A human mesothelioma cell line and its construction method and application

Abstract

This invention provides a stable passaged human mesothelioma cell line MESO-016, its construction method, and its application in tumor research and drug development. The cell line provided by this invention can be continuously passaged in vitro and possesses xenogeneic tumorigenesis ability. Tumors formed in nude mice exhibit the same pathological characteristics as the primary tumor tissue, making it suitable as a cell material for research on mesothelioma-related drugs and diagnostic reagents, as well as for studying the mechanisms of tumor development and progression.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell biology, specifically relating to a stable passaged human epidermal tumor cell line, its construction method, and its application in tumor research and drug development. Background Technology

[0002] Mesothelioma is a rare but highly aggressive and poorly prognostic malignant tumor, with asbestos exposure being the most prevalent causative factor. However, not all mesothelioma cases are associated with asbestos exposure. Besides asbestos, other mineral fibers and novel nanomaterials such as carbon nanotubes have also been shown to have similar tumorigenic potential. Furthermore, chronic inflammation and radiation exposure are also believed to play a role in the development of mesothelioma. Notably, some patients in China have mesothelioma cases without a clear history of asbestos exposure, and certain regions show a localized epidemic trend of peritoneal mesothelioma unrelated to asbestos exposure, suggesting the existence of potentially incompletely elucidated regional or genetic risk factors. A minority of cases are seen in families with BAP1 tumor predisposition syndrome, caused by germline mutations or deletions in the BAP1 gene. Clinically, some pleural mesotheliomas are idiopathic, lacking a clear causative factor. Pericardial mesothelioma, paratesticular mesothelioma, and most female peritoneal mesotheliomas (especially subtypes carrying ALK or EWSR1 gene rearrangements) are generally considered to have no significant association with asbestos exposure.

[0003] In recent years, the incidence of mesothelioma in China has shown a year-on-year upward trend. Analysis of data from my country based on the Global Burden of Disease (GBD) 2017 database revealed approximately 2,900 new cases of mesothelioma in my country in 2017, more than double the number in 1990 (approximately 1,300). The age-standardized incidence rate increased from 0.136 / 100,000 to 0.149 / 100,000. Further trend analysis shows that after a slow increase in new mesothelioma cases in my country until 2005, a relatively rapid increase occurred, with a significant increase in the incidence rate also occurring in 2005. However, the incidence rate began to stabilize during the period of 2012-2017, which may be related to increased awareness and early diagnosis of mesothelioma.

[0004] Based on the location of the tumor, mesothelioma can be divided into pleural mesothelioma and peritoneal mesothelioma, with pleural mesothelioma being the most common. Pleural mesothelioma forms in the inner wall of the lung, and clinical diagnosis is often delayed, with most patients presenting with advanced or metastatic disease. The prognosis is generally poor: in patients with previously untreated advanced or metastatic malignant pleural mesothelioma, the median survival is between 12 and 14 months, and the five-year survival rate is approximately 10%.

[0005] Despite the highly aggressive nature and poor prognosis of mesothelioma, significant gaps remain in research in this field. Due to the rarity of mesothelioma, the scarcity of mesothelioma samples, and the difficulties in establishing and culturing cell lines, the number of isolated mesothelioma cell lines has long been extremely limited. The internationally authoritative cell bank ATCC only includes four mesothelioma cell lines, all derived from Caucasian males. This not only results in a small number of lines but also a lack of racial and gender diversity, making it difficult to represent the heterogeneity of the patient population and thus limiting in-depth exploration of its pathogenesis and targeted therapies. Furthermore, in clinical diagnosis, mesothelioma is often misdiagnosed as lung adenocarcinoma due to similar pathological features, further delaying treatment. Tumor cell lines are crucial research tools for studying the biological characteristics of tumors. In recent decades, a large number of human tumor cell lines have been isolated and used for long-term culture, providing important models for molecular-level tumor research.

[0006] Therefore, establishing research models that more closely reflect real-world conditions, improving diagnostic accuracy, and conducting more in-depth and comprehensive multi-dimensional studies remain major challenges that urgently need to be addressed in the field of mesothelioma. Furthermore, providing a stable human mesothelioma cell line is also essential. Summary of the Invention

[0007] To address the aforementioned technical issues, this invention provides a pleural mesothelioma cell line derived from a Chinese patient, offering a reliable Chinese-derived cell line research model for preclinical studies of mesothelioma. This human mesothelioma cell line (homo sapiens) was deposited on December 25, 2025, at the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number C2025381.

[0008] On the one hand, the present invention provides a human skin tumor cell line, which has the accession number C2025381.

[0009] On the other hand, the present invention provides a method for constructing the aforementioned human skin tumor cell line, the method comprising the following steps: (1) Tissue block processing: The obtained mesothelioma tumor specimens are cleaned and cut into pieces to obtain tumor block specimens; (2) Primary cell culture: After three repeated operations, a cell population with uniform morphology was obtained.

[0010] On the other hand, the present invention provides the use of the aforementioned human mesothelioma cell line for the preparation of mesothelioma diagnostic reagents.

