Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Program Number: B043
Session Name: Poster Session
ETS1 Overexpression is Associated with Increased Immune Infiltration of the Tumor Microenvironment in Head and Neck Squamous Cell Carcinoma
Talia A Wenger, BA1; Chehyun Nam, PhD2; Benjamin Ziman, PhD2; Daniel Arnaudov, BS2; Uttam K Sinha, MD3; De-Chen Lin, PhD2; 1Keck School of Medicine of USC; 2Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, Norris Comprehensive Cancer Center, University of Southern California; 3Caruso Department of Otolaryngology-Head and Neck Surgery, Keck School of Medicine of USCBackground: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 890,000 new cases and almost half a million deaths annually. Despite multi-modal therapy with surgery, chemotherapy, and radiation, survival rates have remained relatively stagnant among HPV-negative cancers. Immunotherapy, particularly PD-1 inhibitors, have shown promise in a subset of HNSCC patients, but additional research is needed to appropriately identify all patients who may benefit. E26 Transformation-Specific 1 (ETS1) is a transcriptional regulator with high expression in HNSCC and is known to contribute to tumor metastasis and invasion. However, its exact mechanism of action remains unknown. Here we study the impact of ETS1 overexpression on the native immune system and tumor immune microenvironment, which may allow for tumorigenesis via immune evasion and provide insight into the value of immune therapy in patients with ETS1-high HNSCC.
Methods: Stable ETS1 overexpression cell lines were developed via lentiviral transfection of an mETS1 overexpression vector into murine oral squamous cell carcinoma cells (MOC2). qRT-PCR was used to evaluate immune target expression after selection. Animal experiments were approved by the USC Institutional Animal Care and Use Committee (IACUC). We did orthotopic injection of MOC2 mETS1 overexpression cells (MOC2 mETS1-OE) and MOC2 control cells into C57BL/6 mice and allowed for tumor growth. Tumors were then collected, dissociated into single-cell suspension, and stained with appropriate antibodies for flow cytometry to identify immune cell types. Cells were gated for live/dead status then FSC-A vs SSC-A and FSC-A vs. FSC-H to include only singlets. Finally, neutrophils, macrophages, T cells, and B cells were identified using LY6G, F4/80, CD3, CD45 antibodies, respectively.
Results: MOC2 mETS1-OE cells had greater than 5-fold higher ETS1 expression compared to control (p<0.05). Immune targets IFI35, TRIM21, STAT1, IFITM3, and PDL1 all had at least 3-fold higher expression in MOC2-mETS1-OE compared to control (p<0.01). Tumor growth in mice was allowed to proceed until tumors reached 1.2cm or mice had excessive tumor-associated morbidity per the IACUC protocol. MOC2-mETS1-OE tumors grew faster than MOC2 control tumors. Preliminary flow cytometry data indicated a trend towards increased CD3+ T cell infiltration in MOC2-mETS1-OE tumors compared to control. Additionally, MOC2-mETS1-OE tumors had eleven-fold higher CD45+ CD19+ B cell infiltration compared to control (p<0.01).
Conclusions: ETS1 is a known contributor to metastasis and invasion in SCC, but its role in regulating immunity is unknown. These data indicate a significant association between high ETS1 expression and increased activation of immune actors known to participate in T and B cell signaling and interferon signaling pathways. Furthermore, flow cytometry data indicate that ETS1 overexpression is sufficient to increase immune infiltration of the tumor microenvironment in an orthotopic murine model. However, the exact mechanism of increased T and B cell infiltration and the downstream effects are not yet well understood. Increased T cell infiltration via ETS1-high expression could be a novel therapeutic target if these immune cells can be activated and harnessed to attack tumor cells.
