Suite version 2

The spheroids (n = 3–8 each condition) were fixed in buffered formalin (Histofix). Formalin-fixed paraffin embedded sections were cut at a thickness of 3 µm. The Ki67 immunohistochemistry was performed on a Ventana BenchMark Ultra system (Ventana Medical Systems, Tucson, AZ, USA) using clone 30-9, a monoclonal mouse IgG antibody (Cell Signaling Technology, Cambridge, UK). An immunohistochemistry analysis was performed with the following protocol: pretreatment cell conditioning 1, 95 °C, 32 min.; incubation 36 °C, 16 min. The visualization was conducted using an OptiView DAB System (Ventana Medical System). Thereafter, the slides were scanned using a Leica DM4000B microscope, Leica DFC290 camera and analyzed using the Leica Suite version 2.8.1 (Leica Microsystems, Wetzlar, Germany). The slides were reviewed by a board-certified pathologist (ST).

Excised tumour spheroids were fixed in buffered formalin (Histofix). Formalin-fixed paraffin-embedded sections were cut at a thickness of 3 µm. The Melan A (clone A103, Roche, Ventana) and SOX10 (clone BC34, Biocare Medical) immunohistochemistry analysis was performed using the Ventana BenchMark ULTRA system (Ventana Medical Systems, Tucson, AZ, USA). The immunohistochemistry (IHC) analysis for Melan A was performed using the following protocol: pretreatment CC1 was performed for 36 min at 95 °C, and incubation occurred for 24 min at 27 °C. For SOX10, CC2 pretreatment was performed for 40 min at 90 °C, and incubation occurred for 32 min at 36 °C. Visualisation was conducted using the OptiView DAB System (Ventana Medical System). Thereafter, slides were scanned using a Leica DM4000B microscope with a Leica DFC290 camera and analysed using the Leica Suite version 2.8.1 (Leica Microsystems, Wetzlar, Germany). The slides were reviewed by a board-certified pathologist.