A human peritoneal mesothelioma cell line, its construction method, and its application

Abstract

This invention provides a human peritoneal mesothelioma cell line, its construction method, and its applications. Addressing the lack of commercially available cell lines and stable in vitro and in vivo models for this disease, this invention successfully extracted and constructed this cell line from the peritoneal tissue of patients with peritoneal mesothelioma using enzymatic digestion. This cell line achieved spontaneous immortalization during in vitro culture, stably undergoing more than 60 passages. It not only maintained its malignant biological characteristics, such as high invasiveness and rapid proliferation, but also highly preserved the core genomic structure and oncogenic driver mutations of the primary tumor. In vivo experiments showed that this cell line possessed high tumorigenicity and peritoneal affinity in immunodeficient mice, with an orthotopic transplantation model success rate exceeding 95%. This invention effectively fills a gap in related fields, providing a stable and reliable cell model for research on the pathogenesis, target discovery, and drug screening of this disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a human peritoneal mesothelioma cell line, its construction method, and its applications. Background Technology

[0002] Malignant peritoneal mesothelioma (MPM) is a highly aggressive malignant tumor originating from the mesothelial cells on the peritoneum, characterized by pancake-like thickening or diffuse nodular formation of the peritoneum. There are currently no reports on the incidence of this disease in China. The gold standard for treating MPM patients is cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy. Although this treatment regimen has shown some prognostic value for MPM patients in some clinical trials, the overall survival remains short, with a 1-year overall survival (OS) rate of approximately 46% and a 5-year OS rate of approximately 20%. Further improving the prognosis of MPM patients remains a major challenge.

[0003] Current treatment strategies for peritoneal mesothelioma (MPM) largely draw upon research findings on pleural mesothelioma. This is partly because peritoneal MPM is rarer than pleural MPM, resulting in insufficient biological samples for complex in vitro and in vivo experiments. Furthermore, the American College of Cell Repository (ATCC), the world's largest cell bank, has not yet registered any MPM cell lines, limiting in-depth mechanistic studies. Although both diseases originate from mesothelial cells, peritoneal and pleural MPMs are entirely different, with significant differences in their pathogenesis. Therefore, exploring new treatment strategies for MPM is urgent. Due to the rarity of MPM, the limited number of research teams, and the lack of registered commercial cell lines and mature animal models both domestically and internationally, establishing immortalized MPM cell lines is of significant clinical importance for studying the disease's development and progression, as well as for screening and developing anti-tumor drugs.

[0004] Based on the above, this invention provides a human peritoneal mesothelioma cell line that conforms to the highly invasive characteristics of this disease. In vitro, this cell line can be passaged multiple times and achieves spontaneous immortalization; the classic protein expression profile of mesothelioma can also be detected in high-passage cells. In vivo, this cell line exhibits good tumorigenicity, with an orthotopic transplantation model success rate exceeding 95%. This invention fills the gap in the field of xenografting of human tumor tissue using clinical samples, where individual differences often lead to difficulties in achieving satisfactory consistency. The xenografting model constructed using this invention has the advantages of uniform growth and easy reproducibility. Summary of the Invention

[0005] Currently, most research on peritoneal mesothelioma, both domestically and internationally, focuses on clinical studies, which leaves our understanding of the disease at a relatively superficial stage. Furthermore, the lack of commercially available cell lines and unstable animal models also limits researchers' ability to delve deeper into the mechanisms of peritoneal mesothelioma development and progression.

[0006] To address the aforementioned problems, the present invention aims to provide an immortalized human peritoneal mesothelioma cell line, its construction method, and its applications. This cell line features low requirements for culture conditions, a short doubling cycle, high in vivo tumorigenesis rate, genetic stability, and the ability to be stably passaged for over 60 generations without affecting the expression of mesothelioma markers. This cell line provides a relatively ideal cell model for conducting in vitro experiments and constructing in vivo animal models.

[0007] To achieve the above objectives, the present invention will adopt the following technical solution: Firstly, this invention provides a human peritoneal mesothelioma cell line and its progeny cell lines.

[0008] The cell line of this invention was obtained from the lesion peritoneum of a female patient with peritoneal mesothelioma who visited Xiangya Hospital, and was named human peritoneal mesothelioma cells MPM-XY01 Homo sapiens. Furthermore, this cell line is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: C202693 and deposit date of April 23, 2026. This cell line has been passaged more than 60 times in vitro, and there is no significant difference in biological characteristics and tumorigenicity between low-passage and high-passage cell lines.

[0009] Secondly, this invention provides a method for constructing a human peritoneal mesothelioma cell line and its culture system.

[0010] In some implementation schemes, the mechanical cutting used in the primary cell extraction process is performed on an operating table, ensuring aseptic operation throughout the process.

[0011] Furthermore, the preferred digestion method is enzymatic digestion, using type II collagenase at a concentration of 2 mg / ml.

