Abstract
Background: Oral squamous cell carcinoma (OSCC) develops in an immune cell-rich environment, where inflammatory cells in the tumour microenvironment establish an anti-tumour response by secreting pro-inflammatory cytokines. At the same time the cancer cells may induce various mechanisms suppressing the anti-tumour response such as regulating a network of suppressive cytokines and the recruitment of suppressive regulatory T cells (Tregs). These escape mechanisms are seen at the local tumour site and similar mechanisms may also occur in regional lymph nodes (LN). In this thesis it is postulated that the escape of malignant oral keratinocytes from the primary site and their metastasis to regional lymph nodes is orchestrated by Tregs and their associated immune repertoire.
Objectives: 1. To determine the nature, composition and relationships of T cell subsets, including Tregs, and other associated immune repertoires that may be involved in creating a tolerogenic state within primary OSCC tissues.
2. To investigate the gene expression profile of immune cells with respect to cytokines involved in immune tolerance (including IL-17, TLR2, IL-10, TGFβ and IL-6) in primary OSCC, metastatic OSCC and in regional lymph nodes with OSCC metastases and to correlate the expression within the different tissues with Tumour, Nodes, Metastasis staging.
Methods:Twenty five formalin fixed paraffin embedded (FFPE) archival cases of OSCC and control tissues were stained via single and/or double immunohistochemistry (IHC) and immunofluorescence (IF) with T cell receptor markers including CD3, CD4, CD8, CD56, CD68, CD25/FoxP3, CD4/FoxP3, CD8/FoxP3 antibodies and other associated immune cell markers including Fas and TLR2/FoxP3 antibodies. For investigation of human T cell anergy and immune tolerance genes expression a further 32 FFPE archival cases of OSCC were divided into the following groups:- 1) primary OSCC without metastasis 2) OSCC with associated metastatic LN and 3) the metastatic lymph nodes. Group 4 was non-specific inflammatory control tissues. The expression of human T cell anergy and immune tolerance genes was determined using focused array technology via RT-PCR.
Results: A reduction in NK and CD8+ T cells with a smaller reduction in CD4+ T cells was observed in primary OSCC tissues. A higher frequency of CD25+FoxP3+ Tregs together with an increase in apoptosis was observed in the infiltrates associated with OSCC compared with controls. The reduction in CD8+ T cells and the presence of Tregs in primary OSCC tissues was confirmed with IF observation, although CD8+FoxP3+ cells were not detected. FoxP3+ Tregs was observed to be closely associated with naïve CD4+ cells. TLR2+ FoxP3+ Tregs were also observed in the OSCC tumour microenvironment. In addition, apparent co-localisation between TLR2+ and FoxP3+ cells was observed. The gene analysis studies demonstrated active regulation of T cell anergy and tolerance genes in primary OSCC and in metastatic lymph nodes. STAT3 gene was significantly down-regulated in all OSCC groups with the highest fold change in the metastatic lymph nodes. This may indicate a greater modulation the anti-tumour response in the nodes compared with the primary tissues. The immune suppression mechanisms were similar in lymph nodes with and without extracapsular (ECS) spread, though the suppression mechanism was stronger in lymph nodes with ECS. Treg expression was elevated in all stages of OSCC in comparison to controls.
Conclusions:Immune suppression appears to occur in the OSCC microenvironment and this suppression becomes more profound as the cancer progresses. In the later stages of OSCC, Tregs appear to exert profound control of the tolerance processes. As the tumour metastasises to cervical lymph nodes suppression appears to be less dependent on Tregs and switches to suppression via the STAT3 pathway, in order to prevent pro-inflammatory revival. Finally, it is concluded that the anti-tumour immune response does not fail entirely. Immune cells were not entirely suppressed by OSCC, particularly in the early stages of the cancer process. This may be an indication that OSCC eradication could be achieved with selected immunotherapy.