Development of an In Vitro Platform for Analyzing the Correlation Between Matrix Stiffness and Cancer Stemness Using In-Bath 3D Bioprinting Technique

Cancer stemness refers to the phenomenon where cancer cells acquire stem cell-like phenotypes, which are known to significantly influence carcinogenesis and contribute to poor prognosis. The stiffness of the extracellular matrix (ECM) surrounding tumor changes during the cancer progression. It has been reported that the increase of ECM stiffness can activate cancer stemness, leading to enhancement of cancer progression and acquisition of drug resistance. Therefore, to better understand the relationship between cancer stemness and matrix stiffness, developing an in vitro platform that elaborately recapitulates the heterogeneity of the mechanobiological environment of cancer is necessary. In this study, we developed an in-bath 3D bioprinting technique and stiffness-tunable hybrid bioink to fabricate an in vitro cancer platform that recapitulates complex tumor microenvironment and effectively stimulates cancer stem cells (CSCs). In the platform, PC3 prostate cancer cell line is printed as a form of spheroid. By adjusting the bioink composition, stiffness gradients are created to determine the optimal mechanical environment in which CSCs are stimulated. We found that PC3 cells are actively stimulated in the stiff matrix, exhibiting dynamic changes in cellular organization from epithelial to mesenchymal phenotypes and acquiring drug resistance. Specifically, cancer cells cultured in a stiff matrix exhibited a significant increase in the expression of the transmembrane receptor integrin and focal adhesion kinase. Moreover, next-generation sequencing revealed elevated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cancer cells within this stiff matrix, with nuclear factor kappa B (NF-κB) being the most significantly activated downstream target of the PI3K/Akt pathway. Through the development of this platform, we were able to confirm stiffness-enhanced prostate cancer cell progression mediated via the PI3K/Akt/NF-κB signaling pathway. These results suggest that the developed platform holds strong potential for understanding the mechanobiological aspects of cancer stemness activation and could be utilized as a drug screening platform to identify optimal drug candidates for personalized cancer treatment.