Defects of basement membrane and hemidesmosome structure correlate with malignant phenotype and stromal interactions in HaCaT-Ras xenografts

Abstract

Benign and malignant HaCaT-ras clones, derived from immortalized HaCaT cells were grown as nude mouse surface transplants rendering a human tumor progression model. Searching for malignancy-related alterations, the deposition, localization and mRNA of basement membrane and hemidesmosome components were analysed by immunofluorescence, in situ hybridization and electron microscopy. Initially, at 1 week epithelia of benign and malignant cells revealed a similarly low polarity and an enlarged ‘activated basal’ compartment, reflected by partial dislocation and extended pericellular staining of the hemidesmosome constituent integrin α6β4 seen by immunofluorescence. Whereas benign grafts eventually normalized, closely resembling grafts of HaCaT cells, malignant growth was correlated with a persisting epithelial activation state and continuing higher expression of α6 (by immunoflourescence and in situ hybridization). The basement membrane components bullous pemphigoid antigen 1, laminin-5 and collagen IV exhibited a largely linear distribution at 1 week. However, in the malignant cell transplants initially minor basement membrane discontinuities became more severe at around 2 weeks, associated with close stromal cell contacts, angiogenesis and invasion. Most striking were basement membrane alterations seen by electron microscopy. At 1 week stretches of basement membrane had developed in malignant transplants, though to a much lesser extent than in benign specimens. With invasion these basement membrane structures mostly disappeared despite persistent although variable immunofluorescence, suggesting high turnover without ultrastructural assembly. The hemidesmosome structures were defective throughout, completely lacking anchoring plaques with keratin filaments, whereas they were still associated with basement membrane deposits. Thus, malignant HaCaT-ras transplants, while initially resembling regenerating wounds, revealed an increasing loss of tissue polarity and basement membrane structures, which seemed to be accelerated upon stromal cell contacts.