Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jun 21;4(5):383-90.
doi: 10.7150/jca.6545. Print 2013.

Plasma Fibronectin Promotes Tumor Cell Survival and Invasion through Regulation of Tie2

Lynn M Knowles  1 , Gunjan Malik, Jan Pilch
Affiliations

Plasma Fibronectin Promotes Tumor Cell Survival and Invasion through Regulation of Tie2

Lynn M Knowles et al. J Cancer. .

Abstract

Our previous research has shown that plasma fibronectin promotes lung metastasis by facilitating tumor cell invasion in clotted plasma. To evaluate the role of clotted plasma for tumor cell survival, we treated B16F1 cells embedded in a 3-dimensional matrix of fibrin with tumor necrosis factor α (TNFα), a cytokine with anti-tumor activity. Under these conditions, TNFα caused significant cytotoxicity, which was prevented when we added plasma fibronectin to the fibrin clot. Fibronectin-mediated TNFα resistance was dependent on PI3-kinase, which also mediated the pro-adhesive and pro-invasive effects of plasma fibronectin on tumor cells. To further investigate the role of plasma fibronectin in tumor cell signaling, we performed a gene array that showed specific upregulation of Tie2 in B16F1 cells embedded in fibrin-fibronectin compared to fibrin. Importantly, inhibition of Tie2 resulted in decreased tumor cell invasion, reduced colony formation and increased tumor cell death in response to TNFα. Together, our findings indicate that plasma fibronectin induces tumor cell invasion and protects tumor cells from the cytotoxic effects of inflammatory mediators through up-regulation of Tie2.

Keywords: Fibrin; Metastasis; Plasma fibronectin; Tie2..

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest: None.

Figures

Fig 1
Fig 1
TNFα is cytotoxic for fibrin-embedded tumor cells. (A), B16F1 cells were analyzed for PI uptake by fluorescence microscopy 16 hours after embedding in fibrin and treatment with 50 ng/ml TNFα, 14,000U/ml IFNγ or vehicle (n.s., not significant). (B-D), B16F1 cells embedded in fibrin were analyzed for PI uptake (B), LDH release (C) or DNA fragmentation (D) after treatment with TNFα (5 or 50 ng/ml) compared to vehicle (Veh). Camptothecin and staurosporin (C/S) were used as a positive control.
Fig 2
Fig 2
Fibrin-fibronectin protects B16F1 cells from TNFα. (A), PI+ B16F1 cells embedded in fibrin (Fib) or fibrin-fibronectin (FibFN) were counted in random optical field after treatment with TNFα (50 ng/ml) for 24 and 48 hours compared to vehicle. PI uptake in fibrin-embedded cells treated with vehicle is set to 1. (B), representative phase contrast microscopy images of B16F1 melanoma cells 96 hours after embedding in fibrin or fibrin-fibronectin (FibFN) and treatment with 50 ng/ml TNFα. Scale bar, 500 µm.
Fig 3
Fig 3
Fibrin-fibronectin mediated B16F1 invadopodia formation, cell adhesion and TNFα-resistance depends on PI3-kinase. (A), representative phase contrast images of B16F1 cells 16 hours after embedding in a 3-dimensional matrix of fibrin (Fib; right image) or fibrin-fibronectin (FibFN; left image). Scale bar, 50 µm. (B), B16F1 cells were analyzed for invadopodia formation by phase contrast microscopy in the presence of inhibitors against PI3-kinase (LY294002, 10 µM), PKC (Gö 6976, 1 µM), JNK (SP600125, 10 µM), and MEK1/2 (U0126, 10 µM) compared to vehicle (DMSO) 16 hours after embedding in Fib or FibFN. (C), B16F1 cells treated with 10 µM LY294002 or vehicle were allowed to attach to plates coated with fibrinogen (FG) or fibrin-fibronectin (FibFN) (ea. 10 µg/ml). Adhesion to FG was induced by adding solubilized fibrin-fibronectin complexes to the binding buffer. (D), PI+ B16F1 cells embedded in fibrin (Fib) or fibrin-fibronectin (+FN) were counted in random optical field after treatment with TNFα (50 ng/ml) and 10 µM LY294002 compared to vehicle.
Fig 4
Fig 4
Tie2 is upregulated in fibrin-fibronectin-embedded B16F1 cells. (A), gene expression analysis showed upregulation of mRNA for TNFα, Tie2 and uPA in FibFN- over Fib-embedded B16F1 cells. (B), invadopodia formation after 16 hours of FibFN-embedded B16F1 cells treated with siRNA against Tie2, uPA and TNFα compared to non-targeted siRNA. (C), invadopodia formation of FibFN-embedded B16F1 cells 16 hours after treatment with a Tie2 kinase inhibitor. (D-E), colony size was analyzed in fibrin-fibronectin embedded B16F1 cells treated with Tie2 and control siRNA for 72 and 120 hours (D) or with a Tie2 kinase inhibitor after 120 hours (E). (F), PI+ B16F1 cells embedded in fibrin-fibronectin were counted in random optical field after treatment with Tie2 siRNA and TNFα (50 ng/ml). *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant; versus control (vehicle or scrambled siRNA).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science. 2011;331:1559–64. - PubMed
    1. Malik G, Knowles LM, Dhir R, Xu S, Yang S, Ruoslahti E. et al. Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion. Cancer Res. 2010;70:4327–34. - PMC - PubMed
    1. Gorelik E, Bere WW, Herberman RB. Role of NK cells in the antimetastatic effect of anticoagulant drugs. Int J Cancer. 1984;33:87–94. - PubMed
    1. Esumi N, Fan D, Fidler IJ. Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor. Cancer research. 1991;51:4549–56. - PubMed
    1. Palumbo JS, Barney KA, Blevins EA, Shaw MA, Mishra A, Flick MJ. et al. Factor XIII transglutaminase supports hematogenous tumor cell metastasis through a mechanism dependent on natural killer cell function. J Thromb Haemost. 2008;6:812–9. - PubMed