Abstract PO-096: Cytokines in the lumen of exosomes are undetectable by immunoassays distorting cytokine profiles in HNC patients and healthy donors

Abstract Background: Exosomes, now called small extracellular vesicles (sEV), play a key role in cell-to-cell signaling. They are produced by all cells, circulate freely and are present in all body fluids. Evidence indicates that biologically active cytokines are present on the surface and/or in the lumen of sEV. The contributions of intra-vesicular cytokines to cytokine levels in plasma of cancer patients are unknown. Methods: sEV were isolated by ultrafiltration/size exclusion chromatography from pre-cleared plasma obtained from 30 patients with head and neck squamous cell carcinoma (HNSCC) and 10 healthy donors (HDs). Multiplex immunoassays were used to measure cytokine levels in paired untreated and detergent treated (0.5% Triton X-100) plasma samples and in isolated detergent-treated sEV. Results: The presence of cytokines on the surface and lumen of sEV isolated from plasma of the patients and HDs was first confirmed by immunoblots and on-bead flow cytometry. sEV-associated cytokines were functional in various in vitro coincubation assays with responder immune or tissue cells. Using Ab microarrays for 80 cytokines, we showed that sEV from HNSCC plasma contained a greater variety of cytokines (71/80; 88.8%) than sEV of HDs (43/80; 53.8%). Patients’ sEV had higher cytokine levels (range p<0.01 to 0.05) than did sEV from HD’s plasma. A highly sensitive Curiox 65-plex platform was then used to compare cytokine levels in paired untreated and detergent-treated plasma of HNSCC patients. 51/65 (78.5%) cytokines in the panel were consistently detected in treated and untreated plasma, and the majority of these (45; 88.2%) were present at significantly higher levels (range p<0.0001-0.05) in detergent-treated plasma. The comparison of cytokine levels between treated plasma, untreated plasma and paired sEV showed that the observed differences were due to cytokines sequestered in sEV. The cytokines released from detergent-treated EVs significantly contributed to the incomplete overall cytokine profile of untreated plasma in HNSCC patients. Further, only cytokines in sEV or in detergent-treated plasma correlated with disease stage in HNSCC. Conclusions: In untreated patient plasma, multiplex immunoassays detected only soluble cytokines and missed cytokines contained in the sEV lumen. Permeabilization of sEV was necessary for the Ab-based detection of total plasma cytokines (i.e., soluble + sEV-bound). As sEV-associated cytokines are biologically active, their absence from the plasma cytokine profiles that are determined in untreated plasma leads to inaccurate cytokine measurements. Underestimated cytokine levels in disease likely contribute to the lack of clinically significant correlations of plasma cytokine levels with disease progression. Citation Format: Chang-Sook Hong, Brenda Diergaarde, Theresa L. Whiteside. Cytokines in the lumen of exosomes are undetectable by immunoassays distorting cytokine profiles in HNC patients and healthy donors [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-096.