High-invasiveness human lung adenocarcinoma brain metastasis cell line and application thereof

Abstract

The invention relates to a high-invasiveness human lung adenocarcinoma brain metastasis cell line and application thereof, the cell line is named as a human lung adenocarcinoma brain metastasis cell line A549-F3, the cell line is preserved in China Center for Type Culture Collection on March 31, 2025, and the preservation number is CCTCC NO: C2025107. The A549-F3 cell line with high invasiveness and stable brain metastasis characteristics is successfully constructed through an in-vivo circulation screening method, the defects of an existing cell line in lung adenocarcinoma brain metastasis research are overcome, and a more suitable cell model is provided for deep exploration of related mechanisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a highly invasive human lung adenocarcinoma brain metastasis cell line and application thereof. BACKGROUND

[0002] Lung cancer is one of the most common malignant tumors in the world. In the world, the incidence of lung cancer has increased significantly, and its incidence has ranked first among various tumors in men, and the incidence of lung cancer in women has also increased significantly. So far, lung cancer has become the main cause of cancer-related deaths in the world. In China, the incidence of lung cancer has increased rapidly year by year, and in the past few decades, the mortality rate of lung cancer has increased by 465%, and the mortality rate ranks first in the city and second in the countryside. Among all lung cancers, non-small cell lung cancer accounts for 75-80%, and lung adenocarcinoma is the main type of non-small cell lung cancer.

[0003] Brain metastasis is a common complication of patients with advanced lung adenocarcinoma, which seriously affects the survival and quality of life of patients. At present, the treatment effect of lung adenocarcinoma brain metastasis is still not ideal, and the main reason is the lack of a cell model that accurately simulates the characteristics of lung adenocarcinoma brain metastasis. The cell lines established in the prior art lack in vivo screening process, and are difficult to simulate the adaptability of the metastatic microenvironment. On the other hand, the in vitro model of lung adenocarcinoma brain metastasis (such as traditional lung adenocarcinoma cell lines or Transwell model) cannot truly simulate the penetration of the blood-brain barrier and the interaction with the brain tissue microenvironment, resulting in inaccurate evaluation of the metastatic potential. In addition, patient-derived xenograft models (PDX) retain tumor heterogeneity, but have a long modeling period (half a year to a year), high cost and unstable metastasis rate. In addition, most brain metastasis models use gene editing (such as TP53 mutation) or overexpression of exogenous genes (such as miR-130b), which may introduce non-natural selection bias. In summary, although there are many lung adenocarcinoma cell lines for research, these cell lines have limitations in invasiveness and brain metastasis ability, and cannot fully meet the needs of scientific research and clinical application. Therefore, it is of great scientific significance and clinical value to develop a lung adenocarcinoma cell line with high invasiveness and stable brain metastasis ability. SUMMARY

[0004] The present application provides a highly invasive human lung adenocarcinoma brain metastasis cell line and application thereof. The human lung adenocarcinoma brain metastasis cell line A549-F3 has high invasiveness and high brain metastasis ability, and can be applied to the research of lung adenocarcinoma brain metastasis mechanism, drug screening and biomarker identification.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a highly invasive human lung adenocarcinoma brain metastasis cell line, which is named human lung adenocarcinoma brain metastasis cell line A549-F3 Homo sapiens and preserved in China Center for Type Culture Collection on March 31, 2025 with a preservation number of CCTCC NO: C2025107.

[0006] In a second aspect, the present application provides an application of the above-mentioned human lung adenocarcinoma brain metastasis cell line in the research of lung adenocarcinoma brain metastasis mechanism.

[0007] In a third aspect, the present application provides an application of the above-mentioned human lung adenocarcinoma brain metastasis cell line in drug screening, which is used for testing and screening drugs capable of inhibiting lung adenocarcinoma brain metastasis.

[0008] In a fourth aspect, the present application provides an application of the above-mentioned human lung adenocarcinoma brain metastasis cell line in biomarker identification, which is used for mining specific biomarkers related to lung adenocarcinoma brain metastasis.

[0009] The present application has the following beneficial effects: (1) The present application successfully constructs the A549-F3 cell line with high invasiveness and stable brain metastasis characteristics through in vivo circulation screening method, which makes up for the shortcomings of existing cell lines in the research of lung adenocarcinoma brain metastasis, and provides a more suitable cell model for in-depth exploration of the related mechanism. The A549-F3 cell line has the following characteristics: High invasiveness: The A549-F3 cell line shows significantly higher invasiveness than the parent A549 cells in the in vitro Transwell invasion experiment.

[0010] High brain metastasis ability: In animal models, the brain metastasis incidence of the A549-F3 cell line is significantly higher than that of the parent A549 cells, showing stronger brain metastasis ability.

