tissue into pieces of approximately 1 mm in volume using sterile scissors. 3 Small pieces of the adherent cells were added to 10 ml of DMEM complete medium, and penicillin / streptomycin was added. The cells were then cultured at 37°C in a 5% CO2 incubator for 48 hours. The adherent cells were then passaged and cryopreserved.

[0047] ⑥ Collect tumor cells in the logarithmic growth phase from the primary cells obtained above using cell passage methods, resuspend the cells in sterile 1×PBS and count them, adjusting the cell concentration to 1×10⁻⁶. 7 / mL. Using a 1ml syringe, 100μL of cell suspension was subcutaneously injected into the back of C57BL / 6 mice to re-establish the subcutaneous tumor-bearing model in C57BL / 6 mice. The above screening procedure was repeated 6 times until the tumor size in the PD-1 antibody treatment group was not significantly different from that in the PBS control group.

[0048] ⑦ Isolate the largest tumor tissue from the PD-1 antibody treatment group, wash twice with sterile 1×PBS, discard the waste liquid, add 1ml of DMEM complete culture medium, and then cut the tumor tissue into pieces of approximately 1mm in volume using sterile scissors. 3 Small pieces of the culture medium were added to 10 ml of DMEM complete medium, and penicillin / streptomycin was added. The cells were then cultured and passaged in a 37°C, 5% CO2 incubator. This yielded the stable PD-1 resistant colon cancer cell line MC38-IR.

[0049] ⑧ Subcutaneous tumor models were constructed using the colon cancer cell line MC38 and the colon cancer PD-1 antibody-resistant cell line MC38-IR, respectively. PD-1 antibody treatment was administered and tumor volume changes were detected. The tumors of the colon cancer PD-1 antibody-resistant cell line MC38-IR tumor-bearing mice still grew rapidly after drug treatment, with no significant difference compared to the PBS-treated group, indicating that this mouse colon cancer cell line has PD-1 antibody resistance.

[0050] Example 1: MC38 cell culture MC38 mouse colon cancer cell line (purchased from the American Type Culture Collection, ATCC) was obtained and cultured under sterile conditions. Cell culture was performed in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and passaged in an incubator at 37°C, 5% CO2, and saturated humidity. Cells were digested with 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid) every 2-3 days, washed with PBS, and then checked for viability (≥95%) by trypan blue staining. Only cells in the logarithmic growth phase were used to ensure consistency and reproducibility.

[0051] Example 2 Lentiviral transfection and screening of stable cell lines MC38 cells in logarithmic growth phase with a density of approximately 30%-50% were infected with lentivirus (optimal multiplicity of infection (MOI) of 150). Approximately 48-72 hours after infection, the medium was replaced with a medium containing 2 μg / ml puromycin for pressure selection. The medium was changed every 2-3 days for approximately 7-10 days until the untransduced control group cells died completely. The resulting resistant cell pool was expanded and cultured, and then monocloned using limiting dilution methods. To maintain stable expression of the exogenous gene long-term, 0.5 μg / ml puromycin was added to the standard culture medium for maintenance culture, and re-selection was performed periodically.

[0052] Example 3: Construction of a C57 mouse subcutaneous tumor model Animal models were performed using 6-8 week old female C57BL / 6 mice (specific pathogen-free, SPF grade). These mice were housed in an SPF-grade animal facility with a temperature of 22-25℃, humidity of 40%-60%, and a 12-hour light-dark cycle, with free access to sterilized feed and water. After one week of acclimatization, the hair on the right hind limb of the mice was shaved using a shaver and depilatory cream, and subcutaneous tumor inoculation was performed: logarithmic growth phase MC38 cells were harvested, digested, centrifuged, and resuspended in PBS, adjusting the cell concentration to 1×10⁻⁶. 7 / mL, use a 1mL syringe (27G needle) to draw 100μL of cell suspension (containing 1×10⁻⁶ cells / mL). 6 (1 cell) was injected subcutaneously into the right back of the mouse to a depth of about 2 mm, avoiding major blood vessels.

