A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [ChIP-seq]

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors. ChIP-seq +/- TGFB1 ChIP-seq on A375s treated with 10ng/mL TGFB1 ligand or vehicle control A375 cells were serum starved for 2 hours, then treated with 10ng/mL human recombinant TGFB1 or 4mM HCl containing 1 mg/mL BSA vehicle control for an additional two hours. Experiment performed in technical triplicate.