Mouse intrahepatic bile duct cancer cell line as well as construction method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a mouse intrahepatic bile duct cancer cell strain and a construction method and application thereof, the cell strain is named as mouse intrahepatic bile duct cancer cell strain KP-ICC and preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C202574; the cell strain is derived from a KrasG12D / Tp53flox / flox C57BL / 6J mouse, a spontaneous tumor model is constructed through high-pressure hydrodynamic tail vein injection of pT3-Cre and a sleep beauty transposase plasmid, and the spontaneous tumor model is obtained through in-vitro culture, passage, monoclonal screening and tumorigenicity verification. The KP-ICC cell strain naturally has chemotherapy-immune combined treatment drug resistance and ferroptosis resistance characteristics, and can be widely applied to the fields of intrahepatic cholangiocarcinoma drug resistance mechanism research, drug resistance reversing, combined treatment drug development, drug resistance animal model establishment and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a mouse intrahepatic bile duct cancer cell line and a construction method and application thereof. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignancy after hepatocellular carcinoma. Its incidence has continued to rise in recent years, particularly in my country, where it has become a significant threat to liver health. Due to the insidious early symptoms of ICC, the vast majority of patients are already in the advanced or late stages of the disease at the time of initial diagnosis, missing the optimal time for surgical treatment. Although chemotherapy-immunotherapy (CIT), such as gemcitabine + cisplatin + the anti-PD-L1 monoclonal antibody durvalumab, has achieved some progress in the treatment of advanced ICC, the incidence of drug resistance in ICC is high, and patients' objective response rate and progression-free survival remain low, necessitating the development of new treatment strategies.

[0003] ICC's tumor heterogeneity, metabolic reprogramming, and immune evasion mechanisms make its drug resistance mechanisms complex and incompletely understood. Enhanced aerobic glycolysis (Warburg effect) and fatty acid metabolism in ICC cells are closely associated with drug resistance, and programmed cell death (PCD) such as ferroptosis may be part of this resistance mechanism. Further investigation of ICC drug resistance mechanisms, particularly those related to ferroptosis and fatty acid metabolism, will provide potential avenues for the development of novel therapeutic strategies. Establishing a representative mouse intrahepatic cholangiocarcinoma cell line is crucial for effectively studying ICC drug resistance mechanisms and exploring new therapeutic targets. Summary of the Invention

[0004] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide a mouse intrahepatic cholangiocarcinoma cell line, a construction method thereof, and an application thereof. The construction of the mouse intrahepatic cholangiocarcinoma cell line KP-ICC provides an important experimental platform for the study of ICC drug resistance mechanisms, and can simulate the metabolic reprogramming characteristics, immune escape mechanisms, and drug resistance in chemotherapy-immunotherapy combination therapy of ICC. The KP-ICC cell line can be used for drug screening and drug resistance mechanism research, providing a key tool for the development of new targeted drugs, and promoting ICC drug resistance research and treatment strategy optimization. Therefore, the establishment of the KP-ICC cell line has laid a solid foundation for basic research on ICC, the construction of a drug screening platform, and the exploration of drug resistance mechanisms.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a mouse intrahepatic bile duct carcinoma cell line, the cell line is named mouse intrahepatic bile duct carcinoma tumor cell KP-ICC Mus musculus, and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202574; it was deposited in the China Center for Type Culture Collection (CCTCC) on April 28, 2025, and the deposit address is Wuhan University, Wuhan, China; the cell line is derived from male Kras G12D / Tp53 flox / flox Isolated from C57BL / 6J mouse model.

[0006] Preferably, the cell line has the following characteristics: a) p53 protein expression is lost, and MAPK and AKT signaling pathways are activated; b) Tumorigenicity verification showed that it could form typical intrahepatic cholangiocarcinoma tissue after subcutaneous inoculation, expressing Ck19 and Sox9 but not Hnf4a.

