KRAS G12D inhibitor drug-resistant bile duct cancer cell line as well as construction method and application thereof

Abstract

The invention provides a KRAS G12D inhibitor drug-resistant bile duct cancer cell line as well as a construction method and application thereof. The KRAS G12D inhibitor drug-resistant bile duct cancer cell line MuICC-YAK-MR is preserved in the China Center for Type Culture Collection on August 20, 2025, and the preservation number of the KRAS G12D inhibitor drug-resistant bile duct cancer cell line MuICC-YAK-MR is CCTCC (China Center for Type Culture Collection) NO: C2025240. The cell line MuICC-YAK-MR can provide a more reliable and clinical experimental model for subsequent clarification of a molecular mechanism of drug resistance of intrahepatic cholangiocarcinoma to a KRAS G12D inhibitor (MRTX1133) and development of targeted drugs, clinical treatment drug screening and drug combination strategies for intrahepatic cholangiocarcinoma patients.

Description

A KRAS G12D inhibitor-resistant cholangiocarcinoma cell line and its construction method and application Technical Field

[0001] This invention relates to the field of cell technology, specifically to a KRAS G12D inhibitor-resistant cholangiocarcinoma cell line, its construction method, and its application. Background Technology

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignant tumor and the second most common primary liver cancer after hepatocellular carcinoma. Radical surgical resection remains the primary and potentially curative treatment. However, due to the complex anatomical location and significant invasive growth characteristics of ICC, surgical procedures are difficult and risky, resulting in a low radical resection rate and a high recurrence rate, leading to a poor overall prognosis. Currently, the commonly used systemic treatment regimen is gemcitabine combined with cisplatin chemotherapy, but this regimen has limited efficacy, low patient sensitivity, and a lack of more effective treatment options.

[0003] With the development of sequencing technology, many new therapeutic targets have been discovered. Among these, 20%-30% of patients with intrahepatic cholangiocarcinoma have KRAS mutations, with G12D being the most common mutation, accounting for as much as 43.3% of all KRAS mutations. This subtype is significantly associated with poor prognosis in cholangiocarcinoma patients. Therefore, research into KRAS G12D-targeted drugs for intrahepatic cholangiocarcinoma and related drug resistance mechanisms is of great significance for improving patient prognosis and quality of life.

[0004] Currently, numerous inhibitors target KRAS, among which the most advanced in basic and clinical research and the most likely to achieve clinical application is the selective non-covalent KRAS G12D inhibitor MRTX1133. Preclinical studies have shown that MRTX1133 can selectively bind to KRAS G12D mutants, effectively inhibiting KRAS-dependent signaling pathways and exhibiting significant in vitro and in vivo anti-tumor effects. MRTX1133 has received FDA approval for clinical trials, and a phase 1 / 2 multi-extension cohort trial (NCT05737706) of MRTX1133 in patients with advanced solid tumors carrying KRAS G12D mutations is also underway. Therefore, exploring the resistance mechanisms of MRTX1133 in the treatment of KRAS G12D intrahepatic cholangiocarcinoma and developing synergistic strategies are of significant clinical importance.

[0005] Drug-resistant cell lines can be constructed in vitro or in vivo. In vivo construction has the advantage of better simulating the clinical treatment environment and tumor microenvironment, including the interaction between tumor and host, providing more comprehensive research on drug resistance mechanisms, and evaluating the efficacy of drug combination therapy.

[0006] In the clinical treatment of intrahepatic cholangiocarcinoma, due to the large individual differences among patients and the heterogeneity of tumors, there is a lack of effective targeted drugs. Therefore, personalized precision treatment is a new direction for the clinical treatment of intrahepatic cholangiocarcinoma.

[0007] Currently, most inhibitors targeting KRAS G12D mutants are still in the preclinical or early clinical research stages, and mature and effective clinical targeted therapy strategies are lacking. Although novel KRAS G12D inhibitors, represented by MRTX1133, have shown strong anti-tumor activity in various tumor models, their research and application in intrahepatic cholangiocarcinoma are still in the preliminary exploratory stage. Furthermore, acquired resistance of tumor cells to KRAS G12D inhibitors is a common problem in the clinical treatment of intrahepatic cholangiocarcinoma. This process involves multiple complex mechanisms, including genetic heterogeneity of tumor cells, alterations in drug metabolism and efflux, secondary mutations of drug targets, and regulation of the tumor microenvironment. Current research on the resistance mechanisms of KRAS G12D inhibitors remains relatively limited, and suitable experimental models for systematic studies are lacking.

