Novel human-derived distal bile duct cancer cell line with TP53 missense mutation and application of novel human-derived distal bile duct cancer cell line

Abstract

The invention provides a novel human distal bile duct cancer cell line with TP53 missense mutation and application, the novel human distal bile duct cancer cell line CBC3T-3 is established, the cell line is preserved in the China Center for Type Culture Collection (the preservation number is CCTCC NO: C202555), the uniqueness and stability of the cell line are proved through STR typing and karyotype analysis, and the TP53 missense mutation novel human distal bile duct cancer cell line has the advantages that the TP53 missense mutation novel human distal bile duct cancer cell line CBC3T-3 can be used for preparing the TP53 missense mutation novel human distal bile duct cancer cell line CBC3T-3; and a plurality of driver gene mutations including TP53 missense mutation are carried. The CBC3T-3 has strong proliferation, invasion and migration capabilities, has high tumor formation rate in immunodeficient mice, is resistant to cis-platinum and sensitive to paclitaxel and gemcitabine, and provides an experimental basis for selection of clinical chemotherapy regimens. According to the model, the TP53 missense mutation type distal bile duct cancer in-vitro model is successfully established, and a key experimental platform is provided for deeply researching the drug resistance mechanism of the TP53 missense mutation type distal bile duct cancer and developing an individualized treatment strategy aiming at the subtype of the TP53 missense mutation type distal bile duct cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine and biology, and particularly relates to a novel human distal cholangiocarcinoma cell line with TP53 missense mutation and application thereof. BACKGROUND

[0002] Distal cholangiocarcinoma, originating from the distal 1 / 3 segment of extrahepatic bile duct, is an important subtype of cholangiocarcinoma, characterized by insidious onset, rapid progression and extremely poor prognosis. Globally, its incidence is increasing year by year, and the disease burden is even heavier in East Asia due to the prevalence of high-risk factors such as viral hepatitis and biliary stones. Due to the lack of specific clinical symptoms in the early stage, most patients are diagnosed at the local advanced stage or with distant metastasis, missing the opportunity for radical surgery, and the 5-year overall survival rate is less than 10%, which is one of the worst types of digestive system malignancies.

[0003] Currently, the clinical diagnosis and treatment of distal cholangiocarcinoma still faces multiple bottlenecks. In the aspect of diagnosis, imaging examinations (such as MRI and CT) can preliminarily locate the bile duct occupation, but it is difficult to accurately distinguish the tumor benignity and pathology subtype; the sensitivity and specificity of serum tumor markers (such as CA199) are limited, and about 20% of patients show false negative results due to Lewis antigen negative, resulting in high misdiagnosis and missed diagnosis rates. In terms of treatment, surgical resection (such as pancreaticoduodenectomy) is the only possible cure, but the operation is difficult, the complication rate is high, and only about 30% of patients meet the surgical indications. For patients who cannot undergo surgery, gemcitabine-based combination chemotherapy (such as gemcitabine + cisplatin, gemcitabine + capecitabine) is the standard treatment regimen, but its objective response rate is less than 30%, and patients are prone to develop resistance in a short period, with a median progression-free survival of only 6-8 months, and more effective treatment strategies are urgently needed.

[0004] Tumor cell lines, as the core model of in vitro research, play an irreplaceable role in revealing the pathogenesis of tumors, screening targeted drugs, and exploring drug resistance mechanisms. At present, the established human cholangiocarcinoma cell lines (such as TFK-1, HuCCT1) are mostly derived from upper bile duct or intrahepatic cholangiocarcinoma, and mostly carry a single gene mutation (such as KRAS, IDH1), which is significantly different from the complex gene mutation spectrum (especially TP53 missense mutation) commonly found in patients with distal cholangiocarcinoma, making it difficult to effectively translate the research results based on existing cell lines to the clinic. TP53 gene, as the "genome guardian", its mutation occurs in 40%-60% of distal cholangiocarcinoma patients, and patients with multiple missense mutations in the same gene often show stronger invasiveness, higher chemotherapy resistance and poorer prognosis. However, there is currently no stable human TP53 missense mutation distal cholangiocarcinoma cell line worldwide, which greatly limits the research on the molecular mechanisms of this subtype of tumor and the development of precision treatment drugs.

