Spontaneous upregulation of stemness and 3D spheroid formation of single cancer cells induced by scaffold-free polypeptide polyelectrolyte multilayer coating.

Abstract

Three-dimensional (3D) cell structures, such as tumor spheroids, have emerged as essential tools in cancer research. However, current scaffold-free planar platforms designed to maintain 3D cell structures, including hydrogel-based low-adherence attachment plates, face limitations in handling rare cell samples and achieving high-throughput spheroid formation. We address these challenges by presenting a polyelectrolyte multilayer (PEM)-based surface coating designed to promote rapid spheroid formation from individual cancer cells. The coating was developed using a layer-by-layer deposition of poly-L-lysine and poly-L-glutamic acid, with the number of layers adjusted to tailor the surface hydrophilicity, charge neutrality, and mechanical dissipation capacity. The human colorectal carcinoma cell line HCT116 was used to assess the effectiveness of spheroid formation on this coating. When both surface charge neutrality and mechanical dissipation capacity were enhanced, we observed nearly 100 % efficiency in spheroid formation from single cells within three days, surpassing conventional scaffold-free techniques. Spheroid growth on this platform relies on EpCAM and exhibits enhanced expression of stemness and mixed epithelial/mesenchymal markers. The rapid expansion of individual cells on this platform can be utilized for high-throughput ex vivo applications, thereby advancing our understanding of cancer biology and facilitating the development of effective therapies.