Lipopolysaccharide-Squamous Cell Carcinoma-Monocyte Interactions Induce Cancer-Supporting Factors Leading to Rapid STAT3 Activation

Abstract

Oral and oro-pharyngeal squamous cell carcinomas (OSCC) exhibit surface breach, and recent studies have demonstrated bacterial contamination of primary and metastatic OSCC. Increasing concentrations of inflammatory products, such as interleukin (IL)-6 and vascular endothelial growth factor (VEGF), correlate with, and contribute to, cancer progression, but their regulation in OSCC is poorly understood. We hypothesized that monocyte-lineage cells and bacterial contamination may contribute important inflammatory products that can support OSCC progression. We found that relative to non-specific chronic mucositis, oral carcinoma-in-situ/superficially-invasive OSCC contained more monocyte-lineage cells. In vitro, we used lipopolysaccharide (LPS) to model bacterial contamination, and evaluated the effects of oral and oropharyngeal (O)SCC-monocyte interactions and of LPS on OSCC cells and on the production of IL-6 and VEGF. OSCC cell lines varied in constitutive cytokine and chemokine production, and OSCC-monocyte interactions in the absence of LPS stimulated IL-6 and VEGF occasionally, while LPS-OSCC-monocyte interactions were always strongly stimulatory. Importantly, LPS independently stimulated some OSCC lines to secrete monocyte-dendritic cell chemoattractants CCL2 and/or CCL20, as well as IL-6 and/or VEGF. While very little constitutive Y705-STAT3 phosphorylation (pY705-STAT3) was detectable in HNSCC lines, IL-6 rapidly induced pY705-STAT3 in OSCC lines that produced little IL-6 constitutively. Supernatants from LPS-OSCC-monocyte co-cultures always rapidly and strongly activated STAT3, which was partly due to IL-6. We conclude that monocytes and microbial contamination have the potential to contribute to OSCC progression, as STAT3 activation in OSCC cells depends on soluble factors, which are consistently available through LPS-OSCC-monocyte interactions.

Dendritic Cells (DC) Facilitate Detachment of Squamous Carcinoma Cells (SCC), While SCC Promote an Immature CD16+ DC Phenotype and Control DC Migration

“…1). Given that most peripheral blood monocytes are CD16-negative, express CCR2 and migrate into inflamed areas and into the HNSCC microenvironment in response to CCL2 [4,6], these monocytes and the monocyte-derived DC could then gain CD16 expression through interactions with HNSCC cells or their products, and possibly by interacting with other mucosal cells. An important reason CD16 + monocytes and monocytederived DC are of special interest is that they are associated with chronic inflammatory states [33,37], and chronic inflammation is consistently present in the microenvironment of HNSCC.…”

“…Monocytes, Dendritic Cells (DC) Monocytes were prepared as described [4,5]. Monocytes were used as controls or differentiated into DC over 6 days with 1000 U/ml each GM-CSF and IL-4 (R&D Systems, Minneapolis, MN) in X-VIVO-15 at 37°C/7% CO 2 , with complete replacement of media and cytokines on alternate days.…”

“…Negative control preparations were matched to primary antibodies for isotype, concentration and buffer. IHC was performed using LabVision kits, LP Value HRP-DAB or One-Step HRP-DAB as described previously [4,5].…”

“…The mechanisms that regulate HNSCC pathogenesis, particularly those pertaining to the tumor microenvironment are poorly defined. The microenvironment of HNSCC is characterized by dense infiltrates of immune system cells, including monocyte lineage cells [4][5][6], such as CD68…”

“…+ [4,6] monocytes, macrophages and dendritic cells (DC). High numbers of CD68 + cells in HNSCC were shown to correlate with lymph node metastasis and advanced stage of disease [6].…”