Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors

Abstract

Little is known about the growth-signaling pathways that govern the proliferation of Ewing tumor (ET) cells either in vitro or in vivo. We have studied signal transduction pathways in ET cell lines and compared kinase expression levels and proliferation rates with primary tumors. Cell lines were studied both as conventional adherent monolayers and as anchorage-independent multi-cellular spheroids. Importantly, we observed signi®cant di erences between these in vitro models and found that ET spheroids were more closely related to primary tumors with respect to cell morphology, cell ± cell junctions, proliferative index and kinase activation. Monolayer ET cells demonstrated serum-dependent phosphorylation of ERK1/2 and AKT and constitutively high serum-independent cyclin D1 protein expression. However, when ET cells were placed in suspension culture, there was immediate serum-independent activation of ERK1/2 and AKT. In addition, cyclin D1 protein expression was completely blocked until stable multicellular spheroids had formed, indicating that cell ± cell adhesion is necessary for the proliferation of anchorage independent ET cells. This reduction in cyclin D1 expression was post-transcriptional and could be mimicked in monolayer cells by treatment with phosphatidyl inositol-3 kinase (PI3K) inhibitors. Moreover, PI3K inhibition signi®cantly reduced ET cell proliferation and, in primary ET samples, cyclin D1 expression correlated with expression of activated AKT. Thus, the PI3K ± AKT pathway appears to be critical for the proliferation of ET cells both in vitro and in vivo and tumor cell growth in vivo may be better represented by the study of anchorage-independent multi-cellular spheroids.

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

“…Phorbol 12-myristate 13-acetatestimulated HT29 cells, expressing high levels of COX-2, resulted to be able to degrade and migrate through ECM components of Matrigel-coated membranes, which indicates their malignant behaviour in vitro, associated with a well-described invasive and metastatic ability in vivo (Chen et al, 2001;Kakiuchi et al, 2002). Moreover, HT29 wild-type cells easily formed colonies in soft agar, which is an accepted criterion for transformation (Aaronson and Todaro, 1968) and an experimental condition that better represents tumour cells growth and invasiveness in vivo (Lawlor et al, 2002). The knockdown of COX-2 enzyme in HT29 cells abrogated their ability to invade Matrigel-coated membranes in a Boyden chamber assay, either in the absence or in the presence of PMA-stimulation, and strongly impaired their anchorageindependent growth in soft-agar basal conditions.…”

Herceptin Down-Regulates HER-2/neu and Vascular Endothelial Growth Factor Expression and Enhances Taxol-Induced Cytotoxicity of Human Ewing's Sarcoma Cells In vitro and In vivo

“…This antiangiogenic effect may in addition lead to enhanced sensitivity to chemotherapeutic agents such as taxol. Additionally, constitutive ERK activation has been shown not only in cells that overexpress Her2 but also in cells expressing EWS/FLI-1 chimeric proteins (35). Interfering with Her2-dependent signaling was shown to interfere with the constitutive activation of ERK-1/2 in EWS/FLI-1-expressing cells.…”

Understanding Micrometastatic Disease and Anoikis Resistance in Ewing Family of Tumors and Osteosarcoma

“…Lawlor and colleagues demonstrated that EFT cell lines readily survive in anchorage-independent conditions, persisting as multicellular spheroid clusters [42]. These spheroids show considerable phenotypic differences from their monolayer counterparts, including dramatically reduced proliferation with associated suppression of cyclin D1 levels, greater dependence on serum stimulation, and hyperactivation of various oncogenic signaling pathways including the PI3K/Akt and Ras-ERK pathways.…”