[0011] On the other hand, the present invention provides the use of the aforementioned human mesothelioma cell line for preparing mesothelioma animal models.

[0012] On the other hand, the present invention provides the use of the aforementioned human mesothelioma cell line as a drug target for the development of drugs to inhibit mesothelioma.

[0013] On the other hand, the present invention provides the use of the aforementioned human mesothelioma cell lines for in vitro studies of mesothelioma.

[0014] On the other hand, the present invention provides the use of the aforementioned human mesothelioma cell lines for screening drugs for the treatment of mesothelioma.

[0015] The technical solution of this invention is summarized as follows: After digesting the shredded tumor tissue blocks using collagenase IV / hyaluronidase, the cells were cultured in a petri dish. Taking advantage of the fact that MESO-016 cells do not adhere firmly to the culture dish, tumor cells and tumor-associated fibroblasts were separated, thus obtaining a tool cell with uniform morphology, xenogeneic tumorigenesis ability, and the same pathological characteristics as the primary tumor tissue.

[0016] Beneficial effects The cell line of this invention can be continuously passaged in vitro and has xenogeneic tumorigenesis ability. The tumors formed in nude mice have the same pathological characteristics as the primary tumor tissue, making it suitable as a cell material for research on mesothelioma-related drugs and diagnostic reagents, as well as for research on the mechanisms of tumor development and progression.

[0017] The cell line provided by this invention is derived from samples of Chinese mesothelioma patients, filling the gap in the racial and regional representativeness of existing research models. It not only overcomes the limitations of currently used international cell lines, which are all derived from Caucasians and cannot reflect the genetic background and etiological characteristics of Chinese patients, but also helps to deeply analyze the molecular mechanisms of mesothelioma occurrence and development in the Chinese population, providing an irreplaceable experimental basis for developing diagnostic methods and treatment strategies that are suitable for the local population. Attached Figure Description

[0018] Figure 1 This is a cell image (10x magnification) of MESO-016, a pleural mesothelioma cell line of the present invention, grown under an inverted optical microscope.

[0019] Figure 2 This is the STR identification result of MESO-016, a pleural mesothelioma cell line of the present invention.

[0020] Figure 3 This is a cell growth curve of MESO-016, a pleural mesothelioma cell line of the present invention.

[0021] Figure 4 This invention presents a tumor growth curve of the MESO-016 pleural mesothelioma cell line after subcutaneous transplantation into BALB / c nude mice.

[0022] Figure 5This invention presents the histopathological analysis results of tumor tissue formed by inoculating MESO-016 pleural mesothelioma cells into the groin of BALB / c nude mice, including HE staining images and immunohistochemical staining results of mesothelioma markers. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0024] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0025] Example 1: Isolation and culture of primary human mesothelioma cells The human mesothelioma cell line MESO-016 provided by this invention is a pleural mesothelioma cell line derived from a population in southern China, specifically from tumor tissue of a 52-year-old female patient with epithelioid pleural mesothelioma. After pathological confirmation, this tissue was used to construct a patient-derived xenograft (PDX) model. Passaged tumor cells from the PDX model were surgically harvested, dissected, and then cultured in vitro.

[0026] The specific steps are as follows: (1) Tissue block processing: The PDX model tumor tissue was thoroughly washed in pre-cooled DPBS buffer to remove residual blood. The washed tissue was transferred to a 5 mL centrifuge tube and cut into pieces of approximately 1-2 mm using sterile surgical scissors. 3 Small tissue fragments were collected. DPBS buffer was added, and the mixture was centrifuged at 1500 rpm for 4-5 min, discarding the supernatant. A digestion solution containing collagenase IV (3.2 mg / mL, 125 U / mg) and hyaluronidase (0.32 mg / mL, >311 U / mg) was added to the pellet, and the mixture was incubated at 37°C for 45 min to 1 h, vortexing for 10-15 s every 10-15 min to promote tissue dissociation. After digestion, the mixture was centrifuged at 1000 rpm for 3 min, discarding the supernatant. RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) was added, and the mixture was centrifuged at 1000 rpm for 3 min, discarding the supernatant; the mixture was washed once more to remove cell debris and enzyme residue. Depending on the volume of the cell pellet, the cells were resuspended in an appropriate amount of RPMI-1640 complete medium, seeded in cell culture dishes, and incubated in a 37°C, 5% CO2 incubator for primary culture.

[0027] Primary cell culture: Minimize disturbance during the initial stages of primary culture. On days 3 and 7 post-inoculation, replenish with appropriate amounts of RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) based on medium consumption to maintain the cell growth environment. Once cell adhesion and spreading are observed under a microscope, replace the medium with fresh medium every two days. During culture, in addition to adherent cells, single or small clusters of suspended cells were consistently present. Trypan blue staining confirmed that most of the suspended cells were viable, ruling out cell detachment due to large-scale death or apoptosis. New suspended cells continued to appear after replacing the medium, suggesting this phenomenon is an inherent biological behavior of the cell line. Further transfer of the medium containing suspended cells to new culture dishes revealed that some cells could re-adhere and proliferate, indicating good survival, adhesion, and proliferation capabilities, suggesting that this cell line is semi-adherent and semi-suspended. Furthermore, after brief exposure to room temperature, some adherent cells shrank, rounded, and detached at the edges; re-adhesion was restored upon re-placement in a 37°C incubator. Utilizing this reversible adhesion property and the more stable adhesion behavior of fibroblasts, selective separation of tumor cells and fibroblasts was achieved through temperature changes. After three repeated operations, a morphologically uniform cell population was obtained. This cell line has now been passaged more than 20 times, exhibiting stable proliferation, and has been named the human skin tumor cell line MESO-016.