[0005] In addition, the existing cholangiocarcinoma cell lines also have the following limitations: part of the cell lines have genetic background drift due to long-term passage, resulting in significant difference between their biological characteristics and primary tumors; part of the cell lines lack the ability to form tumors in vivo or have low tumor formation rate, and cannot construct reliable animal models to simulate the clinical tumor growth and metastasis process; at the same time, most of the cell lines have not been systematically identified by STR typing and chromosome karyotype analysis, and there is a risk of cell cross contamination or unknown identity, which affects the reliability and repeatability of the research results. Therefore, it is a key requirement to establish a cell line with clear origin, clear genetic background, carrying TP53 missense mutation and retaining the biological characteristics of primary tumor, which has become a key requirement to break through the current bottleneck of distal cholangiocarcinoma research and promote the development of precision medicine. SUMMARY

[0006] In view of the core bottleneck that there is a lack of in vitro research model that can accurately simulate the clinical high-risk subtype (especially TP53 missense mutation type) in the current distal cholangiocarcinoma research field, the present application provides a new human distal cholangiocarcinoma cell line with TP53 missense mutation and application, a new human cell line named CBC3T-3 is established and systematically identified, and the pathological characteristics, highly heterogeneous genomic map and malignant phenotype of high invasion and easy drug resistance of the primary tumor are completely retained, which reveals the molecular mechanism of TP53 missense mutation driving tumor progression and chemotherapy resistance (especially cisplatin resistance), screens and verifies the effective treatment strategy for the high-risk subtype, and is expected to greatly promote the development of precision medicine for distal cholangiocarcinoma.

[0007] In one aspect, the present application provides a new human distal cholangiocarcinoma cell line with TP53 missense mutation, which is named CBC3T-3 cell line, the cell line is preserved in China Center for Type Culture Collection (CCTCC NO: C202555), and the genome of the cell line contains multiple missense mutations of TP53 gene; and the cell line has a near triploid karyotype.

[0008] Preferably, the missense mutation causes the 35th, 62nd, 155th and 194th leucine (L) of the encoded protein to be replaced by phenylalanine (F), respectively, to obtain p.L35F, p.L62F, p.L155F and p.L194F.

[0009] Preferably, the STR genotyping data of the CBC3T-3 cell line includes: allele D18S51 is 14, 14; D6S1043 is 12, 12; AMEL is X, Y.

[0010] Preferably, the CBC3T-3 cell line grows in a manner of adherent cluster when cultured in vitro, the cell morphology is fusiform or polygonal, and the cell has a high nuclear-cytoplasmic ratio; under transmission electron microscopy, swollen mitochondria, broken cristae, aggregated heterochromatin and desmosome structures can be observed.

[0011] Preferably, the separation and purification process of the CBC3T-3 cell line requires 3 months and the cell line is subcultured for more than 50 times.

[0012] Preferably, the CBC3T-3 cell line is identified by STR technology.

[0013] Preferably, the STR technology uses 21 alleles of the CBC3T-3 cell line for detection and analysis on an Applied Biosystems 3730XL genetic analyzer.

[0014] Preferably, the 21 alleles include CSF1PO, D5S818, D19S433, D21S11, D18S51, PentaD, AMEL, vWA, D3S1358, D6S1043, D13S317, D7S820, D16S539, D8S1179, D2S441, TPOX, PentaE, TH01, D12S391, D2S1338 and FGA genes.

[0015] Another aspect of the present application provides a novel human distal cholangiocarcinoma cell line with TP53 missense mutation, and the application includes the use in constructing a cholangiocarcinoma animal xenograft model.

[0016] Preferably, the application further includes an experiment of TP53 missense mutation leading to a cholangiocarcinoma chemotherapy resistance mechanism.

[0017] The embodiment of the present application has the following beneficial effects:

[0018] (1) A highly clinically relevant and rare research model is provided: the cell line is directly derived from the primary tumor of a Chinese distal cholangiocarcinoma patient, perfectly reproducing the complex genomic characteristics (such as TP53 missense mutation) and malignant phenotype (high invasion and high metastasis) of this disease subtype, and solving the long-term bottleneck problem of lack of high-quality and clinically relevant in vitro models in this research field.

[0019] (2) Provide a unique platform for studying the carcinogenic mechanism of TP53 missense mutations: CBC3T-3 cell line is the first reported worldwide cholangiocarcinoma model stably carrying multiple TP53 missense mutations (p.L62F; p.L35F; p.L155F; p.L194F); To further explore how these special mutations synergize to completely inactivate TP53 function and further drive tumor occurrence, proliferation, invasion and drug resistance, it provides an invaluable cell tool with important theoretical value. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The clinical pathological features of the patient in Example 1 of the present application are shown in Table 1, wherein, Figure 1 A is the laboratory examination result of CBC3T-3; Figure 1 B is the patient's clinical information feature, imaging examination; Figure 1 C is the white and dense tumor tissue at the lower end of the bile duct seen during surgery, Figure 1 D is the chromosome karyotype analysis map.

