Human lung cancer cell line TRI-LC21 and application thereof
By isolating and purifying the human lung cancer cell line TRI-LC21 from tumor tissue of SMARCA4-UT patients, the problem of the lack of stable in vitro models in the prior art has been solved, providing a key tool for SMARCA4-UT research and achieving long-term passage and stability of biological characteristics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEST CHINA HOSPITAL SICHUAN UNIV
- Filing Date
- 2025-12-17
- Publication Date
- 2026-04-24
AI Technical Summary
The lack of in vitro experimental models with a clear origin, stable genetic background, long-term passage capability, and realistic simulation of the biological characteristics of SMARCA4-deficient undifferentiated thoracic tumors (SMARCA4-UT) in existing technologies has led to a shortage of research tools for this disease.
The human lung cancer cell line TRI-LC21 was isolated and purified from the primary tumor tissue of a SMARCA4-UT patient with pathological diagnosis of BRG1 protein expression deficiency. The cell line was purified by differential adhesion and digestion methods, and was established and stably passaged for more than 30 generations to ensure the genetic stability and biological characteristics of the cell line.
It provides a reliable and phenotypic stable cell model that can be passaged for a long time and has the typical biological characteristics of SMARCA4-UT, which can be used for basic research, drug screening and treatment strategy development of SMARCA4-UT, filling the gap in existing technology.
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Figure CN121914969A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of human tumor cell line technology, specifically relating to a human lung cancer cell line TRI-LC21 and its applications. Background Technology
[0002] SMARCA4-deficient undifferentiated thoracic tumor (SMARCA4-UT) is a distinct disease entity formally established in the 2021 World Health Organization (WHO) classification of lung cancer. This highly aggressive tumor typically originates from thoracic structures such as the lungs, mediastinum, or pleura. Histologically, it exhibits significant dedifferentiation, a complex immunophenotype, and is consistently accompanied by the complete absence of BRG1 protein expression (encoded by the SMARCA4 gene). Epidemiologically, it predominantly affects young male smokers, and is often diagnosed at an advanced stage with a very poor prognosis. The median overall survival is only 4 to 7 months, and the tumor is generally unresponsive to current standard chemotherapy and radiotherapy regimens.
[0003] Currently, scientific research on SMARCA4-UT is still in its early exploratory stages. Its exact pathogenesis, core molecular driving events, the basis of drug resistance formation, and potential effective therapeutic targets remain unclear. A key research bottleneck lies in the lack of reliable and stable in vitro experimental models. Due to the scarcity of clinical samples of this tumor, the frequent presence of extensive tissue necrosis, and the extreme difficulty of in vitro culture, no internationally recognized, long-term stable SMARCA4-UT cell line has been established to date. Although some studies have reported the presence of SMARCA4 deficiency or exhibiting molecular characteristics similar to SMARCA4-UT in some traditional lung cancer or small cell lung cancer cell lines, the tumor origin of these cell lines is often unclear, and there is controversy within the academic community regarding whether they can be accurately classified as SMARCA4-UT models. This reflects the previous lack of clarity in our understanding of this disease entity and highlights the severe shortage of systematically validated standard in vitro models specifically for this unique subtype.
[0004] Therefore, developing a cell line with a clear origin, stable genetic background, long-term in vitro culture capability, and the ability to realistically mimic the key biological characteristics of SMARCA4-UT has become a pressing technical challenge in this field. The establishment of such a model will effectively fill existing technological gaps, providing an indispensable research tool for in-depth analysis of the pathological mechanisms of SMARCA4-UT, screening potential therapeutic drugs, and developing novel treatment strategies, thus possessing significant scientific value and clinical translational implications. Summary of the Invention
[0005] The purpose of this invention is to provide a human lung cancer cell line TRI-LC21 and its applications, in order to solve the technical problem of the lack of a standardized in vitro model with a clear source, stable genetic background, long-term passage capability, and realistic simulation of the biological characteristics of SMARCA4-deficient undifferentiated thoracic tumor (SMARCA4-UT) in the prior art. This fills the model gap in the field of disease research and provides a key tool for its pathogenesis research, drug screening and treatment strategy development.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a human lung cancer cell line TRI-LC21, wherein the accession number of the human lung cancer cell line TRI-LC21 is CCTCC NO: C202581, the classification name is: human lung cancer cell TRI-LC21 Homo sapiens, and it was deposited at the China Center for Type Culture Collection on November 13, 2025.
