| Summary: | A better understanding of how microenvironmental factors regulate cancer dormancy is needed for development of new therapeutic strategies to control metastatic recurrence and disease progression. Modeling cancer dormancy using engineered, in vitro platforms is necessary for investigation under well-defined and well-controlled microenvironments. We present methods and protocols to fabricate, characterize, and implement engineered hydrogels with well-defined biochemical and physical properties for control over breast cancer cell phenotype in three-dimensional (3D) culture. Changes in hydrogel adhesivity, crosslink density, and degradability induce a range of phenotypic behaviors in breast cancer cells including: (1) high growth, (2) moderate growth, (3) single cell, restricted survival dormancy, and (4) balanced dormancy. We describe a method of classifying hydrogel formulations that support each of these phenotypic states. We also describe a method to phenotypically switch cancer cells from single cell dormancy to high growth by dynamically modulating ligand density, thereby recapitulating reactivation and metastatic recurrence. Protocol name: Methods to characterize hydrogel properties, Quantification of cellular metrics, Phenotypic classification of cancer cell states, Keywords: Biomaterials, Tissue engineering, Hydrogel, PEG, Cancer, Dormancy, Metastasis, Relapse, Microenvironment
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