[0012] Furthermore, the primary culture system for the cell line is 10% FBS + 10 μg / ml recombinant human insulin + DMEM / F12 complete medium, and the passage culture system for the cell line (P5 and later) is 10% FBS + DMEM / F12 complete medium. The cell line is cultured under the conditions of a 37°C, 5% CO2 constant temperature cell culture incubator.

[0013] Furthermore, cryopreservation solutions prepared with high concentrations of serum are preferred for cell cryopreservation, resulting in higher cell viability after thawing.

[0014] Thirdly: Providing biological behavioral characteristics of the MPM-XY01 cell line. In some implementation schemes, the cell line MPM-XY01 was tested for proliferation, colony formation and migration capacity, and the changes in migration capacity of different numbers and passages of cells were compared.

[0015] Fourthly: Providing the application of the MPM-XY01 cell line in constructing mouse xenograft models. In some embodiments, the selected mouse strain is preferably a severely immunodeficient mouse. The mice used in this invention are 3-4 week old male NOD SCID mice that exhibit T lymphocyte and B lymphocyte deficiencies.

[0016] Furthermore, anesthesia was administered via intraperitoneal injection of sodium amobarbital, at a dose of 40-60 mg / kg.

[0017] Furthermore, the number of cells injected into the subcutaneous tumorigenesis model should be greater than 10 million, and the number of cells injected into the orthotopic tumorigenesis model should be greater than 5 million. Preferably, the xenograft model is an orthotopic transplantation model with a success rate of over 95%, and the cell line has peritoneal affinity, with tumor tissue growing in a nodular pattern in the parietal peritoneum of mice.

[0018] Furthermore, the immunohistochemical markers are internationally recognized positive indicators for peritoneal mesothelioma, including but not limited to D2-40, CK5 / 6, Calretinin, and WT-1.

[0019] Fifth aspect: Providing genomic characteristics of cell line MPM-XY01 In some implementations, the primary tumor (PT), xenograft model (PDX), and cell line genome detection methods are whole exome sequencing (WES).

[0020] Furthermore, the analytical methods include correlation analysis of variant allele frequencies (VAF) and copy number variations (CNV) and description of mutational characteristics of driver genes.

[0021] Advantages compared to existing technologies: This invention provides a human peritoneal mesothelioma cell line (named MPM-XY01), derived from the diseased peritoneum of a female patient with peritoneal mesothelioma. This cell line effectively overcomes the deficiency of existing in vivo and in vitro models of peritoneal mesothelioma, and has the following significant technical advantages and beneficial effects: (1) Currently, there are no commercially available peritoneal mesothelioma cell lines. Existing studies are mostly limited to primary cells extracted from clinical samples with limited passage counts, or to pleural mesothelioma cell lines. However, peritoneal mesothelioma and pleural mesothelioma are two completely different diseases with significant differences in pathogenesis, gene mutation characteristics, and drug sensitivity studies. This invention provides a stable in vitro model for the study of the pathogenesis of peritoneal mesothelioma, target discovery, and drug screening.

[0022] (2) Most existing methods for establishing immortalized cell lines rely on the introduction of exogenous genes (such as transfection with the tHERT gene or SV40 large T antigen). Although this method can achieve immortalization of cells with a limited number of passages, it also changes the original gene expression characteristics of the cells. The cell line MPM-XY01 provided by this invention achieves spontaneous immortalization during in vitro culture. This phenomenon not only allows the cell line to maintain the molecular biological characteristics and genetic background of the primary tumor, but also gives it the ability to proliferate indefinitely, ensuring the authenticity of the results of subsequent research.

[0023] (3) The MPM-XY01 cell line exhibits highly malignant tumor cell biological behavior in vitro, including rapid proliferation and high invasiveness. Its stable growth rate and superior colony formation ability effectively shorten the time cost for subsequent high-throughput drug screening, drug sensitivity testing and in-depth research on metastasis mechanisms.

[0024] (4) The MPM-XY01 cell line provided by this invention can successfully construct an in vivo xenograft model with a high success rate after intraperitoneal injection in NOD-SCID mice, and the pathological and immunohistochemical characteristics of the obtained tumor samples are highly consistent with the patient's primary tumor. This advantage overcomes the previous limitation of lacking mutual verification between in vivo and in vitro studies for peritoneal mesothelioma, and provides a stable and efficient animal model for preclinical drug efficacy evaluation. Attached Figure Description

[0025] Figure 1 A. Tumor tissue from the patient; B. Immunofluorescence detection of the expression of mesothelioma markers Calretinin (green), D2-40 (red), and WT-1 (orange) in the patient's tumor tissue; C. Cell morphology (100X) of MPM-XY01 cell line at passages P0, P10, P30, and P60; D. Immunofluorescence detection of the expression of mesothelioma markers Calretinin (green), D2-40 (red), and WT-1 (brown) in the MPM-XY01 cell line.

[0026] Figure 2Cell function assays for the MPM-XY01 cell line: A. CCK8 cell proliferation assay; B. Cell colony formation assay; C. Transwell cell migration assay for different cell numbers; D. Quantitative results of Transwell cell migration for different cell numbers; E. Wound healing assay for different cell passages; F. Quantitative results of wound healing assay for different cell passages.

