Organization of cytoskeleton and fibronectin matrix in rous sarcoma virus (RSV)-transformed fibroblast lines with different metastatic potential

Abstract

Metastatic clones growing in 0.6% ‘hard’ agar were selected from the non-metastatic Rous sarcoma virus (RSV)-transformed tumorigenic B77-3T3 mouse fibroblast line. The incidence of spontaneous lung metastases varied among clones around 100%, while it was lower than 5% in the parental tumor line. The organization of microfilaments, microtubules and intermediate filaments as well as the pattern of extracellular fibronectin matrix were analyzed by immunofluorescence in two representative clones (B77-AA6 and B77-AA12) and was compared with the structural features displayed by a highly metastasizing RSV-induced mouse sarcoma line (SR-BALB). In the metastatic clones studied microtubules and intermediate filaments were similarly organized in a pattern not significantly different from that of the non-metastatic parental cell line. The major finding was a marked concentration of actin-containing structures in the periphery of cells and notably at the level of surface protrusions, suggesting a high surface motility. In the same lines the production of fibronectin and its distribution in the cell layer and culture medium were analyzed. Metabolic labelling and immunofluorescence experiments indicated that the nonmetastasizing cells (B77-3T3) retain higher amounts of fibronectin in the cell layer and organize this molecule in extracellular fibers, while the metastatic clones (B77-AA6 and B77-AA12) as well as the metastatic line (SR-BALB) are unable to retain and organize fibronectin at their surface. This paper shows that the progression of tumorigenic cell lines toward a metastatic phenotype involves a redistribution of cytoskeletal actin and a loss of organized fibronectin matrix.