[0007] The present invention further provides a method for constructing the above-mentioned human lung cancer cell line TRI-LC21.
[0008] Preferably, it includes the following steps:
[0009] S1. Obtain tumor samples, and after aseptic extraction, place them in pre-cooled tissue preservation solution and transport them to the laboratory at low temperature;
[0010] S2. Clean the tumor sample, remove non-tumor tissue, and then cut it into small pieces;
[0011] S3. Digest the shredded tumor tissue with digestive juices;
[0012] S4. After digestion, centrifuge to collect the cell pellet, resuspend in DPBS, filter, and centrifuge again to obtain the cell pellet.
[0013] S5. Resuspend the cell pellet in complete culture medium and inoculate for culture;
[0014] S6. Preliminary purification of cells was carried out using differential adhesion to enrich tumor cells;
[0015] S7. Observe the cell growth status, change the culture medium regularly, and passage the cells when the cell confluence reaches more than 80%.
[0016] S8. Remove contaminating cells using cell purification methods. The completely purified tumor cells are then cultured in DMEM medium containing 10% FBS for passage and seed preservation.
[0017] S9. If tumor cells can continue to grow and be passaged for more than 30 generations without showing signs of senescence, then the lineage is successfully established.
[0018] Preferably, the digestion solution in step S3 is a DMEM / F12 culture medium containing 1.25 mg / mL collagenase I and 1.25 mg / mL collagenase IV, and the digestion method is to place it on a constant temperature shaker at 37°C and shake for 1 hour.
[0019] Preferably, the centrifugation speed in step S4 is 1200 rpm and the centrifugation time is 3 min.
[0020] Preferably, the complete culture medium in step S5 is DMEM / F12 containing 2% FBS and 1% penicillin-streptomycin.
[0021] Preferably, the specific steps of the differential adhesion method in step S6 are as follows: after each 20-30 min of static culture, collect the unattached cell suspension and inoculate it into a new culture dish, repeating this operation 2-3 times.
[0022] Preferably, the cell purification method in step S8 is at least one of mechanical scraping, differential adhesion, or digestion and elimination.
[0023] The present invention further provides the application of the above-mentioned human lung cancer cell line TRI-LC21.
[0024] Preferably, the human lung cancer cell line TRI-LC21 is used to construct a model system for lung cancer research.
[0025] Preferably, the human lung cancer cell line TRI-LC21 is used to prepare products for the diagnosis and / or treatment of lung cancer.
[0026] Preferably, the product is at least one of a drug, a diagnostic reagent, or a kit.
[0027] The beneficial effects of this invention are:
[0028] 1. Clear source: The cell line was obtained from the primary tumor tissue of a patient with a pathological diagnosis of BRG1 protein expression deficiency and a tendency for SMARCA4-UT malignant tumor. The cell line was consistent with the patient's tumor tissue by STR analysis, ensuring the reliability of the source.
[0029] 2. Long-term passage and phenotypic stability: TRI-LC21 can be stably passaged in vitro for more than 30 generations, with no significant drift in morphology, proliferation capacity and major phenotype.
[0030] 3. Possesses typical biological characteristics of SMARCA4-UT: complete absence of BRG1 (SMARCA4) protein expression, and has typical undifferentiated morphology and immunophenotypic characteristics of this subtype (such as positive for CD34, SALL4, etc.).
[0031] 4. Can be used for in vivo tumor formation experiments: This cell line can form xenograft tumors in immunodeficient mice.
