Oxidized dextran and thiolated chitosan-based hydrogel driven biomimetic triple negative breast cancer 3D in vitro model for cancer progression and therapeutic studies

Abstract

In the advancing field of in vitro cancer modeling, three-dimensional (3D) culture systems are increasingly recognized for their ability to recapitulate critical tumor-specific characteristics. Given the aggressive nature and high mortality associated with Triple-Negative Breast Cancer (TNBC), there is a pressing need for physiologically relevant 3D in vitro models that effectively simulate key Tumor Promoting Factors (TPFs). This study presents a Modified Dextran-Chitosan (MDC) hydrogel with engineered non-fouling properties that supports the formation of MDA-MB-231-derived 3D tumoroids. The hydrogel facilitated upregulated expression of extracellular matrix markers, including COL1A1 (2.29-fold↑) and FN1 (0.84-fold↑). Cell proliferation within 3D cultures was significantly reduced on days 2 (p < 0.001), 4 (p < 0.0001), and 6 (p < 0.001) compared to 2D cultures. Enhanced hypoxic conditions (based on EF5 adducts’ fluorescence; p < 0.0001), epithelial-to-mesenchymal transition (EMT) traits, and stemness marker expression [e.g., NANOG (3.33-fold↑)] were observed in 3D tumoroids. Additionally, the 3D tumor microenvironment showed elevated activity of key TPFs, including IL6, IL10, TNFA, FGF2, BMP2, and active TGFB (p < 0.0001). The MDC hydrogel, with stiffness mimicking breast tissue (~11 kPa), also promoted mechanotransducive signalling, evidenced by increased YAP1 expression (2.4-fold↑) and a significantly elevated nuclear-to-cytoplasmic YAP1 ratio (p < 0.0001) relative to 2D cultures on TCPS (~3 GPa). Whole transcriptome sequencing and gene set enrichment analyses further validated the enhanced tumorigenic phenotype of the 3D model. Moreover, the 3D tumoroids exhibited significant resistance (p < 0.001) to combined Doxorubicin-Paclitaxel treatment. Thus, the MDC hydrogel-based 3D TNBC model emerges as a robust and scalable platform for anticancer drug screening, evaluating precision medicine and investigating cancer biology.