Additional file 1: of A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells

Figure S1. Transfer of docetaxel-treated tri-cultures leads to massive loss of external melanoma cells. Drawings schematically depicting the general composition of the tri-culture model (A) and the proposed mechanism for the loss of external SK-MEL-28 cells upon docetaxel treatment (B). (A) A core of CCD-Sk1137 fibroblasts (grey) is surrounded by a ring of CK14-positive HaCaT keratinocytes (yellow), and this by CK10-positive HaCaT keratinocytes (red). SK-MEL-28 melanoma cells (green) can be divided in individual ‘internal’ melanoma cells found largely in the fibroblast core, and clustered ‘external’ melanoma cells located on the outer rim of the tri-cultures. (B) In all experiments, spheroid formation was performed in cell repellent plates. In mold experiments (left part), spheroids were then transferred to an agarose mold, where docetaxel treatment was followed by washing and embedding for cryosectioning. Subsequently, cryosections were immunostained. In experiments without mold, docetaxel treatment was also done in the cell repellent plate. Then, treated spheroids were transferred to another standard plastic well for washing and embedding. Presumably, external melanoma cells got loose upon docetaxel treatment and were largely lost upon transfer in the experiments without mold. This is schematically shown by the loosened cells in the pipette on the right side of the scheme. (C) Micrograph of a tri-culture spheroid in the agarose mold. Note, that the agarose does not cover the spheroid, thus, docetaxel can freely access the spheroid as in the standard plastic well. The advantage of the mold is, that it can be directly cryosectioned avoiding further steps of pipetting. (JPG 1420 kb)