[0053] Example 4: PD-1 antibody therapy and screening of drug-resistant tumors in C57 mice Mice were observed daily after inoculation. After 7 days, tumor volume was measured using electronic calipers, and mice with tumor volumes of 200±20 mm were selected. 3 Mice were used for experiments. Tumor-bearing mice were randomly divided into two groups: the experimental group received PD-1 antibody treatment (purchased from BioXcell), and the control group received PBS. Administration was intraperitoneal injection at a dose of 200 μg / mouse, administered every 3 days for 4 consecutive days. During treatment, tumor volume was measured every 3 days, and growth curves were recorded. The specific procedure included: establishing a subcutaneous xenograft model by inoculating MC38 cells into C57BL / 6 mice; after 6 cycles of in vivo passage and selection, tumor-bearing mice were treated with anti-PD-1 antibody in each cycle; subsequently, tumor tissue was harvested for in vitro culture, ultimately obtaining a stable passaged anti-PD-1 resistant cell line (MC38-IR) (treatment procedure see...). Figure 1 A).

[0054] The results showed that the tumor volume of mice in all groups continued to increase with the increase of the inoculation days; the tumor volume growth in the treatment group was relatively slow, indicating that anti-PD-1 treatment had a significant inhibitory effect on parental MC38 cells (e.g., Figure 1 (B and C in the text). The treatment endpoint was set at 20 days post-tumor bearing or a tumor volume ≥2000 mm. 3 (Humanitarian endpoint). The selection criteria were: in the PD-1 antibody treatment group, individuals with the largest tumor volume and no significant reduction compared to the control group (through t-test, p>0.05) were considered to have drug resistance potential. This step simulates clinical drug resistance evolution, enriching drug-resistant clones through immune pressure.

[0055] Example 5: C57 mouse PD-1 antibody-resistant primary cell culture and screening Under aseptic conditions, selected drug-resistant mice were euthanized, disinfected by immersion in 75% alcohol for 5 minutes, and tumor tissue was isolated in a laminar flow hood. The tumor was rinsed three times with PBS to remove necrotic tissue and fascia, and then minced to approximately 1 mm. 3 Small pieces of tissue were digested with 1% collagenase IV in a 37°C incubator for 1 hour. The tissue suspension was filtered through a 70-mesh sterile filter, centrifuged, and the cell pellet was seeded into DMEM medium containing 2 μg / ml puromycin and 10% fetal bovine serum. Penicillin-streptomycin antibiotics were added, and the culture was incubated at 37°C with 5% CO2 for 24 hours. Afterward, non-adherent tissues were discarded, and the cells were washed with PBS and cultured again in DMEM medium containing 2 μg / ml puromycin. The cells were passaged every 3-4 days to obtain primary tumor cells (e.g., ...). Figure 1 E in Figure 1 In this context, D represents the original tumor cell.

[0056] Example 6: Obtaining and Preserving Stable Cell Lines with PD-1 Antibody Resistance in Mouse Colon Cancer Primary cells were digested and resuspended, and the C57BL / 6 mouse subcutaneous tumor model was reconstructed following the steps described above. The PD-1 antibody treatment and screening process was repeated. This was repeated for six rounds until the average tumor volume in the PD-1 antibody treatment group was not statistically different from that in the control group (p>0.05), indicating stable drug resistance phenotype. In each screening, the largest tumor in the treatment group was preferentially selected to ensure enrichment of highly efficient drug-resistant clones. Tumor tissue was isolated from the PD-1 antibody treatment group in the last round of screening, and primary cultured according to the above method. The cells were stably passaged for more than 10 generations to obtain the colorectal cancer immunotherapy-resistant cell line MC38-IR. The morphological characteristics of MC38-IR cells after 24 hours of in vitro culture (200×) showed significant differences compared to parental cells, suggesting that cellular phenotype changes may have occurred during the acquisition of drug resistance (e.g., ...). Figure 1(E in the text). For long-term preservation, the cell line was cryopreserved in liquid nitrogen and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2025358 and deposit date of December 2, 2025.

