Assessment of Doxorubicin Internalization and Cytotoxicity in Primary Tumor Spheroids After Collagen Digestion

The limited penetration of chemotherapeutic agents into solid tumors remains a major obstacle to effective cancer treatment and is strongly influenced by the extracellular matrix (ECM). Three-dimensional (3D) tumor spheroids derived from primary tumor cells (PTCs) represent a valuable in vitro model to study how ECM composition and organization regulate drug distribution and cytotoxicity with accurate physiological relevance. By recapitulating key features of the tumor microenvironment, including endogenous collagen deposition and diffusion-limited drug accessibility, these models enable mechanistic investigation of microenvironment-driven drug resistance. Here, we describe a set of integrated protocols to evaluate the impact of ECM remodeling on chemotherapeutic response in primary breast tumor spheroids. Spheroids are generated from isolated primary tumor cells and subjected to controlled enzymatic degradation of collagen-rich ECM using ultrapure recombinant collagenases. As a model chemotherapeutic agent, doxorubicin is employed thanks to its widespread clinical use, well-characterized cytotoxic mechanism, and intrinsic fluorescence, which allows direct visualization of drug uptake and spatial distribution by confocal microscopy. In parallel, drug-induced cytotoxicity is quantified using a luminescent 3D viability assay. Together, these protocols provide a reproducible and accessible platform for investigating ECM-mediated barriers to drug delivery and toxicity in 3D tumor models. This framework facilitates the implementation of physiologically relevant assays to study drug efficacy and therapeutic resistance in solid tumors. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of breast cancer, isolation of tumor primary cells, and generation 3D spheroids Support Protocol: Culture and storage of PTCs Basic Protocol 2: Evaluation of collagen expression and enzymatic degradation in primary tumor spheroids by confocal microscopy Basic Protocol 3: Enzymatic degradation of the endogenous matrix collagen in primary tumor spheroids using recombinant collagenases Basic Protocol 4: Assessment of doxorubicin uptake in spheroids by confocal microscopy Basic Protocol 5: Assessment of cell viability in doxorubicin-treated spheroids using CellTiter-Glo 3D assay.