Epithelial–Stromal Interactions Modulating Penetration of Matrigel Membranes by HPV 16-Immortalized Keratinocytes

The role of epithelial–stromal interactions in the progression of human papillomavirus-associated squamous intraepithelial lesions to invasive cervical cancer is poorly understood. Using the Matrigel artificial basement membrane assay as a model of keratinocyte invasion, the effects of selected growth factors on penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel were studied. Also studied in this model were the effects of conditioned media from fibroblast lines derived from normal cervical tissues (normal fibroblasts) and adjacent cervical cancer biopsies (tumor-associated fibroblasts) and from primary keratinocytes. Addition of basic fibroblast growth factor, transforming growth factor-α, and hepatocyte growth factor/scatter factor or conditioned media from tumor-associated fibroblasts to the Matrigel resulted in near-doubling of penetration of human papillomavirus 16-immortalized keratinocytes, whereas transforming growth factor-β, platelet derived growth factor-β, or conditioned media from primary keratinocytes decreased penetration 10-fold. Antibodies to basic fibroblast growth factor abrogated the stimulatory effects of conditioned media from tumor-associated fibroblasts on keratinocyte penetration, whereas antibodies to transforming growth factor-β abrogated the inhibitory effects of conditioned media from normal fibroblasts on keratinocyte penetration. S1 nuclease protection and enzyme-linked immunosorbent assay showed increased expression of transforming growth factor-β and decreased expression of basic fibroblast growth factor in normal compared with tumor-associated fibroblasts. Messenger RNA in situ hybridization of five cervical cancer biopsies demonstrated basic fibroblast growth factor expression in stromal cells surrounding nests of invading keratinocytes. Epithelial–stromal interactions mediated by growth factors such as transforming growth factor-β and basic fribroblast growth factor modulate penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel in vitro and these interactions may also be operative in vivo