Decellularized Extracellular Matrix-Based Tunable 3D Hydrogel: An Alternative Methodology for the Development of a Doxorubicin-Independent 3D Breast Cancer Microphysiological Chemoresistance Model

Breast cancer is the most prevalent malignancy among women and the second leading cause of cancer-related mortality worldwide, with 2.26 million cases and 685,000 deaths in 2020. Its complex nature complicates treatment, and emerging evidence shows that extracellular matrix (ECM) biomechanics, particularly stiffness, dynamically change in response to physiological and pathological cues. These alterations influence malignant cell morphology and promote chemoresistance by regulating tumor growth, invasion, and metastasis. Clinically, breast tumor ECM stiffness (2-100 kPa) is markedly higher than that of healthy breast tissue (0.1-1 kPa). Developing 3D models that accurately recapitulate breast cancer ECM stiffness remains challenging, as current systems lack physiologically relevant crosslinking and rheology. Increasing evidence suggests that ECM stiffness alone can drive chemoresistance; thus, precise in vitro replication of tumor matrix biomechanics may induce drug-resistant phenotypes through mechanical cues. A key unresolved question is how matrix rigidity mechanistically regulates therapeutic resistance? Is it possible to create a chemoresistance model without any drug exposure? In this work, Pork Achilles tendon was decellularized using surfactants (SDS-Triton X-100) and enzymes (Collagenase-Dispase and Trypsin-EDTA), preserving ECM structure and biomechanics, and engineered into a customizable photo-cross-linked 3D hydrogel to study the effects of matrix crosslinking and rheology on in vitro chemoresistance. H&E staining and SEM were used to characterize the decellularized tissue. Further, we lyophilized and cryo-milled the decellularized tissue into a fine powder to fabricate a photo-cross-linked 3D hydrogel with tunable matrix crosslinking and rheology. Drug resistance was evaluated by culturing MDA-MB-231 cells encapsulated in 3D hydrogels with variable matrix crosslinking and rheology, and assessing the CD44⁺/CD24^- cancer stem cell-associated phenotype linked to aggressiveness and therapy resistance.