[0027] Figure 3 Results of sex identification at the Amel locus and 23 STR loci.

[0028] Figure 4 Xenograft models derived from cell lines: A. Orthotopic xenograft mouse model; B. Subcutaneous xenograft mouse model; C. Weight change trend of a single xenograft mouse model; D. Overall weight change trend of a xenograft mouse model; E. Diagram of the dissected abdominal wall of a mouse.

[0029] Figure 5 Immunohistochemical staining was used to detect the expression of Calretinin, D2-40, WT-1 and CK5 / 6 proteins, markers of mesothelioma, in primary tumor tissues of patients and xenograft mouse model tumor tissues. Scale bar: 50 μm.

[0030] Figure 6 WES results of primary tumor (PT), xenograft model (PDX), and cell line MPM-XY01 (cell) in patients with peritoneal mesothelioma; A. Scatter plot of variant allele frequency (VAF) for PT and cell; B. Scatter plot of VAF for PT and PDX; C. Scatter plot of VAF for PDX and cell; D. Genome copy number variation (CNV) map of PT, PDX, and cell.

[0031] Figure 7 Driver gene mutation information of primary tumor (PT), xenograft model (PDX), and cell line MPM-XY01 (cell) in patients with peritoneal mesothelioma. Detailed Implementation

[0032] To better understand the purpose, technical solution, and beneficial effects of this invention, detailed explanations will be provided below using specific examples in conjunction with the accompanying drawings. It should be understood that the specific examples described herein are for illustrative purposes only and are not intended to limit the invention.

[0033] Example 1: Construction method of human peritoneal mesothelioma cell line MPM-XY01 1. Experimental Materials DMEM / F12 (Servicebio, G4612), 100x penicillin-streptomycin (MCE, HY-K1006), fetal bovine serum (Umedium, 3022A), PBS solution (KeyGEN BioTECH, KGL2206-500), T25 culture flask (Corning, 430639), 15ml centrifuge tubes, 50ml centrifuge tubes, type II collagenase (Servicebio, GC305014), HBSS buffer (Solarbio, H1025), erythrocyte lysis buffer (Solarbio, R1013), 0.25% trypsin (KeyGEN BioTECH, KGL2102-100), serum-free cell cryopreservation solution (ShareBio, SB-EQ001), scalpel blades, ophthalmic scissors, ophthalmic forceps, 5ml syringe, 70μm cell strainer.

[0034] 2. Patient Information The patient, a 67-year-old female, was diagnosed with malignant peritoneal mesothelioma by pathological examination of peritoneal tissue obtained by peritoneal puncture.

[0035] 3. Experimental Methods (1) Primary culture Obtaining tumor tissue from the peritoneum of a patient with peritoneal mesothelioma from the operating table ( Figure 1 A) Using a sterile scalpel, remove excess tissue such as blood, fat, and retina from the tumor. After rinsing with physiological saline, cut the tumor into tissue blocks approximately 1 mm³ in size. Then, place the blocks in a DMEM / F12 solution containing 1% penicillin-streptomycin and quickly transfer them to the laboratory using an ice pack. Use sterile ophthalmic scissors to cut the tissue blocks into homogenates. Use enzymatic digestion to lyse the tumor tissue and obtain a single-cell suspension.

[0036] Specifically, the digestive solution used in enzymatic digestion consists of a solution containing Ca... 2+ Mg 2+ The HBSS buffer was prepared by dissolving type II collagenase in 5 ml of HBSS buffer, ensuring a collagenase concentration of 1-2 mg / ml. The tumor tissue homogenate was then placed in the digestion solution and digested in a shaker at 37°C for 2 hours. The undigested tissue was filtered twice through a 40 μm cell sieve to obtain a single-cell suspension. An equal volume of DMEM / F12 complete culture medium was then added to stop the digestion.

[0037] Collect the cell suspension into a 15ml centrifuge tube and centrifuge at 800rpm for 5min to obtain a cell pellet. Add 1ml of erythrocyte lysis buffer, mix well by pipetting, and incubate at room temperature for 5min. Centrifuge again at 800rpm for 5min to obtain a cell pellet. The number of erythrocyte lysis cycles can be adjusted according to the degree of erythrocyte lysis in the pellet. Resuspend the lysed cell pellet in 1ml of preheated 37°C DMEM / F12 medium, seed it into a T25 culture flask, and then add complete medium (10% FBS + 10μg / ml recombinant human insulin in DMEM / F12 medium) to a final volume of 4ml. Place the culture flask in a 37°C, 5% CO2 incubator for primary culture. The next day, discard the original medium and add 4ml of fresh complete medium to continue culturing.

