Abstract
IL17 is a pro-inflammatory cytokine with increased gene expression in some cancers. It has been demonstrated to exhibit both pro- and anti-tumour effects. The pro-tumour effects of IL17 are mediated either by inducing the expression of matrix metalloproteinases (MMPs) in tumour cells or stimulating increased tumour angiogenesis. The anti-tumour effects of IL17 are exerted either through increased cytotoxic T (Tc) cell or interferon (IFN)γ activity. Immune cells including Th17 cells, Tc cells, natural killer (NK) cells and macrophages are some of the sources of IL17. The presence of these immune cells has been reported in the microenvironment of oral squamous cell carcinoma (OSCC). It was therefore hypothesized that IL17 expressed by multiple immune cells may play a role in the progression of OSCC.
The initial step to explore this hypothesis was to determine the presence of helper T cells (Th, CD4+), Tc cells (CD8+), NK cells (CD56+) and macrophages (CD68+) in 14 cases of OSCC using immunohistochemistry (IHC). Their presence was quantified in ten defined high power fields using ImageJ software. The cell densities were compared in groups of OSCC classified according to a histological invasive front grading system. This was followed by comparative analysis of the numbers of IL17+ cells in OSCC and inflammatory controls (IC). Additionally, IL17+ cell counts were compared according to their topographical distribution in OSCC (tumoural islands, tumour-stroma interface and distant tumour stroma) and also between the different prognostic groups. Double-labeling immunofluorescence was used to identify the immune cell types that co-expressed IL17. The cytoplasmic similar expression to FGF/IL17R (SEFIR) sequence of the transmembrane IL17 receptors (IL17R) was detected in OSCC tissues using IHC and the mRNA expression of specific receptors, IL17RA and IL17RC was assessed in three OSCC cell lines SCC4, SCC15 and SCC25 using quantitative PCR. Soluble IL17R (sIL17R) was detected in the cell culture supernatants of all OSCC cell lines collected at different time points (24, 48 and 72 hrs) using a sandwich ELISA. Recombinant human IL17 (rhIL17) over a range of concentrations (10, 50, 100 ng/mL) was used to stimulate OSCC cell lines to determine the effect in terms of proliferation, invasion and differential expression of genes associated with tumour metastasis in OSCC cells.
Proliferation was assayed at 0, 24, 48 and 72 hrs using cell titer blue dye and invasion at 48 hrs using a QCM ECMatrix cell invasion assay. The effect of rhIL17 on OSCC cells in terms of differential expression of tumour metastasis genes was determined using a human tumour metastasis gene array by quantitative PCR. The results of the proliferation, invasion and gene array experiments were compared with OSCC cells not treated with rhIL17. The data was analyzed using Mann-Whitney test, unpaired Student’s t test and one-way or two-way ANOVA as appropriate using GraphPad Prism 6 software. The level of statistical significance was set at p<0.05.
Macrophages, Th, Tc and NK cells were seen in OSCC. The mean Th and Tc cell count was higher in the good prognosis group compared with moderate, whereas macrophages and NK cell counts were higher in the moderate group but these differences were not significant statistically. Significantly more Th, Tc and macrophages than NK cells were present in both groups. The Th/Tc ratio was higher in the moderate prognosis group than in the good grade, however this failed to reach significance. Significantly more IL17+ cells were present in OSCC than IC, however IL17+ cell counts were not significantly different in the various sites assessed within the lesions. Double-labeling immunofluorescence studies revealed that Th cells, Tc cells, macrophages and mast cells co-expressed IL17. Cytoplasmic expression of transmembrane IL17R was detected in all OSCC tissues. SIL17R was detected in the cell culture supernatants of all three OSCC cell lines and its concentration increased in a time dependent manner. SCC25 had a significantly higher concentration of sIL17R at 72 hrs compared with 24 and 48 hrs, but this was not the case with SCC4 and SCC15 cells. As well, SCC25 had a significantly higher concentration of sIL17R at 72 hrs compared with SCC4 and SCC15 cells. The rate of proliferation of OSCC cell lines was not affected by the addition of rhIL17. However, IL17 promoted the in vitro invasion of SCC15 and SCC25 cells in a dose dependent manner. At the highest concentration of IL17 (100 ng/mL), SCC25 cells showed significant invasion compared to untreated cells, while the SCC15 cells exhibited significant invasion at both 50 and 100 ng/mL of IL17. In contrast, IL17 failed to stimulate invasion in SCC4 cell line. Genes associated with tumour metastasis were differentially expressed in OSCC cells treated with rhIL17; mRNA expression of MMP2, MMP3, MMP7, MMP10 and MMP11 was up-regulated in SCC15 cells, whereas SCC25 cells overexpressed MMP2, MMP10 and MMP13 when treated with rhIL17. On the other hand, expression of MMP3, MMP9 and MMP13 was down-regulated by rhIL17 in SCC4 cells. At the same time, on stimulation with rhIL17, VEGFA was overexpressed in SCC15 cells and down-regulated in SCC4 cells, whereas no change was observed in SCC25 cells; mRNA expression of numerous other genes affecting the tumour cell growth, proliferation, adhesion, cell cycle regulation and apoptosis was found to be differentially expressed in SCC4 and SCC15 cells but not SCC25 cells.
Therefore, based on the findings of my study, it is postulated that IL17 is co-expressed by multiple cell types in OSCC and facilitates the tumour progression by differential expression of genes associated with tumour metastasis, particularly those associated with extra-cellular matrix proteins and regulation of apoptosis