[0021] Figure 2 The cell morphology of CBC3T-3 in Test Example (1-2) of the present application is shown in Table 2, wherein, Figure 2 A is the morphology of the cells under an optical microscope, magnified by 40 times and 100 times, respectively; Figure 2 B is the image of the cell surface shown by a scanning electron microscope, with red arrows pointing to the tight junctions between cells; Figure 2 C is the image of CBC3T-3 cells shown by a transmission electron microscope, with yellow arrows pointing to secretory granules, blue arrows pointing to heterochromatin aggregation, red arrows pointing to swollen mitochondria and broken cristae, black arrows pointing to mitochondrial vacuoles, and green arrows pointing to desmosome structures.

[0022] Figure 3 The genomic features of CBC3T-3 cells in Test Example (3) of the present application are shown in Table 3, wherein, Figure 3 A is the distribution result of somatic SNV detection on different regions of the genome; Figure 3 B is the distribution result of somatic InDel detection on different regions of the genome; Figure 3 C is the tumor mutation burden result of CBC3T-3 cells; Figure 3 D is the analysis of 50 random points of single sample clonal structure: the horizontal coordinate is the tumor cell frequency of mutation, the vertical coordinate is the gene where the mutation site is located; the cell frequency of mutation is represented by red, the closer the value is to 1, the more it indicates a primary clonal mutation, and the smaller the value is, the more it indicates a subclonal mutation.

[0023] Figure 4 The growth characteristics and in vivo tumorigenicity of CBC3T-3 cells in Test Example (4) of the present application are shown in Table 4, wherein, Figure 4A is the growth curve of CBC3T-3 cells; Figure 4 B is the invasion and migration image collection of CBC3T-3 and TFK-1 cells and its statistical chart, scale, 200 μm; Figure 4 C is the subcutaneous tumor image collection of CBC3T-3 cell xenograft tumor model; Figure 4 D is the growth curve of mouse subcutaneous transplanted tumor; Figure 4 E is the weight change curve of mice; Figure 4 F is the immunohistochemical results of patient primary tumor, mouse subcutaneous transplanted tumor and CBC3T-3, scale, 50 μm.

[0024] Figure 5 A is the drug sensitivity of CBC3T-3 cells in the test example (5) of the present application, Figure 5 A is the cell viability value of CBC3T-3 cells exposed to different concentrations of chemotherapeutic drugs for 48 h and IC50; Figure 5 B is NovoDR drug resistance mutation screening; Figure 5 C is the cell viability value of TFK-1 cells exposed to different concentrations of cisplatin for 48 h and IC50. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. In specific embodiments, the CBC3T-3 cell line was deposited with the China Center for Type Culture Collection on January 20, 2025, and the classification and naming is: human distal bile duct cancer cell line CBC3T-3 Homo sapiens, address: Wuhan University Preservation Center, Wuchang District, Wuhan City, Hubei Province; the preservation number is CCTCC NO: C202555.

[0026] Example 1

[0027] Construction of CBC3T-3 cell line

[0028] (1) A 54-year-old male patient was admitted to the Department of General Surgery, the First Hospital of Lanzhou University in 2022 due to "abdominal pain, abdominal distension with skin and sclera jaundice, skin itching for more than 1 month". As Figure 1A shows: Laboratory examination showed that serum tumor marker CA199 was significantly elevated (109.0 U / mL, reference value: 0-35 U / mL); liver function tests suggested obstructive jaundice, including AST 380 U / L (reference value: 15-40 U / L), ALT 310 U / L (reference value: 9-50 U / L), ALP 195 U / L (reference value: 10-60 U / L) and TBIL 131.2 μmol / L (reference value: 0-23 μmol / L); as Figure 1 B shows: Imaging examination (abdominal MRI and CT) showed lower bile duct space-occupying lesions, considering malignant tumor. The patient had a 10-year history of type 2 diabetes mellitus, had received bladder tumor resection, and had no related family history of genetic diseases. After multidisciplinary consultation, pancreaticoduodenectomy was performed, as Figure 1 C shows: Intraoperative view of the lower end of the bile duct with white dense tumor tissue. Pathologically diagnosed as moderately to poorly differentiated cholangiocarcinoma (AJCC-pTNM stage: T2N0Mx), postoperative gemcitabine combined with capecitabine adjuvant chemotherapy regimen, the patient recovered well. This study was approved by the Medical Ethics Committee of the First Hospital of Lanzhou University (LDYYLL2025-954) and written informed consent was obtained from the patient.