[0032] The cell line of this invention provides an important experimental model for basic research, drug screening, drug resistance mechanism analysis, immunotherapy and targeted therapy research of SMARCA4-UT. Attached Figure Description
[0033] Figure 1 This is a cell morphology analysis diagram of the human lung cancer cell line TRI-LC21 in this invention;
[0034] Figure 2 This is a cell growth curve of the human lung cancer cell line TRI-LC21 in this invention;
[0035] Figure 3 This is the STR typing map of the human lung cancer cell line TRI-LC21 in this invention;
[0036] Figure 4 This is the STR typing atlas of the patient's tumor tissue in this invention;
[0037] Figure 5 This is the growth curve of the human lung cancer cell line TRI-LC21 xenograft tumor in this invention;
[0038] Figure 6 This is a morphological diagram of a solid tumor at the endpoint of xenografting of the human lung cancer cell line TRI-LC21 in this invention;
[0039] Figure 7 This refers to the H&E staining and immunohistochemical (IHC) staining of the human lung cancer cell line TRI-LC21 in this invention. Detailed Implementation
[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0043] The human lung cancer cell line TRI-LC21 of this invention was deposited at the China Center for Type Culture Collection on November 13, 2025, with accession number CCTCC NO: C202581.
[0044] Example 1: Establishment of the human lung cancer cell line TRI-LC21
[0045] S1. Sample Acquisition and Pretreatment: Within 30 minutes after surgical resection of solid tumor patients, tumor tissue samples were acquired under aseptic conditions, placed in 15 mL sterile centrifuge tubes containing 10 mL of HTK tissue preservation solution pre-cooled to 4°C, and immediately transported to the cell culture laboratory on ice.
[0046] S2. Tissue cleaning and mincing: In a biosafety cabinet, the tumor tissue was transferred to a 10 cm cell culture dish and washed three times with 10 mL of pre-cooled DPBS buffer to remove residual blood and non-tumor tissue from the surface. After removing non-target tissues such as fascia, an appropriate amount of DPBS was added to cover the tissue, and it was minced into tissue fragments of about 1 mm³ using sterile instruments.
[0047] S3. Enzymatic digestion: Transfer the minced tissue to a 15 mL centrifuge tube and add 8 mL of preheated 37°C digestion solution, which is DMEM / F12 medium (Gibco, 11320033) containing 1.25 mg / mL collagenase I (Gibco, 17100017) and 1.25 mg / mL collagenase IV (Gibco, 17104019). Then place in a 37°C shaker and shake for about 1 hour for digestion.
[0048] S4. Cell collection and filtration: After digestion, centrifuge at 1200 rpm for 3 minutes to collect the cell pellet; resuspend the pellet in 10 mL DPBS, filter through a 70 μm sterile cell filter, collect the filtrate in a 50 mL centrifuge tube, and centrifuge again at 1200 rpm for 3 minutes to obtain the cell pellet.
[0049] S5. Initial inoculation culture: Resuspend the cell pellet with an appropriate amount of complete culture medium, inoculate evenly into cell culture dishes, and incubate at 37°C in a 5% CO2 incubator; the complete culture medium is DMEM / F12 medium containing 2% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
[0050] S6. Differential Adhesion Preliminary Purification: Utilizing the difference in adhesion rates between fibroblasts and tumor cells, differential adhesion purification is performed. Specifically, after the cells are incubated statically for 20-30 minutes, the unattached cell suspension is collected and transferred to a new culture dish for further culture. This step is repeated 2-3 times to achieve preliminary separation of fibroblasts and tumor cells.
[0051] S7. Culture Maintenance and Observation: Observe cell morphology and growth status under a microscope daily, and replace with fresh complete culture medium every 2-3 days. When the cell confluence reaches 80% or more, perform passage; if the proportion of contaminating cells is high, perform further purification; further purification: remove contaminating cells from the culture system using one or more of the following methods: mechanical scraping, differential adhesion, or digestion and removal.
[0052] S8. Post-purification culture: The purified tumor cells were replaced with DMEM medium containing 10% FBS for continuous culture, and routine passage and cryopreservation were performed.
[0053] S9. Line establishment identification: If the tumor cells can continue to grow and be passaged for more than 30 generations without showing signs of senescence, then the human lung cancer cell line TRI-LC21 is considered to have been successfully constructed.
