ACTIVATION OF MURINE DENDRITIC CELLS THROUGH STING PATHWAY INTERACTIONS WITH GLYCATED CHITOSAN

Abstract

N-dihydrogalactochitosan (Glycated Chitosan, GC) is a promising immunoadjuvant for cancer therapeutics. Synthesized from chitin and galactose, GC stimulates the innate and adaptive antitumor immune responses more potently than its parent molecule, chitosan, giving it great potential for therapeutic applications. However, the cellular mechanisms of GC have yet to be fully investigated. In previous studies, it has been demonstrated that GC has strong interactions with dendritic cells (DCs) where interactions of stimulator of interferon genes (STING) and the stimulation of type I IFN are essential for the antitumor response. The activation of DCs via the STING pathway has been shown to lead to inflammatory cell death that further enhances the antitumor response and recruitment of antigen presenting cells. Herein we investigated STING-GC interactions within murine DCs, using immunofluorescence (IF), western blotting, and bulk RNA sequencing analysis to determine STING-GC colocalization in the lysosome. Using the Western blot, lysates isolating the cellular components of murine dendritic cells allowed for identification of STINGs location following GC stimulation. Within the lysosomal isolate, increased expression of STING was observed, indicating STING trafficking to the lysosome post GC stimulation. IF antibody staining allowed visualization of cellular vesicles, STING, and GC. By tagging lysosomal markers (LAMP 1) and STING, it can be seen that STING and GC are contained within swollen intracellular vesicles indicating cell stress. Through bulk RNA sequencing analysis of wild type and STING knockout (KO) mice, an upregulation of lysosomal genes dependent on STING were detected indicating immunogenic cell death (ICD) via lysosomal activity is occurring. Furthermore, upregulation of genes associated with the NLRP3 pathway was visualized post GC stimulation indicating cross talk between GC, STING, and NLRP3 pathway ICD. Visualizing the GC-STING relation is an important step in identifying the cellular mechanisms of GC, paving the way for further understanding of GC’s immunological mechanism and the optimization of therapeutic applications using GC as an immunostimulant.