Abstract
The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50% conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.
Highlights
Patients with head and neck squamous cell carcinoma (HNSCC) manifest significant immunosuppression
Early cytokine responses involving interferon γ (IFN-γ), vascular endothelial growth factor (VEGF), TNF-α, and IL-4 were assayed following the incubation of human peripheral blood mononuclear cells (PBMCs) with medium conditioned by HNSCC cell lines in order to determine whether tumor-secreted factors can affect PBMCs
Levels of cytokines secreted by the SCC lines used (i.e., KB, KB16, and HEP cells) were determined from 1:2 dilutions of culture media collected at the 0 h timepoint using enzyme linked immunosorbent assays (ELISAs) specific for detection of IFN-γ, VEGF, TNF-α, and IL-4
Summary
Patients with head and neck squamous cell carcinoma (HNSCC) manifest significant immunosuppression. The extent of this immunosuppression is often more profound than that observed in patients with other malignancies,[1] and has been postulated to occur in a hierarchical manner In the latter case, genetic alterations[2] have been found to affect the primary tumor region more than draining lymph nodes.[1] immune effector cells obtained from the peripheral blood of patients with cancer, including HNSCC, have been reported to exhibit. Neutralizing antibodies to VEGF, or granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown the capacity to partially reverse the inhibitory effects of tumor cell supernatants on the differentiation and function of dendritic cells undergoing maturation.[7] In the present study, early cytokine responses involving IFN-γ, VEGF, TNF-α, and IL-4 were assayed following the incubation of human PBMCs with medium conditioned by HNSCC cell lines in order to determine whether tumor-secreted factors can affect PBMCs
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