[0007] The present invention also provides a method for constructing a mouse intrahepatic bile duct carcinoma cell line, comprising the following steps: High-pressure hydrodynamic tail vein injection was used to inject the G12D / Tp53 flox / flox A mixture of pT3-Cre, the Sleeping Beauty transposase plasmid, and normal saline was delivered into C57BL / 6J mice to induce KrasG12D activation and Tp53 gene deletion in liver cells, establishing an intrahepatic cholangiocarcinoma tumor model. isolating primary tumor cells from the tumor model, performing subculture and single cell clone screening to obtain stably growing monoclonal cells; The monoclonal cells are subjected to molecular biological identification and tumorigenicity verification, wherein the molecular biological identification includes Cre gene integration verification, STR identification, p53 protein expression detection, and MAPK and AKT signaling pathway activation status analysis. The tumorigenicity verification is achieved by subcutaneously injecting the cells into mice and detecting tumor marker expression.

[0008] Preferably, the mass ratio of the pT3-Cre to the Sleeping Beauty transposase plasmid is (8-9):1.

[0009] Preferably, the primary tumor cells are subcultured in RPMI 1640 medium containing 10% fetal bovine serum and 1× penicillin / streptomycin to the logarithmic growth phase, and single cell clone screening is performed after 5 subcultures. The cells are diluted into a 96-well plate, one cell is inoculated into each well, and cultured at 37°C and 5% CO2 for 2 weeks.

[0010] Preferably, in the molecular biological identification, Cre gene integration verification is achieved by RT-qPCR method, p53 protein expression detection and MAPK and AKT signaling pathway activation status analysis are achieved by Western blotting WB method, and the detection indicators of the signal pathway activation status include the expression of AKT, AKT pS473, ERK1 / 2 and ERK1 / 2 pT202 / pY204 proteins.

[0011] Preferably, the cell line is identified by short tandem repeat (STR) sequencing, and the loci identified by STR include STR1-1, STR1-2, STR2-1, STR3-2, STR4-2, STR5-5, STR6-4, STR6-7, STR7-1, STR8-1, STR11-2, STR12-1, STR13-1, STR15-3, STR17-2, STR18-3, STR19-2, and STRX-1.

[0012] The present invention also provides an application of the mouse intrahepatic bile duct carcinoma cell line for the mechanism study of intrahepatic bile duct carcinoma, wherein the mechanism study includes exploring the tumor formation mechanism, drug resistance mechanism, metabolic reprogramming mechanism or immune escape mechanism of intrahepatic bile duct carcinoma.

[0013] The present invention also provides an application of the mouse intrahepatic bile duct carcinoma cell line for drug screening of intrahepatic bile duct carcinoma, wherein the drug screening includes screening single-drug therapeutic drugs or combined therapeutic drugs for intrahepatic bile duct carcinoma.

[0014] Preferably, the drug is used to inhibit PCK1 phosphorylation to enhance the efficacy of CIT via the ferroptosis pathway.

[0015] Preferably, the drug comprises PPB or simvastatin, and the PPB or simvastatin enhances the efficacy of CIT through the pPCK1-pLDHA-SPRINGlac metabolic axis. The present invention also provides an application of the mouse intrahepatic cholangiocarcinoma cell line for developing therapeutic targets for intrahepatic cholangiocarcinoma, wherein the therapeutic target development includes screening biomarkers or potential therapeutic targets for intrahepatic cholangiocarcinoma.

[0016] Preferably, the therapeutic target is Pck1, which is a key metabolic regulatory molecule that mediates both KP-ICC tolerance to CIT treatment and ICC resistance to ferroptosis.

[0017] The principle of the present invention is that the present invention adopts high pressure water dynamic tail vein injection technology to inject Kras G12D / Tp53 flox / floxThe pT3-Cre and Sleeping Beauty transposase plasmids were delivered to C57BL / 6J mice, driven by high-pressure fluid dynamics, ensuring that the plasmids entered the body through the tail vein and penetrated into the liver. The liver has abundant blood flow and high vascular permeability, which are conducive to the effective uptake of plasmids and gene expression. Among them, the pT3-Cre plasmid can trigger the activation of KrasG12D and Tp53 - / - The Sleeping Beauty transposase plasmid stably integrates these mutant genes into the host genome through the transposition system, promoting gene expression in liver cells. During liquid injection, physiological saline not only serves as a plasmid solvent, but also ensures rapid penetration of the plasmid into the liver through high-pressure injection, avoiding liquid dilution and improving delivery efficiency. By combining the transposase system with efficient gene delivery technology, the intrahepatic cholangiocarcinoma cell line KP-ICC was successfully constructed, providing an effective experimental model for tumor research and gene function verification.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention successfully constructed and preserved the mouse intrahepatic cholangiocarcinoma cell line KP-ICC, providing an important in vitro model for in-depth research on the pathogenesis, drug resistance mechanism, and molecular targets of intrahepatic cholangiocarcinoma. This cell line can simulate the tumor heterogeneity and metabolic reprogramming characteristics of intrahepatic cholangiocarcinoma, and is suitable for molecular mechanism research of intrahepatic cholangiocarcinoma, anti-tumor drug screening, and efficacy evaluation of combination therapy strategies. It has high scientific research value and potential clinical translational applications.

