Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Oral cancer is the sixth most common cancer worldwide, accounting for approximately 375,000 new cases and 175,000 deaths annually. The average 5-year survival rate is 64% in the United States. The Food and Drug Administration has approved adjuvant immunotherapies for metastatic or recurrent disease in head and neck squamous cell carcinoma. Understanding the tumor microenvironment and the cancer secretome may reveal novel therapeutic targets that improves survival in patients with oral cavity squamous cell carcinoma (OCSCC).
Purpose: To determine OCSCC secretome composition and inhibit the expression of highly secreted cytokines to determine the effect on cell viability and motility.
Methods: We cultured human OCSCC cell lines (UCSF-OT-1109 and SCC9) to 70% confluency. The media was changed to Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. Forty-eight hours later, the supernatant was collected and added to an 80-cytokine array (Aah-CY-T-5-2 Human Array C5, RayBiotech) for overnight incubation. The array was then processed according to the manufactures protocol. Chemiluminescence imaging and ImageJ software were used to quantify protein expression. To assess the role of Interleukin 8 (IL8) on cell viability, cell lines were seeded in 96-well plates and treated with anti-interleukin (IL)-8 neutralizing antibody (1 ug/ml) and mouse IgG1 isotype control (1ug/mL). Then, cell viability was quantified at 24- and 48-hours post-treatment. To determine the effect of anti-IL8 neutralizing antibody on cell migration, we seeded cells in 24 well plates and let them grow to a confluent monolayer. Then, a scratch was introduced, and the media was treated with isotype control or anti-IL8 neutralizing antibody. The rate of gap closure was recorded using Gen5 imaging and microscopy software and images were captured every four hours between 0 – 28 hours post-treatment. We analyzed data using parametric and non-parametric t-tests.
Results: Secretome analysis showed that both UCSF-OT-1109 and SCC9 cells expressed high levels of IL8 and chemokine (C-C motif) ligand 5 (CCL5), while UCSF-OT-1109 and SCC9 cells expressed monocyte chemoattractant protein-1 (MCP-1) and IL-6, respectively. Because both cell lines secreted IL8, we aimed to determine the effect of IL-8 signaling inhibition on mechanisms of disease such as proliferation and migration. We found that anti-IL8 antibody (1 ug/ml) reduced SCC9 cell migration compared to isotype control treated cells, but did not affect SCC9 or USCF-OT-1109 cell viability.
Conclusion: In our pilot study, we demonstrated that IL-8 was highly secreted by OCSCC. Inhibition of IL8 signaling using neutralizing antibody limited cell migration but did not reduce OCSCC cell viability, indicating that IL8 may have an important role in OCSCC migration. Future investigations are warranted to elucidate how IL8 signaling activates downstream pathways that promote cancer cell migration and determine the potential impact of IL-8 inhibitors on tumor control in patients with OCSCC.