cGAS-STING pathway reprograms macrophage polarization and is highly expressed in responding tumors after neoadjuvant immunotherapy in head and neck carcinoma

Abstract

Given the critical role of the cGAS-STING pathway in antitumor immunity, this study investigates the functional role of STING in head and neck squamous cell carcinoma (HNSCC) to evaluate the therapeutic potential of STING agonists. Analysis of the TCGA-HNSC dataset reveals that elevated expression of the STING-encoding gene TMEM173 is significantly correlated with increased M1 macrophage infiltration and enrichment of macrophage polarization-related signaling pathways. In vitro experiments in which RAW 264.7 cells are co-cultured with tumor cell-conditioned medium demonstrate that the STING agonist MSA-2 effectively reprograms tumor-induced M2-polarized macrophages toward the M1 phenotype. This MSA-2-induced M1 polarization is accompanied by increased expressions of IFN-α, IFN-β, IFN-γ, TNF-α, and IL-6, while the STING inhibitor H-151 reverses these effects. Flow cytometry further reveals that MSA-2 treatment reduces PD-1 and increases MHC II expression on macrophages. Immunohistochemical analysis of clinical samples confirms that high STING expression is correlated with increased numbers of CD68⁺ and CD80⁺ (M1-like) macrophages. In support of translational relevance, analysis of single-cell RNA-seq data from HNSCC patients receiving neoadjuvant immunotherapy indicates that TMEM173 is expressed primarily in T cells and macrophages and that the cGAS-STING pathway score is significantly higher in patients who respond to treatment. Collectively, these findings provide systematic clinical and experimental evidence supporting the potential of STING agonists, such as MSA-2, to enhance antitumor immunity in HNSCC, particularly when combined with immunotherapy.

1. Introduction

Head and neck squamous cell carcinoma (HNSCC) is characterized as a highly immune-infiltrated malignancy, with its etiology and progression linked to profound immunodeficiency, including immune cell dysregulation, diminished cytokine production, and impaired antigen presentation [1,2]. Tumor-associated macrophages (TAMs) are key contributors to the immunosuppressive microenvironment in HNSCC [3] and are primarily classified into two functional subsets: M1 TAMs, which mediate antitumor immune responses, and M2 TAMs, which exhibit immunosuppressive and pro-tumorigenic properties [4]. Increased TAM infiltration has been associated with lymph node metastasis and advanced-stage HNSCC [5]. Notably, M2 TAMs predominate in the HNSCC tumor microenvironment (TME), and their interaction with T cells—particularly regulatory T cells—underlies immune cell dysfunction and defective antigen presentation in HNSCC [6].

The cGAS-STING pathway plays a pivotal role in orchestrating antitumor immune activation and viral clearance [7,8]. Upon recognition of tumor-derived double-stranded DNA, cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which subsequently activates STING. This activation triggers a transcriptional cascade involving interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), ultimately inducing type I interferon expression [9]. Additionally, cGAS-STING signaling enhances the expression of costimulatory molecules and promotes endogenous antigen presentation via major histocompatibility complexes (MHCs) [10]. In tumor cells, activation of the cGAS-STING pathway stimulates cytokine and chemokine secretion, thereby augmenting the immune system’s capacity to eliminate tumor cells [11].

Given its role in regulating antitumor immunity, targeting the cGAS-STING pathway—specifically via STING stimulation in the TME—is promising as a therapeutic strategy in combination with immunotherapy. STING activators are categorized into two main classes: cGAMP-based nucleotide analogues and non-nucleotide STING agonists. Nucleotide analogues such as ADU-S100 (MIW815) and MK-1454 induce immune cell activation, antigen presentation, cytokine release, and CD8⁺ T-cell proliferation, with enhanced efficacy when combined with immune checkpoint inhibitors [12–15]. Non-nucleotide agonists, including amino... [truncated for brevity]