[0008] A mouse intrahepatic cholangiocarcinoma model can realistically simulate the interaction between mouse immune cells, stromal cells, and intrahepatic bile duct tumors, offering significant advantages for studying the tumorigenesis and development mechanisms of intrahepatic cholangiocarcinoma, particularly the tumor-immune interaction. Among existing technologies, Chinese invention patent application (CN202210824732.7) discloses a mouse intrahepatic cholangiocarcinoma cell line mIC. 22 and its application: This patent describes the construction of a mouse intrahepatic cholangiocarcinoma model by hydrodynamic injection of NICD / AKT plasmid via tail vein, and the isolation and acquisition of mIC 22 cell lines were used, however, these cell lines do not contain KRAS-related targets and are not suitable for KRAS-related research; Chinese invention patent application (CN202410238357.7) discloses a mouse biliary system tumor cell line MBTC. K01 and its applications: This technology induces cholangiocarcinoma in mice carrying the KRAS mutation gene through the AAV system, obtaining the MBTC-K01 cell line of biliary system tumors containing the KRAS mutation. However, the cell line has a complex origin, including various tissue components such as intrahepatic and extrahepatic bile ducts and gallbladder. The model construction cost is high, and it is not suitable for specific research on KRAS G12D intrahepatic cholangiocarcinoma.

[0009] Furthermore, the construction of existing drug-resistant cell lines usually adopts the method of in vitro drug concentration gradient induction. Although this method has the advantages of simple operation and low cost, it is separated from the complex tumor microenvironment in vivo, and the drug exposure mode is significantly different from the in vivo pharmacokinetic characteristics. Therefore, the drug resistance mechanism of the obtained cell line may deviate from the actual clinical situation and cannot fully reflect the real process of tumor drug resistance formation in vivo.

[0010] Therefore, there is an urgent need to construct a mouse cell line for intrahepatic cholangiocarcinoma that can stably maintain the MRTX1133 resistance characteristics. This is of great research value and application prospects for elucidating the molecular mechanism of MRTX1133 resistance, evaluating combination therapy strategies, and exploring the interaction between drug resistance and the tumor immune microenvironment. Summary of the Invention

[0011] To address the lack of experimental models in existing technologies for studying the resistance mechanism of KRAS G12D inhibitors, and the fact that KRAS G12D inhibitor-resistant cholangiocarcinoma cell lines constructed using existing methods for building resistant cell lines cannot fully reflect the actual process of tumor resistance formation in vivo, this invention provides a KRAS G12D inhibitor-resistant cholangiocarcinoma cell line, its construction method, and its applications.

[0012] According to a first aspect of the present invention, a KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR is provided, which was deposited at the China Center for Type Culture Collection on August 20, 2025, with accession number CCTCC NO: C2025240.

[0013] The inventors of this application constructed a spontaneous tumorigenesis mouse model of intrahepatic cholangiocarcinoma carrying the KRAS G12D mutation by injecting a plasmid solution obtained from a mixture of YAP plasmid, AKT plasmid, KRAS G12D plasmid, and SB100 transposase plasmid into C57BL / 6J mice via hydrodynamic high-pressure tail vein injection. By continuously administering the drug to the mice, simulating the occurrence and development of drug resistance during clinical treatment, a tumor model with drug resistance characteristics was successfully obtained. From the tumor tissue of this stably passaged tumor model with KRAS G12D mutation characteristics and acquired resistance to the KRAS G12D inhibitor (MRTX1133), a primary mouse cholangiocarcinoma cell line was isolated and named MuICC-YAK-MR, which is classified as the mouse intrahepatic cholangiocarcinoma cell line MuICC-YAK-MR. The *Musmusculus* cell line, MuICC-YAK-MR, was deposited on August 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: C2025240. This cell line, MuICC-YAK-MR, provides a more reliable and clinically relevant experimental model for elucidating the molecular mechanisms of resistance to the KRAS G12D inhibitor (MRTX1133) in intrahepatic cholangiocarcinoma, as well as for developing targeted drugs, screening clinical treatments, and combining therapy strategies for patients with intrahepatic cholangiocarcinoma.

