Decitabine co-operates with the IL-33/ST2 axis modifying the tumor microenvironment and improving the response to PD-1 blockade in melanoma.

Background: IL-33 is an epithelial-derived alarmin with various roles in cancer. In melanoma, endogenous and exogenous IL-33 exert anti-tumor effects through the stimulation of several immune effector cells. In this study, we explored the combination of IL- 33 with Decitabine (DAC), a DNA methylation inhibitor that promotes immune recognition by re-activating silenced genes, for melanoma treatment.

Methods: Multicellular spheroids, organ-on-chip technology and in vivo models were used to test the anti-tumor effects of IL-33 combined with DAC against mouse and human melanoma. Mice deficient for the IL-33 receptor ST2 (ST2 mice) were employed to address the role of endogenous IL-33 signaling in DAC therapeutic response and tumor-immune crosstalk.

Results: In multicellular spheroids of mouse and human melanoma cells, DAC alone inhibited tumor cell aggregation, suggesting its direct effect on tumor cells. In vivo, DAC combined with IL-33 reduced tumor growth and prolonged the survival of mice transplanted with melanoma cells, outperforming single treatments. Moreover, the combined DAC/IL-33 treatment was the most efficient in promoting immune recruitment (i.e., T cells and eosinophils) at the tumor site and induced the up-regulation of PD-1 resulting in better therapeutic response to PD-1 blockade in vivo. In a microfluidic-based competitive migration assay, DAC/IL- 33 treatment generated the strongest chemotactic response, attracting spleen cells from naïve wild-type, but not ST2 mice, indicating that IL-33 signaling was crucial for immune cell recruitment. Accordingly, DAC failed to induce tumor immune infiltration and was ineffective in reducing tumor growth in ST2 mice. In vivo, DAC increased the expression of ST2 and IL-33 at the tumor site, suggesting it may enhance endogenous IL-33 production. Methylation studies indicated that DAC increased the expression of IL-33 in mouse and human melanoma cells through demethylation of a transcription factor binding site located inside the IL33 gene.

Conclusions: Our findings indicate that DAC effectively co-operates with IL-33/ST2 axis against melanoma through immune cell recruitment and epigenetic regulation of gene expression, thus remodeling the tumor immune microenvironment to overcome resistance to PD- 1 inhibition.