[0019] (2) The application of the mouse intrahepatic cholangiocarcinoma cell line KP-ICC in high-throughput sequencing and other technologies can provide researchers with an ideal experimental platform to help them deeply explore the pathogenesis and drug resistance of intrahepatic cholangiocarcinoma from a higher dimension.

[0020] (3) The mouse intrahepatic cholangiocarcinoma cell line KP-ICC constructed by the present invention can be widely used in the fields of studying the drug resistance mechanism of intrahepatic cholangiocarcinoma, reversing drug resistance, developing combination therapeutic drugs, and establishing drug resistance animal models. This cell line provides a valuable cell research model for screening and evaluating effective drugs and treatments for reversing drug resistance in intrahepatic cholangiocarcinoma and has good application prospects.

[0021] In summary, the present invention not only provides a new experimental tool for the study of the drug resistance mechanism of intrahepatic cholangiocarcinoma, but also provides important support for the development of new treatment strategies and solving the problem of drug resistance. It has far-reaching scientific significance and clinical application value for improving the treatment effect of intrahepatic cholangiocarcinoma and prolonging the survival of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Figures depict the construction and characterization process of the mouse intrahepatic cholangiocarcinoma cell line KP-ICC, described in Example 1 of the present invention. (A) is a schematic diagram of the KP-ICC cell line construction process; (B) is a graph showing the tumor mutation burden (TMB) analysis results for KP-ICC and KPPC (unit: mutations / million bases, muts / Mb); (C) is a graph showing the similarity of gene expression profiles between KP-ICC, KPPC, and normal liver organoids assessed by calculating the Spearman correlation coefficient; (D) is a graph showing the molecular validation of Tp53 loss and Kras activation; (E) is a graph showing the morphological and immunohistochemical analysis of subcutaneous tumors; and (F)-(H) are representative tumor images, tumor volume, and tumor weight, respectively, demonstrating the in vivo efficacy of the CIT group in the KP-ICC mouse model. Data are presented as individual data points and mean ± standard deviation (SD). Statistical Analysis: Two-sided Student's t-test or two-sided Mann-Whitney U test (nonparametric) was used for comparisons between two groups. Two-way ANOVA was used for comparisons between multiple groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: not significant.

[0023] Figure 2 Figure 2 shows the KP-ICC cell-based mouse metabolic gene CRISPR screening system and its results in studying the CIT resistance mechanism in Example 2 of the present invention. (A) is a flowchart of the KP-ICC cell-mediated in vivo metabolic gene CRISPR screening experiment, (B) is a diagram showing the functional ranking analysis results of KP-ICC in vivo screening of metabolic genes, (C) is a diagram showing the results of identifying KP-ICC / CIT resistance-specific genes, (D) is a diagram showing the design of in vitro screening for KP-ICC ferroptosis-sensitive genes, (E) is a diagram showing the results of in vitro CRISPR screening of KP-ICC cells under RSL3 treatment, and (F) is a diagram showing the strategy for identifying key molecules regulating CIT resistance and ferroptosis using KP-ICC as a background.

[0024] Figure 3Figures show the anti-tumor efficacy and immune microenvironment changes of CIT combined with inhibition of PCK1 phosphorylation in Example 3 of the present invention. (A) shows a gross image of the tumor; (B) shows tumor weight; (C) shows tumor volume; (D) shows the concentration of MDA, a marker of intratumoral lipid peroxidation; (E) shows the ratio of intratumoral CD3⁺CD8⁺ T cells; (F) shows the ratio of intratumoral TNFα⁺ / IFNγ⁺CD8⁺ T cells; and (G) shows the density of CD8⁺ T cell infiltration as determined by immunohistochemistry (IHC). Data are presented as individual data points and mean ± standard deviation (SD). Statistical analysis: Two-sided Student's t-test or two-sided Mann-Whitney U test (nonparametric) was used for comparisons between two groups. Two-way ANOVA was used for comparisons between multiple groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: not significant.

