Basement membrane and TGF-b1 modulate the behavior of human tumor cells in organotypic cultures

The role of basement membrane (BM) proteins and stroma-derived growth factors, such as transforming growth factor B-1 (TGF-B1), on neoplastic progression of oral epithelium is not well understood. Objectives: The goal of this study was to begin to characterize how such stromal factors control the behavior of human keratinocytes at varying stages of malignant transformation in human tissues with three-dimensional tissue architecture. Methods: To study this dynamic cross-talk, normal keratinocytes (NK), and three related cell lines, immortalized keratinocytes (HaCaT), early stage (HaCaT-II-4) and late-stage (RT-3) malignant keratinocytes were grown in organotypic cultures in the presence or absence of TGF-B1 (5ng/ml). To accomplish this, organotypic cultures were grown on 1 - contracted Type I collagen gel without BM, 2 - de-epidermalized human dermis with preserved BM organization (AlloDerm) and 3 – polycarbonate membranes coated with specific BM components. The behavior of tumor cells was assayed by immunohistochemical (IH) stains for integrin receptors (a2,a3,a6), laminin 5, proliferation (BrdU incorporation) and markers of keratinocytes differentiation, as well as by Western analysis. Results: We found that BM was required to normalize the synthesis and deposition of laminin 5 to the BM zone and induce proteolytic processing of it’s GAMMA 2 chain as seen by Western analysis. Furthermore, we found that only polycarbonate membranes coated with laminin 1 were able to induce such normalization of laminin 5. While the presence of BM was not sufficient to normalize expression of a6 integrin and cell proliferation, addition of TGF-B1 restricted a6 expression and proliferation to the BM zone. This suggested that BM rendered II-4 cells responsive to TGB-1. Conclusion: These findings demonstrated that keratinocytes at an early stage of transformation were sensitive to microenvironmental control through interactions with stromal factors. We are currently studying if keratinocytes at a more advanced stage of neoplastic progression are also modulated by these environmental factors.