Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) activate innate immunity to increase tumor immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including inefficient transport to the cytosol where STING is localized. We recently developed STING-activating nanoparticles (STING-NPs), polymer vesicles designed for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2’3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumorally-administered (IT) STING-NPs significantly enhance the therapeutic efficacy of cGAMP and improve response to immune checkpoint inhibition (ICI) in established murine B16 melanoma tumors. However, the immunologic effects of STING-NPs have not been well characterized in tumors or lymphatic tissue.

We utilized Digital Spatial Profiling (DSP) to identify immunologic responses in the injected tumor, a distal tumor, and both tumor draining lymph nodes (TDLN) 48 hours after a single IT injection of STING-NP or three injections of STING-NP co-administered with systemic ICI (anti-PD-1/CTLA-4). DSP analysis permits simultaneous detection of over 30 protein markers in distinct spatial regions of interest (ROI; n=2-4 per tumor/TDLN) or cell types in tissue sections.

After a single STING-NP injection of B16 tumors, B7-H3, an immunosuppressive T cell checkpoint, was significantly induced in tumors compared to PBS or free cGAMP. More strikingly, in T cell-rich regions of the TDLN, STING-NP dramatically increased B7-H3, GZMB, and S100A9 while suppressing VISTA and CD73 expression. After multiple injections of STING-NP, B7-H3, S100A9, CD73, Foxp3, and beta-catenin were substantially upregulated in the injected tumor, with more modest fold changes in distal tumors. Co-treatment with systemic ICI in part abrogated this effect. In contrast, STING-NP-induced changes in GZMB, CD4, and CD8a expression were observed regardless of ICI treatment, and were generally equivalent in both treated and distal tumors, suggesting an abscopal effect. Both the treated and distal TDLN showed striking S100A9 increases which was preferential, but not exclusive, to T cell-rich vs. B cell regions. This effect was exacerbated with the combination of STING-NP and ICI therapy. B7-H3 upregulation in response to STING-NP (regardless of ICI) was exclusive to treated TLDNs vs. distal TDLNs. B7-H3 was expressed in both B and T cell regions, but was nearly two-fold higher in B cell-rich regions. Upregulation of GZMB (5-10 fold over free cGAMP alone) was observed in both treated and distal TDLNs.

Markers of T cell activation were observed after STING-NP treatment, including moderate abscopal effects in synchronous distal tumors. In TDLNs, STING-NP robustly induced S100A9 and B7-H3, which may be immunosuppressive escape mechanisms that could be targeted to enhance responses.

Citation Format: John T. Wilson, Daniel Shae, Paula I. Gonzalez-Ericsson, Violeta Sanchez, JingJing Gong, Yan Liang, Douglas Hinerfeld, Joseph M. Beechem, Justin M. Balko. Digital spatial profiling of molecular responses to nanoparticle STING agonists identify S100A9 and B7-H3 as possible escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4978.