[0029] (2) Collect fresh sterile bile duct cancer primary lesion tissue during surgery, immediately place in 10% double-antibiotic (penicillin-streptomycin) pre-cooled DMEM / F-12 (Gibco, USA) culture medium to maintain tissue activity. Rinse with 10% double-antibiotic PBS for 3 times, remove fibrous envelope, fat and visible blood vessels with sterile forceps. Cut the tissue into 1-2 mm 3 tissue blocks, transfer to DMEM / F-12 containing 200U collagenase II for 10 min to obtain single cell suspension; evenly inoculate the single cell suspension in a 6-well plate, replace the medium after 48h and observe the growth state of the cells. Remove fibroblasts using mechanical scraping method, and use differential digestion method to separate and purify tumor cells when the cell confluence reaches 80%.

[0030] (3) Cell culture

[0031] 1) Experimental preparation: Human extrahepatic bile duct cancer cell line TFK-1 was purchased from China National Biomedical Experimental Cell Resource Bank (Beijing, China); culture medium: RPMI 1640 (BI, Israel); 10% fetal bovine serum (ABW, USA); 1% Penicillin-Streptomycin Solution (BI, Israel); cell culture box environment: 37℃, 5% CO2 humidified incubator.

[0032] 2) In the super-clean bench, add 10% volume of fetal bovine serum and 1% volume of Penicillin-Streptomycin Solution solution to RPMI-1640 basic medium, mix well. Put the prepared complete medium into a 37°C water bath for preheating.

[0033] 3) Cell recovery:

[0034] a. Thawing: quickly take the frozen TFK-1 cell cryopreservation tube from the liquid nitrogen tank and immediately put it into a 37°C water bath, gently shake it without stopping, so that it completely melts within 1 minute;

[0035] b. Transfer and dilution: after thoroughly spraying and wiping the outer wall of the cryopreservation tube with 75% ethanol, move it into the super-clean bench; use a sterile pipette to slowly transfer the cell suspension into a 15 mL centrifuge tube containing 5-7 mL of preheated medium, gently blow and mix;

[0036] c. Centrifugation: centrifuge at 1000 rpm for 5 min to precipitate the cells;

[0037] d. Resuspension: carefully discard the supernatant, avoid touching the cell precipitate, add 1 mL of fresh complete medium to the centrifuge tube, gently resuspend the cells, transfer the cell suspension to the culture bottle (containing 5 mL of medium), shake the culture bottle crosswise before and after, so that the cells are evenly distributed;

[0038] e. Culture: tightly cover the bottle cap, put it into a 37°C, 5% CO2 incubator for static culture.

[0039] 4) Medium replacement and subculture: when the cell confluence reaches 80-90%, perform cell subculture.

[0040] (4) STR identification: The cell strain was subjected to STR typing identification by China Typical Culture Collection Center (Wuhan, China), and at least 13 detection sites were compared according to the identification standard of ANSI / ATCC ASN-0002-2021 to confirm the cell identity; first, the DNA of CBC3T-3 cells was extracted, and the 21 alleles (CSF1PO, D5S818, D19S433, D21S11, D18S51, Penta D, AMEL, vWA, D3S1358, D6S1043, D13S317, D7S820, D16S539, D8S1179, D2S441, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA) of CBC3T-3 cells were amplified by using the STR amplification scheme, and the 21 alleles were detected and analyzed on an Applied Biosystems 3730XL genetic analyzer; by comparing the STR data in the ATCC, DSMZ and CELLOSAURUS databases, the matching degree of the new cell line's profile with the existing data was determined.

