An immortalized cell line for isolated fibrous tumors of the central nervous system and its applications

Abstract

This invention relates to the field of biotechnology, specifically, to an immortalized cell line for solitary fibrotic tumors of the central nervous system (CNS SFT). This cell line exhibits stable, unlimited proliferation, maintaining tumor cell characteristics even after more than 20 passages in vitro. The cell line expresses the characteristic CNS SFT fusion gene NAB2-STAT6 and has been identified as originating from CNS SFT tumors. This cell line, named TT-SFT-6569, was deposited on July 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46559. This cell line can serve as an in vitro model for CNS SFT, used for screening candidate drugs and studying tumorigenesis mechanisms, providing an important tool for drug development and pathological research of CNS SFT.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an immortalized cell line of isolated fibrous tumors of the central nervous system and its applications. Background Technology

[0002] Solitary fibrous tumors (SFTs) are rare mesenchymal tumors that can occur anywhere in the body, but those originating in the central nervous system (CNS) are extremely rare, accounting for approximately 0.4% of all CNS tumors. These tumors are closely related to the meninges and commonly occur at the skull base, sagittal sinus, or parafacial region of the falx cerebri and tentorium cerebelli. Preoperative imaging diagnosis can easily be confused with meningiomas. SFTs are borderline tumors, and surgery is the best treatment option. If complete resection is not possible during surgery, postoperative radiotherapy is necessary to control tumor growth, development, and spread. Compared to meningiomas, CNS SFTs are more aggressive, prone to recurrence, and can metastasize. Therefore, finding comprehensive treatment methods in addition to surgery is crucial for improving the treatment outcomes of CNS SFTs. In vitro studies of the cell function and mechanisms of CNS SFTs are important tools for seeking new treatments for malignant tumors and are also crucial for translating basic research into clinical applications. However, currently, there are very few reports on primary culture of CNS SFTs both domestically and internationally. Summary of the Invention

[0003] The technical problem this invention aims to solve is the lack of stable in vitro models for research on solitary fibrous tumors of the central nervous system (CNS SFT). CNS SFT is a very rare type of central nervous system tumor, and there is a lack of effective drug screening models and mechanistic research tools for this tumor in clinical practice.

[0004] Therefore, this invention provides an immortalized cell line derived from human CNS SFT tissue to fill the gap in the prior art. To achieve the above objective, this invention provides the following technical solution: The first aspect of this invention provides a human CNS SFT immortalized cell line. This cell line is obtained from fresh tumor specimens from CNS SFT patients through primary cell culture and immortalized by introducing the human telomerase reverse transcriptase (hTERT) gene. The cell line has the following characteristics: (1) it can proliferate and be passaged continuously in vitro for more than 20 generations without showing obvious signs of senescence, indicating that it has acquired immortalized proliferative capacity; (2) it maintains the typical morphological characteristics of CNS SFT tumor cells, with cells growing in a long spindle or polygonal shape adherent to the wall, and some cells arranged in bundles or whorls; (3) in terms of molecular markers, the cells show positive expression of the characteristic NAB2-STAT6 fusion gene of CNS SFT, and short tandem repeat (STR) sequence analysis confirms that the cell origin is consistent with the original tumor; (4) the cell line is formally deposited, named TT-SFT-6569, and deposited at CGMCC (accession number CGMCC No. 46559).

[0005] A second aspect of the invention further provides its application in drug screening and tumor mechanism research: researchers can use this cell line to screen potential therapeutic drugs targeting CNS SFT and evaluate the effects of drugs on tumor cell proliferation and survival.

[0006] A third aspect of the invention further provides the application of this cell line in studying the occurrence, development and malignant transformation mechanisms of CNS SFT, such as studying the pathogenic role of NAB2-STAT6 fusion genes, FER genes, etc. in tumors. Compared with the prior art, the beneficial effects of the present invention are as follows: This invention marks the first time an immortalized cell line derived from solitary fibrous tumors of the human central nervous system (CNS) has been constructed, filling a gap in this field both domestically and internationally. This cell line exhibits long-term stable passage and retains tumor molecular characteristics, providing a reliable in vitro research model for CNS SFT. Drug sensitivity testing and screening can be performed on this cell model, aiding in the discovery of effective therapeutic drugs; simultaneously, it allows for research into the mechanisms of tumorigenesis and progression, deepening our understanding of the biological characteristics of CNS SFT. In summary, this invention provides an important tool for basic research and new drug development in CNS SFT, demonstrating significant application value. Attached Figure Description

