Complexity of VEGF responses in skin carcinogenesis revealed through ex vivo assays based on a VEGF-A null mouse keratinocyte cell line.

Vascular endothelial growth factor (VEGF-A) is a critical player in cutaneous angiogenesis. However, the relative contribution of VEGF-A from different sources including epithelial and mesenchymal cells has not been fully characterized during skin repair and tumorigenesis. Moreover, the actual involvement of other vascular-specific acting molecules has remained elusive in part due to the masking and/or overlapping effects of VEGF-A. To shed light on these uncertainties we generated and characterized a clonal VEGF-null mouse keratinocyte cell line, through in vitro adenoviral gene transfer of Cre recombinase to VEGF-LoxP primary keratinocytes followed by repeated cell passaging under controlled conditions and cloning. In vitro and in vivo assays demonstrated that VEGF-null keratinocytes were nontumorigenic and expressed normal differentiation markers after calcium switch. Hras-induced tumorigenesis of immortalized VEGF-null keratinocytes upon subcutaneous injection was markedly reduced but not fully suppressed. However, the metastatic ability of Hras-transformed VEGF-null keratinocytes was abolished. These ex vivo approaches suggest the existence of VEGF-dependent and independent angiogenic stimuli in skin carcinogenesis. The VEGF-null mouse keratinocyte cell line arises as an important tool to assess the actual contribution of keratinocyte-derived VEGF with respect to other angiogenic factors in skin homeostasis and malignancy.