Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.

Fibroblast-directed expression and localization of 92-kDa type IV collagenase along the tumor-stroma interface in an in vitro three-dimensional model of human squamous cell carcinoma.

The malignant dissemination of tumors has been shown to require expression of one or more members of the matrix metalloprotease (MMP) enzyme family, whose function is to catalyze degradation of extracellular matrix proteins. In human squamous cell carcinoma (SCC) of the skin, expression of the MMP 92-kDa type IV collagenase (MMP-9), was previously shown to localize to malignant keratinocytes residing along the tumor/stromal interface. The purpose of the study presented here was to determine whether this localized expression pattern is due to interactions between SCC cells and adjacent stromal fibroblasts. To examine this question, SCC cells were grown as organotypic skin cultures, an in vitro three-dimensional model of reconstructed human epidermis in which keratinocytes are grown on a type 1 collagen gel embedded with human dermal fibroblasts. In this study, MMP-9 expression was compared in organotypic cultures (constructed with SCC cells or the non-tumorigenic keratinocyte cell line HaCaT), in which human dermal fibroblasts were either included or excluded from the underlying stromal layer. In the absence of fibroblasts, expression of MMP-9 was slightly higher in SCC than HaCaT cultures. In cultures constructed with fibroblasts, however, induction of MMP-9 mRNA was observed in SCC but not HaCaT cultures. This induction of MMP-9 mRNA was accompanied by high levels of MMP-9 protein expression along the SCC/stromal interface. These data provide strong evidence that interactions between malignant keratinocytes and adjacent stromal fibroblasts are critical in directing expression of MMP-9 to the tumor-stroma interface in human SCC tumors.

Integrin and basement membrane normalization in mouse grafts of human keratinocytes--implications for epidermal homeostasis.

Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains alpha 2, alpha 3, beta 1 and alpha 6, while beta 4 already showed a preferential basal location; (2) de novo expression of alpha 5 and alpha v; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular alpha 2 beta 1 and alpha 3 beta 1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin alpha 6 beta 4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.