[0011] (2) The A549-F3 cell line of the present application provides an ideal cell model for analyzing the molecular mechanism of lung adenocarcinoma brain metastasis, and can be used as an efficient drug screening platform for testing and screening drugs capable of inhibiting lung adenocarcinoma brain metastasis, and can also be used for mining specific biomarkers related to lung adenocarcinoma brain metastasis, providing potential markers for early diagnosis and prognosis evaluation. The application in drug screening and tumor biomarker identification expands its value in the field of biomedicine, helps to accelerate the research and development process of lung adenocarcinoma brain metastasis treatment drugs and improve the diagnosis level, and provides strong support for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 : Comparison chart of in vitro invasiveness of A549-F3 cells and parent A549 cells (Transwell experiment results).

[0013] Figure 2 : Proliferation curves of two cell lines (CCK-8 assay results).

[0014] Figure 3 : Comparison chart of brain metastasis incidence in animal models.

[0015] Figure 4 : Schematic diagram of the construction process of A549-F3 cell line. DETAILED DESCRIPTION

[0016] To further illustrate the technical means and effects adopted by the present application to achieve the predetermined inventive purpose, the specific embodiments, structures, features, and effects thereof according to the present application are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0017] The present application injects the parent A549 human lung adenocarcinoma cells into animals after being treated by a specific method to simulate the in vivo circulation process, then isolates and screens cells with brain metastasis ability, and finally obtains the cell line A549-F3 with high invasiveness and stable brain metastasis characteristics after multiple rounds of screening and expansion culture. On the one hand, the biological characteristics of the A549-F3 cell line are described, including its high invasiveness in in vitro experiments (such as the number of cells penetrating the matrix membrane in the Transwell invasion experiment is significantly higher than that of the parent cells), stronger proliferation ability (indicated by the results of CCK-8 or cell colony formation experiments), and higher brain metastasis incidence in in vivo animal experiments. On the other hand, the application of the cell line in the study of lung adenocarcinoma brain metastasis mechanism is described, such as its application in the study of key molecules, signal pathways, and the interaction between cells and brain microenvironment during cell invasion and metastasis. In addition, the application of the A549-F3 cell line in drug screening is introduced, providing an efficient screening platform for testing and screening drugs or compounds that can inhibit lung adenocarcinoma brain metastasis, and providing a basis for the development of new treatment strategies. The application of the cell line in identifying tumor biomarkers is also proposed, which can be used to compare and analyze the differences between A549-F3 cells and parent cells or other cell lines, to explore specific biomarkers related to lung adenocarcinoma brain metastasis, and to provide potential marker candidates for early diagnosis and prognosis evaluation.

[0018] (I) Cell line construction process Select the logarithmic growth phase of human lung adenocarcinoma cell line A549, after trypsin digestion, resuspended with RPMI 1640 medium containing 10% fetal bovine serum, adjust the cell concentration to 2x106 / mL. Inject 2x103 cells per mouse into 6-8 week old immunodeficient mice (such as BALB / c nude mice) by left ventricular injection. When the experiment, the mouse is in a supine position, the limbs are fixed, and the left ventricular positioning is performed. The sign of successful positioning is that the injector can see obvious arterial blood pulsation when it is pulled back. When injecting cells, control the speed and push slowly.

[0019] After injection, observe the health status and behavior of the mice regularly. After waiting for 4 weeks, detect the formation of brain metastasis by small animal live imaging technology. Dissect the mouse, collect the brain tissue, cut the brain metastasis tissue, and then digest it with a solution containing collagenase type IV and DNase I. Collect the single cell suspension by centrifugation, resuspend it with RPMI 1640 medium, and then inoculate it in a culture bottle, and culture it at 37℃, 5% CO2. Change the culture medium regularly during the culture process, and observe the growth of the cells.

[0020] The isolated and cultured brain metastasis cells are injected into new immunodeficient mice through left ventricular injection, and the above steps are repeated. After 3 rounds of screening (A549, A549-F1, A549-F2), the A549-F3 cell line with high invasiveness and stable brain metastasis ability is obtained.

[0021] Figure 4 The complete process of obtaining the A549-F3 cell line from the parent cells through in vivo circulation screening at each step is described in detail, including cell injection, animal in vivo circulation, brain metastasis cell isolation, in vitro expansion culture, and multiple rounds of screening. Key links such as help to understand the construction method of the cell line.

[0022] (II) Cell property identification: Cell morphology observation: Use an inverted microscope to observe the morphology of A549-F3 cells and compare them with parent A549 cells.

