Granzyme B, HIF-1α, VEGFA and SerpinB9 in the local tumour immune system of immune-stimulated slice cultures of head and neck cancer.

backgroundEven when T-cell activation is successful, head and neck squamous cell carcinoma (HNSCC) often becomes resistant to immunotherapy. This study investigated the molecular factors that influence immune-induced tumour cell death in organotypic HNSCC slice cultures (SCs).

methodsSC samples were obtained from 23 patients (22 with HNSCC and one tumour-free control). Immune stimulation was performed using a T-cell activator kit. The protein levels of granzyme B (GrB), cleaved caspase-3 (CC3), Slug, hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor alpha (VEGFA) and SerpinB9 were assessed using immunohistochemistry (IHC) and immunofluorescence (IF). The patient samples were stratified according to Slug expression and HPV status. Slug, VEGFA, HIF-1α, HPV positivity and baseline SerpinB9 were considered input determinants, whereas GrB, CC3 and induced SerpinB9 were considered response factors.

resultsThe baseline intensity of HIF-1α was low in most tumour tissue samples, except in two patients who had high levels. SerpinB9 levels were higher in tumour cells from five patients than in a single tumour-free control sample. VEGFA immunofluorescence (IF) levels were clearly higher in several tumour samples, including HPV-positive cases, compared to the single tumour-free control sample. A significant correlation was observed between HIF-1α and VEGFA IF sum intensities. Following immune stimulation, the intensity of GrB increased in 14 out of 22 tumour samples. CC3 was induced in tumour cells in 12 out of 19 samples, particularly in five out of six HPV-positive samples, while SerpinB9 was induced in nine out of 18 samples. Interestingly, this occurred in only two out of six HPV-positive samples. The induction of CC3 and SerpinB9 was significantly correlated (r = 0.67, p < 0.01). No difference in GrB induction was observed between Slug-positive and Slug-negative tumours. However, lower levels of CC3 and SerpinB9 induction were observed in Slug-positive tumours.

conclusionsThe Slug-HIF-1α-VEGFA axis may enable tumour cells to evade the primary immune response by inhibiting apoptosis induced by the immune system. Extensive immune activation induced both the caspase cascade and the protective factor SerpinB9 in tumour cells of HPV-negative HNSCC mainly. GrB, CC3 and SerpinB9 exhibited reduced spatial gradients at increasing distances from the immune-stimulating beads.