Abstract 35: Chemo-radiotherapy induces tolerogenic STAT3 signaling in circulating myeloid-derived suppressor cells in patients with head and neck squamous cell carcinoma (HNSCC)
Authors: Won Haejung , Sampath Sagus , Massarelli Erminia , Maghami Ellie , Kortylewski Marcin 
Published: December 2017

Abstract
Background and Objectives: While radiation therapy (RT) and certain types of chemotherapy are potentially immunogenic, they rarely generate antitumor immune responses. Our previous studies in mice showed that effects of RT on solid tumors are limited by the activation of tumor-promoting and immunosuppressive protein STAT3 in tumor-associated myeloid cells. Current study focused on determining whether STAT3 plays similar role in promoting immune evasion and therapeutic resistance in patients with head and neck squamous cell carcinoma (HNSCC) undergoing chemo-radiation therapy.
Patients, Materials, and Methods: 15 patients with stage III-IV HNSCC who received CRT with curative intent consented to collect blood and tissue biopsy (IRB#14255). Before and during week 2 of RT, tissue biopsies were collected of a cancer-involved lymph node that received full dose radiation. Blood specimens were collected from patients before and during RT on weeks 1, 2, 4, 6, and 7, as well as 6-12 weeks after treatment completion. After isolation, PBMCs and plasma samples were cryopreserved and stored in liquid nitrogen for no longer than 12 months. For flow-cytometric analysis, PBMC were thawed in 37°C water bath, washed and pre-incubated in RPMI1640 media supplemented with 20% of autologous plasma for 2 h. Cells were then stained with fluorescently-labeled antibodies specific for CD14, CD15, CD33, and HLA-DR (eBioscience). For intracellular staining of phosphorylated-STAT3 (pSTAT3), cells were fixed, permeablized and then stained with fluorescently-labeled antibodies specific to tyrosine 705-phosphorylated STAT3 (pSTAT3; BD Biosciences) or an isotype control. mRNA was isolated from PBMC from baseline and Week 2 draw during RT from 6 patients. Expression of 730 genes selected for immune profiling was evaluated by nCounter Immune Cell Profiling Assay (Nanostring) and data was analyzed using nSolver software (Nanostring).
Results: CRT resulted in the accumulation of potently immunosuppressive polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs; HLA-DRloCD14-CD15+) in circulation of HNSCC patients. The PMN-MDSCs had high levels of activated STAT3, which correlated with the expression of immune checkpoint inhibitor, programmed death-ligand 1 (PD-L1). The analysis of gene expression in patients' PBMC indicated increased expressions of multiple protein mediators that promote immune suppression and MDSC functions such as IL-1β (20.7 fold, p=1.59E-5), IDO (3.6 fold, p=0.1) and VEGF (13.2 fold, p=7.97E-6) during RT. Pathway analysis using KEGG data base predicted that RT enhanced NF-κB signaling in PBMC. In contrast, number of circulating CD3+ T cell was significantly reduced and expression of genes involved in T-cell cytotoxicity including Granzymes A/K/M and Perforin-1 were decreased by greater than twice (p&lt;0.05) during RT. In addition, significant increase in expression of immune checkpoint molecules; PD-L1 (2.7 fold, p=0.05) and TIM-3 (10.7 fold, p=2.45E-6), was observed.
Conclusion: The data suggest that RT induces accumulation of PMN-MDSC in circulation and activation of STAT3 which positively regulates suppressive function of MDSC. Correspondingly, the percentage of T cells in circulation and the expression level of genes regulating cytotoxic functions were significantly reduced during RT. Taken together, the results suggest that RT could induce tolerogenic effects in HNSCC patients. Our further studies will test the feasibility of using the TLR9-targeted STAT3 inhibitors, alone or combination with checkpoint inhibitors, to overcome immune suppression and improve RT efficacy in syngeneic mouse models of HNSCC.
Citation Format: Haejung Won, Sagus Sampath, Erminia Massarelli, Ellie Maghami, Marcin Kortylewski. Chemo-radiotherapy induces tolerogenic STAT3 signaling in circulating myeloid-derived suppressor cells in patients with head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 35.