[0038] (2) Subculture About one week after inoculation, epithelial-like clonal cells will appear. Discard the original culture medium and wash the adherent cells twice with PBS. For the first passage, digest with 0.25% trypsin-EDTA for 1 min. During digestion, continuously monitor the degree of digestion. Because the digestion time for fibroblasts is significantly shorter than that for peritoneal mesothelioma cells, the differential adhesion method can be repeatedly used to purify the primary peritoneal mesothelioma cell line. After the first digestion, gently pipette away any cells that are not fully adherent and have a morphology more similar to fibroblasts, discard these cells, and add 1 ml of fresh trypsin to continue digesting the epithelial-like peritoneal mesothelioma cells. When the peritoneal mesothelioma cells become rounded, add an equal volume of complete culture medium to stop the digestion. After digestion, centrifuge and wash twice with PBS, then re-inoculate into new T25 culture flasks. Subculture every 2-3 days thereafter, performing this purification procedure three times during this period. After the fibroblasts are completely removed, culture again until the cell density reaches 90%, and then passage using the above method. In this invention, the cell line is typically passaged at a 1:2 ratio and achieves over 90% confluence within 48 hours. If passaged at a 1:3 ratio, the cell line will reach over 90% confluence between 72 and 96 hours.

[0039] (3) Cell cryopreservation and thawing The cell line provided by this invention can be preserved or further cultured using cell cryopreservation and thawing methods well known to researchers in the art. For example, when cryopreserving cells, digest the cells with 0.25% trypsin-EDTA for 90 seconds. When the cells become rounded under a microscope, gently tap the culture flask to detach the cells, then add an equal volume of complete culture medium to stop the digestion. Transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 800rpm for 5min to obtain a cell pellet. Discard the supernatant, add 1ml of PBS to resuspend the cells, and centrifuge again. Discard the supernatant and repeat the process twice. Add 1ml of cell cryopreservation solution (commercial cryopreservation solution or 90% FBS + 5% DMSO + 5% complete culture medium) to the cell pellet, resuspend the cells, transfer them to cryovials, and place them directly into a -80°C gradient cryopreservation box for storage. The next day, transfer them to liquid nitrogen for long-term storage.

[0040] During cell thawing, place the cells cryopreserved using the above method into a preheated 37°C water bath, and continuously agitate the cryovials during the thawing process. After thawing, transfer the cells to 15ml centrifuge tubes, add at least 5 volumes of complete culture medium, and centrifuge to obtain a cell pellet. Resuspend the cells in 1ml of complete culture medium, seed them into new T25 culture flasks, and then add complete culture medium to a final volume of 4ml. Incubate at 37°C in a 5% CO2 incubator.

[0041] The cell line provided by this invention can be stored in a -80°C refrigerator for more than 3 months. The survival rate of cells revived within 1 month is usually more than 90%, and the survival rate of cells revived after more than 3 months is more than 70%. If long-term storage is required, the cells should be stored in liquid nitrogen.

[0042] 4. Experimental Results The human peritoneal mesothelioma cell line MPM-XY01 constructed using the above method is in good growth condition, with cell morphology mainly cobblestone-like, consistent with the morphology of epithelioid cells, and has been passaged more than 60 times.

[0043] Example 2: Morphological observation and mesothelial marker detection of cell line MPM-XY01 Continuous tracking observation under an inverted microscope showed that ( Figure 1 The MPM-XY01 cell line (C, 100X) maintained stable and uniform adherent growth characteristics throughout primary culture (P0), 10th generation (P10), 30th generation (P30), and 60th generation (P60). The overall morphology of the cells was mainly typical polygonal or irregular epithelial-like, with a relatively dense arrangement and good refractive properties. No obvious cell senescence, morphological variation, or decline in growth status was observed during continuous in vitro culture for up to 60 generations, indicating that the cell line has excellent phenotypic stability and continuous proliferative activity during long-term passage.

[0044] To demonstrate that MPM-XY01 is a cell line derived from peritoneal mesothelioma, immunofluorescence assays were performed on the primary tumor of peritoneal mesothelioma patients and the established MPM-XY01 cell line. The three markers detected—Calretinin, D2-40, and WT-1—are recognized positive markers for peritoneal mesothelioma. Immunofluorescence is a routine experiment familiar to technicians, and no other special procedures were performed; therefore, the specific experimental steps will not be described further. In the primary tumor ( Figure 1 B), Calretinin (green), D2-40 (red) and WT-1 (orange) were all significantly expressed, and their expression levels were similar to those of the MPM-XY01 cell line ( Figure 1 The fact that D) is largely the same indicates that MPM-XY01 is a cell line derived from peritoneal mesothelioma.

[0045] Example 3: Cell function detection of MPM-XY01 cell line 1. Experimental Methods: (1) Cell proliferation experiment: Cells in the logarithmic growth phase were digested and centrifuged, and then counted using an automated cell counter (Thermo Fisher Scientific, Countess 3). The cell concentration was diluted to 20,000 cells / ml with DMEM / F12 complete medium. 100 μl of cell suspension was added to each well of a 96-well plate to achieve a cell count of 2000 cells per well, with five groups (0h, 24h, 48h, 72h, and 96h), each with five replicates. On the second day, 100 μl of CCK8 reagent was added to each well. Specifically, the CCK8 reagent was prepared according to the manufacturer's instructions, using DMEM / F12 and CCK reagent at a 10:1 ratio. This time point was designated as h0 of the cell proliferation experiment. After 2 hours of incubation, the absorbance of the cells at 450 nm was measured using a spectrophotometer. Subsequently, the cell absorbance at 450 nm was continuously measured at 24h, 48h, 72h and 96h using the same method. The data were analyzed using GraphPad Prism (Version 11) and continuous cell proliferation curves were plotted.