[0014] According to a second aspect of the present invention, a method for constructing the above-mentioned KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR is provided, comprising the following steps: S1. preparing a plasmid solution using YAP plasmid, AKT plasmid, KRAS G12D plasmid, and SB100 transposase plasmid; S2. preparing an inhibitor solution using KRAS G12D inhibitor; S3. injecting the plasmid solution into mice via hydrodynamic high-pressure tail vein injection, followed by injecting the inhibitor solution into mice via intraperitoneal injection, thereby constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR.

[0015] Preferably, in S1, the mass ratio of YAP plasmid, AKT plasmid, KRAS G12D plasmid, and SB100 transposase plasmid is (0.5~2):(0.5~2):(0.5~2):(0.1~0.5).

[0016] Preferably, the AKT plasmid is pT3-EF1α-HA-myr-Akt, the YAP plasmid is pT3-EF1α-YAPS127A, the KRASG12D plasmid is pT3-EF1α-KRAS G12D, and the SB100 transposase plasmid is PCMV-SB100.

[0017] Preferably, in S2, the inhibitor solution is prepared by the following steps: dissolving the KRAS G12D inhibitor in dimethyl sulfoxide (DMSO) to prepare a clear solution with a concentration of 150-250 mg / mL; sequentially adding polyethylene glycol 300 (PEG 300), Tween 80 (Tween-80), and physiological saline to the clear solution to prepare an inhibitor solution with a final concentration of 15-25 mg / mL, wherein the mass fraction of DMSO in the inhibitor solution is 5-15%, the mass fraction of PEG 300 in the inhibitor solution is 35-45%, and the mass fraction of Tween-80 in the inhibitor solution is 3-7%.

[0018] Preferably, in S2, the KRAS G12D inhibitor is MRTX1133.

[0019] Preferably, in S3, the injection mass of the plasmid solution is 8-12% of the mouse's body weight.

[0020] Preferably, in S3, the inhibitor solution is continuously injected into mice via intraperitoneal injection twice daily, with each injection dose of the inhibitor solution for mice being 10-20 mg / kg.

[0021] Preferably, in S3, the plasmid solution is injected into mice via hydrodynamic high-pressure tail vein injection, and after being fed normally for 5-10 days, the inhibitor solution is injected into the mice via intraperitoneal injection.

[0022] In the construction method of the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR involved in this protocol, YAP, AKT, KRAS G12D, and SB100 plasmids were jointly delivered into the liver of C57BL / 6J mice using hydrodynamic high-pressure tail vein injection to induce spontaneous formation of intrahepatic cholangiocarcinoma. The resulting tumor tissue was collected, paraffin-embedded, and sectioned. Immunohistochemical detection of the cholangiocarcinoma marker keratin 19 (CK19) confirmed the constructed model as an intrahepatic cholangiocarcinoma model. Based on this model, MRTX1133 drug intervention was continuously administered to simulate the development of drug resistance during clinical treatment. The acquired drug-resistant tumor tissue was isolated and cultured to obtain the mouse intrahepatic cholangiocarcinoma drug-resistant cell line MuICC-YAK-MR. This method can obtain drug-resistant cells under conditions of a complete immune system and tumor microenvironment. The method is simple, highly reproducible, and feasible. The obtained cell lines are of a single, well-defined, and genetically stable origin, all derived from KRAS. G12D-mutated intrahepatic cholangiocarcinoma, and the resulting cell line better reflects the true biological characteristics of tumor resistance to KRAS G12D inhibitor (MRTX1133). It can provide a stable, reliable and more clinically relevant experimental model for studying the occurrence and development of KRAS G12D intrahepatic cholangiocarcinoma and the molecular mechanism of KRAS G12D inhibitor (MRTX1133) resistance, drug screening, and combination therapy strategies.

[0023] Furthermore, the mouse intrahepatic cholangiocarcinoma drug-resistant cell line MuICC-YAK-MR, constructed using the method provided in this protocol, can be used to study the sensitivity and resistance mechanisms of KRAS G12D targeted therapy in intrahepatic cholangiocarcinoma, the development of drug resistance reversal strategies, and the interaction between the tumor microenvironment (including immune cells and stromal cells) and drug resistance formation.

[0024] According to a third aspect of the present invention, the application of the above-mentioned KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR in cholangiocarcinoma drug resistance mechanisms, cholangiocarcinoma drug resistance reversal strategies, cholangiocarcinoma targeted therapy drugs, and cholangiocarcinoma clinical treatment drug screening studies is provided.

