Lung adenocarcinoma cell strain, lung adenocarcinoma animal model and application

Abstract

The invention relates to the technical field of biology, in particular to a lung adenocarcinoma cell strain, a lung adenocarcinoma animal model and application. The provided lung adenocarcinoma cell strain carries at least one of I-type gene mutation or II-type gene mutation with clinical significance. In-vitro researches (such as drug sensitivity test and signal path analysis) carried out by the cell strain can directly and truly reflect biological behaviors and drug reactions of lung adenocarcinoma with a specific gene mutation background; and a highly clinically related and standardized in-vitro research tool is provided for targeted drug research and development, drug combination strategy exploration and drug resistance mechanism research.

Description

[0001] This application claims priority to Chinese Patent Application No. 2025115092860, filed on October 21, 2025, entitled "A cell line derived from EGFR-mutant lung adenocarcinoma pleural effusion and its luciferase-labeled strain and application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of biotechnology, and in particular relates to lung adenocarcinoma cell lines, lung adenocarcinoma animal models and their applications. Background Technology

[0003] Human epidermal growth factor (EGFR) is one of the key driver genes in lung adenocarcinoma. The proportion of EGFR mutations is high among Chinese lung adenocarcinoma patients, and EGFR-TKI resistance and tumor metastasis are the core challenges in current diagnosis and treatment. While currently marketed human epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) can significantly improve patient prognosis, in clinical practice, most patients inevitably develop resistance after a period of treatment, leading to treatment failure. Simultaneously, tumor metastasis, especially brain metastasis, is another key factor affecting the prognosis of lung adenocarcinoma patients, further exacerbating the difficulty of clinical treatment. These two factors together constitute the core challenges that urgently need to be overcome in the current field of lung adenocarcinoma diagnosis and treatment.

[0004] Currently, commonly used lung adenocarcinoma cell lines carrying EGFR mutations in scientific research, such as NCI-H1975, HCC827, NCI-H1650, and NCI-820, are all derived from European and American populations. Considering the significant differences that may exist among different populations in tumor gene mutation profiles, drug sensitivity, and disease progression characteristics, these European and American cell lines cannot fully mimic the disease characteristics of Chinese lung adenocarcinoma patients and cannot meet the needs of preclinical research on EGFR-TKI resistance mechanisms and metastasis patterns in Chinese patients. Although the PC9 cell line also carries EGFR mutations and is derived from East Asian populations (Japanese patients), sharing some similarities with the EGFR mutation background of Chinese patients, this cell line originates from orthotopic lung cancer tissue and lacks metastatic characteristics, thus presenting significant limitations in research on lung adenocarcinoma metastasis-related mechanisms.

[0005] To address the need for cell lines in lung adenocarcinoma metastasis research, existing technologies have attempted to develop cell lines targeting metastatic characteristics. Currently, a high-potential lung cancer cell line for brain metastasis, H1975luc, has been publicly disclosed. BM4. This cell line was obtained by transfecting the NCI-H1975 cell line from Europe and the United States with the luciferase gene, followed by repeated inoculation into the left ventricle of nude mice and in vitro culture screening. Although it exhibits high brain metastasis characteristics, its basal cell line, NCI-H1975, did not originate from the metastatic sites of patients. Furthermore, the process of constructing a metastasis model through artificial left ventricular injection differs significantly from the actual pathological process of natural invasion and metastasis of cancer cells in clinical patients, making it difficult to accurately reflect the biological characteristics of clinical tumor metastasis. More importantly, the development of this cell line did not establish the association between metastatic characteristics and EGFR-TKI resistance, failing to meet the current research needs for studying the synergistic mechanism of EGFR-TKI resistance and tumor metastasis, and greatly limiting its application value in the combined study of targeted therapy resistance and metastasis in lung adenocarcinoma. Summary of the Invention

[0006] The purpose of this application is to provide lung adenocarcinoma cell lines, lung adenocarcinoma animal models and their applications, aiming to solve the problem of the lack of a comprehensive model that can be used to study both EGFR-TKI resistance mechanisms and tumor metastasis mechanisms in lung adenocarcinoma research originating from the Chinese population.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a lung adenocarcinoma cell line carrying a gene mutation, the gene mutation including at least one of clinically significant class I or class II gene mutations.

[0008] In some embodiments, class I gene mutations include deletion mutations carrying the EGFR gene. In some embodiments, class II gene mutations include at least one of a nonsense mutation carrying the TP53 gene or a nonsense mutation carrying the PTEN gene.

