CD44 alternative splicing in gastric cancer cells is regulated by culture dimensionality and matrix stiffness.

Two-dimensional (2D) cultures often fail to mimic key architectural and physical features of the tumor microenvironment. Advances in biomaterial engineering allow the design of three-dimensional (3D) cultures within hydrogels that mimic important tumor-like features, unraveling cancer cell behaviors that would not have been observed in traditional 2D plastic surfaces. This study determined how 3D cultures impact CD44 alternative splicing in gastric cancer (GC) cells. In 3D cultures, GC cells lost expression of the standard CD44 isoform (CD44s), while gaining CD44 variant 6 (CD44v6) expression. This splicing switch was reversible, accelerated by nutrient shortage and delayed at lower initial cell densities, suggesting an environmental stress-induced response. It was further shown to be dependent on the hydrogel matrix mechanical properties and accompanied by the upregulation of genes involved in epithelial-mesenchymal transition (EMT), metabolism and angiogenesis. The 3D cultures reported here revealed the same CD44 alternative splicing pattern previously observed in human premalignant and malignant gastric lesions. These findings indicate that fundamental features of 3D cultures - such as soluble factors diffusion and mechanical cues - influence CD44 expression in GC cells. Moreover, this study provides a new model system to study CD44 dysfunction, whose role in cancer has been in the spotlight for decades.

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology.

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.

Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium.

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through ß4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6ß4 integrin clustering into hemidesmosomes.