Synergistic Effect of Co‐Culturing Breast Cancer Cells and Fibroblasts in the Formation of Tumoroid Clusters and Design of In Vitro 3D Models for the Testing of Anticancer Agents

Breast cancer is still the leading cause of women's death due to relapse and metastasis. In vitro tumor models are considered reliable tools for drug screening and understanding cancer‐driving mechanisms due to the possibility of mimicking tumor heterogeneity. Herein, a 3D breast cancer model (3D‐BCM) is developed based on enzymatically‐crosslinked silk fibroin (eSF) hydrogels. Human MCF7 breast cancer cells are encapsulated into eSF hydrogels, with and without human mammary fibroblasts. The spontaneously occurring conformational change from random coil to β‐sheet is correlated with increased eSF hydrogels’ stiffness over time. Moreover, mechanical properties analysis confirms that the cells can modify the stiffness of the hydrogels, mimicking the microenvironment stiffening occurring in vivo. Fibroblasts support cancer cells growth and assembly in the eSF hydrogels up to 14 days of culture. Co‐cultured 3D‐BCM exhibits an upregulated expression of genes related to extracellular matrix remodeling and fibroblast activation. The 3D‐BCM is subjected to doxorubicin and paclitaxel treatments, showing differential drug response. Overall, these results suggest that the co‐culture of breast cancer cells and fibroblasts in eSF hydrogels allow the development of a mimetic in vitro platform to study cancer progression. This opens up new research avenues to investigate novel molecular targets for anti‐cancer therapy. Breast cancer patients often develop resistance to standard treatments. 3D in vitro tumor models can help investigate its underlying molecular mechanisms. These results indicate the importance of cancer‐stroma interaction, as well as matrix stiffness, which influence chemotherapeutic drug treatment response. The herein developed breast cancer model will allow to develop more patient‐specific therapeutic regimens in the future.