Abstract 3152: NF-κB-mediated pro-inflammatory cytokine release and neutrophil recruitment in head and neck squamous cell carcinoma cell lines
Authors: Shakeel Hina , Bilwani Fareena , Iqbal Kiran , Ghias Kulsoom 
Published: July 2021

Abstract
Introduction: The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. IL-8 and GM-CSF, whose secretion is regulated by NF-κB, are pro-inflammatory cytokines that promote extravasation of neutrophils into tissue and play an important role in the proliferation, invasion, and metastasis of HNSCC. The onset and progression of HNSCC is also associated with genetic alterations that affect several cellular signaling pathways such as transforming growth factor-β (TGFβ) pathway. TGF-β is responsible for polarization of neutrophils towards anti-tumor (N1) versus pro-tumor (N2) phenotype.
Objective: The role of neutrophils in HNSCC and the possible molecular mechanisms of pro-tumor neutrophil recruitment is being investigated.
Methods and Results: IL-8 and GM-CSF released at various time points by FaDu (ATCC® HTB-43™), SCC-9 (ATCC® CRL-1629™) and Cal-27 (ATCC® CRL-2095™) was measured by ELISA. Conditioned media from FaDu and Cal-27 cells exhibited consistent expression of IL-8 (115 - 117pg/mL and 103 - 110pg/ml, respectively) at 6 - 72 hours. SCC-9 conditioned media exhibited sustained levels of IL-8 expression (103 - 110pg/ml) at 6 - 24 hours that then declined at 48 - 72 hours. GM-CSF was not significantly expressed in the FaDu conditioned media; even with increasing cell density, the mean expression remained 0.142pg/ml. SCC-9 and Cal-27 expressed GM-CSF at 6 - 72 hours (16 - 20pg/mL and ~15pg/ml, respectively). In all cell lines, IL-8 expression decreased in a dose and time dependent manner after treatment with the NF-κB inhibitor, BAY 11-7082, while effect on GM-CSF expression was negligible. Expression of phosphorylated IκBα in FaDu and SCC-9 cell lysates decreased in a time and dose dependent manner, indicating inhibition of the NF-κB pathway by BAY11-7082. Expression of phosphorylated NF-κB also decreased as compared to control in these cells. In nuclear extracts, BAY 11-7082 dose-dependent alteration in NF-κB expression was observed in FaDu and Cal-27 cells at earlier time points. Conditioned media from SCC-9 and Cal-27 cells also exhibited sustained baseline expression of TGF- β (~30pg/ml) over 6 - 72 hours.
Conclusion: HNSCC cells derived from tongue and hypopharyngeal squamous cell carcinoma release pro-inflammatory cytokines. The NF-κB signaling pathway appears to be involved in the release of IL-8, a cytokine that is in turn likely to be involved in recruitment of neutrophils to the cancer site that may be polarized towards the N2 pro-tumor phenotype by TGF-β secreted by HNSCC cells.
Citation Format: Hina Shakeel, Fareena Bilwani, Kiran Iqbal, Kulsoom Ghias. NF-κB-mediated pro-inflammatory cytokine release and neutrophil recruitment in head and neck squamous cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3152.