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).

Integrin α11 is overexpressed by tumour stroma of head and neck squamous cell carcinoma and correlates positively with alpha smooth muscle actin expression.

BACKGROUND: Cancer-associated fibroblasts (CAFs) were shown to be important for tumour progression in head and neck squamous cell carcinomas (HNSCCs). Their heterogeneity and lack of specific markers is increasingly recognized. Integrin α11 was recently shown to be expressed by CAFs and might serve as a specific CAF marker. AIM: To investigate integrin α11 expression and its correlation with the expression of a well-known marker of CAF, alpha smooth muscle actin (α-SMA), in HNSCC. METHODS: Fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples from healthy volunteers (n = 24), oral lichen planus (OLP) (n = 32) and HNSCC (n = 106) were collected together with clinical data after ethical approval. Immunohistochemistry to detect integrin α11 and α-SMA was performed on FF and FFPE samples. qPCR for integrin α11 (ITGA11) and α-SMA (ACTA2) was performed on FF samples. Data were analysed using chi-square test and Kaplan-Meier survival analysis. RESULTS: Significantly higher levels of integrin α11 and α-SMA at both protein and mRNA levels were found in HNSCC vs. normal controls and OLP. A strong correlation was found between integrin α11 and α-SMA expression, and double staining showed their colocalization. Both integrin α11 and α-SMA were detected surrounding metastatic islands. Expression of α-SMA at tumour front but not tumour centre correlated with patient survival. CONCLUSION: Integrin α11 was overexpressed in HNSCC stroma and colocalized with α-SMA. Expression of α-SMA at tumour front but not tumour centre had prognostic value for survival, pinpointing the importance of assessing tumour front when evaluating stromal molecules as prognostic biomarkers.

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.

Gene expression profiles of head and neck carcinomas from Sudanese and Norwegian patients reveal common biological pathways regardless of race and lifestyle.

PURPOSE: To explore possible range of gene expression profiles in head and neck squamous cell carcinomas (HNSCC) and pairwised normal controls from Sudanese (n = 72) and Norwegian (n = 45) patients using a 15K cDNA microarray and to correlate the findings with clinicopathologic variables. EXPERIMENTAL DESIGN: Samples from Sudan were grouped according to anatomic location/patients' habit of toombak (snuff) use, and 37 pools of 2 to 11 tumors matched to 37 pools of their normal controls from the same patients, respectively, were prepared. For Norway, eight pools of 3 to 11 tumors matched to eight pools of their normal controls from the same patients, respectively, were prepared according to anatomic location. Pools (n = 45) were hybridized to microarrays. For controls, 33 of the pools were hybridized against Human Reference RNA. Scanned array images were recorded, and data analysis was done in groups. For verification, results for selected genes were analyzed using quantitative real-time PCR/immunohistochemistry. RESULTS: We identified 136 genes from Sudan and 154 from Norway as differentially expressed between tumors and controls. Changes of the genes found were confirmed in >70% of the pools by hybridization against Reference RNA. Seventy-three genes and three main pathways (signal transduction, cell communication, and ligand-receptor interaction) were of relevance to the HNSCCs from both countries. Hierarchical clustering of the 73 genes identified subclasses of mixed tumors from the two populations, two independent subgroups for Norwegian tumors by their anatomic sites, and five subgroups for Sudanese tumors by their toombak habits. Quantitative real-time PCR/immunohistochemistry validated the microarray-based data. CONCLUSIONS: Differences in gene expression between tumor and nontumor tissues were identified in HNSCCs. Analysis of the two population groups revealed a common set of 73 genes within three main biological pathways. This indicates that the development of HNSCCs is mediated by similar biological pathways regardless of differences related to race, ethnicity, lifestyle, and/or exposure to environmental carcinogens. Of particular interest, however, was the valuable association of gene expression signature found with toombak use and anatomic site of the tumors.