[0025] According to a fourth aspect of the present invention, the application of the above-mentioned KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR in the study of the interaction between cholangiocarcinoma immunity, matrix microenvironment and drug tolerance is provided.

[0026] The KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR provided by this invention can be applied to the study of drug sensitivity and acquired resistance mechanisms and drug reversal strategies of intrahepatic cholangiocarcinoma to KRAS-targeted inhibitors, the evaluation of the efficacy of KRAS G12D-targeted therapy for intrahepatic cholangiocarcinoma, and the study of the interaction between tumor immunity, matrix microenvironment and drug tolerance. Attached Figure Description

[0027] Figure 1 shows the structures of the YAP plasmid, AKT plasmid, KRAS G12D plasmid and SB100 transposase plasmid provided in Example 1.

[0028] Figure 2 shows the construction method of the KRAS G12D primary intrahepatic cholangiocarcinoma mouse model and the MRTX1133 in vivo drug resistance model provided in Example 1.

[0029] Figure 3 shows the tumor tissue observation results, HE staining, and CK19 immunohistochemical staining results of the YAK mouse model provided in Example 1.

[0030] Figure 4 shows the survival curves of mice in the control group and the MRTX1133-treated group provided in Example 1.

[0031] Figure 5 shows the cell growth morphology observation results of the cell lines MuICC-YAK and MuICC-YAK-MR provided in Example 2.

[0032] Figure 6 shows the cell viability curves of the cell lines MuICC-YAK and MuICC-YAK-MR provided in Example 3 under different concentration gradients of MRTX1133.

[0033] Figure 7 shows the STR typing map of the cell line MuICC-YAK-MRSTR provided in Example 4.

[0034] Figure 8 shows the STR identification results of the cell line MuICC-YAK-MRSTR provided in Example 4.

[0035] Figure 9 shows the observation results of tumor tissue after subcutaneous tumor formation in male C57BL / 6J mice by the MuICC-YAK-MR cell line provided in Example 5, as well as the results of HE staining, CK19 and α-SMA immunohistochemical staining. Detailed Implementation

[0036] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Construction of a KRAS G12D Primary Intrahepatic Cholangiocarcinoma Mouse Model, Evaluation of MRTX1133 Efficacy, and Construction of an In Vivo Drug Resistance Model. This example aims to construct a KRAS G12D primary intrahepatic cholangiocarcinoma mouse model, evaluate the efficacy of MRTX1133, and construct an in vivo drug resistance model. The specific experimental steps are as follows: 1. Construct expression vectors containing the YAP gene, AKT gene, KRAS G12D gene, and SB100 gene, respectively. The obtained expression vectors are named YAP plasmid (pT3-EF1α-YAPS127A), AKT plasmid (pT3-EF1α-HA-myr-Akt), and KRAS G12D plasmid (pT3-EF1α-KRAS1133), respectively. The structures of the G12D and SB100 transposase plasmids (PCMV-SB100) are shown in Figure 1. In Figure 1, A represents the structure of the YAP plasmid, B represents the structure of the AKT plasmid, C represents the structure of the KRAS G12D plasmid, and D represents the structure of the SB100 transposase plasmid. All plasmids—YAP, AKT, KRAS G12D, and SB100—use the pT3 plasmid backbone and the EF1α promoter to construct the target genes YAP, Akt, and KRAS, respectively. The expression vectors obtained from the G12D and SB100 transposase genes, the AKT plasmid (pT3-EF1α-HA-myr-Akt) contains the hemagglutinin tag HA, the myristic acylation signal sequence myr, and the Luciferase sequence, while the YAP plasmid (pT3-EF1α-YAPS127A) and the KRASG12D plasmid (pT3-EF1α-KRAS G12D) do not contain the above sequences found in the AKT plasmid.

[0038] 2. Dissolve 8 μg YAP plasmid, 8 μg AKT plasmid, 8 μg KRAS G12D plasmid and 2.4 μg SB100 transposase plasmid in 2 mL physiological saline (0.9% NaCl) to prepare a plasmid solution for injection.

[0039] 3. Construction of KRAS G12D primary intrahepatic cholangiocarcinoma mouse model and evaluation of MRTX1133 efficacy and in vivo drug resistance model. Refer to Figure 2 to construct the KRAS G12D primary intrahepatic cholangiocarcinoma mouse model and the MRTX1133 in vivo drug resistance model.