[0041] (5) Chromosome karyotype analysis: After the cells were cultured to the logarithmic growth phase, 0.2 μg / mL of colchicine was added to the culture system to inhibit spindle formation, so that the chromosomes were condensed but not separated; after 2 h of colchicine treatment, the cells were collected and treated with 0.075 M KCl hypotonic solution to swell the cells, so that the chromosomes could be fully expanded in the subsequent preparation process. Freshly prepared fixing solution (methanol: glacial acetic acid = 3:1) was used to fix the cells, maintain the cell morphology and remove the residual hypotonic solution. The fixed cell suspension was dropped onto a clean glass slide and placed in a 70°C oven for drying for 2 h; Giemsa staining was used to stain the chromosomes, and the prepared chromosome specimens were observed under an optical microscope and images were taken for karyotype analysis. The karyotype analysis spectrum of CBC3T-3 cells is shown in FIG. 6D: CBC3T-3 cells have a near 3-ploid karyotype, with complex chromosome number and structural aberrations, and the classic karyotype number is 67. Figure 1 D shown: CBC3T-3 cells have a near 3-ploid karyotype, with complex chromosome number and structural aberrations, and the classic karyotype number is 67.

[0042] (6) Cell growth curve: CBC3T-3 cells were seeded in 6-well plates at a density of 5×10 3Cells were seeded in 96-well plates at a uniform density of cells / well. At 6h, 1 day, 2 days, 3 days and 4 days after seeding, Cell Counting Kit-8 (CCK-8) reagent (APExBio, USA) was added to each well. After incubation for 2h in the dark, the optical density (OD) value of each well at 450nm wavelength was measured using a microplate reader. All experimental data were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA), and cell growth curves were plotted to evaluate the proliferation kinetics.

[0043] (7) Transwell migration and invasion experiment: Matrigel (R&D Systems, USA) was diluted with serum-free medium at a ratio of 1:8, and 50μL was evenly spread on the 8μm pore size Transwell upper chamber (Corning, USA). The chamber was transferred to a 37℃, 5% CO2 cell incubator for 30min. After the matrigel completely polymerized, the uncoagulated gel was gently aspirated using a pre-cooled sterile syringe tip. 200μL of serum-free medium containing 8×10 4 cells was added to the upper chamber of the Transwell, and 700μL of medium containing 20% fetal bovine serum (Cell-Box, China) was added to the lower chamber of the Transwell to construct a chemical chemotactic gradient system. The culture was continued in a 37℃, 5% CO2 incubator for 48h, and the cell state was observed every 12h during the incubation. The upper chamber was fixed with 4% paraformaldehyde for 30min and stained with 0.1% crystal violet for 30min. Three non-overlapping fields were randomly selected under a fluorescence inverted optical microscope, and the invasion area was quantified using the threshold analysis method of image J software. The statistical analysis was performed using GraphPad Prism 8.0 software.

[0044] (8) Transmission electron microscopy: Cells were fixed with electron microscopy fixative (Servicebio, G1102, China) for 2-4h, and then rinsed with 0.1 mol / L phosphate buffer solution for 3 times. The samples were embedded in 1% agarose and fixed with 1% osmium acid solution prepared with 0.1 mol / L phosphate buffer solution at room temperature for 2h in the dark. After rinsing with 0.1 mol / L phosphate buffer solution for 3 times, the samples were dehydrated with ethanol in a gradient, permeated, embedded and polymerized. The samples were sectioned using an ultramicrotome (Leica, UC7, Germany). The sections were stained with 2% uranyl acetate saturated alcohol solution in the dark for 8min, and then dried overnight. The samples were observed and imaged using a transmission electron microscope (HITACHI, HT7700 80kV, Japan).

[0045] (9) Scanning electron microscope: cells were fixed with electron microscope fixing solution (Servicebio, G1102, China) for 2 h, and then washed with 0.1 mol / L phosphate buffer solution for 3 times. Ethanol was added for gradient dehydration, and the sample was dried using a cold dryer. A small amount of conductive glue was applied to the sample stage, and the image was observed and collected using a high-resolution cold field emission scanning electron microscope (HITACHI, SU8100, Japan).

[0046] Example 2

[0047] Drug sensitivity experiment

[0048] (1) The CBC3T-3 or TFK-1 cells in the logarithmic growth phase were uniformly plated in a 96-well plate (5000 cells / well) for 24 h, and then different concentrations of drugs (gemcitabine, oxaliplatin, cisplatin, fluorouracil and paclitaxel) were added for intervention for 48 h. The culture medium in the wells was discarded, and 200 μL of CCK8 reagent was added to each well. Incubation was performed in a 37°C cell incubator for 2 h, and the absorbance at 450 nm was determined using an enzyme-linked immunoassay instrument (BioTek, synergy H1, USA). GraphPad Prism 8.0 software was used for statistical analysis and plotting.