[0025] Figure 4 The figures show the anti-tumor effect and immune response results of C57BL / 6J mice subcutaneously inoculated with KP-ICC cells and receiving different treatment regimens after 2 weeks of treatment in Example 3 of the present invention. Among them, the treatment regimens (A)-(G) include placebo group (Placebo), PPB, Simv, CIT, CIT+PPB, CIT+Simv, (A) is a gross image of the tumor, (B) is a tumor weight result graph, (C) is a tumor volume result graph, (D) is a tumor malondialdehyde (MDA) concentration result graph, (E) is a tumor CD3⁺CD8⁺T cell ratio result graph, and (F) is a tumor TNFα⁺ / IFNγ⁺CD8⁺T cell ratio graph. Ratio results are shown. (G) shows the density of CD8⁺ T cell infiltration detected by IHC. (H)-(L) show the placebo, CIT, Simv, CIT+Simv, and CIT+Simv+MVA-Li groups. (H) shows a gross tumor image, (I) shows tumor weight, (J) shows tumor volume, (K) shows intratumoral malondialdehyde (MDA) concentration, and (L) shows the ratio of intratumoral CD8⁺ T cells. Data are presented as individual data points and mean ± standard deviation (SD). Statistical analysis: Two-sided Student's t-test or two-sided Mann-Whitney U test (nonparametric) was used for comparisons between two groups. Two-way ANOVA was used for comparisons between multiple groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: not significant. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the accompanying drawings, embodiments and experimental examples. Of course, the scope of protection of the present invention is not limited to the following examples. Professionals and technicians in this field will understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention provides a general and / or specific description of the materials and test methods used in the experiments. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible here. The following examples further illustrate the present invention, rather than limit the present invention. Any equivalent transformation that is merely formal and not substantial based on the concept of the present invention should be regarded as the scope of the technical solution of the present invention.

[0027] Unless otherwise specified, the experimental methods or test methods described in the following examples are all conventional methods; the reagents and materials described are all obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] Kras used in the examples G12D / Tp53 flox / flox C57BL / 6J mice were provided by Professor Zhong Bo of Wuhan University and are known mice. The literature source is: HILL MA, ALEXANDER WB, GUO B, et al. Kras and Tp53 Mutations Cause Cholangiocyte- and Hepatocyte-Derived Cholangiocarcinoma [J]. Cancer Res, 2018, 78(16): 4445-51. The pT3-Cre and Sleeping Beauty transposase plasmids used in the examples were provided by Addgene.

