Tumor-mimetic hydrogel stiffness regulates cancer stemness properties in H-Ras-transformed cancer model cells.

Abstract

Cancer stem cells (CSCs) cause therapy-resistance and recurrence, therefore an establishment of therapeutic approaches targeting CSCs is essential for eradicating cancers; however, a lot of aspects of the mechanisms of CSCs generation remain unclear. We previously demonstrated that human glioblastoma cell lines cultured on double-network (DN) hydrogel were rapidly reprogrammed into CSCs. To elucidate molecular mechanisms underlying CSCs generation, we here focused on the elastic modulus of hydrogels mimicking the stiffness of tumor tissues. Mouse NIH3T3 fibroblasts transformed with representative oncogenes H-Ras and Src were employed as cancer model cells, and cultured on (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel with different elastic modulus. The H-Ras-, but not Src-, transformed NIH3T3 cells induced stem cell properties on the PAMPS gel, particularly with elastic modulus of approximately 10 kPa that mimics general tumor tissues. On the gels, expression levels of stemness markers such as Sox2, Nanog, and Oct4 were increased in the parental and H-Ras-transformed cells. Pull-down assay demonstrated that multiple proteins in H-Ras-transformed cells bound to the PAMPS gels with 9.3 kPa stiffness, and desmoplakin was identified as one of the gel-bound proteins by LC-MS/MS analysis. Among Ca²⁺ channels functioning as mechanosensors, the expression levels of TRPC and TRPV families were efficiently increased on the gel with approximately 10 kPa, as well as stemness markers. These data suggest that tumor tissues with the stiffness of 10 kPa effectively trigger the signals for generating CSCs through alterations of membrane structures and Ca²⁺ influx, which will be beneficial for developing novel therapeutic applications targeting CSCs.