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Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion - PubMed

  • ️Fri Jan 01 2010

Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

Deshui Jia et al. BMC Cancer. 2010.

Abstract

Background: The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms.

Methods: The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT) makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis.

Results: A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line.

Conclusions: We have successfully established a new human lung cancer cell line with highly metastatic potentials, which is subject to EMT and possibly mediated by increased fibronectin expression. This cell line and its reproducible s.c. mouse model can further be used to identify underlying mechanisms of lung cancer metastasis.

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Figures

Figure 1
Figure 1

In vivo selection of highly metastatic human lung cancer cells. A, In vivo selection scheme. Parental human SPC-A-1 lung cancer cells were subcutaneously injected into NOD/SCID mice. When the subcutaneous tumour developed, small pieces of tumour tissue were implanted into the s.c. sites of new recipient mice in the first generation of mouse models, and primary tumours were excised 4 weeks later. Fourteen weeks after resection, visual lung metastases were isolated and re-implanted into the s.c. region of new recipient mice for in vivo selection. Finally, after three rounds of in vivo seleciton, tumour nodules were isolated from the lungs harboring massive metastatic lesions and s.c. implanted into new recipient mice, after which the primary tumour was removed to initiate in vitro culture and the SPC-A-1sci cell line was derived. B, Representative images of visual inspection of one mouse lungs for the presence of gross tumour nodules (arrows indicate) in the fourth generation. C, Representative bioluminescence images taken from the ventral side of the mice by in vivo BLI (upper) for primary tumour growth, and fluorescence images taken from the lungs of one sacrificed mouse by ex vivo BFI (lower) for spontaneous metastasis at three sequential time points from week 7 to 9, at 1-week intervals, after s.c. injection with GFP-Luc transduced SPC-A-1sci cells.

Figure 2
Figure 2

SPC-A-1sci cells display distinct phenotype consistent with EMT. A, Representative phase-contrast images of SPC-A-1sci and SPC-A-1 cells are shown. Magnification, 200×. B, Immunofluorescence staining of α-tubulin, E-cadherin, ZO-1, Vimentin and Fibronectin in SPC-A-1sci cells and SPC-A-1cells. The green/red signal represents the staining of the indicated proteins, and the blue signal represents the nuclear DNA staining by DAPI. Magnification, 600×. C, Quantitative-PCR analysis of the mRNA levels of E-cadherin, Vimentin and Fibronectin in SPC-A-1sci and SPC-A-1 cells. Results are expressed as mean ± SD; **, P < 0.01; *, P < 0.05. D, Expression of E-cadherin and vimentin were examined by immunohistochemical staining of serial sections from s.c. primary tumours of SPC-A-1 cells, SPC-A-1sci cells and also pulmonary metastatic nodules of SPC-A-1sci cells. Magnification, 200×.

Figure 3
Figure 3

SPC-A-1sci cells acquire increased potentials of migration and invasion. A, In vitro growth curves of SPC-A-1sci and SPC-A-1 cells, cells (2 × 104/well) were cultured in 24-well plates, after which cells were trypsined and counted at indicated times. Results are expressed as mean ± SD; *, P < 0.05. B, Soft agar colony formation of SPC-A-1sci and SPC-A-1 cells, cells (1 × 103/well) were cultured in 24-well plates, colonies were photographed and counted after three weeks. Results are expressed as mean ± SD; *, P < 0.05. C, In vivo growth curve of SPC-A-1sci and SPC-A-1 cells, cells (2.0 × 106 per mouse) were s.c. implanted into NOD/SCID mice, after which the tumour volume was measured over time. Results are expressed as mean ± SD. C, Motility activities of SPC-A1sci and SPC-A-1 cells were determined by wound healing assays Confluent monolayers of SPC-A1sci and SPC-A-1 cells in fibronectin coated 24-well plates were wounded by scratching, after which the wells were washed, incubated with conditioned medium for 24 h and photographed (lower). Motility was assessed on the basis of percentage of wounded area filled in (upper). Results are expressed as mean ± SD; **, P < 0.01. D and E, Trans-well migration and invasion assays of SPC-A-1sci and SPC-A-1 cells For migration assay (D), cells (2.5 × 104/well) were seeded into non-coated trans-well plates and culture for 16 h at 37°C; for invasion assays (E), cells (1.0 × 105/well) were seeded into Matrigel-coated trans-well plates and cultured for 24 h at 37°C, after which cells that had migrated or invaded to the underside of the inserts were stained with H&E and the cells on each insert were photographed (lower) and quantified (upper) at 400× magnification. All experiments were repeated thrice; Results are expressed as mean ± SD; *, P < 0.05.

Figure 4
Figure 4

Fibronectin promotes SPC-A-1sci cell invasion and metastasis. A, Representative phase-contrast images of SPC-A-1sci and SPC-A-1 cells 4 hours after seeded on fibronectin coated or uncoated plates in cell spreading assays. Magnification, 200×. B, Immunoblotting detection of fibronectin in SPC-A-1sci cells infected with shRNAs against fibronectin (shRNA-Fn1) or negative control (shRNA-NC) as indicated. β-actin was used as a loading control. C and D, Trans-well migration and invasion assays for SPC-A-1sci cells infected with shRNAs against fibronectin (shRNA-Fn1) or negative control (shRNA-NC) as indicated. All experiments were repeated thrice; Results are expressed as mean ± SD; *, P < 0.05.

Figure 5
Figure 5

Knockdown of fibronectin inhibits the metastatic ability of SPC-A-1sci cells in vivo. A, Representative images of histological inspection of one mouse lungs for the presence of microscopic lesions (Magnification, 50×; insert, 200×) four weeks after the tail vein injection with SPC-A-1sci cells stably expressing shRNA against negative control (shRNA-NC; Upper) or fibronecetin (shRNA-Fn1; Lower). B, Quantification of microscopic nodules in the lungs of each mouse expressed as mean ± SD of numbers obtained from eight animals in each group (shRNA-NC versus shRNA-Fn1); P = 9.27641E-07.

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References

    1. Gibbons DL, Lin W, Creighton CJ, Zheng S, Berel D, Yang Y, Raso MG, Liu DD, Lozano G. Expression signatures of metastatic capacity in a genetic mouse model of lung adenocarcinoma. PLoS ONE. 2009;4(4):e5401. doi: 10.1371/journal.pone.0005401. - DOI - PMC - PubMed
    1. Tanaka E, Yamashita J, Hayashi N, Kato S, Kondo K, Ogawa M. A pulmonary metastatic model of human non-small cell lung carcinoma cells that produce a neutrophil elastase-like molecule in severe combined immunodeficiency mice. Chest. 2003;123(4):1248–1253. doi: 10.1378/chest.123.4.1248. - DOI - PubMed
    1. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000;406(6795):532–535. doi: 10.1038/35020106. - DOI - PubMed
    1. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436(7050):518–524. doi: 10.1038/nature03799. - DOI - PMC - PubMed
    1. Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology. Pathology. 2007;39(3):305–318. doi: 10.1080/00313020701329914. - DOI - PubMed

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