[0030] Example 1: Construction and drug resistance study and drug evaluation of mouse intrahepatic cholangiocarcinoma cell line KP-ICC This example provides a method for constructing a mouse intrahepatic cholangiocarcinoma cell line KP-ICC and subsequent genetic and transcriptome similarity analysis, aiming to provide an experimental platform for studying the pathogenesis, drug resistance mechanism, and drug screening of intrahepatic cholangiocarcinoma. Figure 1 A. The construction process of mouse intrahepatic cholangiocarcinoma cell line KP-ICC is shown as follows: First, 4-5 week old male Kras G12D / Tp53 flox / floxC57BL / 6J mice were injected with a mixture of pT3-Cre and Sleeping Beauty transposase plasmids via tail vein high pressure injection. The plasmid solution included 2 mL of normal saline, 20 μg of pT3-Cre plasmid and 2.4 μg of Sleeping Beauty transposase plasmid, ensuring that the injection process was completed within 7 seconds. Six months after the injection, the mouse tumors were harvested, and the tumor cells were isolated from them for further primary culture. The isolated primary tumor cells were then cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1× penicillin / streptomycin to the logarithmic growth phase. After 5 passages, the single-cell clone screening method was used to screen and amplify surviving and stably growing monoclonal cells, and RT-qPCR was used to verify whether the Cre gene was successfully expressed in the corresponding cell clones. Through in-depth analysis of TMB and transcriptome data ( Figure 1 B). STR identification was performed to confirm the identity of the cell line (Table 1). Further analysis of the transcriptome expression similarity between KP-ICC and existing ICC cells KPPC with the same background showed that the transcriptome characteristics of KP-ICC and KPPC were significantly similar, and at the same time different from normal liver organoids ( Figure 1 C). Successfully expanded clones were then injected subcutaneously into 5-6 week-old male C57BL / 6J mice, and tumor growth was observed. One week after injection, subcutaneous tumors were harvested for IHC analysis to detect the expression of HNF4A, CK19, and SOX9. Immunohistochemical staining revealed that KP-ICC tumors were positive for CK19 and Sox9, while negative for Hnf4a, confirming the tumor identity and tumorigenicity of the cell line. Figure 1 E). Western blot was used to examine the expression of p53 and the activation status of the MAPK and AKT pathways. WB assays showed that Tp53 protein was completely absent in KP-ICC and KP2 cells, while Kras downstream signaling molecules (such as phosphorylated AKT and ERK1 / 2) were significantly activated. No such changes were observed in primary mouse hepatobiliary cells (normal control). The expression of AKT, AKT pS473, ERK1 / 2, and ERK1 / 2 pT202 / pY204 proteins was confirmed ( Figure 1D). When tumors reached 0.5 cm in diameter, they were harvested and subjected to IHC analysis to detect Hnf4a, Ck19, and Sox9 expression to confirm tumorigenicity. DNA and RNA were co-extracted using the FastPure DNA / RNA Mini Kit V2 and submitted to GenePlus Technology for whole-exome sequencing (WES) and transcriptome sequencing. WES libraries were prepared using the VAHTS Universal Plus DNA Library PrepKit for MGI V2, and transcriptome libraries were prepared using the VAHTS Universal V10 RNA-seq Library PrepKit for MGI. Sequencing was performed on the MGISEQ-2000 platform using paired-end sequencing. Alignment was performed using STAR v2.7, duplicate reads were removed using Picard, and variant calling was performed using GATK and Mutect2. Transcriptome data from the KP-ICC cell line and KPPC model were aligned. Gene counts were extracted using the FeatureCounts function in the Subread package, normalized using the limma package, and batch effects were corrected using the sva package. The transcriptome similarity between different samples was calculated using the Spearman correlation coefficient. This cell line can be used as an important platform for studying drug resistance, screening drug resistance markers, and drug development in intrahepatic cholangiocarcinoma.

[0031] like Figure 1 F- Figure 1 As shown in H, the KP-ICC mouse model showed significant tolerance to chemotherapy (GP, gemcitabine + cisplatin), immunotherapy (anti-PD-L1 monoclonal antibody) and their combined therapy (CIT). The specific experimental steps are as follows: KP-ICC cells (1×10 6 cells / mouse) were inoculated subcutaneously into 4-5 week old male C57BL / 6J mice. After 7 days, the mice were randomly divided into 4 groups (n=5 / group): placebo group, GP chemotherapy group (gemcitabine + cisplatin), anti-PD-L1 monoclonal antibody group (anti-mPD-L1 monoclonal antibody) and CIT group (GP + anti-PD-L1 monoclonal antibody). After 2 weeks of treatment, tumor samples were collected and tumor volume and weight were measured. The data showed that the KP-ICC model showed significant drug resistance under all treatment regimens. Among them Figure 1 F shows the comparison of tumor volumes in different treatment groups. Figure 1 G and Figure 1 H shows the changes in tumor weight.

[0032] Table 1 STR identification results of KP-ICC locus KP-ICC allele locus KP-ICC allele STR1-1 17 STR11-2 16 STR1-2 18,19 STR12-1 17 STR2-1 16 STR13-1 17 STR3-2 14 STR15-3 22.3 STR4-2 20.3 STR17-2 16 STR5-5 18 STR18-3 16 STR6-4 18 STR19-2 13 STR6-7 17 STRX-1 27 STR7-1 27.2 D16S539 NA STR8-1 16 D3S1358 NA In summary, this example established the mouse intrahepatic cholangiocarcinoma (KP-ICC) cell line through efficient gene delivery technology. Through genetic and transcriptomic analysis, this model provides a powerful experimental model for studying the molecular mechanisms of intrahepatic cholangiocarcinoma, drug screening, and drug resistance mechanisms. Furthermore, the KP-ICC model exhibits significant tolerance to chemotherapy (gemcitabine + cisplatin), immunotherapy (anti-PD-L1 monoclonal antibody), and their combination therapy (CIT), making it an ideal platform for exploring drug resistance mechanisms and developing new treatment strategies for intrahepatic cholangiocarcinoma.