[0049] (2) Cell subcutaneous tumorigenicity: 4-week-old non-obese diabetic-severe combined immunodeficiency mice (NOD-SCID) were subcutaneously injected with 100 μL of PBS-Matrigel (1:1) mixed suspension containing 5 x 10 6 CBC3T-3 cells; the animal state was monitored every 4 days after injection, and the tumor long diameter (L) and short diameter (W) were measured using a vernier caliper after tumor formation, and the tumor volume was calculated; the mice were sacrificed after 10 weeks, and the subcutaneous tumors were removed for photography and weighing, part of the tumor tissue was fixed for HE and immunohistochemical staining, and the rest of the tissue was stored in a -80°C refrigerator.

[0050] (3) H&E staining and immunohistochemical staining: the tissue was fixed with formalin, embedded with paraffin and sectioned; after dehydration, antigen repair and serum blocking, the section was incubated with primary antibodies at 4°C overnight; antibodies against Ki67 (GB111499, dilution 1:500), TP53 (GB12626, dilution 1:1000), CK19 (GB11197, dilution 1:500) were purchased from Servicebio (Wuhan, China); the section was washed with PBS for 3 times, and the secondary antibody was added for incubation for 50 min, then DAB developing solution was added for color development, and the cell nucleus was stained with hematoxylin. The image was observed using a microscope (Nikon, Japan).

[0051] (4) Whole-exome sequencing (WES): Genomic DNA of the patient's tumor tissue adjacent normal tissue and CBC3T-3 cells was extracted for library construction, Agilent SureSelect Human All Exon V6 capture platform of Novogene Company (Beijing, China) was used to capture human coding region genomic sequence, and sequencing was performed based on Illumina NovaSeq 6000 platform; after BWA alignment and GATK variant detection, a variant annotation report including SNP, InDel, CNV was generated.

[0052] (5) Statistical analysis: The experiments of this example were performed in three biological replicates (n = 3), all statistical analyses were performed using GraphPad Prism 8 (San Diego, CA, USA), and P < 0.05 was considered statistically significant.

[0053] Test Example

[0054] Identification of CBC3T-3 cell line

[0055] (1) A cell line was extracted from the primary tumor tissue of the patient in Example 1 and named CBC3T-3; the separation and purification process of the cell line requires about 3 months, and a stable cell line (passage > 50 times) can be established in about 1 year; STR technology was used for cell identification, no cross contamination of human cells was found in CBC3T-3 cells, the STR profile of CBC3T-3 cells and primary tumor tissue was similar, and did not match the STR data in the existing database, indicating that CBC3T-3 cells were a new cell line, as shown in Table 1:

[0056] Table 1 Alleles of 21 sites in CBC3T-3 cells and tumor tissue

[0057] Marker CBC3T-3 cell Tumor tissue D19S433 14，14.2 14，14.2 D5S818 10，11 10，11 D21S11 30 30 D18S51 14，14 12，14 D6S1043 12，12 12，19 AMEL X, Y X, X D3S1358 15，16 15，16 D13S317 8，8 8，8 D7S820 11，11 11，11 D16S539 10，11 10，11 CSF1PO 10，13 10，13 Penta D 12，12 12，12 D2S441 11，12 11，12 vWA 16，18 16，18 D8S1179 12，13 12，13 TPOX 8，11 8，11 Penta E 11，15 11，15 TH01 9，9 9，9 D12S391 19，21 19，21 D2S1338 18，23 18，23 FGA 19，25 19，25

[0058] (2) The morphological characteristics of the cells were observed by optical microscope, as shown in Figure 2 A: Adherent growth of cells in groups was observed, and the cells were spindle-shaped or polygonal, with large nuclei, high nuclear-cytoplasmic ratio, and vacuoles in the cytoplasm; as shown in Figure 2 B: Scanning electron microscope showed that the cell surface was rich in microvilli, pseudopods or irregular protrusions, and there were tight junctions and intercellular bridges between cells; as shown in Figure 2C shows: Transmission electron microscopy shows that the nuclear membrane of CBC3T-3 cells is irregular, the ratio of nucleus to cytoplasm is increased, heterochromatin is aggregated, a large number of mitochondria can be seen in the cytoplasm, part of the mitochondria is swollen, the cristae are broken or vacuolated, the number of endoplasmic reticulum and Golgi apparatus is less. The number of secretory granules in cells increases, and there are bridge structures between cells.