[0054] Example 2 Detection
[0055] 1. Cell morphology detection
[0056] 1.1 Method
[0057] The growth of the human lung cancer cell line TRI-LC21 was observed under a light microscope after passage and culture.
[0058] 1.2 Results
[0059] The results showed that the cells grew in an adherent monolayer, with polygonal cell morphology and large, prominent nuclei. Figure 1 ).
[0060] 2. Cell growth curve detection
[0061] 2.1 Methods
[0062] Cell Counting Kit-8 (CCK-8) is a widely used assay for cell proliferation based on WST-8 (water-soluble tetrazolium salt). In the presence of the electron carrier 1-methoxy-5-methylphenazineonium sulfate dimethyl ester (1-MethoxyPMS), WST-8 is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product. The amount of formazan produced is directly proportional to the number of viable cells. The absorbance measured at 450 nm using a microplate reader indirectly reflects the number of viable cells, thus allowing for direct cell proliferation analysis.
[0063] CCK8 assay for cell proliferation includes the following steps:
[0064] (1) Take human lung cancer cell line TRI-LC21 cells in good growth condition, digest them with trypsin to prepare a cell suspension, count them with a cell counting chamber, and adjust the cell concentration to 3×10⁻⁶.5 / mL.
[0065] (2) Seed the cell suspension into a 96-well plate, add 100µL of cell suspension to each well to make the cell count per well 3000, and make 5 replicates for each cell type. Add 100µL of PBS to the edge wells to prevent the liquid from evaporating from the experimental wells.
[0066] (3) CCK8 was measured at different time points (0h, 24h, 48h, 72h, 96h): 10µL of CCK8 was added to each well and a blank control was set up (100µL of culture medium was added to 10µL of CCK8, 5 replicates).
[0067] (4) After incubating at 37℃ for 1-2 hours, the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.
[0068] 2.2 Results
[0069] Use GraphPad software to plot growth curves ( Figure 2 The results showed that the population doubling time of TRI-LC21 cells was approximately 75 hours.
[0070] 3. Identification of cell origin
[0071] 3.1 Method
[0072] Short tandem repeats (STRs) are a class of DNA tandem repeat sequences widely found in eukaryotic genomes. They consist of a core sequence of 2-6 bases repeated n times, typically 5-50 times. The number of repetitions of the core sequence is highly polymorphic among individuals; therefore, STRs are also known as the DNA fingerprint of a cell. The number of repetitions of a set of STR sequences is almost unique among different individuals, making it a primary method for identifying cell identity and origin. STR testing was performed on human lung cancer cell line TRI-LC21 cells and cryopreserved patient tumor tissues by General Biotechnology (Anhui) Co., Ltd., and an STR identification report was issued.
[0073] 3.2 Results
[0074] As shown in Table 1, the typing results of the human lung cancer cell line TRI-LC21 ( Figure 3 ) and the patient's tumor tissue STR ( Figure 4 The atlas comparison results showed a high degree of consistency, confirming that both originated from the same patient. Furthermore, the STR profile of TRI-LC21 did not match any registered cell lines in the public database, indicating that it is a newly established and unique patient-derived lung tumor cell line.
[0075] Table 1 STR typing results
[0076]
[0077] 4. Subcutaneous tumorigenesis assay in nude mice to detect tumorigenesis in cell lines.
[0078] 4.1 Methods
[0079] The human lung cancer cell line TRI-LC21, which is in good growth condition, was digested with trypsin to prepare a cell suspension. The cells were counted using a cell counting chamber, and the cell concentration was adjusted to 2 × 10⁻⁶. 7 / mL. 100 μL of each cell type was seeded into the axillae of five nude mice, and xenograft tumors formed after approximately 3 weeks. Tumor growth curves were plotted using Graph Pad software.
[0080] 4.2 Results
[0081] The results of subcutaneous tumor growth curve analysis of the human lung cancer cell line TRI-LC21 in nude mice showed that palpable tumors appeared in the mice three weeks after inoculation. Figure 5 The solid tumors harvested at the experimental endpoint confirmed the tumorigenic potential of this cell line. Figure 6 ).