[0033] Example 2: KP-ICC cell-based mouse metabolic gene CRISPR screening system and its application in the study of CIT resistance mechanisms This example provides a method for in vitro and in vivo CRISPR high-throughput screening using the mouse intrahepatic cholangiocarcinoma cell line KP-ICC, which is used to systematically analyze the metabolic mechanism of intrahepatic cholangiocarcinoma resistance to CIT and identify potential therapeutic targets. This experiment used a CRISPR knockout library containing 2,865 mouse metabolism-related genes (#No.160129, Addgene). After infecting KP-ICC cells with a lentiviral system, in vitro (RSL3-induced ferroptosis) and in vivo (subcutaneous modeling + drug treatment) screening were performed. Figure 2 A). The infected KP-ICC cells were selected with puromycin and used in subsequent experiments, and some samples were retained as controls. In the in vivo experiment, the virus-infected KP-ICC cells were subcutaneously injected into 4-5 week old male C57BL / 6J mice and randomly divided into groups for Placebo, GP chemotherapy, anti-PD-L1 immunotherapy (aPd-l1) or CIT combined treatment. Tumor tissues were collected two weeks after treatment, and DNA was extracted for sgRNA abundance sequencing and analysis. The MAGeCK-MLE β score results showed that compared with the control group, the CIT, GP, aPd-l1 and other treatment groups all had specific gene depletion spectra, especially in the CIT group, genes such as Pck1 were significantly deleted, which is a potential driver of drug resistance ( Figure 2 B). Further comparison of the β-score ratios between the CIT and monotherapy groups (CIT vs. GP / aPd-l1) is shown in the scatter plot, with genes (red or blue) with β-score ratios higher than 1.5. This confirms that Pck1 is specifically depleted in the CIT combination group and is a potential driver of resistance under CIT combination therapy ( Figure 2 C). At the same time, in in vitro experiments, KP-ICC was used as a model and treated with RSL3 (ferroptosis inducer) and DMSO, respectively. sgRNA NGS analysis was performed to identify key metabolic genes that regulate ferroptosis. Combined with CRISPR screening ( Figure 2D) Based on MAGeCK-MLE analysis, the scatter plot showed that multiple genes including Pck1 were also significantly depleted in the RSL3-treated group, among which Pck1 was the most prominent, suggesting that it has a ferroptosis resistance function in KP-ICC ( Figure 2 E). Integrate in vitro and in vivo screening data and combine CIT treatment response with ferroptosis sensitivity analysis ( Figure 2 F), ultimately confirming that Pck1 is a key metabolic regulatory molecule that mediates both KP-ICC tolerance to CIT treatment and resistance to ferroptosis, providing a theoretical basis and potential target support for subsequent targeted interventions.

[0034] In summary, KP-ICC cells are not only suitable for viral infection and in vivo modeling, but also provide an ideal screening environment and can be efficiently applied to CRISPR loss-of-function platforms. This platform can be widely used for ICC-related drug sensitivity studies, analysis of resistance mechanisms, and target discovery, providing a powerful research tool for the development of more precise and effective treatment strategies. In particular, the identified Pck1 is a key regulator of ICC resistance to combined ferroptosis and immune therapy and is expected to serve as a candidate drug target for reversing resistance, with significant potential for clinical translation.

[0035] Example 3: Validation of CIT resistance mechanisms and intervention strategies based on the KP-ICC model This study systematically explores the mechanisms of ferroptosis resistance in the mouse intrahepatic cholangiocarcinoma cell line KP-ICC in CIT resistance and validates novel therapeutic strategies that intervene in this mechanism. This model not only reveals the regulation of ferroptosis by pPCK1-mediated metabolic reprogramming, but also evaluates the in vitro and in vivo anti-tumor effects of strategies based on pPCK1 intervention and MVA metabolic pathway inhibition.

