Loss of the tumorigenic phenotype with in vitro, but not in vivo, passaging of a novel series of human bronchial epithelial cell lines: possible role of an alpha 5/beta 1-integrin-fibronectin interaction.

We established an immortalized, nontumorigenic human bronchial epithelial cell line by transfection with the origin of replication-defective SV40 large T plasmid. This line spontaneously became tumorigenic at passage 184 (NL20T), although subsequent passages (passages 189, 200, and 205) failed to form tumors. The tumorigenic cell line NL20T was reinoculated back into athymic nude mice, and the two subsequently derived cell lines (NL20T-A and NL20T-B) have been passaged 85 times in vitro and remain tumorigenic. However, late-passage NL20T cells consistently lose their tumorigenicity when passaged in vitro on tissue culture plastic dishes (NL20T-n cells). Thus, two of the cell lines, NL20 and NL20T, reverted to the nontumorigenic phenotype reproducibly and spontaneously following serial passage on plastic tissue culture plates, whereas cells passaged in mice (NL20T-A and -B) did not. We used these nontumorigenic (NL20 and NL20T-n) and tumorigenic (early passage NL20T, NL20T-A, and NL20T-B) cells to study the role of the alpha 5/beta 1-integrin and attachment to fibronectin in tumorigenicity. The two nontumorigenic cell lines (NL20 and NL20T-n) attached slower to fibronectin-coated plates than the two tumorigenic cell lines in a cellular-extracellular matrix adhesion assay. Attachment was abrogated by exposure to a blocking antibody to the alpha 5/beta 1-integrin, the fibronectin receptor, in the two tumorigenic cell lines. Cell surface expression of the alpha 5/beta 1 cell surface protein by flow cytometry was highest in the tumorigenic NL20T and NL20T-A cells. NL20T-A cells were cultured with an antibody to alpha 5/beta 1 and inoculated s.c. into athymic nude mice; tumorigenicity of the NL20T-A cells was inhibited in a dose-dependent manner. Tumorigenicity was also inhibited partially with monoclonal antibodies to either alpha 5 or beta 1. A mixture of 10% tumorigenic NL20T-A cells and 90% nontumorigenic NL20 cells was cultured on plastic, type IV collagen, laminin, and fibronectin for 9 weeks. Only cells cultured on fibronectin formed tumors when inoculated s.c. into athymic nude mice. We conclude that these data are consistent with the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was selected subsequently in vivo but was lost with in vitro culture in NL20 cells, and that alpha 5/beta 1-integrin interaction with the extracellular matrix may play a role in tumorigenicity in our system.