Cancer remains the leading cause of death globally, with nearly 20 million new cases and 10 million fatalities in 2022. The majority (90%) are solid tumors, which are strongly influenced by the tumor microenvironment (TME)—a complex network of blood vessels, connective tissues, immune cells, and the extracellular matrix (ECM). The ECM, rich in collagen, fibronectin, elastin, and laminin, is constantly remodelled, driving tumor progression and drug resistance while increasing tissue stiffness. Breast cancer, the most common malignancy in women, causes 500,000 deaths annually. Its ECM, particularly collagen composition, varies depending on tumor type and prognosis. Recent research has emphasized 3D tumor spheroid models, which replicate in vivo tumor conditions better than traditional 2D cultures. These models allow more accurate drug testing and better understanding of tumor biology. To improve treatment, researchers have developed a 3D breast cancer spheroid model using rat-derived primary cells with an endogenous ECM structure. By applying collagenases to break down ECM barriers, they demonstrated that ECM degradation enhances drug penetration and chemotherapy effectiveness. This study highlights the critical role of ECM in cancer progression and treatment resistance. Targeting the ECM within advanced 3D models could improve therapeutic outcomes and lead to more effective anticancer strategies.