[0007] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The immortalized cell line TT-SFT-6569 is shown under a light microscope. Figure 2To identify the expression of the NAB2-STAT6 fusion gene characteristic of CNS SFT by Q-PCR, immortalized cell lines still showed positive expression of the NAB2-STAT6 fusion gene; Figure 3 The drug dose-response curves were obtained by treating TT-SFT-6569 cells with drugs such as Pryotinib dimaleate, Certinib dihydrochloride, Tucatinib, E260, and DS21360717. Figure 4 To inhibit FER gene expression (A), the proliferation (B), invasion (C), and migration (D) abilities of TT-SFT-6569 cells were reduced. Detailed Implementation

[0008] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0009] In this embodiment, the reagents and instruments used are as follows: Main reagents: Accutase digestive enzymes (Biolegend, 423201), DMEM / F12 medium (Gibco, C11330500BT), penicillin-streptomycin (Solarbio, P8420 / S8290), erythrocyte lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., C3702), fetal bovine serum (Gibco, 16000044), EGF (Novoprotein, C029-500 μg), bFGF (ORIGENE, TP750002), RWD tissue dissociation tubes (Shenzhen Ruiwode Life Science Technology Co., Ltd., DSC-410), Matrigel (CORNING). 354248), Trypsin / EDTA, pCDH-EF1a-mTagBF2-T2A-hTERT-MCS plasmid, PMD2 plasmid, pspax2 plasmid, PEI (Shanghai Beyotime Biotechnology Co., Ltd., Y268769), HEPES (Shanghai Beyotime Biotechnology Co., Ltd., Y170403), sodium butyrate (Shanghai Beyotime Biotechnology Co., Ltd., ST1636), PEG6000 (Shanghai Beyotime Biotechnology Co., Ltd., Y268733), Polybrene (Shanghai Beyotime Biotechnology Co., Ltd., C0351).

[0010] Main instruments: RWD tissue cell dissociation instrument (Shenzhen Ruiwode Life Technology Co., Ltd.); Microscopic scissors (Shanghai Jinzhong); Microscopic metal forceps (Shanghai Jinzhong); Clean bench (Sujing Antai Co., Ltd., Suzhou, China); Benchtop high-speed centrifuge (HITACH, Japan); Low-temperature centrifuge (Thermal Fisher Scientific, USA); Optical microscope (Nikon, Japan); Liquid nitrogen tank (CBS, USA); Pure water system (Millipore, USA); Ice maker (Anchuang Instruments, Suzhou, China); Constant temperature metal bath (Bori, Hangzhou, China); CO2 incubator (Thermo Fisher, USA); Vortex apparatus (Haimen Qilinbei Instrument Co., Ltd.); Cell counter (Countstar, Shanghai, China).

[0011] Example 1: Isolation, identification, and preservation of primary cells from solitary fibrous tumors of the central nervous system. Tumor tissue was obtained from fresh SFT specimens surgically removed by the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, between September 2023 and December 2024. Based on preoperative imaging, 10 cases of highly suspicious CNS SFT were selectively cultured, and postoperative pathology confirmed CNS SFT. A total of 8 primary CNS SFT cells were cultured. All patients signed informed consent forms, and all tumor tissue collection and use complied with the Declaration of Helsinki.

[0012] 1. Primary isolation and culture of CNS SFT cells: (1) Under aseptic conditions, fresh CNS SFT tissue removed by microsurgery was immediately placed in sterile tissue preservation solution for culture within 6 hours; (2) CNS SFT tissue was removed from the laminar flow hood and placed in a culture dish. Blood components on the CNS SFT tissue were washed away with sterile cold PBS, and fibrous tissues such as dura mater, blood clots, charred tissue and arachnoid membrane were cut off with microscissors. The tissue was washed three times with cold PBS. (3) CNS SFT tissue was cut into uniform small pieces of 1 cubic millimeter in the culture dish with curved microscissors. Accutase cell digestion solution was added 10 times more than the total amount of tissue, and all of the tissue was transferred to RWD tissue dissociation tubes and mixed thoroughly. (4) RWD tissue dissociation tubes were placed in a tissue cell suspension preparation instrument. The solution was placed at 37°C and digested for 16 minutes. The solution was automatically mixed to ensure that the tissue and digestion solution were in full contact until the tissue turned white and loose. Three times the volume of PBS was added to stop the digestion. (5) Centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add 5 mL of erythrocyte lysis buffer, mix well by pipetting, incubate at room temperature for 5 minutes, add 15 mL of PBS to stop lysis, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. (6) Add DMEM / F12 culture medium containing 10% fetal bovine serum, add 20 ng / mL EGF + 20 ng / mL EGF + 1% penicillin-streptomycin antibiotic to the culture medium, seed in Matrigel-coated 6 cm culture dishes, and incubate in a 37℃, 5% CO2 cell incubator. Because CNS SFT cells grow slowly, the cell density must be high when seeding cells, otherwise it will be difficult for them to grow to confluence and they will gradually undergo apoptosis.

