Abstract
Using MDA-MB-231 cells as a model of triple negative breast cancer (TNBC) and its metastatic sub-cell lines that preferentially metastasize to lung, bone or brain, we found that the mRNA and protein levels of fibronectin (FN) are increased in MDA-MB-231 cells and its lung metastatic derivative, when cultivated in three-dimensional (3D) suspension cultures. The increase of FN expression in 3D was dependent on p38 mitogen-activated protein kinase (MAPK) because it was prevented by treatment of cells with SB203580, an inhibitor of p38MAPK. The up-regulated FN was converted into fibrils, and it enhanced cell spreading when cells cultured in 3D were transferred to two-dimensional (2D) culture. The arginine-glycine-aspartate (RGD) peptides and siRNAs targeting of integrin β-5 inhibited spreading of cells regardless of the presence of FN on 2D culture dishes. In addition, the levels of phosphorylated Src were found to be increased in 3D and the treatment of cells with SU6656, an inhibitor of Src, decreased the rate of cell spreading on FN. Collectively, these studies demonstrate that increased cellular FN in 3D suspension culture facilitates cancer cell attachment and spreading via integrin β-5 and Src, suggesting that the increased FN promotes initial attachment of cancer cells to secondary organs after circulation during metastasis.
Highlights
Conventional 2D culture systems have provided many insights into cell biology and therapeutic treatment of cancer, the development of new culture systems to better mimic in vivo situations provides additional insights into cancer cell behavior
Considering that FN, which is a marker of epithelial-mesenchymal transition (EMT) and a crucial component of extracellular matrix (ECM), is not expressed in normal adult breast tissues and its expression is up-regulated during breast cancer development[14,15], we investigated the role of increased cellular FN in 3D suspension cultures when cells encounter 2D surfaces
To investigate changes induced by different culture conditions, a triple negative breast cancer (TNBC) cell line, MDA-MB-231 cells and its derivatives that prefer to metastasize to lung (LM2), bone (BoM2), or brain (BrM2) were cultured in 3D ultra-low attachment plates or 2D plates for 48 hours
Summary
Conventional 2D culture systems have provided many insights into cell biology and therapeutic treatment of cancer, the development of new culture systems to better mimic in vivo situations provides additional insights into cancer cell behavior. Tumor cells undergo metastasis which consists of multiple steps including invasion through tissues via penetration of the basement membrane, intravasation to the circulatory system to move through the blood or lymph, and extravasation from the circulation system, followed by colonization in the second organ as a new niche[8]. During this process, tumor cells in the circulatory system inevitably remain detached from the scaffolding structures of tissue. We found that increased cellular expression of FN in 3D conditions facilitates cancer cell attachment and spreading via integrin β-5 and Src, suggesting that increased FN promotes initial attachment of cancer cells to secondary organ after circulation during metastasis
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