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. 2025 Sep-Oct;39(5):2669-2680.
doi: 10.21873/invivo.14067.

Development and Characterization of a Murine Lung Adenocarcinoma Cell Line With High Thoracic Pleural Metastatic Potential

Liwei Liao #  1 , Weidong Xu #  1 , Jia Li #  1 , Jiaye Li #  1 , Rui Li #  1 , Ruixia Li  1   2 , Chang Li  1 , Ziwen Zheng  1 , Mingming Deng  1 , Jinrui Miao  1   2 , Zilin Wang  1 , Qin Zhang  1 , Yiding Bian  1 , Kai Wang  1 , Han Wang  1   2 , Gang Hou  3
Affiliations

Development and Characterization of a Murine Lung Adenocarcinoma Cell Line With High Thoracic Pleural Metastatic Potential

Liwei Liao et al. In Vivo. 2025 Sep-Oct.

Abstract

Background/aim: Pleural metastasis and malignant pleural effusion (MPE) are common complications of lung adenocarcinoma. Patients with MPE have poor outcomes, with overall survival ranging from 5 to 11.4 months. The lack of established cell lines and stable animal models of pleural metastasis has limited studies on the underlying mechanisms of MPE development. In this study, we aimed to develop a murine lung adenocarcinoma cell line with high thoracic pleural metastatic potential.

Materials and methods: Luciferase-tagged Lewis lung carcinoma (LLC) cells were implanted into the pleural cavity of C57bl/6 mice, with five rounds of subsequent extraction from pleural foci and reinjection into the pleural cavity. The metastatic properties of the established cell line were verified in vivo by evaluating the metastatic burden and MPE volume (n=5). In vitro, the metastatic ability of cell lines was assessed by scratch assay, transwell migration assay, cell-matrix adhesion assay and cell-cell adhesion assay (3-5 replicates). The transcription profile was characterized by mRNA sequencing. Differential analysis and KEGG enrichment were performed to show their distinctions. Differential genes were verified by quantitative real-time PCR (qPCR).

Results: An LLC subpopulation with high thoracic pleural metastatic potential was generated and named LLC-PLM. In vivo, compared with parental LLC (LLC-P), LLC-PLM demonstrated a greater incidence of MPE and greater MPE volumes. In vitro, LLC-PLM demonstrated increased metastatic capacity and augmented adhesion capacities, compared to LLC-P. Transcriptomic analysis revealed that pathways related to adhesion, migration, and membrane signaling were notably enriched and activated in LLC-PLM cells. Relative genes were obviously activated, including Lamc2, Col4a3, Col6a3, Col1a1, Itga2 and Itga1.

Conclusion: We successfully established a murine cell line LLC-PLM that can serve as a valuable tool for studying pleural metastasis and MPE.

Keywords: Lewis lung carcinoma (LLC); Lung adenocarcinoma; malignant pleural effusion (MPE); murine tumor model; pleural metastasis.

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Conflict of interest statement

The Authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1
Figure 1
Process of the construction of a murine lung adenocarcinoma cell line with high thoracic pleural metastatic potential (LLC-PLM).
Figure 2
Figure 2
Assessment of the thoracic pleural metastatic ability of Lewis lung carcinoma cells with high thoracic pleural metastatic ability (LLC-PLM) in vivo. (A) In vivo bioluminescence-based imaging of pleural metastases formed by LLC-P and LLC-PLM cells. (B) Metastatic growth curve determined by in vivo imaging. (C) Comparison of the tumor burdens after 10 days of LLC-P and LLC-PLM implantation (values represent mean±SD, n=5). (D) Gross image of pleural metastatic lesions (The white dashed curve delineates the MPE, and the white arrow highlights the pleural metastatic lesions). (E) Comparison of the MPE volumes 10 days after implantation of LLC-P and LLC-PLM cells (values represent mean±SD, n=5). (F) Morphology of LLC-P and LLC-PLM cells under optical microscopy.
Figure 3
Figure 3
Assessment of the metastatic and adhesive abilities of LLC-PLM in vitro. (A, B) Representative images and the quantified results (mean±SD, n=3) of the scratch assay with LLC-P and LLC-PLM cells. (C, D) Representative images and the quantified results (mean±SD, n=5) of the transwell assay of LLC-P and LLC-PLM cells. (E, F) Representative images and the quantified results (mean±SD, n=3) of the cell–matrix adhesion assay of LLC-P and LLC-PLM cells. (G, H) Representative images and the quantified results (mean±SD, n=3) of the cell-cell adhesion assay for LLC-P and LLC-PLM cells.
Figure 4
Figure 4
Cisplatin sensitivity of LLC-PLM cells. (A) Activity of LLC-PLM and LLC-P cells treated with 0, 1, 2, or 4 μg/ml cisplatin for 2 days. (B) Comparisons of activities of LLC-PLM and LLC-P cells treated with 0 cisplatin for 2 days (data presented as mean±SD, n=3).
Figure 5
Figure 5
Transcriptomic characterization of LLC-PLM. (A) PCA of the transcriptomes of LLC-P and LLC-PLM. (B) Volcano plot of identified genes. (C) Differentially expressed genes between LLC-P and LLC-PLM. (D) Protein-protein network of the differentially expressed genes. (E) KEGG pathway enrichment of DEGs between LLC-P and LLC-PLM. (F, H) Gene Ontology enrichment analysis of DEGs between LLC-P and LLC-PLM, involving biological process, cellular component, and molecular function categories.
Figure 6
Figure 6
Activation of adhesion-related signaling in LLC-PLM. (A) Activation of adhesion-related signaling in LLC-PLM cells, as revealed by mRNA sequencing. (B) Upregulation of adhesion-related genes in LLC-PLM cells, as measured by qRT-PCR.
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