[0046] (2) Cloning experiment Cells were counted and diluted to concentrations of 500 cells / ml, 1000 cells / ml, and 2000 cells / ml. 1 ml of the diluted cell suspension and 1 ml of fresh complete culture medium were added to each well of a 6-well plate, resulting in 500, 1000, and 2000 cells per well, respectively. The plates were incubated for 14 days, with the medium changed every 3 days. Culture was stopped when visible cell clones appeared in the wells (more than 50 individual clones under a microscope) and before large-scale confluence between clones. The culture medium was then aspirated, and the cells were gently washed twice with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15-20 minutes. After fixation, the cells were washed twice with PBS to remove residual paraformaldehyde, and then 1 mL of 0.1% crystal violet staining solution was added and stained at room temperature for 10-15 minutes. The staining solution was recovered, and excess background stain was washed away with running water (slow flow, avoiding direct contact with the bottom of the wells). Invert the culture plate onto filter paper and air dry at room temperature.

[0047] (3) Cell scratch test Cells in the logarithmic growth phase were seeded into 6-well plates, so that each well contained 3*10^3 cells. 6 This number of cells reached 90% confluence by the second day. On the second day, three scratches were made in a 6-well plate using a 200 μl pipette tip. After washing away the detached cells with PBS, fresh serum-free medium was added, and the plate was placed in a cell culture incubator. The scratch time was selected as h0 of this experiment, and scratch images were taken at this time point using an inverted microscope. Subsequently, scratch healing images were taken at 6h, 12h, and 24h.

[0048] (4) Transwell migration experiment Cells in the logarithmic growth phase were digested and counted. 200 μl of serum-free cell suspension was added to the upper chamber of the Transwell, bringing the cell count to 2 * 10^6. 4 4*10^ 4 and 8*10^ 4 Cells were cultured in the lower chamber with 500 μl of 10% FBS complete medium for 24 h. The medium in the lower chamber was then discarded, and the cells were fixed and stained using the same method as above. After washing away the staining solution, the cells on the upper layer of the chamber were carefully wiped with a cotton swab. The chamber was then air-dried and five fields of view were randomly selected for photographing under an inverted microscope. This experiment utilizes the chemotactic effect of serum growth factors on tumor cells, causing them to migrate from the upper chamber to the lower chamber of the Transwell, and is commonly used to study cell migration.

[0049] 2. Experimental Results The experimental results comprehensively confirmed that this cell line possesses typical and stable highly malignant biological characteristics in vitro. Cell viability assay ( Figure 2 A) shows the changes in cell viability of the MPM-XY01 cell line over 96 hours. Cell viability increased significantly linearly over time, as demonstrated in the plate colony formation assay (PCL). Figure 2 B) showed that with the increase of the number of seeded cells, the number and density of clonal plaques formed increased significantly, indicating that the cell line has a very strong in vitro proliferation capacity; Transwell migration assay ( Figure 2 D) indicates that it has significant invasiveness, with the number of cells penetrating the chamber increasing in a typical number-dependent manner with seeding density; in addition, the scratch test ( Figure 2 C) It was confirmed that the cell line migrates extremely fast, with a scratch healing rate of nearly 100% within 24 hours. Furthermore, the healing kinetic curves of the 15th generation (P15) and the 30th generation (P30) cells were highly consistent. This not only highlights its strong migration activity but also fully demonstrates that the cell line can maintain a highly stable malignant phenotype during continuous passage culture, providing a very reliable in vitro model for downstream applications such as pharmacodynamic evaluation of antitumor drugs and tumor mechanism research.

[0050] Example 4: Identification of short tandem repeat (STR) sequences in cell line MPM-XY01 1. Experimental Methods: Cells in the logarithmic growth phase were digested, centrifuged, resuspended, and counted to obtain the cell number (≥ 10^6). 6 Cell line authentication for MPM-XY01 was performed via short tandem repeat (STR) analysis. In short, genomic DNA was extracted from cultured cells, and PCR amplification of 23 distinct human STR loci and the sex-determining Amel locus was performed using the GenePrint® 24 system (Promega). Thermal cycling was conducted on a MiniAmp thermal cycler (Thermo Fisher Scientific), and PCR products were prepared using Hi-Di™ formamide (Thermo Fisher Scientific). Finally, the genotyping of the cell line was analyzed using an ABI 3730XL genetic analyzer (Applied Biosystems) to determine the cell line's unique genetic characteristics and rule out the possibility of cross-contamination between or within species. The obtained STR locus information was then compared with existing cell banks (ATCC, Cellosaurus, and DMSZ databases).

