Abstract
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, accounting for approximately 6% of all cancer cases, and has a high rate of recurrence. Tumors generally possess a rare subpopulation of tumor initiating cells, with stem cell-like properties, often referred to as cancer stem cells (CSCs). CSCs are resistant to chemotherapy and radiotherapy, and consequently are believed to be responsible for tumor recurrence. One of the key properties of CSCs is their ability to evade immune surveillance through the promotion of a pro-tumor microenvironment. Tumor-derived exosomes have been demonstrated to promote tumor growth, drug resistance, and angiogenesis. A growing body of evidence has also established that tumor-derived exosomes possess potent immunoregulatory properties that may facilitate tumor progression by inducing apoptosis of cytotoxic CD8+ T cells, differentiation of regulatory T cells, and polarization of macrophages to the M2 phenotype. To date, most studies investigating the immunoregulatory effects of CSCs have focused on canonical secretory proteins, such as cytokines and growth factors, as the mediators of these properties. However, a current gap in knowledge that remains to be resolved is whether CSC-derived exosomes possess immunoregulatory properties, which we evaluated here. We hypothesized that HNSCC-derived CSC’s immunomodulatory properties are, in part, mediated by the expression of immune checkpoint proteins on both the cell surface and their derived exosomes. Freshly resected tumor tissue samples were used to generate subcultured CSC lines. To assess whether our cell lines were CSCs, we used flow cytometry to measure expression of the canonical stem cell markers. Our results suggest that our lines are positive for CD44, CD90, and Nestin. To assess whether the CSC lines expressed immune checkpoint proteins, we used flow cytometry to measure expression of the immune checkpoint proteins. Our data indicates that the primary CSC lines varyingly express immune checkpoint proteins and the expression is modulated by inflammatory cytokine signaling. To assess whether CSCs are immunomodulatory, we stimulated peripheral blood mononuclear cells (PBMCs) from a healthy donor, stimulated them with phytohemagglutinin (PHA), and added them to CSCs with and without IFN-g. Our results indicate that the CSC lines modulate the proliferation of immune cells. To assess whether exosomes secreted from the CSC lines express immune checkpoint proteins, we isolated exosomes from the HN2 line by exposing the cells to hypoxia and serum starvation conditions and then isolated exosomes from the conditioned media after 48hrs, followed by monoclonal antibody staining. The expression of the immune checkpoint proteins listed were then assessed via flow cytometry. Preliminary data demonstrates that CSC derived exosomes express immune checkpoint proteins on their surface and indicates a different expression profile to that of the cell lines. Taken together, our data indicate that further investigation of the functional properties of CSCs and CSC-derived exosomes is warranted. Such data may yield discovery of novel druggable CSC targets to limit rates of recurrence in HNSCC.