[0040] (1) Construction of KRAS G12D primary intrahepatic cholangiocarcinoma mouse model: 4-5 week old C57BL / 6J mice weighing about 18-20 grams were selected. High-pressure hydrodynamic injection technology (injection time is very short, less than 10 seconds, rapid intravenous injection of a large volume of liquid) was used to inject the prepared plasmid solution into the mice via the tail vein at a dose of 10% of the mouse body weight to construct spontaneously tumorigenic KRAS. In the G12D primary intrahepatic cholangiocarcinoma mouse model (hereinafter referred to as "YAK mouse model"), mice were sacrificed 30 days after injection of plasmid solution. Extensive tumor nodules were observed on the liver surface. Tumor tissue was taken from the tumor nodules, paraffin-embedded, and sectioned. Hematoxylin-eosin (HE) staining and CK19 immunohistochemical staining were performed, respectively. The results are shown in Figure 3. In Figure 3, A shows the tumor tissue observation results of the YAK mouse model, B shows the HE staining results of the tumor tissue of the YAK mouse model, and C shows the CK19 immunohistochemical staining results of the tumor tissue of the YAK mouse model.

[0041] As shown in Figure 3, the tumors in the YAK mouse model exhibited typical irregular glandular structures with significant fibrous stroma hyperplasia, demonstrating high cellular atypia. The above histopathological analysis (HE staining) results showed that the tumor tissues of the YAK mouse model had typical tumor pathological features and were positive for CK19 expression. These results indicate that the YAK mouse model constructed in this embodiment is a spontaneous tumorigenesis model specific to intrahepatic cholangiocarcinoma.

[0042] (2) Evaluation of the efficacy of MRTX1133 and construction of an in vivo drug resistance model. Based on the above-mentioned YAK mouse model, MRTX1133 was administered in vivo starting on day 7 after the tail vein injection of the plasmid solution. The specific method was as follows: MRTX1133 was dissolved in DMSO to prepare a clear solution with a concentration of 200 mg / mL. PEG 300, Tween-80 and physiological saline were added to the clear solution in sequence to prepare an inhibitor solution with a final concentration of 20 mg / mL (the mass fractions of each component in the inhibitor solution were as follows: 10 wt% DMSO, 40 wt% PEG 300, 5 wt% Tween-80, 45 wt% physiological saline). One week after the plasmid solution was injected into the mice via the tail vein using high-pressure hydrodynamic technology, the mice were randomly divided into a control group and an MRTX1133 administration group. The inhibitor solution was continuously injected into the mice in the MRTX1133 administration group via intraperitoneal injection. The injection dose of the inhibitor solution to the mice was 15 mg / mL. Mice were administered mg / kg twice daily, while control mice were intraperitoneally injected with an equal volume of a mixed solvent (excluding MRTX1133, prepared by mixing 10 wt% DMSO, 40 wt% PEG 300, 5 wt% Tween-80, and 45 wt% physiological saline). The continuous administration period was until the experiment ended, to simulate the process of drug resistance development during clinical treatment. The survival time and tumor growth of mice in the control group and the MRTX1133 administration group were compared to evaluate the antitumor activity of MRTX1133 and the construction of the MRTX1133 drug resistance model. The results are shown in Figure 4.

[0043] As shown in Figure 4, compared with the control group mice, the survival time of the MRTX1133-treated group mice was significantly prolonged. However, as the tumors developed drug resistance, the tumor progression in the MRTX1133-treated group mice reached the experimental endpoint.

[0044] Example 2 Isolation of primary mouse cholangiocarcinoma cell lines MuICC-YAK and MuICC-YAK-MR After the control group and MRTX1133-treated group mice in Example 1 reached the experimental endpoint, tumor tissues were taken from the control group and MRTX1133-treated group mice, respectively. The tumor tissues were digested and mechanically separated in a clean bench. Single-cell suspensions were prepared from the digested and separated tissue blocks and cultured in vitro. The specific experimental steps are as follows: 1. The tumor tissue was aseptically washed and chopped into 1-2 mm pieces. 3The tumor tissue was sized and then placed in a buffer solution containing antibiotics, 1 mg / mL collagenase II and 1 mg / mL collagenase IV and digested at 37°C for 45 minutes to obtain a digestion solution. The digestion solution was filtered through a 100 μm filter to remove incompletely digested tissue pieces. Then, the red blood cells were lysed to remove them, resulting in a single-cell suspension of the tumor tissue. The single-cell suspension was centrifuged at 300 g for 5 minutes to obtain a cell pellet.

