Cytokeratin 5

Formalin‐fixed, paraffin‐embedded tissue sections (5 μm) were derived from primary melanomas from each cohort. The IHC methodology for AMBRA1 (Abcam, Cambridge, U.K.), loricrin (Abcam) and cytokeratin 5 (Novocastra; Leica Biosystems, Milton Keynes, U.K.) are detailed in Appendix S1 (see Supporting Information).
Semi‐quantitative analysis of epidermal AMBRA1 expression was undertaken using Leica Digital Image Hub software (Leica Biosystems). Up to 10 representative ×200 microscope fields were analysed for mean positive pixel intensity of AMBRA1 expression levels and compared to the mean AMBRA1 expression in the epidermis directly above the melanoma, allowing a relative percentage expression change to be calculated with the normal epidermis considered as 100% expression.

CAFs were isolated as previously described.^{45 (link)} Briefly, tissue from early-stage IDC (stage 1) that was less than 10 mm in diameter was sliced and then digested overnight with a collagenase preparation (ISU ABXIS; Seoul, South Korea). Digested tissue was filtered through a 70 μm cell strainer (SPL Life Science; Pocheon-si, South Korea). Cells were separated by Ficoll gradients, washed with PBS, resuspended with DMEM/F12 cell culture medium containing 20% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL; Grand Island, NY, USA) and cultured at 37 °C in a humidified incubator containing 5% CO₂. The fibrotic nature of the isolated cells was confirmed by microscopic determination of morphology and immunofluorescence characterization using the antibodies against vimentin (Abcam; Cambridge, UK), cytokeratin (Dako; Glostrup, Denmark) and cytokeratin 5 (Novocastra; Newcastle upon Tyne, UK). Breast cancer cell lines (BT-474, MCF7, SK-BR-3, MDA-MB-231) were purchased from Korean Cell Line Bank (Seoul, South Korea) (authenticated using morphology and STR profiling) and cultured with DMEM cell culture medium containing 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator containing 5% CO₂.

CAFs were isolated as previously described [46 (link)]. Briefly, IDC tissues were minced and then digested overnight in a collagenase cocktail (ISU ABXIS; Seoul, South Korea). Digested tissues were filtered through a 70-μm cell strainer (SPL Life Science; Pocheon-si, South Korea). Cells were separated using Ficoll gradients, washed with phosphate-buffered saline (PBS), resuspended in Dulbecco's Modified Eagle medium (DMEM)/F12 medium containing 20% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL; Grand Island, NY, USA), and cultured at 37°C in a humidified atmosphere of 5% CO₂. The fibrotic characteristics of the isolated cells were confirmed by microscopic examination of morphology and immunofluorescence analysis using antibodies against vimentin (Abcam; Cambridge, UK), cytokeratin (Dako; Glostrup, Denmark), and cytokeratin 5 (Novocastra; Newcastle upon Tyne, UK).