Effect of the premalignant and tumor microenvironment on immune cell cytokine production in head and neck cancer

Abstract

Head and neck squamous cell carcinoma (HNSCC) is marked by immunosuppression, a state in which the established tumor escapes immune attack. However, the impact of the premalignant and tumor microenvironments on immune reactivity has yet to be elucidated. The purpose of this study was to determine how soluble mediators from cells established from carcinogen-induced oral premalignant lesions and HNSCC modulate immune cell cytokine production. It was found that premalignant cells secrete significantly increased levels of G-CSF, RANTES, MCP-1, and PGE2 compared to HNSCC cells. Splenocytes incubated with premalignant supernatant secreted significantly increased levels of Th1-, Th2-, and Th17-associated cytokines compared to splenocytes incubated with HNSCC supernatant. These studies demonstrate that whereas the premalignant microenvironment elicits proinflammatory cytokine production, the tumor microenvironment is significantly less immune stimulatory and may contribute to immunosuppression in established HNSCC.

Figure 1

Premalignant lesion cells release significantly increased levels of pro-inflammatory mediators compared to HNSCC cells in vitro. Cells were grown to confluency and supernatant was collected after 48 h for cytokine/chemokine analysis. Data shown represent three independent experiments performed in duplicate. * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001.

Figure 2

Splenocytes cultured with premalignant supernatant release significantly increased levels of innate proinflammatory mediators compared to splenocytes cultured with HNSCC supernatant or media alone. Splenocytes were incubated with media, premalignant cell-conditioned supernatant, or HNSCC cell-conditioned supernatant, respectively, and supernatant was collected for cytokine/chemokine analysis after 72 h. Data shown represent 3 independent experiments performed in duplicate. * p < 0.05 ** p < 0.01.

Figure 3

Splenocytes cultured with premalignant supernatant secrete significantly increased levels of Th1-, Th2-, and Th17-associated cytokines upon stimulation compared to splenocytes cultured with HNSCC supernatant or media alone. Splenocytes were incubated with media, premalignant cell-conditioned supernatant, or HNSCC cell-conditioned supernatant, respectively, and supernatant was collected for cytokine analysis after 72 h. Data shown represent 3 independent experiments performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.