[0045] 2. Prepare mouse primary tumor cell culture medium by adding the following components to Advanced DMEM / F12 medium: 1.6 mg / mL bovine serum albumin (BSA), 10 mM nicotinamide, 50 ng / mL epidermal growth factor (EGF), 150 nM all-trans retinoic acid (ATRA), 150 nM dexamethasone, 10.5 μM selective ROCK1 inhibitor Y27632, 100 μg / mL primary cell antibiotic (Primocin), and 2 mM glutamine.

[0046] 3. After resuspending the cell pellet in the primary tumor cell culture medium prepared above, a single-cell suspension was obtained. After cell counting, approximately 100,000 cells were seeded into 6-well plates for in vitro expansion culture. Subsequently, through continuous passage (passing every two days) and selection of single clones, two stable passaged mouse intrahepatic cholangiocarcinoma cell lines were finally obtained and named MuICC-YAK and MuICC-YAK-MR (drug-resistant strain), respectively. The MuICC-YAK cell line was isolated and screened from the tumor tissue of control mice, while the MuICC-YAK-MR cell line (drug-resistant strain) was isolated and screened from mice in the MRTX1133 administration group. The MuICC-YAK-MR cell line was deposited on August 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC. NO: C2025240. The cell morphology of the two cell lines, MuICC-YAK and MuICC-YAK-MR, was observed under a microscope. The results are shown in Figure 5, where the scale bar is 200 μm.

[0047] As shown in Figure 5, both the MuICC-YAK and MuICC-YAK-MR cell lines adhered to the wall and exhibited an elliptical cell morphology.

[0048] Example 3 In vitro drug sensitivity test This example aims to study the in vitro drug resistance sensitivity of the mouse intrahepatic cholangiocarcinoma cell lines MuICC-YAK and MuICC-YAK-MR obtained in Example 2. The dose-gradient inhibitory effect of the KRAS G12D inhibitor (MRTX1133) on the cell proliferation of MuICC-YAK and MuICC-YAK-MR was detected using a CCK-8 assay kit. The specific experimental procedures are as follows: MuICC-YAK and MuICC-YAK-MR cell lines were seeded in 96-well plates and cultured. Different concentrations (nM) of MRTX1133 were set up and different concentration gradients of MRTX1133 were added to the culture system. After culturing for 48 hours, the cell viability (%) was detected. The results are shown in Figure 6.

[0049] As shown in Figure 6, the cell viability of both MuICC-YAK and MuICC-YAK-MR cell lines decreased with the increase of MRTX1133 concentration in the culture system.

[0050] Furthermore, the half-maximal inhibitory concentration (IC50) of the two cell lines MuICC-YAK and MuICC-YAK-MR against MRTX1133 was calculated using dose-response curves, and the results are shown in Table 1.

[0051] Table 1. Calculation results of the half-maximal inhibitory concentration (IC50) of MRTX1133 by the MuICC-YAK and MuICC-YAK-MR cell lines.

[0052] As shown in Table 1, the half-maximal inhibitory concentrations (IC50 values) of the two cell lines, MuICC-YAK and MuICC-YAK-MR, against MRTX1133 were 184.3 nM and 2333 nM, respectively. The resistance index (RI) of the MuICC-YAK-MR cell line was calculated to be 12.66. These results indicate that the MRTX1133-resistant strain MuICC-YAK-MR cell line was successfully constructed.

[0053] Example 4: STR Identification of the MuICC-YAK-MR Cell Line. STR gene loci consist of short tandem repeats of 3–7 base pairs in length. These repeat sequences are widely distributed in the human genome and can serve as highly polymorphic markers, detectable by PCR (polymerase chain reaction). Alleles at STR gene loci can be distinguished by differences in the copy number of repeat sequences within the amplified region, and can be identified by fluorescence detection after capillary electrophoresis separation. Subsequently, using a specific calculation method, the cell line to which the sample belongs or the name of any potentially cross-contaminated cell lines can be deduced by comparing the obtained STR typing results with a professional cell STR database.