[0057] Example 7: In vitro proliferation and cell cycle analysis of mouse colon cancer PD-1 antibody-resistant cell lines The clonogenic assay is used to assess single-cell proliferation capacity. The specific steps are as follows: First, MC38-IR cells in the logarithmic growth phase and parental MC38 cells are collected, digested with 0.25% trypsin-EDTA, centrifuged, resuspended in PBS, and counted by trypan blue staining. The cell concentration is then adjusted to 5 × 10⁻⁶ cells / year. 2 Cells / mL. Add 1 mL of cell suspension to each well of a 6-well plate, then add 800 μL of DMEM medium and 200 μL of fetal bovine serum to achieve a final serum concentration of 10%. Gently mix and incubate at 37°C with 5% CO2 for 7 days. Replace half the medium every 3 days during culture to maintain nutrient supply. After 7 days, discard the medium, gently wash twice with PBS, and fix with 4% paraformaldehyde for 30 minutes. After discarding the fixative, stain with 1% crystal violet for 30 minutes, carefully rinse with water to remove residual stain, and air dry at room temperature. Finally, use a scanner to acquire clone images. ImageJ software is used to count cell clusters with a diameter >50 μm as valid clones, and the colony formation rate (clone number / inoculated cell number × 100%) is calculated.

[0058] The results showed that, based on crystal violet staining, the clonal morphology of MC38-IR cells and parental MC38 cells after 10 days of culture was observed. Both groups of cells were able to form typical cell clones, and no significant morphological differences were observed. Figure 2 A); Quantitative analysis showed that the average clone number of the MC38-IR cell group was not significantly different from that of the parental MC38 cell group (p>0.05), indicating that the in vitro proliferation capacity of MC38-IR cells was comparable to that of the parental cells. Figure 2 B).

[0059] Cell cycle assays primarily utilize flow cytometry to analyze the fluorescence intensity of propidium iodide (PI) binding to DNA to determine cell cycle phase distribution. First, cells in the logarithmic growth phase are collected and digested with 0.25% trypsin-EDTA to prepare a single-cell suspension. After washing with pre-chilled PBS, pre-chilled 70% ethanol (-20°C) is slowly added while continuously mixing via pipetting. The cells are then fixed overnight at 4°C. After fixation, the cells are centrifuged to remove the ethanol, washed with PBS to remove residual fixative, and then resuspended in PI staining solution (final concentration 50 μg / mL) containing RNase A (final concentration 100 μg / mL). The cells are incubated at 37°C in the dark for 30 minutes to degrade RNA and ensure PI specifically binds to DNA. The stained cell suspension is filtered through a 300-mesh nylon screen into flow cytometry tubes to remove cell clumps. Before flow cytometry analysis, flow cytometry parameters are set (PI excitation wavelength 488 nm, emission wavelength 617 nm), and fluorescence compensation is adjusted using unstained and single-stained cells. More than 10,000 cell signals were collected for each sample, and the DNA content histogram was analyzed using FlowJo software to calculate the percentage of cells in the G2 / M phase.

[0060] The results showed that propidium iodide (PI) staining analysis revealed that the distribution ratio of MC38-IR cells in the G2 / M phase was basically the same as that of parental MC38 cells, indicating that the cell cycle progression of drug-resistant cells was not significantly altered. Figure 2 C), there was no statistically significant difference between the two groups (p>0.05), further confirming that MC38-IR cells are similar to parental cells in maintaining basic cell functions. Figure 2 D).

[0061] according to Figure 2 It can be seen that the in vitro proliferation capacity of the mouse colon cancer PD-1 antibody-resistant cell line MC38-IR is no different from that of the control cell line MC38. Figure 2 A and B, p > 0.05). Meanwhile, the cell cycle of the mouse colon cancer PD-1 antibody-resistant cell line MC38-IR was not different from the control cell line MC38 (…). Figure 2 (C and D, p > 0.05).

[0062] Example 8: In vivo proliferation capacity of mouse colon cancer PD-1 antibody resistant cell lines Based on a C57BL / 6 mouse subcutaneous xenograft model, the in vivo biological characteristics of the MC38-IR (immunogenic resistance) cell line were evaluated through systematic experiments. Six- to eight-week-old female C57BL / 6 mice were randomly divided into an MC38-IR experimental group and a parental MC38 control group, with eight mice in each group. Logarithmic growth phase cells were digested with trypsin, resuspended in PBS, and the cell concentration was adjusted to 1 × 10⁻⁶. 7 / mL, 100μL of cell suspension (containing 1×10⁻⁶ cells) was subcutaneously injected into the right back of each mouse. 6 (Number of cells). Starting on day 7 post-inoculation, the long and short diameters of the tumor were measured every 3 days using electronic calipers, and the tumor volume was calculated. Monitoring continued until day 23 post-inoculation. At the experimental endpoint, mice were sacrificed, the tumor tissue was completely dissected, washed with PBS, and photographed.