[0082] 5. H&E staining and immunohistochemical (IHC) detection of human lung cancer cell line TRI-LC21 xenograft tumors
[0083] 5.1 Method
[0084] H&E staining: Fixed tumor tissue was dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Continuous sections with a thickness of 4 μm were cut using a microtome, placed on glass slides, and baked. Hematoxylin-eosin (H&E) staining was used to observe the morphological characteristics of the tissue.
[0085] Immunohistochemical (IHC) staining: performed using a BOND RX Fully Automated Research Stainer (Leica). The stained sections were mounted and observed under a microscope.
[0086] 5.2 Results
[0087] H&E staining results: H&E staining results of human lung cancer cell line TRI-LC21 xenograft tumors showed diffusely proliferating epithelioid cells. These cells were relatively homogeneous in morphology, with varying intercellular adhesion and prominent nucleoli. Figure 7 ).
[0088] Immunohistochemical (IHC) staining results: IHC staining of human lung cancer cell line TRI-LC21 xenograft tumors showed that SMARCA4 (BRG1), TTF-1, SMARCA2, p40, and CD56 were negative, while SMARCB1, SOX2, SALL4, CD34, and Vimentin were positive. Figure 7 The Ki-67 proliferation index is approximately 50%, consistent with the typical immunophenotype of SMARCA4-UT.
[0089] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.
Claims
1. A human lung cancer cell line TRI-LC21, characterized in that: The human lung cancer cell line TRI-LC21 has the accession number CCTCC NO: C202581.
2. The method for constructing the cell line according to claim 1, characterized in that: Includes the following steps: S1. Obtain tumor samples, and after aseptic extraction, place them in pre-cooled tissue preservation solution and transport them to the laboratory at low temperature; S2. Clean the tumor sample, remove non-tumor tissue, and then cut it into small pieces; S3. Digest the shredded tumor tissue with digestive juices; S4. After digestion, centrifuge to collect the cell pellet, resuspend in DPBS, filter, and centrifuge again to obtain the cell pellet. S5. Resuspend the cell pellet in complete culture medium and inoculate for culture; S6. Preliminary purification of cells was carried out using differential adhesion to enrich tumor cells; S7. Observe the cell growth status, change the culture medium regularly, and passage the cells when the cell confluence reaches more than 80%. S8. Remove contaminating cells using cell purification methods. The completely purified tumor cells are then cultured in DMEM medium containing 10% FBS for passage and seed preservation. S9. If tumor cells can continue to grow and be passaged for more than 30 generations without showing signs of senescence, then the lineage is successfully established.
3. The method according to claim 2, characterized in that: The digestion solution in step S3 is a DMEM / F12 medium containing 1.25 mg / mL collagenase I and 1.25 mg / mL collagenase IV, and the digestion method is to place it on a constant temperature shaker at 37°C and shake for 1 hour.
4. The method according to claim 2, characterized in that: In step S4, the centrifugation speed is 1200 rpm and the centrifugation time is 3 min.
5. The method according to claim 2, characterized in that: The complete culture medium in step S5 is DMEM / F12 containing 2% FBS and 1% penicillin-streptomycin.
6. The method according to claim 2, characterized in that: The specific steps of the differential adhesion method described in step S6 are as follows: After each 20-30 min of static culture, collect the unattached cell suspension and inoculate it into a new culture dish, repeating this operation 2-3 times.
7. The method according to claim 2, characterized in that: The cell purification method described in step S8 is at least one of mechanical scraping, differential adhesion, or digestion and elimination.
8. The application of the human lung cancer cell line TRI-LC21 according to claim 1, characterized in that: The human lung cancer cell line TRI-LC21 was used to construct a model system for lung cancer research.
9. The application of the human lung cancer cell line TRI-LC21 according to claim 1, characterized in that: The human lung cancer cell line TRI-LC21 is used to prepare products for the diagnosis and / or treatment of lung cancer.
10. The application according to claim 9, characterized in that: The product is at least one of a drug, a diagnostic reagent, or a kit.