Fibroblast in vitro stimulation with TGFβ1 and bFGF; αSMA expression and S100A4 immunohistochemistry staining in oral cancer

Abstract

Oral squamous cell carcinoma (OSCC) is an aggressive cancer associated with high mortality rates. The objective of this master project was to test the following hypotheses: (1) transforming growth factor β1 (TGFβ1) activates fibroblasts in vitro and basic fibroblast growth factor (bFGF) deactivates them while both increase fibroblast proliferation in cancer associated fibroblasts (CAFs); (2) the expression of anti-smooth muscle actin (αSMA) increases in presence of TGFβ1 and decreases upon treatment with bFGF; and (3) the calcium binding protein S100A4, also known as specific fibroblast protein 1 (FSP1), is a reliable epithelial to mesenchymal transition (EMT) biomarker for OSCC. A fibroblast cell line (MRC5) was treated with TGFβ1 (2 ng/ml) and bFGF (1, 10 and 100 ng/ml) to examine the effects on fibroblast morphology and proliferation. The impact of TGFβ1 and bFGF treatments on αSMA expression was tested through Western Blot analysis. The accuracy of S100A4 as an OSCC biomarker was tested through immunohistochemistry (IHC) staining on mouse tongue tissues with tumor as literature suggests that in invasive OSCC, S100A4 appears to be overexpressed. The fibroblast cell line was stimulated with TGFβ1 and bFGF which both increased proliferation. TGFβ1 resulted successfully in activated fibroblast morphology with dense fiber formation. Supplementing the microscopy observation, total protein quantification, showed that TGFβ1 treated fibroblasts, had higher total protein content than untreated fibroblasts for all incubations (24, 48 and 72h). For bFGF treated fibroblasts, the average total protein content displayed higher values than controls in all incubations, however, the treated groups had very similar total protein content despite the bFGF treatment concentration. TGFβ1 group was expected to enhance αSMA expression with inconclusive results and bFGF treated cells presented a reduction of αSMA expression compared to the negative control, in agreement with literature references. There was no noticeable variation on αSMA expression among the different bFGF treatments, indicating that bFGF indeed reduces the expression of αSMA, without a bFGF concentration-dependent effect. The S100A4 staining experiment conducted on mouse tongue tissues with tumor and the results seemed to be unclear as S100A4 displayed a lot of staining in various areas outside of the tumor. The positive stains outside of the tumor could be related to other cell types than cancer cells, like fibroblasts, but due to heavy staining it was not possible to identify the cell types and attribute the out-of-tumor locations that correspond to cancer cells. Hence, S100A4 is not a very sensitive biomarker for S100A4.