Gravitational forces and matrix stiffness modulate the invasiveness of breast cancer cells in bioprinted spheroids

The progression of breast cancer is influenced by the stiffness of the extracellular matrix (ECM), which becomes stiffer as cancer advances due to increased collagen IV and laminin secretion by cancer-associated fibroblasts. Intriguingly, breast cancer cells cultivated in two-dimensions exhibit a less aggressive behavior when exposed to weightlessness, or microgravity conditions. This study aims to elucidate the interplay between matrix stiffness and microgravity on breast cancer progression. For this purpose, three-dimensional spheroids of breast cancer cell lines (MCF-7 and MDA-MB-231) were formed. These spheroids were subsequently bioprinted in hydrogels of varying stiffness, obtained by the mixing of gelatin methacrylate and poly(ethylene) glycol diacrylate mixed at different ratios. The constructs were printed with a custom stereolithography (SLA) bioprinter converted from a low-cost, commercially available 3D printer. These bioprinted structures, encapsulating breast cancer spheroids, were then placed in a clinostat (microgravity simulation device) for a duration of seven days. Comparative analyses were conducted between objects cultured under microgravity and standard earth gravity conditions. Protein expression was characterized through fluorescent microscopy, while gene expression of MCF-7 constructs was analyzed via RNA sequencing. Remarkably, the influence of a stiffer ECM on the protein and gene expression levels of breast cancer cells could be modulated and sometimes even reversed in microgravity conditions. The study's findings hold implications for refining therapeutic strategies for advanced breast cancer stages - an array of genes involved in reversing aggressive or even metastatic behavior might lead to the discovery of new compounds that could be used in a clinical setting.