[0023] Cell invasion experiment: Transwell chamber is used for invasion experiment to detect the ability of cells to cross the matrix glue. The results show that the invasion ability of A549-F3 cells is significantly higher than that of parent A549 cells.

[0024] Figure 1 It is shown that the number of A549-F3 cells crossing the matrix membrane in the Transwell invasion experiment is significantly more than that of parent cells, which directly reflects its higher invasion ability (**** P<0.0001).

[0025] Cell proliferation experiment: CCK-8 assay was used to detect the proliferation ability of cells. The results showed that the proliferation rate of A549-F3 cells was higher than that of the parent cells.

[0026] Figure 2 The proliferation curves showed that the proliferation rate of A549-F3 cells at different time points was higher than that of the parent cells, which reflected its stronger proliferation characteristics (*** P<0.001).

[0027] Animal model verification: A549-F3 cells and parent A549 cells were injected into immunodeficient mice, respectively. The incidence of brain metastasis and the size of metastatic foci were detected by in vivo imaging. The results showed that the incidence of brain metastasis and the size of metastatic foci of A549-F3 cells were significantly higher than those of the parent cells.

[0028] Figure 3 The animal experiment showed that A549-F3 cells had a higher incidence of brain metastasis in vivo, further confirming the advantage of its brain metastasis characteristics.

[0029] (Three) Application in the study of lung adenocarcinoma brain metastasis mechanism Molecular mechanism research: Using A549-F3 cell line, RNA sequencing technology was used to analyze the gene expression difference between it and the parent A549 cells, and the differentially expressed genes related to brain metastasis were screened out. CRISPR / Cas9 gene editing technology or RNA interference technology was used to verify the function of these differentially expressed genes, and the mechanism of their role in lung adenocarcinoma brain metastasis was studied.

[0030] Cell and microenvironment interaction research: A549-F3 cells were co-cultured with cells in brain microenvironment (such as brain endothelial cells, astrocytes, etc.), simulating the in vivo brain metastasis microenvironment. The influence of cell interaction on lung adenocarcinoma brain metastasis was studied, and the dynamic regulation mechanism of cell and brain microenvironment was revealed.

[0031] (Four) Application in drug screening Drug screening platform construction: A high-throughput drug screening platform based on A549-F3 cell line was established. Various candidate drugs were added to the cell culture medium. Cell invasion experiment, cell proliferation experiment and cell apoptosis experiment were used to detect the inhibitory effect of drugs on cells.

[0032] Drug screening experiment: Transwell chamber was used to detect the invasion ability of cells after drug treatment, and drugs that could significantly inhibit cell invasion were screened out. CCK-8 assay was used to detect the inhibitory effect of drugs on cell proliferation. Flow cytometry was used to detect the ability of drugs to induce cell apoptosis.

[0033] Animal model verification: the screened candidate drugs are applied to animal models, and the inhibitory effect of the drugs on brain metastases is detected by imaging, to further evaluate the in vivo anti-brain metastasis activity of the drugs.

[0034] (Five) Application in biomarker identification Proteomics analysis: proteomics technology is used to analyze the protein expression profiles of A549-F3 cells and parent A549 cells, and to screen differentially expressed proteins. Further bioinformatics analysis is used to screen candidate proteins that may serve as specific biomarkers for lung adenocarcinoma brain metastasis.

[0035] Clinical sample verification: clinical samples (such as brain metastasis tissues, serum, etc.) of lung adenocarcinoma brain metastasis patients are collected, and the expression levels of candidate biomarkers in clinical samples are verified by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and other methods. The correlation between biomarkers and patient prognosis, treatment response is studied, to provide basis for early diagnosis and prognosis evaluation of lung adenocarcinoma brain metastasis.

[0036] In summary, the A549-F3 cell line and its construction method and application have wide prospects and important significance in the field of lung adenocarcinoma brain metastasis research, and are expected to contribute to improving the prognosis of lung adenocarcinoma brain metastasis patients and improving the treatment effect.

[0037] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any brief introduction, modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A highly invasive human lung adenocarcinoma brain metastasis cell line, characterized in that: The cell line was named human lung adenocarcinoma brain metastasis cell line A549-F3 and was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCCNO: C2025107.

2. The application of the human lung adenocarcinoma brain metastasis cell line described in claim 1 in the study of the brain metastasis mechanism of lung adenocarcinoma.

3. The application of the human lung adenocarcinoma brain metastasis cell line of claim 1 in drug screening, used to test and screen drugs that can inhibit lung adenocarcinoma brain metastasis.

4. The application of the human lung adenocarcinoma brain metastasis cell line of claim 1 in biomarker identification, used to discover specific biomarkers related to lung adenocarcinoma brain metastasis.

Images