Culture dimensionality controls CD44 alternative splicing

摘要

CD44 and its alternative spliced variants have been broadly associated with cancer, from resistance to oxidative stress to metastatic behavior. In the context of gastric cancer (GC) we have previously shown that de novo expression of CD44 variant 6 (CD44v6) accompanies malignant progression. Two-dimensional (2D) cell culture is commonly used to study cancer in vitro, but fails to mimic key architectural and physical features of the tumor microenvironment. This study determined how more physiologically relevant three-dimensional (3D) culture impacts CD44 alternative splicing in epithelial GC cells. In 3D culture, GC cells gradually lost expression of the standard CD44 isoform (CD44s), while simultaneously gaining CD44v6 expression that was virtually absent in 2D monolayers. This switch in CD44 spliced isoforms was reversible, accelerated by nutrient shortage and delayed with lower initial cell densities, suggesting that it is a stress-induced response. Loss of CD44s in 3D culture was further shown to be dependent on the matrix mechanical properties and accompanied by the upregulation of key epithelial-mesenchymal transition (EMT) markers, such as SNAIL and ZEB1, along with several genes involved in aberrant metabolic pathways, cellular stress, angiogenesis and antisenescense cellular programs. Human gastric premalignant and malignant lesions revealed that 3D cultures, but not 2D monolayers, were able to capture aberrant CD44 splicing patterns observed in vivo. Altogether, these findings suggest that cells modulate CD44 expression in response to culture dimensionality and physical cues, and could translate into clinical strategies to locally modulate CD44 expression in GC, the 4th most common cancer in the world.