Abstract 951: Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity
Authors: Marie-Nicole Theodoraki, Ravi Muthuswamy, Jamie Voyten, Francesmary Modugno, Natasa Obermajer, Robert P. Edwards, Pawel Kalinski
Journal: Cancer Research (2017)

Abstract Background: Infiltration of cytotoxic T-cells (CTL) in tumors is known to be associated with improved patient’s clinical outcomes. In contrast, local infiltration with regulatory T-cells (Treg) predicts accelerated progression and shorter overall survival. The above observations highlight the need for new measures to promote selective accumulation of CTLs but not Tregs in tumor microenvironments (TME). Here we compare the TME-modulating impact of poly-I:C with a selective TLR3 ligand, rintatolimod showing an induction of CCL22, CXCL12 and expression of COX-2 and downstream suppressive factors, selectively by poly-I:C. Finally, we report on the optimized combinatorial adjuvant to reprogram tumor microenvironment. Materials and Methods: Twelve ovarian cancer specimens were cultured in the presence of IFNα, indometacin (COX-1/2 inhibitor) or/and one of two synthetic TLR3 ligands, poly-I:C (non-selective activator of TLR3 and helicases) or rintatolimod (selective TLR3 ligand). Biopsies were harvested for mRNA measurements and culture supernatants were analyzed for CCL5, CXCL10 and CCL22 concentrations. Alternatively, established ovarian cancer cell lines, monocyte-derived macrophages, THP1 cells or adult fibroblasts were used in analogous experiments. Chemotaxis assays were performed using pre-activated CD8+ T-cells (top chamber) and supernatants from the differentially treated ovarian cancer specimens. Western blot experiments were used to evaluate the impact of NFκB. Blocking experiments using soluble TNFα receptor inhibitor were performed. Results: Both, Poly-I:C and rintatolimod, induced CTL attractant CXCL10. Unexpectedly, poly-I:C but not rintatolimod promoted the expression of the MDSC/Treg attractant CXCL12, which was reversed by addition of indometacin. Furthermore, poly-I:C induced expression of COX-2 and COX-2-dependent suppressive factors (IDO, IL-10). Evaluating the COX-2 cascade, TNFα was found to be increased in the presence of poly-I:C but not rintatolimod. Additionally, Western Blot analysis showed I-κB degradation (as a marker of NF-κB activation), selectively in response to poly-I:C. We observed that the combination of both TLR-3 ligands with IFNα and indomethacin selectively induced the desirable chemokines CCL5 and CXCL10 and suppressed CCL22 in tumor samples with similar results in the macrophages and fibroblasts. The ovarian cancer cell lines showed only minimal expression of chemokines. Conclusion: We demonstrate the feasibility of selective modulation of TME, using different clinically applicable factors and their combinations. We show for the first time, that poly-I:C, but not a selective TLR3 ligand, induces NFκB and undesirable COX2-dependent suppressive factors, which though can be eliminated through addition of a COX-1/2 inhibitor. These effects may be avoided using a selective TLR3 ligand. Citation Format: Marie-Nicole Theodoraki, Ravi Muthuswamy, Jamie Voyten, Francesmary Modugno, Natasa Obermajer, Robert P. Edwards, Pawel Kalinski. Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2017-951