[0051] 2. Experimental Results: like Figure 3As shown, the MPM-XY01 cell line exhibited homozygosity at a large number of STR loci, and heterozygosity only at the D16S539 and Penta E loci, without showing any other trielelic or tetraelelic abnormalities. No cell lines with a matching degree >80% were found in any of the three cell banks for the MPM-XY01 cell line.

[0052] Table 1. Genotyping results of Amel at STR and sex loci in cell line MPM-XY01.

[0053] Example 5: Mouse xenograft model based on MPM-XY01 cell line 1. Experimental materials: PBS solution, EP tubes, disposable insulin syringes, isoflurane.

[0054] 2. Mouse breed: 4-6 week old NOD SCID male mice.

[0055] 3. Experimental methods: MPM-XY01 cell lines in logarithmic growth phase were digested, resuspended in PBS, and diluted to a concentration of 10^6. 7 / ml or higher, aspirate the cell suspension into 1.5ml EP tubes and place them on an ice box for later use. Complete the relevant experiments within 1 hour after cell digestion. Anesthetize mice with pre-prepared sodium amobarbital via intraperitoneal anesthesia at a dose of 40-60mg / kg. After anesthesia, inject 200μl of cells subcutaneously and intraperitoneally using an insulin injection. The total cell volume injected is (1-10)*10^ 6 The mice were then measured for body weight and subcutaneous tumor size every three days. After about two months of culture, the mice began to lose body weight. Generally, the experiment reached its endpoint when the mice lost more than 20% of their body weight or the largest diameter of the tumor exceeded 1.5 cm. The mice were then euthanized and the tumor tissue was collected.

[0056] Specifically, the euthanasia method was cervical dislocation. The baseline level of mouse weight loss should be the current original weight of the mouse. Since the orthotopic mouse xenograft model cannot objectively detect tumor size, the level of mouse weight loss was selected as the evaluation index of the orthotopic model.

[0057] Preferably, the amount of cells injected into subcutaneous xenograft mice is 1*10^ 7 The amount of cells injected into an orthotopic tumor-bearing mouse model should be at least 5*10^ 6 .

[0058] When dissecting mice, the fur and subcutaneous fat in the abdominal region are first removed with scissors to expose the abdominal wall. Then, the entire abdominal wall is completely dissected along both sides of the abdomen, the costal arch, and the line connecting the pubic symphysis. Because peritoneal mesothelioma is characterized by diffuse thickening of the peritoneum and the formation of multiple peritoneal nodules, the abdominal wall of mice is also a common area for tumor implantation in orthotopic tumor models.

[0059] Specifically, the anatomical extent of the mouse abdomen extends from the line connecting the bilateral pubic symphysis to the line connecting the xiphoid process and the costal arch, with the mid-axillary line on both sides.

[0060] The obtained tumor tissue was prepared into paraffin blocks, and the expression of peritoneal mesothelioma markers (D2-40, CK5 / 6, Calretinin, WT-1) in the tumor tissue was detected by immunohistochemistry and compared with the expression level of the primary tumor. Paraffin embedding, sectioning, HE staining, and IHC staining are all experiments well known to those skilled in the art, and therefore will not be described in detail.

[0061] 4. Experimental Results During the observation period of the animal model, the weight of all mice was in a slow growth phase for the first 40 days. From day 40 to 50, the weight of the mice reached a plateau and stopped growing or even decreased slightly. From day 50 to 70, the weight of the mice dropped sharply, and the mice showed severe cachexia. Figure 4 (C and D).

[0062] (1) General appearance After intraperitoneal injection of MPM-XY01 cell line into mice, extensive tumor seeding occurred in the peritoneum. White tumor tissue was found in organs and tissues such as the peritoneum, perigastric region, spleen, kidneys, liver, and mesentery. Figure 4 A) In addition, in the subcutaneous tumorigenesis model ( Figure 4 (B) It was found that the tumor volume increased significantly compared to the initial injection stage. This animal model confirmed that the MPM-XY01 cell line constructed in this invention possesses in vivo tumorigenicity, and the tumorigenicity of in situ implantation is superior to that of subcutaneous implantation. In this invention, the tumorigenicity rate in the mouse in situ model reached 100%, and the tumorigenicity rate in the subcutaneous model reached 80%. In addition, a large number of implanted tumor tissues were also found on the dissected mouse abdominal wall (including the parietal peritoneum). Figure 4 E), this phenomenon of multiple nodules on the abdominal wall is related to the lesioned peritoneum / greater omentum in patients with peritoneal mesothelioma ( Figure 1 A) Extremely similar, this biological behavior indicates that the cell line has a very high affinity for the peritoneum and is an excellent cell model for developing an in vivo model of peritoneal mesothelioma.

[0063] (2) Immunohistochemistry Immunohistochemistry was used to detect the expression of mesothelioma markers in primary tumor tissues of peritoneal mesothelioma patients, subcutaneous tumor tissues of mice, and intraperitoneal tumor tissues. The results are as follows: Figure 5 As shown, the expression of the four mesothelioma markers (D2-40, CK5 / 6, Calretinin, WT-1) was completely consistent in the three samples, further indicating that the MPM-XY01 cell line was constructed based on the tumor samples of patients and that the cell line had good genetic stability during passage, with the expression of the marker protein profile remaining basically unchanged.