[0009] In some embodiments, deletion mutations in the EGFR gene include exon 19 deletion mutations.

[0010] In some embodiments, a deletion mutation in exon 19 results in the deletion of glutamic acid from position 746 to position 750 alanine in the protein it encodes.

[0011] In some embodiments, nonsense mutations in the TP53 gene cause the protein it encodes to prematurely form a stop codon at amino acid position 91.

[0012] In some embodiments, nonsense mutations in the PTEN gene cause the protein it encodes to prematurely form a stop codon at amino acid position 319.

[0013] In some embodiments, the cell line includes at least one sex locus.

[0014] In some embodiments, the genotyping of a single sex locus is Amelogenin: X, X; In some embodiments, the cell line includes at least 8 core STR sites; In some embodiments, the eight core STR loci include vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0015] In some embodiments, the cell line includes at least one of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0016] In some embodiments, the cell line includes all of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; The TH cell line also includes at least one of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24.

[0017] In some embodiments, the cell line further includes all of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24.

[0018] In some embodiments, the cell line is derived from Chinese patients with lung adenocarcinoma.

[0019] In some embodiments, the cell lines were derived from pleural effusion samples from Chinese patients with lung adenocarcinoma. O1: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0020] In some embodiments, the lung adenocarcinoma cell line is human lung adenocarcinoma cell line SDL; the accession number is CCTCC NO:C2025235, it is deposited at the China Center for Type Culture Collection, the deposit date is August 13, 2025, the deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, and the classification name is human lung adenocarcinoma cell line SDL Homo sapiens.

[0021] Secondly, this application provides a lung adenocarcinoma cell line, which is isolated from the aforementioned lung adenocarcinoma cell line.

[0022] Thirdly, this application provides a modified cell line, which includes the cell line described above.

[0023] In some embodiments, the cell line also includes exogenous nucleic acids.

[0024] In some embodiments, the exogenous nucleic acid includes at least one of plasmid DNA or at least a partial fragment thereof, siRNA or at least a partial fragment thereof, mRNA or at least a partial fragment thereof, miRNA or at least a partial fragment thereof, and viral vector or at least a partial fragment thereof.

[0025] In some embodiments, exogenous nucleic acids include at least one of a marker gene or at least a fragment thereof, a therapeutic gene or at least a fragment thereof, and a functional regulatory gene or at least a fragment thereof.

[0026] In some embodiments, the marker gene includes at least one of the following: luciferase gene, fluorescent protein gene, and β-galactosidase gene.

[0027] In some embodiments, the modified cell line includes human lung adenocarcinoma cells SDL-luci; accession number CCTCCNO:C2025236, deposited at the China Center for Type Culture Collection, deposited on August 13, 2025, at the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, and classified as human lung adenocarcinoma cells SDL-luci Homosapiens.

[0028] Fourthly, this application provides an animal model of lung adenocarcinoma, wherein the tumor in the animal model is formed by the aforementioned lung adenocarcinoma cell line.

[0029] In some embodiments, the animal model is constructed by inoculating the above-described cell lines into immunodeficient mice.

[0030] Fifthly, this application provides the application of the above-mentioned lung adenocarcinoma cell lines or the above-mentioned lung adenocarcinoma animal models in the preparation of kits or reagents for drug research or screening, the application of kits or reagents for studying the mechanism of lung adenocarcinoma development, or the application of components in in vitro or in vivo model systems for drug screening or mechanism research.

[0031] The first aspect of this application provides a lung adenocarcinoma cell line carrying clinically relevant gene mutations; in vitro studies of this cell line (such as drug sensitivity testing and signaling pathway analysis) can more directly and realistically reflect the biological behavior and drug response of lung adenocarcinoma with a specific gene mutation background, providing a highly clinically relevant and standardized in vitro research tool for targeted drug development, exploration of combination drug strategies, and research on drug resistance mechanisms.

[0032] The second aspect of this application provides lung adenocarcinoma cells, which are isolated from the aforementioned lung adenocarcinoma cell line. Therefore, the obtained cells provide a highly clinically relevant and standardized in vitro research tool for the development of targeted drugs related to lung adenocarcinoma, the exploration of combination drug strategies, and the study of drug resistance mechanisms.

[0033] The third aspect of this application provides a modified cell line, which is made more suitable for clinical research by modifying lung adenocarcinoma cell lines carrying clinically relevant gene mutations.

[0034] The fourth aspect of this application provides an animal model of lung adenocarcinoma, in which the tumor is formed by the aforementioned lung adenocarcinoma cell line. This method is a standardized and highly reproducible mature technology for constructing human tumor animal models, which is convenient for promotion and application in different laboratories.