[0054] This embodiment aims to identify the constructed and screened mouse intrahepatic cholangiocarcinoma cell line MuICC-YAK-MR using STR typing technology to determine whether it has been contaminated by other cell lines and the names of any potentially cross-contaminated cell lines. The specific experimental steps are as follows: MuICC-YAK-MR cell line was digested with trypsin, and the cell pellet was collected. Whole-genome DNA was extracted from MuICC-YAK-MR cell line using a genomic DNA extraction kit (Aige Biotechnology). DNA concentration was detected using a micro-UV-Vis spectrophotometer (Thermo Fisher NanoDrop 2000c) with QC. Simultaneously, STR multiplex fluorescence amplification was performed using an STR-MUS typing kit (ABI 9700 PCR System), and the corresponding signals were detected. Capillary electrophoresis and fragment separation were performed using an ABI 3730XL genetic analyzer (ABI Corporation, USA), and GeneMapper® ID software (version ID-X) was used. 1.5) Genotyping was performed, and the STR typing results of the cell line MuICC-YAK-MR were obtained after detecting the corresponding signals. The results are shown in Figure 7, Figure 8 and Table 2. Figure 7 is the STR typing map of the cell line MuICC-YAK-MRSTR, Figure 8 is the STR identification results of the cell line MuICC-YAK-MRSTR, and Table 2 is the STR genotyping results of the cell line MuICC-YAK-MRSTR. Alleles represents the chromosomal location of the detection site, and X is the sex chromosome X.

[0055] It should be noted that in mouse STR genotyping, STR loci are usually named using a numbered system (such as 18-3, 4-2, 6-7, etc.). This numbering is an internal definition of a specific STR marker in the STR-MUS system, used to distinguish different polymorphic STR amplification sites. Each site corresponds to a fixed STR region in the mouse genome, and its alleles are represented by the copy number of the repetitive sequence (or the corresponding fragment length), used for cell line identification and contamination screening. The mouse STR loci (such as 18-3, 4-2, 6-7, etc.) mentioned in Table 2 are numbered STR markers defined in the STR-MUS system, used to distinguish different mouse STR polymorphic sites.

[0056] Table 2. STR genotyping results of the MuICC-YAK-MRSTR cell line.

[0057] As shown in Figures 7 and 8 and Table 2, after extracting the whole genome DNA of the MuICC-YAK-MR cell line, the results showed that no site had more than two allele peaks at any of the eight STR sites detected, meaning no multiple allele sites were found. These results indicate that the MuICC-YAK-MR cell line is a single cell and there is no contamination from other cells.

[0058] According to the US National Standard ASN-0002-2011, a cell line with a matching rate of not less than 80% may be the cell line to which the tested cell belongs, or a derivative of that cell line, or it may originate from the same donor as that cell line; a cell line with a matching rate of less than 56% is generally considered to be unrelated to the tested cell; and a cell line with a matching rate between 56% and 80% requires further study to confirm its identity.

[0059] This embodiment also compares the cell line MuICC-YAK-MR with cell lines in the Aiji Bio mouse cell line identification database. The results are shown in Table 3. Table 3 lists the five cell lines with the highest percent match rate (Percent Match) with the MuICC-YAK-MR cell line in the database. Among them, the two alleles Alleles 1 and Alleles 2 in the STR locus are separated by ","; NA means "Not Applicable", which means that there is no reference STR type profile for this cell line in the database; "N°=Number of" is the French / international standard notation.

[0060] Table 3. Comparison results of cell line MuICC-YAK-MR with the Aiji Bio mouse cell line identification database

[0061] As shown in Table 3, the cell line MuICC-YAK-MRSTR is not included in the databases of the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Japan Research Biobank (JCRB), and the RIKEN Institute (RIKEN). No matching sites were found in these databases, indicating that MuICC-YAK-MRSTR is a novel cell line.

[0062] Example 5: In vivo tumorigenicity and histological characteristics of the MuICC-YAK-MRSTR cell line. This example aims to detect the in vivo tumorigenicity and histological characteristics of the MuICC-YAK-MRSTR cell line. The specific experimental procedures are as follows: 1. In vivo tumorigenicity detection: The logarithmically growing MuICC-YAK-MR cell line was digested and centrifuged, and the cell density was adjusted to obtain a cell pellet. The pellet was then mixed with sterile phosphate-buffered saline (PBS) and Matrigel at a mass ratio of 1:1. The cell pellet was resuspended in 100 μL of the mixture and then subjected to a 2×10⁻⁶ folding process. 6 One cell was injected subcutaneously into 5-week-old male C57BL / 6J mice.

