Research ArticleAnchorage-independent growth of breast carcinoma cells is mediated by serum exosomes
Introduction
The in vitro two-dimensional growth of cultured cells including tumor cells is well characterized, particularly the growth signals emanating from the integrin/extracellular matrix interaction [1]. For a number of years, ideas have bounced back and forth as to whether the growth signaling mechanisms in two-dimensional tissue culture can be extrapolated to the growth cues in vivo [2]. A number of in vitro culture systems have been developed including organotypic culture system, that takes into account the three-dimensional in vivo growth of tumor cells [3]. Suffice to say, the in vivo growth of tumor cells is complex because not only is it three-dimensional in character, there are numerous variables including stromal factors that affect growth [4]. Apart from the limitations imposed on it, the anchorage-independent growth of tumor cells in soft agar bears the closest resemblance to the in vivo growth when compared to the two-dimensional growth on plastic [5].
Serum, particularly fetal bovine serum is routinely used to supplement cell growth media. Serum has a number of adhesion or attachment factors which allow the cells to adhere and spread on the bottom of the culture flask to initiate growth signals. Apart from the well characterized adhesion proteins such as fibronectin, laminin, and collagen [6], serum also contains a myriad of factors whose role in cell growth is either defined or yet to be determined [7]. There are over 1000 proteins in serum, where some are at nanomolar or lower concentrations and others at micro molar or higher concentrations. In studies designed to measure the contribution of a particular growth factor, it is customary to use serum free medium supplemented with graded doses of the protein or factor. Such studies have been done to determine the growth factor most suited to support anchorage-independent growth [8]. Interestingly, in majority of such studies, in addition to the growth factor of interest, the medium was also supplemented with fetal bovine serum [9]. Serum proteins appear to be particularly important requirement for anchorage-independent growth. We have made an effort in the present study to identify the protein or group of proteins “platform” associated with anchorage-independent growth in serum.
We hypothesized that fetuin-A or a group of proteins which form a complex with or co-purify with it is/are responsible for anchorage-independent growth in serum. We had demonstrated that fetuin-A is one of the serum proteins that could support anchorage-independent growth in breast carcinoma cells [10]. Unfortunately fetuin-A easily associates with other serum proteins making it difficult to single it out as the principal driver of growth. Recent studies indicated that exosomes (nanovesicles) purified from HIV infected monocyte-derived macrophages and urine, contain fetuin-A among other serum proteins [11], [12]. We hereby present data that demonstrate that serum proteins or factors (including fetuin-A) that confer anchorage-independent growth are associated with exosomes.
Section snippets
Materials and methods
Cells: A sub-clone of the breast cancer cell line BT-549 stably transfected with galectin-3 and re-named BT-549Gal-3, and MDA-MB-435, were kindly donated by Dr. Avraham Raz, Karmanos Cancer Research Institute. MCF-10A and MDA-MB-231, were purchased from ATCC. The cells were routinely cultured in DMEM/F12 supplemented with essential and non-essential amino acids, 100 μg/ml penicillin–streptomycin, 2.5 μg/ml Fungizone, 20 ng/ml epidermal growth factor, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone,
Role of serum exosomes on anchorage dependent and independent growth of breast carcinoma cells
Our objective here was to determine the serum fraction (exosome-free or enriched) that supports anchorage-independent growth of breast carcinoma cells. The breast carcinoma cells were suspended in soft agar and allowed to grow either in the absence or presence of serum exosomes for at least 3 weeks. The data clearly show that AIG is robust only in those wells containing exosomes (Fig. 1). This serum fraction which we have named exosome enriched medium (EEM) in addition to common serum proteins
Discussion
In the present studies, we have re-visited a long standing question regarding the identity of factor(s) in fetal bovine serum that mediates anchorage-independent growth of tumor cells. A number of growth factors such as IGF, EGF, TGF-β, βFGF and PDGF have individually and as a group, been shown to play a role in anchorage-independent growth of tumor cells [8], [32], [33]. However, even after all these growth factors were included as a cocktail in serum free medium and mixed with soft agar,
Acknowledgments
Data analysis was performed by the Meharry Medical College Microarray and Bioinformatics Core (MMBC), which is supported in part by NIH grants G12RR03032-19 and P20RR011792. (url: http://www.mmc.edu/bioinformatics). Mass spectrometry and experimental design were conducted by the Vanderbilt University Mass Spectrometry Core Lab (MSRC). (url: http://www.mc.vanderbilt.edu/msrc/massspec/index.php).
The work was supported by 1SC1CA134018-01 (J.O.); DOD W81XWH-07-1-0254 (J.O).
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