[0035] The cell line and model provided in the fifth aspect of this application can serve as a core tool for constructing a high-throughput drug screening and efficacy evaluation platform, accelerating the development of new drugs targeting specific mutations such as EGFR and TP53 / PTEN. Simultaneously, as a standardized research model, it can deeply reveal the molecular mechanisms by which related gene mutations drive tumor progression and drug resistance. Its application spans from basic research to clinical translation, providing crucial support for validating biomarkers, predicting combination therapies, and developing personalized treatment strategies, thus possessing significant scientific and industrial value. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1This is a schematic diagram of the method for constructing a human lung adenocarcinoma pleural effusion-derived cell line provided in Embodiment 1 of this application; Figure 2 This is a schematic diagram of the method for constructing cells stably expressing luciferase provided in Example 2 of this application; Figure 3 These are the morphologies of SDL under a microscope (A) and SDL-luci under a microscope (B) provided in the embodiments of this application.

[0038] Figure 4 Tumor growth curves were obtained to construct an in vivo model by subcutaneous injection of SDL cells in Balb / c-nude mice.

[0039] Figure 5 These are in vivo imaging images on day 0 and day 21 of a transfer model established by tail vein injection of SDL-luci cells in Balb / c-nude mice. Detailed Implementation

[0040] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0041] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0042] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0043] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0044] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0045] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0046] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0047] The first aspect of this application provides a lung adenocarcinoma cell line carrying gene mutations, including at least one of clinically significant class I or class II gene mutations.

[0048] The first aspect of this application provides a lung adenocarcinoma cell line carrying clinically relevant gene mutations. In vitro studies of this cell line (such as drug sensitivity testing and signaling pathway analysis) can more directly and realistically reflect the biological behavior and drug response of lung adenocarcinoma with a specific gene mutation background, providing a highly clinically relevant and standardized in vitro research tool for targeted drug development, exploration of combination drug strategies, and research on drug resistance mechanisms.

[0049] In some embodiments, the cell line is derived from pleural effusion samples from Chinese patients with lung adenocarcinoma.

[0050] Pleural effusion is a common metastasis-related fluid sample in patients with advanced lung adenocarcinoma. The cells from which it originates directly reflect the biological characteristics of tumor cells during the metastasis process in vivo. Compared with the limitations of the PC9 cell line, which is "originating from situ tissue and lacks metastatic characteristics", this cell line can more accurately simulate the cell phenotype of the metastatic stage of clinical lung adenocarcinoma, providing a cell model that is closer to clinical reality for the study of metastasis mechanisms.

[0051] The tumor cells in the pleural effusion sample were directly derived from the patient, fully preserving the tumor heterogeneity of Chinese lung adenocarcinoma patients, rather than being artificially induced or modified cell lines. Research results based on this cell line are more easily translated into clinical treatment plans, reducing the risk of a disconnect between laboratory research and clinical application, and providing more reliable preliminary data for the subsequent development of targeted drugs or treatment strategies for Chinese patients.

[0052] The cell line provided in this application embodiment was derived from a pleural effusion sample of a 51-year-old Chinese female (Han nationality) patient with lung adenocarcinoma. The patient was diagnosed with stage IV lung adenocarcinoma, had no history of smoking, and her treatment history is as follows:

[0053] Type I gene mutations directly promote the survival, proliferation, and growth of tumor cells and are a major driving force in cancer development. In some embodiments, type I gene mutations include deletion mutations carrying the EGFR gene. In some embodiments, deletion mutations in the EGFR gene include exon 19 deletion mutations.

[0054] In some embodiments, a deletion mutation in exon 19 results in the deletion of glutamic acid from position 746 to position 750 alanine in the protein it encodes.

[0055] The deletion of glutamate from position 746 to alanine from position 750 (E746-A750) in the EGFR gene is one of the most common EGFR activating mutations in Chinese lung adenocarcinoma patients, accounting for approximately 40%-50% of all EGFR mutations. This mutation highly coincides with the first-line therapeutic targets of EGFR-TKIs (such as gefitinib and erlotinib) in clinical practice. This cell line carrying this specific mutation can be directly used for EGFR-TKI sensitivity testing and resistance mechanism research, solving the problem of "mutation type mismatch with clinically high-frequency mutations" in some existing cell lines, and significantly improving the clinical relevance of the research.