[0013] 2. Primary culture of CNS SFT cells: CNS SFT cells were placed in DMEM / F12 medium containing 10% fetal bovine serum. The medium was supplemented with 20 ng / mL EGF + 20 ng / mL EGF + 1% penicillin-streptomycin antibiotics. The cells were cultured routinely at 37°C in a 5% CO2 incubator. After 24 hours, the medium was changed, and observation continued. Only when the cells reached over 80% confluence could they be passaged with Trypsin / EDTA digestive enzymes. Since primary CNS SFT cell cultures often contain fibroblasts, and CNS SFT cells are relatively sensitive to digestive enzymes, and fibroblasts adhere firmly, the cells should not be digested for too long to purify them. During digestion, the culture medium was continuously agitated to ensure sufficient contact between the digestive solution and the cells. When cytoplasmic shrinkage and increased intercellular spaces were observed under a microscope, complete culture medium was immediately added to stop the digestion. The digestion time was generally 1–2 minutes. Undigested CNS SFT cells were then removed by pipetting.

[0014] 3. Immortalization of primary CNS SFT cells: Take 500 μL of DMEM, add 4.8 µg PMD2, 7.2 µg pspax2, and 12 µg pCDH-EF1a-mTagBF2-T2A-hTERT-MCS. Vortex for 5-10 seconds, then quickly add 60 µL PEI (1 mg / mL), vortex for 5-10 seconds, let stand for 20 minutes, and drop into culture plates. Change the complete medium every 12-24 hours. After 36 hours, discard the supernatant and replace it with DMEM / F12 (10% FBS + 1% HEPES + 1% sodium butyrate). After 12 hours, collect the supernatant and replace it with DMEM / F12 (10% FBS + 1% HEPES + 1% sodium butyrate). Sodium butyrate was added, and the supernatant was collected after 12 hours. The cells were then incubated at 4°C, 4500 rpm for 10 minutes, or directly filtered through a 0.22 μm filter to remove cell debris. 5*PEG6000 was added, and the cells were incubated at 4°C for at least overnight. The supernatant was then removed, and the cells were resuspended in 600 µl of DMEM / F12 medium. In the first generation of primary cells, Polybrene transfection reagent was added to infect them with hTERT-MCS lentivirus. The cells were cultured routinely and passaged repeatedly until they grew well and were stably passaged to more than 20 generations.

[0015] Results: Morphological observation of CNS SFT cells: After 6 hours of primary culture, adherence to the culture vessel was observed. Within 24 hours, elongated spindle-shaped cells showed adherent growth. Most CNS SFT cells exhibited bipolar growth, with some cells clustering together. After approximately 48 hours, the number of cultured cells significantly increased, exhibiting typical bipolar and polygonal adherent growth, good refractive properties, abundant cytoplasm, and relatively small nucleoli. After four days, the cells covered the bottom of the flask, forming a tightly packed monolayer. Initially, passaged cells were spherical; after 24 hours, most cells adhered and spread out in a bipolar pattern. After passage, the bipolar and multipolar phenomena became more pronounced, the cytoplasm became more abundant, and with increasing passage number, the nucleoli enlarged, and nuclear division was observed. After the fifth passage, the cells exhibited typical morphology, significantly increased cell size, and bipolar or multipolar growth. Cells were interconnected in a network, with abundant cytoplasm, large nuclei, clearly visible nucleoli, and marked mitotic activity. Numerous dense inclusion bodies appeared in the cytoplasm, but cell proliferation remained vigorous, and the cells were identified as CNS SFT cells. However, as the number of passages increased, cell growth gradually slowed. Proliferation was most vigorous from passages 1 to 4, with cells arranged regularly, growing in bundles or whorls, exhibiting vigorous metabolism and strong proliferative capacity. Passages typically occurred every 24 to 48 hours. After the fifth passage, cell proliferation gradually slowed, cell morphology showed obvious signs of aging, and the number of intracellular inclusion bodies gradually increased. WHO Grade 1 primary CNS SFT cells can generally be passaged to 5-6 passages; WHO Grade 2 primary CNS SFT cells can generally be passaged to 8-9 passages; and WHO Grade 3 primary CNS SFT cells can generally be passaged to 15-17 passages. Using the above methods, four immortalized primary CNS SFT cell lines were successfully constructed (3 WHO Grade 1 lines and 1 WHO Grade 2 line). STR identification of the cultured cells showed that all cases were positive for the NAB2-STAT6 fusion gene, and were identified as CNS SFT cells. One WHO Grade 1 CNS SFT cell line (named TT-SFT-6569) was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2025, with accession number CGMCC NO. 46559.