[0028] Example 2: Identification of the human skin tumor cell line MESO-016 1. Morphological observation The main observations include cell growth patterns, cell morphology, and nucleo-cytoplasmic ratio.

[0029] Observation of the human skin tumor cell line MESO-016 under an inverted optical microscope Figure 1 As shown, the cells exhibit typical semi-adherent, semi-suspended growth characteristics. Some cells are suspended in the culture medium as individuals or small clusters, while others adhere to the surface of the culture dish and grow in a long spindle to fusiform shape, with some having protrusions or pseudopodia-like structures. The cell edges are clear, and the cells are relatively uniform in size; the nuclei are prominent and oval, with a moderate nucleoplasm-to-nucleus ratio; the intercellular connections are loose, and the cells are arranged sparsely and disorderly.

[0030] 2. Cell line identification using short tandem repeat (STR) sequence analysis. STR identification was commissioned to Hangzhou Meisen Cell Biotechnology Co., Ltd. The SiFaSTR™ 23 multiplex assay kit was used to amplify 21 short tandem repeat (STR) loci and the Amelogenin locus. Detection was performed using an ABI Prism® 3130 XL genetic analyzer, and data were analyzed using GeneMapper® ID v3.2 software (Applied Biosystems). Positive and negative controls were included in each batch. Results are as follows: Figure 2As shown, this cell line has unique human genetic characteristics, no cross-contamination with known cell lines, and does not match any known cell line in the public cell bank, thus confirming it as a new cell line.

[0031] 3. Plotting cell growth curves using the CCK-8 assay The cell lines to be tested were digested with 0.25% trypsin, resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the cell density was adjusted to 2 × 10⁶ cells / year. 4 Cells / mL. 100 μL of cell suspension was seeded into 96-well plates, with 6 replicates per group. Starting from day 0 of seeding, CCK-8 solution was added every 24 hours at a CCK-8 reagent to culture medium volume ratio of 1:10. After incubation at 37°C for 1 hour, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 ). Continuous monitoring continued until day 7, with the incubation time (days) as the x-axis, OD 450 The value is used as the ordinate to plot a cell growth curve, such as... Figure 3 As shown.

[0032] 4. In vivo tumorigenesis and pathological verification of human skin tumor cell lineage Cell suspensions were inoculated into xenogeneic animals to assess their tumorigenic capacity. Subsequently, the resulting tumors underwent detailed pathological characterization and were compared with the characteristics of primary tumors in patients.

[0033] Cells in the logarithmic growth phase were digested into a single-cell suspension using 0.25% trypsin, followed by cell counting. The cells were then mixed with an equal volume of old culture medium and matrix gel (1:1) to adjust the cell concentration to 3.5 × 10⁻⁶ cells / cells. 7 Cells / mL. 100 μL of cell suspension was subcutaneously injected into both groins of 4-8 week old BALB / c nude mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with 3.5 × 10⁶ cells injected at each site. 6 One cell was injected at two sites. Starting from the second week after inoculation, tumor volume was measured weekly, and tumor growth curves were plotted. The results are as follows: Figure 4 As shown. By week seven, one tumor had reached a volume of approximately 200 mm. 3 Nude mice were euthanized by carbon dioxide inhalation, and tumor tissue was removed. The tissue was fixed, embedded in paraffin, sectioned, and then subjected to immunohistochemical staining. The results are as follows: Figure 5 As shown, the tumor's immunophenotype was: CR (scattered +), CK (-), WT1 (-), D2-40 (+), GATA3 (few +), BAP1 (+); which matched the patient's tumor tissue pathology results.

[0034] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A human skin tumor cell line, with accession number C2025381.

2. The method for constructing the human dermatoma cell line according to claim 1, the method comprising the following steps: (1) Tissue block processing: The obtained mesothelioma tumor specimens are cleaned and cut into pieces to obtain tumor block specimens; (2) Primary cell culture: After three repeated operations, a cell population with uniform morphology was obtained.

3. The use of the human mesothelioma cell line of claim 1 for the preparation of mesothelioma diagnostic reagents.

4. The use of the human mesothelioma cell line according to claim 1 for preparing an animal model of mesothelioma.

5. The use of the human mesothelioma cell line of claim 1 as a drug target for the development of drugs to inhibit mesothelioma.

6. Use of the human mesothelioma cell line of claim 1 for in vitro studies of mesothelioma.

7. The use of the human mesothelioma cell line of claim 1 for screening drugs for the treatment of mesothelioma.

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