[0056] The E746-A750 deletion mutation leads to a conformational change in the EGFR protein, thereby continuously activating downstream signaling pathways, and its biological effects differ from other EGFR mutations. This cell line exhibits a single, clearly defined mutation type, eliminating interference from multiple mutations. This helps researchers accurately elucidate the signaling pathway mechanisms associated with the E746-A750 deletion mutation and the differences in response to different EGFR-TKIs, providing a precise research model for developing novel targeted drugs against this specific mutation.

[0057] In some embodiments, class II gene mutations include at least one of a nonsense mutation carrying the TP53 gene or a nonsense mutation carrying the PTEN gene.

[0058] In some embodiments, nonsense mutations in the TP53 gene cause the protein it encodes to prematurely form a stop codon at amino acid position 91.

[0059] In some embodiments, nonsense mutations in the PTEN gene cause the protein it encodes to prematurely form a stop codon at amino acid position 319.

[0060] This cell line carries a nonsense mutation that prematurely terminates at amino acid 91 of the TP53 gene, realistically simulating the disease state of "EGFR+TP53 co-mutation" in clinical practice. It overcomes the limitations of some existing EGFR mutant cell lines (such as PC9) that "lack TP53 mutation and cannot simulate the clinical phenotype of co-mutation", providing a key model for studying the impact of co-mutation on the efficacy of EGFR-TKIs and developing combination therapy strategies.

[0061] As a "genomic guardian," the loss of TP53 function leads to decreased DNA damage repair capacity and enhanced apoptosis resistance in tumor cells, thereby affecting EGFR-TKI resistance and metastatic potential. The TP53 mutation in this cell line is well-defined and stable, and can be used to elucidate key scientific questions such as "how TP53 inactivation regulates EGFR pathway resistance" and "the association between TP53 mutation and tumor cell invasion and metastasis," providing a theoretical basis for developing drugs to reverse resistance or inhibit metastasis targeting TP53 mutations.

[0062] The PTEN gene is a negative regulator of the PI3K / Akt signaling pathway. Mutations or deletions in the PTEN gene lead to overactivation of the PI3K / Akt pathway, a key mechanism of EGFR-TKI resistance. This cell line carries a nonsense mutation with premature termination at amino acid position 319 of the PTEN gene, coexisting with EGFR and TP53 mutations, perfectly replicating the complex clinical scenario of "multi-gene mutation-driven drug resistance." For PI3K / Akt pathway activation caused by PTEN deletion, combination therapy with EGFR-TKIs and PI3K inhibitors is often considered in clinical practice. The PTEN mutation characteristics of this cell line can be directly used to test the synergistic efficacy of "EGFR-TKI + PI3K inhibitor," evaluate the inhibitory effect of combined therapy on drug-resistant cells, provide preliminary experimental data support for the development of multi-target combination therapy regimens, and accelerate the translation and application of drug resistance treatment strategies.

[0063] In some embodiments, the cell line includes at least one sex locus. The genotyping of this sex locus is Amelogenin:X,X; In some embodiments, the cell line includes at least eight core STR sites. The core STR sites include vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0064] In some embodiments, the cell line includes at least one of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0065] In some embodiments, the cell line includes all of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

[0066] In some embodiments, the cell line further includes at least one of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24; In some embodiments, the cell line further includes all of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24.

[0067] STR spectroscopy is the "gold standard" for cell line identification. It details the typing results of 21 STR loci. By comparing the STR spectra of other cell lines, the uniqueness and purity of the cell line can be ensured, avoiding deviations in research results due to cell contamination and guaranteeing the reliability of scientific research data.

[0068] Allele 1 and Allele 2: In normal diploid cells (the vast majority of human cells), any STR locus should have two alleles, one from the father and one from the mother. When Allele 3 (i.e., three numbers) appears at a locus, this is called "trieles." This is extremely rare in normal tissues but very common in tumor cells. The genome of tumor cells is unstable, which may lead to trisomy on a chromosome, meaning that a chromosome has three copies instead of the normal two. The STR locus located on this chromosome will naturally have three alleles. Tumor cells may locally replicate / amplify the genomic region containing this STR locus, resulting in an increased copy number of that locus, thus allowing the detection of the additional alleles.

[0069] The cell lines described above have a short tandem repeat sequence profile, which can be described in more detail using a table, as shown in Table 1: Table 1

[0070] Note: The bolded sites in Table 1 refer to the genotyping of the 8 core STR sites and 1 sex site that are required to be tested according to the US National Standard (ASN-0002-2011), which are internationally recognized testing sites.

[0071] In some embodiments, the cell line is derived from pleural effusion samples from Chinese patients with lung adenocarcinoma.