[0064] Example 6: Analysis of WES sequencing results of cell line MPM-XY01 1. Experimental Methods: Peripheral blood (PB), primary tumor (PT), xenograft model (PDX) tumor tissue, and MPM-XY01 cell line were collected from patients and stored at -80°C. DNA extraction, quality control, and WES sequencing of blood, tissue, or cells were performed at Changsha Fuyin Biotechnology Co., Ltd. Variant allele frequency (VAF), copy number variation (CNV), and driver gene mutation characteristics were derived from the analysis of the raw sequencing data. Specific steps are as follows: (1) Raw data quality control and preprocessing The quality of the raw Fastq files was assessed using FastQC, and then the FastP tool was used to remove sequencing adapters and low-quality bases to obtain Clean Fastq files.

[0065] (2) Sequence alignment and BAM file processing When analyzing PDX model data, the Xenome tool was used to simultaneously align PDX reads to both the human reference genome (GRCh38) and the mouse reference genome (mm10), removing contaminating reads from the mouse host genome and retaining pure human reads. PB, PT, and cell data were directly compared with the human reference genome to ultimately obtain the BAM file.

[0066] (3) Somatic mutation detection and annotation Peripheral blood was used as a control. Mutect2 was used to compare the results with PT, PDX, and cell sequences to remove germline mutations and sequencing artifacts, obtaining VCF files. bcftools and GATK VariantsToTable were used to parse the FORMAT field to obtain sequencing depth information for each sample. For each identified somatic mutation site, the total sequencing depth (DP) and the sequencing depth (AD) supporting the mutant alleles were extracted. ALT Variant allele frequency (VAF) is calculated according to the formula. Calculations were performed. To ensure the accuracy of variant detection, unreliable sites with excessively low total sequencing depth (DP<20) in any sample were removed. Finally, the filtered high-confidence variant dataset was integrated into a structured matrix containing genomic coordinates and VAF values ​​for multiple samples, and data visualization analysis was performed using the R package ggplot2.

[0067] (4) Copy number variation analysis Using PB as the normal diploid baseline, the copy number log ratio of PT, PDX and cell samples was calculated after correcting for sequencing depth bias using CNV analysis software (CNVkit). After segmentation processing using the CBS algorithm, continuous fragment data containing the exact chromosome location and signal ratio were generated. Finally, the R package ggplot2 was used to map these fragment data into continuous genomic coordinates in chromosomal order.

[0068] (5) Analysis of driver gene mutation characteristics The annotated VCF files were converted to MAF format using the R package maftools, and a waterfall plot was drawn to show the mutation types of high-frequency mutant genes in PT, PDX, and cell samples, comparing the consistency and differences of driver gene mutations in these three samples.

[0069] 2. Experimental Results (1) VAF correlation analysis VAF correlation analysis of PT, PDX, and cell revealed that PT vs cell ( Figure 6 The Pearson correlation coefficient for A) is 0.881, PT vs PDX ( Figure 6 B) was 0.875, indicating that the cell line and PDX successfully preserved a large portion of the core mutation spectrum of PT. Furthermore, Figure 6 The slope of A is 1.288. Figure 6 A B value of 1.231 and a slope greater than 1 indicate that the VAF of the same mutation is generally higher than PT in both cells and PDXs. A comparison of VAF between PDXs and cells ( Figure 6 C) The tumor exhibits extremely high consistency: the Pearson correlation coefficient is as high as 0.974, with a slope of 1.019, representing an almost perfect 1:1 distribution. This phenomenon indicates that the clonal selection pressure exerted on the tumor by the in vitro culture system and the in vivo immunodeficient mouse environment is very similar. The subclonal composition of both remains stable during passage. However, because the tumor tissue contains a large number of normal stromal cells, immune cells, and vascular endothelial cells, the wild-type DNA of these normal human cells "dilutes" the mutated VAF of the tumor, causing the VAF of the tumor tissue to shift towards the true clonal ratio, resulting in an overall downward shift in VAF.

[0070] (2) CNV Result Analysis Figure 6 D presents copy number variation (CNV) maps of the primary tumor (PT), PDX model, and in vitro cell lines. Specifically, all three samples exhibited highly consistent and significant amplifications in regions of chromosomes 5, 7, 12, 19, and 20 (e.g., the characteristic full-arm amplification of chromosome 7); simultaneously, they also showed deep fragment deletions in regions of chromosomes 9, 14, 18, and 22. This high degree of conservation demonstrates that, whether in vivo in mice or in vitro culture dishes, the established cell lines and PDX models stably inherited the core genomic structure of the primary tumor, without undergoing chromosomal remodeling deviating from the primary lesion during passage amplification.