[0036] First, a PCK1S90 site mutation model was constructed in KP-ICC cells, and it was verified that pPCK1 can promote glycolysis and cooperate with the MVA metabolic pathway to drive ferroptosis resistance. Mechanistically, pPCK1 can enhance lactate production by phosphorylating LDHA T248, further promoting lactation modification at the SPRINGK82 site, thereby driving the transport of SCAP to the Golgi apparatus and activating SREBP2, thereby enhancing MVA metabolism and synthesizing anti-ferroptosis factors (such as CoQ10H2 and MK4). In in vitro studies, the use of PPB peptide to inhibit pPCK1, the use of LDHA inhibitors or the knockout of SPRING significantly enhanced the sensitivity of KP-ICC to ferroptosis inducers such as RSL3, and induced enhanced lipid peroxidation, suggesting that intervening in this axis can effectively relieve ferroptosis tolerance. In vivo, treatment verification experiments were carried out using KP-ICC tumor-bearing C57BL / 6J mice. Two weeks after subcutaneous inoculation of KP-ICC cells, Placebo, CIT, PPB or CIT+PPB were given. Gross appearance of the tumor ( Figure 3 A) Weight ( Figure 3 B) and volume ( Figure 3 C) showed that CIT alone had limited effect, while the tumors in the CIT+PPB combination treatment group were significantly reduced, suggesting that inhibition of pPCK1 can enhance the sensitivity of CIT treatment. The MDA level in the tumor was significantly increased in the CIT+PPB combination group, indicating enhanced ferroptosis ( Figure 3 D). Flow cytometry detection of CD3⁺CD8⁺T cells ( Figure 3 E) and TNFα⁺ / IFNγ⁺CD8⁺T cells ( Figure 3 F), were significantly increased in the CIT+PPB combination group, indicating an enhanced immune effect. IHC detection of CD8⁺T cell infiltration density was also significantly increased in the CIT+PPB combination group, further verifying that inhibition of pPCK1 can enhance immune clearance ability ( Figure 3 G). KP-ICC tumor-bearing mice were treated with PPB, Simvastatin (Simv), CIT, CIT+PPB, and CIT+Simv for 2 weeks, and the gross appearance of the tumors was evaluated ( Figure 4 A), tumor weight ( Figure 4 B) and volume ( Figure 4 C). The results showed that monotherapy (PPB, Simv or CIT) had limited anti-tumor effects, while combination therapy (CIT+PPB or CIT+Simv) significantly inhibited tumor growth. The MDA concentration increased in the CIT combined with metabolic intervention group (especially CIT+Simv), indicating enhanced lipid peroxidation, consistent with ferroptosis activation ( Figure 4 D). Flow cytometry analysis showed that combined treatment with CIT+PPB and CIT+Simv could significantly increase the proportion of CD8⁺T cell infiltration in tumors ( Figure 4E) and TNFα⁺ / IFNγ⁺CD8⁺T cell activity ( Figure 4 F), IHC showed an increase in the density of CD8⁺ immune cells ( Figure 4 G), indicating enhanced immune clearance. Exogenous supplementation of MVA-Li on the basis of CIT+Simv treatment showed that the tumor gross appearance ( Figure 4 H), tumor weight ( Figure 4 I), tumor volume ( Figure 4 J) all showed “partial recovery”, i.e., the anti-tumor effect was weakened, suggesting the importance of MVA pathway in anti-ferroptosis. MDA concentration ( Figure 4 K) and CD8⁺T cell infiltration levels ( Figure 4 L) also decreased significantly in the MVA-supplemented group, further confirming that the anti-tumor effect of CIT+Simv is highly dependent on the inhibition of the MVA pathway.

[0037] In summary, KP-ICC cells highly express activated pPCK1, accompanied by enhanced glycolysis, lactate accumulation, and activation of the MVA pathway, constructing a dual metabolic biological characteristic of "ferroptosis tolerance + CIT resistance." Its metabolic reprogramming is closely coupled with the immunosuppressive state, providing a complete and controllable experimental platform for studying the pPCK1-pLDHA-SPRINGlac metabolic axis. Through this model, the complete resistance mechanism from pPCK1 phosphorylation, metabolic pathway reprogramming, to immune microenvironment remodeling can be clearly analyzed, and targeted intervention verification can be carried out on this basis. The KP-ICC model is not only an ideal tool for studying ICC ferroptosis and CIT resistance, but also provides strong in vitro and in vivo experimental support for the development of anti-resistance strategies based on metabolic targets, which is of great significance for promoting new drug screening and clinical translation.