[0072] In some embodiments, the lung adenocarcinoma cell line is human lung adenocarcinoma cell line SDL; the accession number is CCTCC NO:C2025235, it is deposited at the China Center for Type Culture Collection, the deposit date is August 13, 2025, and the deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

[0073] The provided lung adenocarcinoma cell line carrying EGFR mutations is deposited at the China Center for Type Culture Collection (CCTCC NO: C2025235). Its EGFR mutation characteristics highly match the clinical features of high EGFR mutation prevalence in Chinese lung adenocarcinoma patients, completely solving the problem that European and American cell lines such as NCI-H1975 and HCC827 cannot simulate the disease characteristics of Chinese patients. This provides a precisely tailored cellular tool for preclinical studies such as EGFR mutation-related pathway analysis and drug sensitivity testing, effectively avoiding research bias caused by differences in population genetic background. Furthermore, the clear deposit information and provenance pathway facilitate direct acquisition from authoritative institutions, avoiding cumbersome primary culture procedures and significantly reducing research costs. Meanwhile, its stable biological characteristics provide a solid guarantee for the consistency and comparability of research results, and strongly promote the standardization process of EGFR-related research in lung adenocarcinoma. Furthermore, this cell line is more conducive to realistically reproducing clinical drug resistance scenarios, providing an ideal model for the study of EGFR-TKI resistance mechanisms, and providing an efficient tool for exploring the metastasis mechanism of lung adenocarcinoma. This cell line also has the characteristics of acquired resistance to EGFR-TKI and high metastasis, providing a key breakthrough for revealing the linkage mechanism between drug resistance and metastasis, and laying a core foundation for the subsequent construction of luciferase stably expressing cell lines and in-depth research on drug resistance / metastasis mechanisms.

[0074] In some specific embodiments, a method for constructing the above-mentioned lung adenocarcinoma cell line carrying EGFR mutations is provided, comprising the following steps: Pleural fluid samples were obtained from patients with lung adenocarcinoma, and cell pellets were obtained by centrifugation. The cell pellet was washed with phosphate buffer; the cells were resuspended in advanced DMEM / F12 medium containing 10% fetal bovine serum and growth factors for primary culture. When the cells reach 80%-90% polymerization, they should be passaged. After 10 passages, the culture medium was changed to DMEM containing 10% fetal bovine serum and cultured for another time to obtain a stable passaged lung adenocarcinoma cell line.

[0075] Secondly, embodiments of this application provide a lung adenocarcinoma cell line, which is isolated from the aforementioned lung adenocarcinoma cell line.

[0076] Thirdly, embodiments of this application provide a modified cell line, which includes the cell line described above.

[0077] In some embodiments, the cell line also includes exogenous nucleic acids; In some embodiments, the exogenous nucleic acid includes at least one of plasmid DNA or at least a partial fragment thereof, siRNA or at least a partial fragment thereof, mRNA or at least a partial fragment thereof, miRNA or at least a partial fragment thereof, and viral vector or at least a partial fragment thereof; In some embodiments, exogenous nucleic acids include at least one of a marker gene or at least a partial fragment thereof, a therapeutic gene or at least a partial fragment thereof, and a functional regulatory gene or at least a partial fragment thereof; In some embodiments, the marker gene includes at least one of the following: luciferase gene, fluorescent protein gene, and β-galactosidase gene.

[0078] In some specific embodiments, a lung adenocarcinoma cell line stably expressing luciferase is also provided. The lung adenocarcinoma cell line is obtained by stable transfection of the above-mentioned lung adenocarcinoma cell line with the luciferase gene. The lung adenocarcinoma cell line is human lung adenocarcinoma cell line SDL-luci; the accession number is CCTCC NO:C2025236, it is deposited at the China Center for Type Culture Collection, the deposit date is August 13, 2025, and the deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

[0079] The provided lung adenocarcinoma cell line stably expressing luciferase, compared to the original cell line not expressing luciferase, allows for real-time, non-invasive tracking of cell survival, proliferation, and metastasis in in vitro and in vivo experiments using a luciferase-luciferin luminescence system through stable transfection of the luciferase gene. Furthermore, luciferase activity detection is characterized by high sensitivity, convenient operation, and strong quantification of results. It can rapidly assess cell status simply by detecting the luminescence signal, significantly shortening the experimental cycle and reducing detection costs, making it applicable to specific research processes.

