In vitro mechanisms of primary dermal fibroblast activation

To address this, primary human dermal fibroblasts were activated toward an inflammatory-like CAF (iCAF) or myofibroblast-like CAF (myCAF) phenotype using four independent induction strategies: (1) treatment with lipopolysaccharide (LPS, iCAF-like) or TGF-beta (myCAF-like); (2) CRISPR-dCas9-mediated transcriptional activation of IL6 (iCAF-like) or ACTA2 (myCAF-like); (3) direct co-culture with unstimulated or LPS-stimulated peripheral blood mononuclear cells (PBMCs); and (4) culture in conditioned medium from SK-MEL-28 melanoma cells.

Activation states were validated by RT-qPCR and Western blot for the canonical markers IL6 and ACTA2 prior to RNA sequencing (Illumina NovaSeq 6000, paired-end 150 bp). Bulk RNA-seq was performed on fibroblasts from each condition and matched controls to define the transcriptome-wide gene expression changes associated with iCAF- and myCAF-like activation, enabling derivation of subtype-specific gene signatures for downstream analysis of stromal reprogramming in melanocytic malignancy.