Introduction and purpose of the study: Cyclic dinucleotides (CDNs), which signal through the STING (Stimulator of Interferon Gene) pathway, are a novel class of adjuvants that have been shown to induce anti-tumor responses in vivo. Preclinical studies will provide the necessary rationale for immunotherapy clinical trials for metastatic/recurrent HNSCC patients. In this study, we examined the role of CDNs in stimulating human dendritic cells and monocytes. We further examined the anti-tumor immune responses generated by natural and synthetic CDNs on SCCVII squamous cell tumors in a murine model.
Experimental design and setup: Preclinical animal study and in vitro human sample experiments performed at an academic animal research facility. Cultured human dendritic cells (DCs) were stimulated with CDNs to examine cytokine response profiles and activity levels. We further attempted to model the effects of CDNs in a murine system. SCCFVII tumor bearing C3H/HeOUJ mice (n = 5 per group) were treated with three intratumoral injections of RR-CDG (synthetic cyclic-di-guanine), CDG (natural cyclic-di-guanine), R848 (TLR 7/8 agonist), or PBS (phosphate buffered saline, control). In addition, a group of RR-CDG treated mice was also treated with anti-CD8 T-cell depletion antibody to evaluate the role of CDNs in stimulating CD8+ T-cells. In vivo tumor growth rate was followed for 23 days, after which spleens were harvested and splenocytes were analyzed for cytokine profile using flow cytometry. In addition to measuring the anti-tumor immune response, the direct toxicity of the CDNs was measured by performing in vitro dose response live/dead cell viability assays.
Findings or new outcome: The primary outcome measure was tumor volume. The secondary outcome measure was comparison of relative expression of Th1 vs. Th2 cytokine profiles from splenocytes in disparate murine treatment groups and from cultured human dendritic cells. In cultured human DCs, CDN stimulation resulted in higher levels of monocyte activation and increased IFNg and IL-12 levels when compared to PBS controls. In the murine model, RR-CDG and CDG treatment groups showed significantly decreased tumor size when compared to the R848, T-cell deficient RR-CDG, and PBS treatment groups (p<0.05). Splenocytes from the RR-CDG treated mice showed an enhanced Th1 response, as determined by increased expression of IFNγ, when compared to the PBS treatment group. In vitro tumor cell killing assay showed that there was no direct tumor cytotoxicity effect in response to CDNs.
Conclusions: Synthetic and natural CDN treated mice had significantly reduced tumor volumes when compared to R848 (TLR7/8 agonist) and control treated mice. Mice treated with synthetic and natural CDNs also showed a predominant Th1 response when compared to mice treated with R848 (TLR7/8) agonists or controls. A pre-dominant Th1 cytokine response and increased monocyte activation in human cell studies demonstrate the translational nature of the animal studies and strongly support the future implementation of intratumoral injection of CDN into HNSCC patients.
Citation Format: Shekhar K. Gadkaree, Rupashree Sen, Juan Fu, Clint Allen, Young J. Kim. Cyclic dinucleotide: A novel adjuvant for squamous cell carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B087.
- ©2016 American Association for Cancer Research.