[0080] In some specific embodiments, the method for obtaining lung adenocarcinoma cell lines stably expressing luciferase described above includes the following steps: Lung adenocarcinoma cells were infected using a lentiviral vector containing the luciferase gene. Cells stably expressing luciferase were obtained through puromycin selection; The expression activity of luciferase was verified, and a lung adenocarcinoma cell line with stable luciferase expression was obtained.

[0081] The provided method for constructing a stable luciferase-expressing lung adenocarcinoma cell line employs the classic combination of "lentiviral vector infection + puromycin screening." The lentiviral vector integrates the luciferase gene into the cell genome, significantly improving the efficiency of obtaining stable expression cell lines and reducing screening time and labor costs. This ensures that the final cell line not only "stablely expresses" but also possesses "effective activity," avoiding failures in in vitro and in vivo tracking experiments due to insufficient luciferase activity and guaranteeing the accuracy of subsequent metastasis studies and drug resistance tests.

[0082] The fourth aspect of this application provides a lung adenocarcinoma animal model, wherein the tumor in the animal model is formed by the aforementioned lung adenocarcinoma cell line.

[0083] The fourth aspect of this application provides a lung adenocarcinoma animal model, in which the tumor is formed by the aforementioned lung adenocarcinoma cell line. This method is a standardized and highly reproducible mature technology for constructing human tumor animal models, which is convenient for promotion and application in different laboratories.

[0084] In some embodiments, the animal model is constructed by inoculating the above-described cell lines into immunodeficient mice.

[0085] The fifth aspect of this application provides the application of the above-described lung adenocarcinoma cell line or the above-described lung adenocarcinoma animal model in the preparation of kits or reagents for drug research or screening, the application of kits or reagents for studying the mechanism of lung adenocarcinoma development, or the application of components in in vitro or in vivo model systems for drug screening or mechanism research.

[0086] The cell line and model provided in the fifth aspect of this application can serve as a core tool for constructing a high-throughput drug screening and efficacy evaluation platform, accelerating the development of new drugs targeting specific mutations such as EGFR and TP53 / PTEN. Simultaneously, as a standardized research model, it can deeply reveal the molecular mechanisms by which related gene mutations drive tumor progression and drug resistance. Its application spans from basic research to clinical translation, providing crucial support for validating biomarkers, predicting combination therapies, and developing personalized treatment strategies, thus possessing significant scientific and industrial value.

[0087] The following description is based on specific embodiments.

[0088] Example 1 This embodiment provides a method for constructing a cell line derived from pleural effusion of human lung adenocarcinoma, the construction process of which is as follows: Figure 1 As shown, the specific steps are as follows: 1. Primary cell isolation a) Transfer the patient's pleural effusion sample to a 50 mL centrifuge tube and centrifuge at 800 rpm for 10 minutes to obtain cell pellet.

[0089] b) Add 10 mL of PBS to wash the cells and continue centrifuging at 800 rpm for 10 minutes.

[0090] c) Repeat step b) 10 times. d) Resuspend the cell pellet in advanced DMEM / F12 (Gibco) medium containing 10% FBS and other growth factors (EGF: Proteintech, 20 ng / mL; Noggin: PrepoTech, 100 ng / mL; R-Spondin: PrepoTech, 100 ng / mL), transfer to T75 culture flasks, and incubate in a 37°C incubator containing 5% carbon dioxide.

[0091] 2. Cell culture and passage a) The primary cells obtained in step 1 were cultured in advanced DMEM / F12 medium containing 10% FBS and other growth factors (as described above) in a 37°C incubator with 5% carbon dioxide, and the medium was changed every three days.

[0092] b) When the cells reach 80%-90% polymerization, passage the cells: discard the culture medium using a vacuum pump, add PBS to rinse the cells, discard the PBS, add 1X trypsin, digest at 37°C for 5 minutes, stop digestion with complete culture medium and transfer the cell suspension to a centrifuge tube, centrifuge at 500 rpm for 5 minutes, resuspend the cell pellet with culture medium, take a certain volume of cell suspension and transfer it to a new culture flask, and continue culturing under the conditions in a).

[0093] c) After ten passages of primary cell culture, the culture medium was replaced with DMEM medium containing 10% FBS. Testing showed that the cells could be stably passaged for more than 30 generations.

[0094] 3. STR testing Whole-genome DNA was extracted from cell pellet using a genomic extraction kit (Aige Biotechnology), and DNA concentration was measured by QC using a micro-UV-Vis spectrophotometer (Thermo Fisher NanoDrop 2000c). Multiplex amplification using an IGE-STR20A multi-fluorescence system (ABI 9700 PCR System) was performed, followed by capillary electrophoresis and fragment separation using an ABI 3730XL genetic analyzer (ABI Corporation, USA), and genotyping analysis using GeneMapper® ID software (version ID-X 1.5). STR locus information is as described above. The SDL of the cells obtained in this application differs from those recorded by ATCC, DSMZ, JCRB, and RIKEN.