[0059] (3) By WES on tumor cells and normal tissues, somatic mutations, copy number variations, etc. are found, so as to reveal the genetic mechanism of tumor and provide basis for targeted therapy; Somatic Single nucleotide variant (SNV) site analysis results show that, for example, Figure 3 A shows: This cell line has a total of 902 SNV mutations, mainly concentrated in the intronic, CDS and missense_SNP regions; for example, Figure 3 B shows: Somatic Insertion and Deletion (InDel) refers to small fragment insertion and deletion, and this cell line has 197 insertions or deletions in different regions of the genome; for example, Figure 3 C shows: Tumor Mutation Burden (TMB) is calculated by using Somatic Mutation (SNV, InDel) non-synonymous mutations in patient tumor tissues, which can be used to evaluate the sensitivity of tumor to immune checkpoint inhibitors: the TMB value of CBC3T-3 cells is 3.2903, which belongs to low mutation load, indicating that the patient may not be sensitive to immune checkpoint inhibitors; for example, Figure 3 D shows: Single-sample clonal structure analysis results show that the tumor has obvious heterogeneity; compare the screened Somatic Mutation with the known driver genes in the Cancer Gene Census database (Cancer Gene Census (sanger.ac.uk)), as shown in Table 2, to identify the driver genes in the tumor sample. Among them, TP53 has multiple missense mutations (p.L62F; p.L35F; p.L155F; p.L194F), TP53 mutation is one of the most common gene mutations in cholangiocarcinoma, loss of TP53 function contributes to tumor occurrence and development, and is associated with poor prognosis of cholangiocarcinoma patients.

[0060] Table 2 Tumor driver genes of CBC3T-3 cells

[0061] Gene Chrom Position Ref Alt Classification AAChange PPFIA4 1 203015444 G T Missense Mutation p.M358I NRAS 1 115256529 T A Missense Mutation p.Q61L PBRM1 3 52668805 C T Missense Mutation p.E372K; p.E358K; p.E390K; p.E393K TP53 17 7578269 G A Missense Mutation p.L62F; p.L35F; p.L155F; p.L194F; ELF4 X 129203494 G C Missense Mutation p.S323C PLEC 8 145004325 C A Nonsense Mutation p.E853X; p.E845X; p.E1004X; p.E835X; p.E867X; p.E871X; p.E894X BIRC6 2 32770834 G C Missense Mutation p.L4238F; p.L4239F ARID1B 6 157100301 C A Nonsense Mutation p.S496X NOTCH2 1 120471614 G A Missense Mutation p.R1293C ERCC2 19 45867002 T C Missense Mutation p.R349G; p.R373G EP300 22 41572503 G T Nonsense Mutation p.E1652X; p.E1678X

[0062] (4) By analyzing the cell growth curve of CBC3T-3, for example, Figure 4A shows: using cell doubling time calculation formula: DT = (t-t0) x log(2) / log(N / N0) to calculate the cell doubling time is about 29h, the cell grows rapidly; as Figure 4 B shows: the invasion and migration image collection of CBC3T-3 and TFK-1 cells and its statistical chart, compared with TFK-1 cells, the cell has better migration and invasion ability; transplant CBC3T-3 cells into NOD-SCID mice to observe its tumor formation ability, as Figure 4 C shows: 3 NOD-SCID mice all have palpable tumor nodules within 4 weeks, as Figure 4 D shows: the growth curve of mouse subcutaneous transplanted tumor, as Figure 4 E shows: the mouse weight change curve, and the mouse weight does not decrease significantly, indicating that the cell line has good in vivo tumorigenicity and can be used for in vivo research experiments of tumor; as Figure 4 F shows: the immunohistochemical results of patient primary tumor, mouse subcutaneous transplanted tumor and CBC3T-3, the immunohistochemical results of mouse subcutaneous transplanted tumor are consistent with the pathological characteristics of primary tumor tissue, the H&E staining of primary tumor tissue shows that tumor cells are arranged in cord-like or glandular tube-like, infiltrated in rich collagen fiber interstitium, cells are cuboidal-cylindrical, with significant nuclear heteromorphism and high nuclear-cytoplasmic ratio, pathological mitotic figures and nerve infiltration phenomenon can be seen, the immunohistochemical results show that CK19 (+), TP5 (+), and Ki-67 proliferation index is 30-40%; the H&E staining of mouse tumor graft shows that tumor cells are pyknosis, necrosis and liquefaction, arranged in flaky or nest-like, cell volume is large, size is different, cytoplasm is red, tumor cell necrosis can be seen in some areas, immunohistochemistry shows that tumor cells TP53 (+), CK19 (+) and Ki-67 (20-30%); wherein, *P<0.05; **P<0.01; ***P<0.001.