[0063] according to Figure 3 The experimental results showed that the tumor volume of PD-1 antibody-resistant MC38-IR cell line tumor-bearing mice was significantly larger than that of control MC38 cell line tumor-bearing mice, and the cells exhibited significantly enhanced proliferative capacity in vivo (e.g., Figure 3 (A in the middle).

[0064] Tumor growth curves showed that tumor proliferation in mouse colon cancer PD-1 antibody-resistant cell line MC38-IR cells was continuously accelerated. Tumor volume changes at different time points (7-23 days) after inoculation showed that the MC38-IR group (black curve) exhibited a rapid upward trend, while the MC38 group (gray curve) grew relatively slowly. By day 23, the average tumor volume in the MC38-IR group was significantly larger than that in the MC38 group. (e.g.) Figure 3 (B in the original text). Statistical analysis showed that on day 23, the tumor volume in the MC38-IR group was significantly larger than that in the MC38 group (***p<0.001), which was statistically significant. This result confirms that MC38-IR cells have a stronger proliferative capacity in the in vivo microenvironment, consistent with their immune resistance phenotype.

[0065] Example 9: Verification of the drug resistance effect of the mouse colon cancer PD-1 antibody-resistant cell line MC38-IR To verify the drug resistance effect of the mouse colon cancer PD-1 antibody-resistant cell line MC38-IR, 6-8 week old female C57BL / 6 mice were randomly divided into four groups: MC38-IR + anti-PD-1 antibody group, MC38-IR + PBS control group, MC38 + anti-PD-1 antibody group, and MC38 + PBS control group, with 6 mice in each group. MC38-IR and MC38 cells in the logarithmic growth phase were harvested, digested with trypsin, and single-cell suspensions were prepared, with the cell concentration adjusted to 1×10⁻⁶. 7 / mL, 100μL of cell suspension (containing 1×10⁻⁶ cells) was subcutaneously injected into the right back of each mouse. 6(cells). Starting on day 7 post-inoculation, the long and short diameters of the tumor were measured every 3 days to calculate the tumor volume, and treatment was administered simultaneously: the anti-PD-1 antibody group and the PBS control group were given 200 μg / mouse of the corresponding antibody via intraperitoneal injection, once every 3 days, for a total of 4 times. The experiment was continued until day 23 post-inoculation, and subcutaneous tumor tissue from each group of mice was isolated and photographed for recording. Figure 4 During the period, tumor volume changes and mouse survival status were recorded in detail, and mouse tumor proliferation curves were plotted. Figure 4 (B). The survival time of subcutaneous tumor-bearing mice in each group was recorded and statistically analyzed. Figure 4 (E).

[0066] Experimental design flowchart as follows Figure 4 Figure A shows the complete experimental procedure for MC38 / MC38-IR cell inoculation into C57BL / 6 mice, followed by anti-PD-1 treatment and tumor isolation. The key time points and treatment regimens from day 0 to day 23 after inoculation are clearly marked.

[0067] The tumor growth dynamic curves of different treatment groups showed that tumor growth was significantly inhibited in the MC38+Anti-PD-1 group (dark gray curve), while the growth trends of the MC38-IR+Anti-PD-1 group (medium gray curve) and the MC38-IR+PBS group (light gray curve) were basically consistent, indicating that MC38-IR cells are not sensitive to PD-1 antibody treatment. Figure 4 (B).

[0068] The tumor volume statistical bar chart at the 23-day endpoint visually shows that the tumor volume in the MC38+Anti-PD-1 group was significantly smaller than that in the MC38+PBS group (p<0.0001), while there was no significant difference between the MC38-IR+Anti-PD-1 group and the MC38-IR+PBS group (ns). Figure 4 (C).

[0069] Statistical differences between different treatment groups were verified. Direct comparison using bar charts showed that MC38-IR cells maintained tumor growth capacity comparable to the control group under anti-PD-1 treatment, confirming their acquired drug resistance characteristics. Figure 4 (D).

[0070] Survival analysis curves showed that the survival time of mice in the MC38+Anti-PD-1 group was significantly prolonged, while the survival curves of the MC38-IR+Anti-PD-1 group and the corresponding PBS control group basically overlapped, further validating the PD-1 antibody resistance of MC38-IR cells from the perspective of animal survival. Figure 4 (E).