[0095] Example 2 This embodiment uses SDL cells as the parent cell line to provide a method for constructing cells that stably express luciferase. The construction process is as follows: Figure 2 As shown, the specific steps are as follows: 1. Lentiviral transfection and screening a) Culture 293T cells to 40%-50% polymerization, transfer plasmids containing the luciferase gene into 293T cells, change the culture medium within 12 hours, collect the culture medium 24 and 48 hours after changing the medium, and filter the culture medium through a 0.22 μM filter membrane to obtain the virus solution.

[0096] b) Once the SDL cells enter the logarithmic growth phase, replace the culture medium with the virus solution obtained in 1.a), and add polybrene at a final concentration of 5 μg / mL for staining. Change the medium 12 hours after infection. When the cell aggregation degree reaches 60%-70%, add 5 μg / mL of puromycin (Taosu Biotechnology) for treatment. Remove puromycin 48 hours later and culture until passage.

[0097] c) Once the cells from b) have entered the logarithmic growth phase, infect them again with a viral solution containing 5 μg / mL polybrene. Change the medium after 12 hours. When the cell aggregation reaches 60%-70%, treat with 10 μg / mL puromycin. Remove puromycin after 48 hours and culture until passage to obtain SDL-luci cells.

[0098] 2. Detection of luciferase expression a) Seed the cells selected by puromycin into 96-well plates. After the cells have grown to confluence, add 0.15 mg / mL luciferase substrate (APExBIO) prepared with PBS and incubate at 37°C in the dark for 15 minutes.

[0099] b) Bioluminescence was detected using a SpectraMax iD3 microplate reader, confirming that the cells obtained through lentiviral transfection and puromycin selection successfully expressed luciferase; results are as follows. Figure 3 As shown, Figure 3 The morphology of SDL, a cell line derived from pleural effusion in human lung adenocarcinoma, under a microscope. Figure 3 B represents the morphology of SDL-luci under a microscope.

[0100] Example 3 This embodiment uses the cell SDL from Example 1 to establish an in vivo animal model. The specific steps are as follows: a) SDL cells cultured under the aforementioned conditions were digested with trypsin and centrifuged to obtain a cell pellet. The cells were then resuspended in PBS and matrix gel (1:1) to achieve a cell concentration of 1×10⁻⁶. 7 / mL, place the cell suspension on an ice box to maintain cell viability.

[0101] b) Select 4-6 week old Balb / c nude mice as experimental subjects. Inject the cell suspension subcutaneously into the root of the left hind limb on the back of the mouse using a syringe. Each mouse receives 1×10⁻⁶ cells. 6 cell.

[0102] c) Begin recording observations one week after injection. Measure the size of the tumor using calipers and record the results as follows: Figure 4 As shown, the tumors inoculated gradually increased in size over three weeks of continuous observation, indicating that the cell line has good tumorigenicity.

[0103] Example 4 This embodiment uses the SDL-luci cells from Example 2 to establish an in vivo transfer model. The specific steps are as follows: 1. Tail vein injection a) SDL-luci cells were digested with trypsin and centrifuged to obtain a cell pellet. The cells were then resuspended in pre-cooled PBS to achieve a cell concentration of 1×10⁻⁶. 7 / mL, place the cell suspension on an ice box to maintain cell viability.

[0104] b) Select 4-6 week old Balb / c nude mice as experimental subjects, and inject 100 μL of cell suspension into the mice via the tail vein using an insulin injector.

[0105] 2. In vivo imaging a) Half an hour after tail vein injection, 100 μL of luciferase substrate (APExBIO, 15 mg / mL) was injected into mice via intraperitoneal injection.

[0106] b) Anesthetize mice with isoflurane and fix them in a live imaging system (PerkinElmer) for imaging.

[0107] c) Twenty-one days after tail vein injection, mice were subjected to in vivo imaging again as described in a) and b), and the results were as follows. Figure 5 As shown, the colonization rate of SDL-luci cells via tail vein injection was 100%.

[0108] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A lung adenocarcinoma cell line, characterized in that, The lung adenocarcinoma cell line carries gene mutations, including at least one of clinically significant class I or class II gene mutations.

