Abstract PO-097: Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway
Authors: Xu Zhongping , Earland Noah , Harris Peter K. , Gerndt Sophie P. , Ramirez Ricardo , Semenkovich Nicholas P. , Chaudhuri Aadel A. , Zevallos Jose P. 
Published: September 2023

Abstract
Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPC) is increasing rapidly in incidence and is now the most common HPV-associated malignancy in the U.S. Our group previously demonstrated that lymph-enriched fluid from surgical drains in HPV+ OPC patients is rich in tumor-derived cell-free DNA, with prognostic implications. In this study, we characterize extracellular vesicles (EVs) derived from cervical lymph in HPV+ OPC patients and explore the impact on innate immune response. The cyclic GMP-AMP synthase (cGAS) pathway plays a key role in immune response by detecting pathogenic or damaged host DNA. We propose that HPV DNA-containing EVs uptake by HPV-negative cells modulates their immune responses via activation of cGAS-STING signaling. Methods: Surgical lymphatic fluid samples were collected from 31 HPV(+) and 10 HPV(-) OPC patients 24 hours post neck dissection, along with supernatant from HPV(+) and HPV(-) OPC cell lines. EVs were isolated from both biofluids and characterized using immunoblotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). EVs were then treated with DNase to eliminate external DNA before extracting EV DNA. Lymph cell-free DNA (cfDNA) was also extracted. We used qPCR to quantify HPV16 levels in supernatant and lymph EVs. Next, we incubated HPV(-) OPC cells with EVs that were isolated from the supernatant from HPV (+) or HPV (-) cells. Finally, we compared cGAS-STING activation induced by HPV (+) EVs to the STING agonist 2,3-GAMP (Sigma) through IFNβ mRNA expression and phosphor-TBK1 immunoblotting. Results: Lymphatic EVs showed typical morphology with diameters ranging 70–150 nm, and were CD63 and TSG101 positive by immunoblot. 27 HPV (+) OPC patients (87%) had detectable HPV16 DNA in their postoperative lymph EVs, while all HPV(-) lymph EVs were negative for HPV16. Comparison of bulk lymph cfDNA and lymph EV DNA showed a significant proportion (39.5±24.4%) of lymph HPV16 DNA is contained by EVs. Further, an EV uptake assay showed HPV16 DNA was trafficked into HPV(-) cells through EVs, and blocking EV internalization with heparin reduced recipient cell HPV16 levels. Crucially, HPV (-) cells cultured with HPV (+) EVs for 8 hours increased IFNβ1 expression, while HPV(-) EVs did not increase IFNβ1 expression. Lastly, we observed TBK1 phosphorylation, a reporter of STING activation, was elevated in these cells treated with HPV(+) EVs. Conclusions: Our study is the first to characterize tumor-associated EVs in lymphatic fluid. We showed many of these tumor-associated lymph EVs in HPV(+) patients harbor HPV16. Additionally, we demonstrate lymph EVs containing HPV16 activate cellular cGAS-STING intermediates. Ongoing studies are investigating the impact of EVs on the tumor microenvironment.
Citation Format: Zhongping Xu, Noah Earland, Peter K. Harris, Sophie P. Gerndt, Ricardo Ramirez, Nicholas P. Semenkovich, Aadel A. Chaudhuri, Jose P. Zevallos. Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-097.