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Introduction: The lack of an appropriate in vitro model of the tumor microenvironment is one of the largest obstacles in evaluating preclinical cancer drug screenings.1 Cancer cell monolayers do not effectively mimic the limited drug penetration properties of the complex tumor structures found in cancer patients. 3-D multicellular tumor spheroids (MCTS) serve as a more effective model as they better resemble cancer in structure as well as limited drug penetration. In our experiments, we created heterospheroids composed of 4T1 breast tumor cells and 3T3 fibroblasts, as well as homospheroids of each cell type. Tumors feature stromal and extracellular matrix components in addition to cancer cells in ratios that vary between different types of cancer. Fibroblasts are the major component of cancer stroma as well as producers of extracellular matrix. Since heterospheroids feature 3T3 fibroblasts, they may better model the diverse tumor microenvironment.2 We also synthesized fluorescent PLGA nanoparticles that were added to our spheroid cultures. Using confocal microscopy and ImageJ’s fluorescence measuring tools, we qualitatively and quantitatively evaluated the drug penetration properties of our spheroids.