2. The lung adenocarcinoma cell line according to claim 1, characterized in that, The Class I gene mutations include deletion mutations in the EGFR gene; and / or, The Class II gene mutations include at least one of the nonsense mutations of the TP53 gene or the nonsense mutation carrying the PTEN gene.

3. The lung adenocarcinoma cell line according to claim 2, characterized in that, The deletion mutations in the EGFR gene include deletion mutations in exon 19.

4. The lung adenocarcinoma cell line according to claim 3, characterized in that, The deletion mutation in exon 19 results in the deletion of glutamic acid from position 746 to position 750 alanine in the protein it encodes.

5. The lung adenocarcinoma cell line according to claim 2, characterized in that, The nonsense mutation in the TP53 gene causes the protein it encodes to prematurely form a stop codon at amino acid position 91.

6. The lung adenocarcinoma cell line according to claim 2, characterized in that, The nonsense mutation in the PTEN gene causes the protein it encodes to prematurely form a stop codon at amino acid position 319.

7. The lung adenocarcinoma cell line according to any one of claims 1-6, characterized in that, The cell line includes at least one sex locus; Optionally, the genotyping of the one sex locus is Amelogenin: X, X; Optionally, the cell line includes at least 8 core STR sites; Optionally, the eight core STR loci include vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10; Optionally, the cell line includes at least one of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10; Optionally, the cell line includes all of the following STR sites: Amelogenin: X, X; vWA: 17, 17; D7S820: 10, 11; CSF1PO: 11, 11; D16S539: 11, 11; TH01: 9, 9; D13S317: 8, 9; TPOX: 8, 8; D5S818: 9, 10.

8. The lung adenocarcinoma cell line according to claim 7, characterized in that, The cell line further includes at least one of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24; Optionally, the cell line further includes all of the following STR sites: D3S1358: 16, 16; PentaE: 5, 18, 19; D8S1179: 10, 14, 15; D21S11: 31.2, 32.3; D2S1338: 21, 22, 23; PentaD: 10, 15; D19S433: 13, 13; D18S51: 14, 14; D6S1043: 10, 10; D1S1656: 14, 16; D12S391: 20, 21; FGA: 23, 24.

9. The lung adenocarcinoma cell line according to any one of claims 1-6, characterized in that, The cell line was derived from Chinese patients with lung adenocarcinoma. Optionally, the cell line is derived from pleural effusion samples from Chinese patients with lung adenocarcinoma.

10. The lung adenocarcinoma cell line according to any one of claims 1-6, characterized in that, The lung adenocarcinoma cell line includes human lung adenocarcinoma cell line SDL; its accession number is CCTCC NO:C2025235, it is deposited at the China Center for Type Culture Collection, the deposit date is August 13, 2025, and the deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

11. A lung adenocarcinoma cell line, characterized in that, The lung adenocarcinoma cells were isolated from the lung adenocarcinoma cell line according to any one of claims 1-10.

12. A modified cell line, characterized in that, The modified cell line includes the cell line according to any one of claims 1-10; Optionally, the modified cell line may also include exogenous nucleic acids; Optionally, the exogenous nucleic acid includes at least one of plasmid DNA or at least a partial fragment thereof, siRNA or at least a partial fragment thereof, mRNA or at least a partial fragment thereof, miRNA or at least a partial fragment thereof, and viral vector or at least a partial fragment thereof; Optionally, the exogenous nucleic acid includes at least one of a marker gene or at least a fragment thereof, a therapeutic gene or at least a fragment thereof, and a functional regulatory gene or at least a fragment thereof; Optionally, the marker gene includes at least one of the following: luciferase gene, fluorescent protein gene, and β-galactosidase gene; Optionally, the modified cell line includes human lung adenocarcinoma cell line SDL-luci; accession number CCTCC NO:C2025236, deposited at the China Center for Type Culture Collection, deposited on August 13, 2025, at the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

13. An animal model of lung adenocarcinoma, characterized in that, The tumors in the animal model were formed from any one of the lung adenocarcinoma cell lines according to claims 1-10.

14. The lung adenocarcinoma animal model according to claim 13, characterized in that, The animal model is constructed by inoculating the lung adenocarcinoma cell line of any one of claims 1-10 into an immunodeficient animal.

15. The use of the lung adenocarcinoma cell line according to any one of claims 1-10 or the lung adenocarcinoma animal model according to claim 13 in the preparation of kits or reagents for studying or screening drugs, in the study of the mechanism of lung adenocarcinoma development, or in the construction of in vitro or in vivo model systems for drug screening or mechanism studies.

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