[0016] Example 2: Morphological and functional identification of TT-SFT-6569 cells The morphological and cellular function of the isolated TT-SFT-6569 immortalized cell line were further identified using the following methods: 1. Morphological observation: After seeding CNS SFT cells, the growth process and morphological changes of the cultured cells were observed and photographed daily using an inverted microscope.

[0017] 2. Identification of CNS SFT cells (quantitative polymerase chain reaction (qPCR) analysis): Following the kit instructions, total RNA was extracted from immortalized CNS SFT cells and corresponding CNS SFT tumor tissues using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, catalog number RC112-01). RNA concentration and purity were determined using a NanoDrop spectrophotometer. Subsequently, 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using the HiScript III All-in-one RT SuperMix (Vazyme, catalog number R333). Quantitative PCR (qPCR) was performed on a CFX96 real-time quantitative PCR detection system (Bio-Rad) using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, catalog number Q712). Relative transcript expression levels were calculated using the comparative Cq method (2^-ΔΔCq), with GAPDH as an internal reference gene.

[0018] 3. Drug Inhibition Assay: The small molecule compounds used included E260 (catalog number HY-112097), DS21360717 (catalog number HY-128576), ceritinib dihydrochloride (catalog number HY-15656A), and tucatinib (catalog number HY-16069) from MedChemExpress, and pyrotinib dimaleate (catalog number T12594) from Shanghai Tigermed Biotechnology. The relative survival rate of each treatment group was calculated as a percentage change compared to the solvent control group. The dose-response curves were fitted in GraphPad Prism (v7.0, La Jolla, USA) using a "logarithmic inhibitor concentration vs. normalized response" model.

[0019] 4. Cell function experiments: (1) FER gene knockdown: The expression of the FER (Ensembl:ENSG00000151422) gene was knocked down using lentiviral-mediated transfection. Lentiviral particles were prepared by co-transfecting HEK293T cells with a lentiviral expression plasmid, psPAX2, and pMD2.G in a ratio of 5:3:2. The shRNA sequence targeting FER was cloned into the pLKO.1-Puro vector (Addgene). Freshly prepared lentivirus was added to primary SFT cell lines and HCT116 cells in 6-well plates at a volume of 5 μL per well, and 5 μg / mL of polybrene (Beyotime Biotechnology, C0351) and 10 μg / mL of protamine sulfate were added simultaneously to improve transfection efficiency. 24 hours after transfection, the medium was replaced with fresh medium; 48 hours later, the medium was replaced with complete medium containing 0.625 μg / mL puromycin to screen for successfully infected cells, and screening continued for 2 days. Cells were then divided into a negative control group (shCon, transfected with control shRNA) and a FER knockdown group (shFER). Knockdown efficiency was assessed using Western blot.

[0020] (2) Western blotting: Cell lysis buffer (Beyotime, P0013B) containing protease inhibitor (Bimake, B14001) and phosphatase inhibitor (Merck Millipore, 524625) was used to prepare cell lysis buffer, and protein concentration was determined using a BCA protein quantification kit (Thermo Scientific, 23227). After separation by 7.5% SDS-PAGE, the protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, ISEQ00010). The membrane was blocked at room temperature for 2 hours, followed by overnight incubation at 4°C with the following primary antibodies: FER (Proteintech, 25287-1-AP) and α-tubulin (Proteintech, 11224-1-AP). The sample was washed four times (10 minutes each) with TBS (TBST) containing 0.1% Tween-20, and then incubated with peroxidase-labeled rabbit anti-mouse IgG (Proteintech) or mouse anti-rabbit IgG H&L (Proteintech) secondary antibody. Chemiluminescence was used for color development, and grayscale analysis was performed using ImageJ software. α-tubulin was used as a loading control.