[0071] according to Figure 4The results showed that the tumors in the PD-1 antibody-bearing mice of the drug-resistant cell line MC38-IR continued to grow rapidly after treatment with PD-1 antibody, with no significant difference compared to the PBS-treated group, indicating that this drug-resistant cell line exhibits PD-1 antibody resistance. Figure 4 (B, C, D), and the survival time of CMT167-R tumor-bearing mice treated with PD-1 antibody was not significantly different from that of the isotype control IgG group. Figure 4 (E).

[0072] This invention successfully constructed a mouse colon cancer cell line, MC38-IR, exhibiting stable PD-1 antibody resistance. Obtained through in vivo screening, this cell line maintained the in vitro proliferation and migration capabilities of its parent cells while displaying significant in vivo drug resistance characteristics: in the C57BL / 6 mouse model, PD-1 antibody treatment of MC38-IR cells did not significantly inhibit tumor volume, and the survival time was not statistically different compared to the control group (p>0.05), while the parental MC38 cells showed a significant treatment response. This stable drug resistance phenotype makes the MC38-IR cell line an ideal tool for studying the mechanisms of immunotherapy resistance in colon cancer.

[0073] The MC38-IR cell line has broad application prospects in the biomedical field. This cell line can be used to screen anti-tumor drugs that reverse immune resistance, identify resistance-related biomarkers, and develop combination therapy strategies. By comparing the gene expression profiles and signaling pathways of MC38-IR cells with their parental counterparts, the mechanisms of PD-1 antibody resistance can be analyzed in depth, providing new insights for overcoming immunotherapy resistance in clinical practice. Furthermore, this model can also be used to evaluate the efficacy of novel immune checkpoint inhibitors, accelerating the development of tumor immunotherapy drugs.

[0074] The innovation of this invention lies in establishing a standardized method for constructing mouse colon cancer immune-resistant cell lines. Through multiple rounds of in vivo screening pressure, the clinical drug resistance evolution process was successfully simulated, resulting in cell lines with stable drug resistance phenotypes. This method overcomes the limitations of traditional in vitro induction models, better reproduces the immune editing process in the tumor microenvironment, and provides a reliable platform for studying the interaction between tumors and the immune system.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mouse colon cancer immunotherapy resistant cell line MC38-IR, characterized in that, The drug-resistant cell line MC38-IR is deposited at the China Center for Type Culture Collection on December 2, 2025, with accession number CCTCC NO:C2025358.

2. The method for preparing the mouse colon cancer immunotherapy resistant cell line MC38-IR according to claim 1, characterized in that, Includes the following steps: (1) The puromycin resistance gene was stably integrated through lentiviral transduction; (2) A C57BL / 6 mouse subcutaneous tumor model was constructed using the colon cancer cell line MC38, and the mice were treated with immune checkpoint PD-1 antibody. The largest tumors after treatment were selected. (3) The tumor tissue obtained in step (1) was separated under aseptic conditions, cut into pieces, cultured and passaged in DMEM medium containing 10% fetal bovine serum to obtain primary tumor cells. (4) Use the primary cell replica in vivo model obtained in step (2) to screen for PD-1 antibody therapy until the tumor volume of the PD-1 antibody treatment group is not significantly different from that of the control group; (5) The tumor tissue obtained in step (3) was isolated under sterile conditions, minced and cultured in DMEM medium and stably passaged to obtain the colorectal cancer immunotherapy resistant cell line MC38-IR.

3. The preparation method according to claim 2, characterized in that, In step (1), the colon cancer cell line is a murine colon cancer cell line.

4. The preparation method according to claim 2, characterized in that, In step (4), the culture conditions are 37°C and CO2 volume concentration is 5%.

5. The application of the MC38-IR cell line for colorectal cancer immunotherapy resistance as described in claim 1 in screening anti-colorectal cancer drug targets.

6. The use of the MC38-IR cell line for colorectal cancer immunotherapy resistance as described in claim 1 in screening and / or preparing anti-colorectal cancer drugs.

7. The use of the colorectal cancer immunotherapy resistant cell line MC38-IR as described in claim 1 in screening and / or preparing detection reagents for evaluating the anti-colorectal cancer efficacy.

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