[0063] (5) The sensitivity of CBC3T-3 cells to several commonly used chemotherapeutic drugs for cholangiocarcinoma is beneficial to provide individualized treatment for patients, at the same time, by constructing drug sensitive cell lines or drug resistant cell lines, the precise treatment of cholangiocarcinoma can be promoted; as Figure 5 A shows: the cell viability value and IC50 of CBC3T-3 cells exposed to different concentrations of chemotherapeutic drugs (5-FU, gemcitabine, paclitaxel, oxaliplatin and cisplatin) for 48h, the cell is most sensitive to paclitaxel, followed by gemcitabine; as Figure 5B shows: comparison of Somatic Mutation obtained by WES sequencing screening with database NovoDR drug resistance gene database, which is important for evaluating patient prognosis and guiding clinical drug use. TP53 mutation is related to chemotherapy resistance of drugs Etoposide, Nitrogen, Mustard and Cisplatin; further determination of the sensitivity of TFK-1 to drug Cisplatin, such as Figure 5 C shows: cell viability value and IC50 of TFK-1 cells exposed to different concentrations of cisplatin for 48 h, wherein IC50 is 29.98 μM; compared with TFK-1, the sensitivity of CBC3T-3 cells to drug Cisplatin is lower, so that the TP53 mutation of this cell line leads to the non-response of patients to Cisplatin treatment.

[0064] In summary, CBC3T-3 cells have strong proliferation, invasion and migration ability, and have high tumorigenicity in immunodeficient mice, which can better simulate the pathological characteristics of primary tumors. Drug sensitivity analysis shows that this cell line is resistant to cisplatin, but sensitive to paclitaxel and gemcitabine, providing experimental basis for clinical chemotherapy regimen selection. In addition, this cell line reveals its genomic characteristics through whole exome sequencing (WES), including somatic mutation distribution, tumor mutation burden (TMB) and clonal structure, which provides a valuable in vitro model for further study of the role of TP53 missense mutation in the occurrence, development and drug resistance mechanism of cholangiocarcinoma. The establishment of CBC3T-3 cell line fills the gap in the research field of TP53 missense mutation type distal cholangiocarcinoma, and provides a key platform for mechanism exploration, drug screening and development of individualized treatment strategies.

[0065] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and any equivalent changes and improvements made within the scope of the present application should still be attributed to the patent coverage of the present application.

Claims

1. A novel human distal cholangiocarcinoma cell line with TP53 missense mutation, characterized in that, The cell line is named as CBC3T-3 cell line, and the preservation number is CCTCC NO: C202555; the genome of the cell line comprises multiple missense mutations of TP53 gene; and the cell line has a near triploid karyotype.

2. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 1, characterized in that, The missense mutations result in that the 35th, 62nd, 155th and 194th leucines (L) of the encoded protein are replaced by phenylalanines (F), respectively p.L35F, p.L62F, p.L155F and p.L194F.

3. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 1, characterized in that, The STR genotyping data of the CBC3T-3 cell line comprises: allele D18S51 is 14, 14; D6S1043 is 12, 12; AMEL is X, Y.

4. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 1, wherein, The CBC3T-3 cell line grows in an adherent cluster when cultured in vitro, and has a cell morphology of fusiform or polygonal shape and a high nucleus-cytoplasm ratio; under transmission electron microscopy, swollen mitochondria, broken cristae, aggregated heterochromatin and desmosome structures can be observed.

5. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 1, wherein, The separation and purification process of the CBC3T-3 cell line requires 3 months and more than 50 passages.

6. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 1, wherein, The CBC3T-3 cell line is identified by STR technology.

7. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 6, characterized in that, The STR technology uses amplification of 21 alleles of the CBC3T-3 cell to perform detection analysis on an Applied Biosystems 3730XL genetic analyzer.

8. The novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 5, characterized in that, The 21 alleles comprise CSF1PO, D5S818, D19S433, D21S11, D18S51, Penta D, AMEL, vWA, D3S1358, D6S1043, D13S317, D7S820, D16S539, D8S1179, D2S441, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA genes.

9. Use of a novel human distal cholangiocarcinoma cell line with a TP53 missense mutation, characterized in that, The application comprises application in construction of an animal transplanted tumor model of cholangiocarcinoma.

10. Use of the novel human distal cholangiocarcinoma cell line with TP53 missense mutation according to claim 9, characterized in that, The application also comprises experiments of chemo-resistance mechanism of cholangiocarcinoma caused by TP53 missense mutations.

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