[0063] 2. Histological and Immunohistochemical Analysis: On day 30 after subcutaneous cell inoculation in male C57BL / 6J mice, the mice were sacrificed and subcutaneous tumor tissue was collected. The tumor tissue was embedded, sectioned, and stained with hematoxylin and eosin (HE). The structure of the tumor tissue was observed under an optical microscope. Simultaneously, the tumor tissue was subjected to immunohistochemical staining for CK19 and α-SMA. The results are shown in Figure 9. In Figure 9, A is the observation result of the mouse subcutaneous tumor tissue, B is the HE staining result of the mouse subcutaneous tumor tissue, C is the CK19 immunohistochemical staining result of the mouse subcutaneous tumor tissue, and D is the α-SMA immunohistochemical staining result of the mouse subcutaneous tumor tissue.

[0064] As shown in Figure 9, after subcutaneously inoculating the MuICC-YAK-MR cell line into male C57BL / 6J mice for 30 days, the subcutaneous tumor tissue exhibited the typical nest-like aggregation growth structure of cholangiocarcinoma. Furthermore, the subcutaneous tumor cells expressed the epithelial marker CK19 (i.e., the tumor tissue showed a significant positive CK19 characteristic marker of cholangiocarcinoma) and widely distributed α-SMA-positive fibroblasts (indicating tumor matrix components), with abundant infiltration of α-SMA-positive tumor-associated fibroblasts. These results demonstrate that the MuICC-YAK-MR cell line can stably form tumors in C57BL / 6J mice and maintain the clinical histological characteristics and tumor microenvironment composition of cholangiocarcinoma, demonstrating good in vivo research application value.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A KRAS G12D inhibitor-resistant cholangiocarcinoma cell line, MuICC-YAK-MR, was deposited at the China Center for Type Culture Collection on August 20, 2025, with accession number CCTCC NO: C2025240.

2. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 1, characterized in that, Includes the following steps: S1. A plasmid solution was prepared using YAP plasmid, AKT plasmid, KRAS G12D plasmid, and SB100 transposase plasmid; S2. An inhibitor solution was prepared using a KRAS G12D inhibitor; S3. The plasmid solution was injected into mice via hydrodynamic high-pressure tail vein injection, followed by intraperitoneal injection of the inhibitor solution into mice to construct the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR.

3. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that: In S1, the mass ratio of the YAP plasmid, the AKT plasmid, the KRAS G12D plasmid, and the SB100 transposase plasmid is (0.5~2):(0.5~2):(0.5~2):(0.1~0.5).

4. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that: The YAP plasmid is pT3-EF1α-YAPS127A, the AKT plasmid is pT3-EF1α-HA-myr-Akt, the KRAS G12D plasmid is pT3-EF1α-KRAS G12D, and the SB100 transposase plasmid is PCMV-SB100.

5. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that, In step S2, the inhibitor solution is prepared by the following steps: dissolving the KRAS G12D inhibitor in DMSO to prepare a clear solution with a concentration of 150-250 mg / mL; sequentially adding PEG300, Tween-80, and physiological saline to the clear solution to prepare an inhibitor solution with a final concentration of 15-25 mg / mL, wherein the mass fraction of DMSO in the inhibitor solution is 5-15%, the mass fraction of PEG300 in the inhibitor solution is 35-45%, and the mass fraction of Tween-80 in the inhibitor solution is 3-7%.

6. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that: In S2, the KRAS G12D inhibitor is MRTX1133.

7. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that: In S3, the injection mass of the plasmid solution is 8-12% of the mouse's body weight, and / or, in S3, the inhibitor solution is continuously injected into the mouse via intraperitoneal injection twice daily, with each injection dose of the inhibitor solution to the mouse being 10-20 mg / kg.

8. The method for constructing the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 2, characterized in that: In step S3, the plasmid solution is injected into mice via hydrodynamic high-pressure tail vein injection. After being fed normally for 5-10 days, the inhibitor solution is injected into the mice via intraperitoneal injection.

9. The application of the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 1 in the study of cholangiocarcinoma drug resistance mechanisms, cholangiocarcinoma drug resistance reversal strategies, cholangiocarcinoma targeted therapy drug research, and cholangiocarcinoma clinical treatment drug screening research.

10. The application of the KRAS G12D inhibitor-resistant cholangiocarcinoma cell line MuICC-YAK-MR as described in claim 1 in the study of the interaction between cholangiocarcinoma immunity, matrix microenvironment, and drug tolerance.

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