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.

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.

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.

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.