[0021] (3) Proliferation capacity assessment: Cell proliferation was further evaluated using the BeyoClick EdU-647 cell proliferation assay kit (Beyotime Biotechnology, catalog number C0081S). 2000 cells were seeded per well in a 96-well plate and cultured for 24, 48, and 72 hours. The culture medium was then replaced with complete EdU-containing medium, and incubation continued for 2 hours. After incubation, cells were fixed with methanol for 15 minutes, washed with BSA, and permeabilized with Triton X-100 for 15 minutes. Click-iT reaction mixture was added, and the cells were incubated in the dark for 30 minutes. Cell nuclei were stained with Hoechst 33342 (incubated in the dark for 30 minutes). Images were captured using a fluorescence microscope, and the number of EdU-positive cells and total cell nuclei were counted in three random fields of view to calculate the EdU positivity rate.

[0022] (4) Migration and Invasion Assays: The migration and invasion abilities of primary SFT cells were assessed using Transwell chambers (Corning, catalog number 3422). In the migration assay, 3 × 10^4 cells were seeded in the upper chamber, and 600 μL of medium containing 10% FBS was added to the lower chamber. In the invasion assay, the upper chamber was pre-coated with Matrigel (Matrigel:serum-free medium = 1:4, Corning, catalog number 356234). After 48 hours of culture, the cells were fixed with methanol, stained with crystal violet, and rinsed. Microscopic images were taken, and three low-power fields were randomly selected to count the total number of migrating or invading cells that had passed through the membrane pores.

[0023] result: Cell morphology: Under a light microscope, TT-SFT-6569 cells showed typical morphology, significantly enlarged cell size, bipolar or multipolar growth, and interconnected cells forming a network. Figure 1 See page 10 (). Figure 1 A), P20 ( Figure 1 B), P30 ( Figure 1 (C) All of these represent maintaining a relatively good cell state.

[0024] Cell identification: qPCR was used to identify the NAB2-STAT6 fusion gene, a characteristic feature of CNS SFT, in TT-SFT-6569 cells. Compared with corresponding CNS SFT tumor tissues, the expression of the NAB2-STAT6 fusion gene did not show a significant decrease. Figure 2 (n=3).

[0025] Drug inhibition assay: TT-SFT-6569 cells were treated with Pryotinib dimaleate, Certinib dihydrochloride, Tucatinib, E260, and DS21360717. The drug dose-response curves showed that E260 and DS21360717 were comparable to Pryotinib dimaleate and Certinib dihydrochloride in their ability to inhibit TT-SFT-6569 cell viability. Figure 3 (n=3).

[0026] Cellular function experiments: Western blotting results showed that shFER could reduce FER gene expression in TT-SFT-6569 cells. Specifically, shFER2 significantly reduced FER gene expression in TT-SFT-6569 cells compared to the control group (shCon). Figure 4A). In the TT-SFT-6569 cell proliferation assay, compared with the shCon group, the shFER1 and shFER2 groups significantly reduced the number of EdU-positive cells after 72 hours of culture. Figure 4 B, n=3), indicating that inhibiting Fer gene expression can reduce the proliferation ability of TT-SFT-6569 cells. Furthermore, reducing Fer expression significantly inhibits the migration ability of TT-SFT-6569 cells. Figure 4 C) and invasiveness ( Figure 4 D). Among them, the shFER2 group showed a more significant effect compared to the shCon group.

[0027] Therefore, we have confirmed the successful construction of primary CNS SFT cells using tumor specimens from SFT patients. This provides a reliable research model for studying the occurrence, development, evolution, and even malignant transformation of CNSSFT.

[0028] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An immortalized cell line for solitary fibrous tumors of the central nervous system (CNS SFT), characterized in that: The cell line was named TT-SFT-6569 and was deposited at the China General Microbiological Culture Collection Center on July 1, 2025, with accession number CGMCC No. 46559.

2. The cell line according to claim 1, characterized in that, The cell line is derived from human CNS SFT tumor tissue and is immortalized after the introduction of the hTERT gene. It can be stably passaged in vitro for more than 20 generations while maintaining the morphology and molecular characteristics of CNS SFT cells.

3. The cell line according to claim 1, characterized in that, The cell line expresses the NAB2-STAT6 fusion gene of CNS SFT.

4. Use of the cell line according to claim 1 in screening drugs for the treatment of CNS SFT.

5. The use of the cell line according to claim 1 in the study of the occurrence and development mechanism of CNS SFT.

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