[0071] At a detailed level, significant biological differences were observed in the signal amplitudes of the three data sets. Compared to cells and PDX, the PT samples exhibited smaller fluctuations in amplification peaks and deletion troughs. This is primarily due to the dilution effect caused by "tumor purity": the large number of normal human stromal cells (with a normal signal of 0) mixed in the PT tissue blocks neutralized and compressed the extreme signals of the tumor; while the constructed cells and PDX, having eliminated interference from normal human cells, served as high-purity tumor clone populations, realistically reflecting the abnormal copy number state of the tumor itself. In summary, these results confirm the genomic fidelity of the constructed cell and PDX models during passage and highlight their unique advantages as high-purity tools for subsequent molecular mechanism and pharmacodynamic studies.

[0072] (3) Analysis of driver gene mutation characteristics Figure 7 This study depicts the molecular variation landscape of primary tumors (PT), PDX models, and in vitro cell lines. In both the mouse xenograft model and in vitro cell culture settings, PDX and cells exhibited the core oncogenic features of PT. For example, homozygous deletion of the CDKN2A gene (HOMDEL) and key truncating mutations in tumor suppressor genes such as PTEN and FBXW7 showed extremely high conservation across the three samples, confirming the successful preservation and stable passage of the dominant tumor clone. However, the atlas also revealed a significant intergroup difference: many variants undetectable in PT samples (such as copy number amplification of EGFR and KRAS) showed clear high-frequency enrichment in PDX and cells. This is primarily attributed to changes in "tumor purity" rather than new mutations induced by model passage. The effective elimination of the normal human cell background during culture allowed previously masked tumor subclonal mutational features to be truly revealed. This atlas confirms that PDX and cells, while preserving the oncogenic driver genes of PT, eliminated the stromal interference-induced mutational background, accurately reflecting the oncogenic mutational information of the tumor.

Claims

1. A human peritoneal mesothelioma cell line, characterized in that, The cell line, named MPM-XY01, has been deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO:C202693 and deposit date of April 23, 2026.

2. The human peritoneal mesothelioma cell line as described in claim 1, characterized in that, The cell line has at least one of the following biological characteristics: Morphological characteristics: The cell morphology is mainly cobblestone-like, polygonal or irregular epithelial-like, tightly arranged and with good refractive properties; Proliferation and passage characteristics: It can spontaneously immortalize itself in vitro, pass it up to 60 times or more, and maintain stable and uniform adherent growth characteristics during long-term passage. Biomarker expression: Positive expression of peritoneal mesothelioma biomarkers, including Calretinin, D2-40 and WT-1; Genetic characteristics: In the identification of short tandem repeat (STR) sequences, the sex locus Amel is X; the D16S539 locus shows heterozygotes of 9.3 and 11.3, the Penta E locus shows heterozygotes of 12 and 13, and most other loci show homozygotes.

3. The method for constructing a human peritoneal mesothelioma cell line as described in claim 1 or 2, characterized in that, Includes the following steps: Tissue Acquisition and Processing: Tumor tissue from patients with peritoneal mesothelioma was obtained from the operating table. Residual fat and omental tissue were removed with a scalpel, and the tumor was cut into 1 mm tissue blocks. The blocks were placed in DMEM / F12 basal culture medium containing 1% penicillin-streptomycin and quickly transferred to the laboratory. Tissue digestion: The tissue block was placed in a digestion solution and digested on a constant temperature shaker at 37°C for 2 hours. The digestion solution was filtered through a 40μm cell sieve to obtain a single-cell suspension. Primary culture: After centrifuging the single-cell suspension and discarding the supernatant, add 1 ml of red blood cell lysis buffer to remove red blood cells. Then, resuspend the red blood cell-free cells in culture medium and seed them into the primary culture system for culture. Passaging and purification: Primary cells were purified multiple times using differential adhesion to remove fibroblasts, resulting in purified human peritoneal mesothelioma cell lines, which were then passaged.

4. The construction method according to claim 3, characterized in that, In the tissue digestion step, the digestive enzyme is preferably type II collagenase; the concentration of type II collagenase is 1-2 mg / ml.

5. The construction method according to claim 3, characterized in that, The specific conditions for the primary culture system and the subculture system are as follows: Primary culture system: complete culture medium containing 10% FBS, 10µg / ml recombinant human insulin and DMEM / F12; Subculture system: From the 5th generation (P5) onwards, the culture system is DMEM / F12 complete medium containing 10% FBS.

6. The application of the human peritoneal mesothelioma cell line according to claim 1 or 2 in constructing an in vitro cell model or an in vivo animal model of peritoneal mesothelioma.

7. The application of the cell line according to claim 6 in an in vitro cell model, characterized in that, The applications should include cell proliferation assays, colony formation assays, scratch assays, and Transwell assays.

8. The application of the cell line according to claim 6 in an in vivo animal model, characterized in that, The method for constructing the in vivo animal model includes the following limiting conditions: Animal strain: Preferred 3-4 week old male NOD SCID mice; Inoculation method and dosage: When constructing a subcutaneous tumorigenesis model, the number of injected cells should be greater than 10 million; when constructing an in situ tumorigenesis model, the number of injected cells should be greater than 5 million.

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