Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive <i>in Vitro</i> Model for Breast Cancer Metastasis

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic <i>in vivo</i> signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment <i>in vitro</i> to study breast cancer metastasis. Porous poly­(ε-caprolactone) (PCL) scaffolds of modulus 7.0 ± 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency <i>in vitro</i> and increased lung metastasis <i>in vivo</i>. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell–cell and cell–matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIF1 signaling pathwaysall of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the <i>in vivo</i> tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive <i>in vitro</i> model to investigate the manifold mechanisms of breast cancer metastasis