Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and ß1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).

Identification of two distinct carcinoma-associated fibroblast subtypes with differential tumor-promoting abilities in oral squamous cell carcinoma.

Heterogeneity of carcinoma-associated fibroblasts (CAF) has long been recognized, but the functional significance remains poorly understood. Here, we report the distinction of two CAF subtypes in oral squamous cell carcinoma (OSCC) that have differential tumor-promoting capability, one with a transcriptome and secretome closer to normal fibroblasts (CAF-N) and the other with a more divergent expression pattern (CAF-D). Both subtypes supported higher tumor incidence in nonobese diabetic/severe combined immunodeficient (NOD/SCID) Ilγ2(null) mice and deeper invasion of malignant keratinocytes than normal or dysplasia-associated fibroblasts, but CAF-N was more efficient than CAF-D in enhancing tumor incidence. CAF-N included more intrinsically motile fibroblasts maintained by high autocrine production of hyaluronan. Inhibiting CAF-N migration by blocking hyaluronan synthesis or chain elongation impaired invasion of adjacent OSCC cells, pinpointing fibroblast motility as an essential mechanism in this process. In contrast, CAF-D harbored fewer motile fibroblasts but synthesized higher TGF-ß1 levels. TGF-ß1 did not stimulate CAF-D migration but enhanced invasion and expression of epithelial-mesenchymal transition (EMT) markers in malignant keratinocytes. Inhibiting TGF-ß1 in three-dimensional cultures containing CAF-D impaired keratinocyte invasion, suggesting TGF-ß1-induced EMT mediates CAF-D-induced carcinoma cell invasion. TGF-ß1-pretreated normal fibroblasts also induced invasive properties in transformed oral keratinocytes, indicating that TGF-ß1-synthesizing fibroblasts, as well as hyaluronan-synthesizing fibroblasts, are critical for carcinoma invasion. Taken together, these results discern two subtypes of CAF that promote OSCC cell invasion via different mechanisms.

Cancer progression is associated with increased expression of basement membrane proteins in three-dimensional in vitro models of human oral cancer.

BACKGROUND: Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front. OBJECTIVE: This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial-mesenchymal interactions play on it, by using an in vitro oral cancer progression model. MATERIAL AND METHODS: In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin. RESULTS: Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium-biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin. CONCLUSIONS: These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial-mesenchymal environment, respectively the existence of fibroblasts.