A new cell-laden 3D Alginate-Matrigel hydrogel resembles human breast cancer cell malignant morphology, spread and invasion capability observed "in vivo"

Purpose of this study was the development of a new cell-laden material to be used as substrate for 3D breast cancer cell culture, more realistic than 2D ones, allowing cells expressing some key mechanisms typical of their in vivo behaviour. This necessity comes from the known poor reliability of 2D and animal models, especially in pathological conditions, such as breast cancer. We designed and tested three different materials, each one constituted by different concentrations of Alginate (A) and Matrigel (M), in order to obtain a structurally stable-in-time and biologically active substrate. Highly metastatic breast cancer cells (i.e. MDA-MB-231) were cultured and embedded within the gels. Known the strong link between cell morphology and their malignant potential, cells growing within the gels were morphologically characterized and their invasiveness cross-correlated through an innovative bioreactor based invasion assay: cell capability to migrate out of the 3D tumour models was investigated by hosting the tumour tissues within the device, on an engineered membrane mimicking the vascular wall. A particular type of gel (i.e. 50% Alginate, 50% Matrigel) emerged as suitable substrate thanks to a series of significant results: 1. cells exhibited peculiar cytoskeleton shapes and poly-nuclei organization characteristic of their malignancy; 2. cells expressed the formation of the so-called invadopodia, actin-based protrusion of the plasma membrane through which cells anchor to the extracellular matrix and degrade it. This feature, never observed in a 3D in vitro environment, was expressed by MDA-MB-231 regardless of time point; 3. cells were able to migrate through the gels, escaping from those and attach to the engineered membrane hosted within a multi-organ bioreactor-based set-up by React4life S.r.l., finally providing a totally revolutionary 3D in vitro model of the very precursor steps of metastasis. Our data show that the here proposed material is suitable for being used in breast cancer studies, since it provides a 3D environment able to enhance cell viability, proliferation, malignancy expression and migration capability.