[0038] The embodiments described above are merely some preferred implementations of the present invention and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A mouse intrahepatic bile duct carcinoma cell line, characterized in that: The cell line is named as mouse intrahepatic bile duct carcinoma cell line KP-ICC, which is deposited in China Center for Type Culture Collection with the deposit number CCTCCNO: C202574; the cell line is isolated from male Kras G12D / Tp53 flox / flox Isolated from C57BL / 6J mouse model.

2. The mouse intrahepatic bile duct carcinoma cell line according to claim 1, characterized in that The cell line has the following characteristics: a) p53 protein expression is lost, and MAPK and AKT signaling pathways are activated; b) Tumorigenicity verification showed that it could form typical intrahepatic cholangiocarcinoma tissue after subcutaneous inoculation, expressing Ck19 and Sox9 but not Hnf4a.

3. A method for constructing a mouse intrahepatic bile duct carcinoma cell line according to claim 1, characterized in that: include: High-pressure hydrodynamic tail vein injection was used to inject the G12D / Tp53 flox / flox A mixture of pT3-Cre, the Sleeping Beauty transposase plasmid, and normal saline was delivered into C57BL / 6J mice to induce KrasG12D activation and Tp53 gene deletion in liver cells, establishing an intrahepatic cholangiocarcinoma tumor model. isolating primary tumor cells from the tumor model, performing subculture and single cell clone screening to obtain stably growing monoclonal cells; The monoclonal cells are subjected to molecular biological identification and tumorigenicity verification, wherein the molecular biological identification includes Cre gene integration verification, STR identification, p53 protein expression detection, and MAPK and AKT signaling pathway activation status analysis. The tumorigenicity verification is achieved by subcutaneously injecting the cells into mice and detecting tumor marker expression.

4. The method for constructing a mouse intrahepatic bile duct carcinoma cell line according to claim 3, characterized in that: The mass ratio of the pT3-Cre and the Sleeping Beauty transposase plasmid is (8-9):

1.

5. The method for constructing a mouse intrahepatic bile duct carcinoma cell line according to claim 3, characterized in that: The primary tumor cells were subcultured in RPMI 1640 medium containing 10% fetal bovine serum and 1× penicillin / streptomycin until the logarithmic growth phase. After 5 subcultures, single cell clone screening was performed, and the cells were diluted into 96-well plates, with one cell inoculated per well, and cultured at 37°C and 5% CO2 for 2 weeks.

6. The method for constructing a mouse intrahepatic bile duct carcinoma cell line according to claim 3, characterized in that: In the molecular biological identification, Cre gene integration verification was achieved by RT-qPCR, p53 protein expression detection and MAPK and AKT signaling pathway activation status analysis were achieved by Western blotting WB method, and the detection indicators of the signaling pathway activation status included the expression of AKT, AKT pS473, ERK1 / 2 and ERK1 / 2 pT202 / pY204 proteins.

7. The method for constructing a mouse intrahepatic bile duct carcinoma cell line according to claim 3, characterized in that: The cell line is identified by short tandem repeat (STR) sequencing, and the STR-identified loci include STR1-1, STR1-2, STR2-1, STR3-2, STR4-2, STR5-5, STR6-4, STR6-7, STR7-1, STR8-1, STR11-2, STR12-1, STR13-1, STR15-3, STR17-2, STR18-3, STR19-2, and STRX-1.

8. A use of the mouse intrahepatic bile duct carcinoma cell line according to claim 1, characterized in that: Used for mechanistic research on intrahepatic cholangiocarcinoma, including exploring the tumorigenic mechanism, drug resistance mechanism, metabolic reprogramming mechanism or immune escape mechanism of intrahepatic cholangiocarcinoma.

9. A use of the mouse intrahepatic bile duct carcinoma cell line according to claim 1, characterized in that: Used for drug screening for intrahepatic bile duct carcinoma, the drug screening includes screening single-drug therapeutic drugs or combined therapeutic drugs for intrahepatic bile duct carcinoma.

10. A use of the mouse intrahepatic bile duct carcinoma cell line according to claim 1, characterized in that: Used for the development of therapeutic targets for intrahepatic cholangiocarcinoma, wherein the therapeutic target development includes screening biomarkers or potential therapeutic targets for intrahepatic cholangiocarcinoma.

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