Synthesis and Characterization of 3-D Tissue Engineered Tumor Models for Melanoma Study and Extracellular Matrix Investigation
Introduction: Melanoma is the deadliest form of skin cancer, projected to claim over 9,700 American lives and necessitate $3.3 billion in care and treatment costs in 2017. In order to reduce the socioeconomic burdens and lessen cancer-related mortality and morbidity, advancements in early-stage diagnostics and treatments are essential. Two-dimensional (2-D) tissue culture treated plastics (TCTP) are reductionist in vitro models that are commonly used in drug development and preclinical drug evaluation. These models poorly recapture the native tumor microenvironment (TME), providing unrealistic growth conditions, and are thus poorly suited to measure the efficacy of newly emerging therapies. We have developed reproducible three-dimensional (3-D) tissue engineered tumor models in order to assess the differential phenotypes expressed by melanoma cells grown on our models of varying stiffness. The primary objective of this work is to investigate and decouple the influence of the extracellular matrix (ECM) physicochemical properties on the metastatic phenotype of melanoma. Further, we aim to provide more robust and relevant models for investigating the TME in response to therapeutics. Methods: Bovine-derived collagen type 1 was used to prepare soft 3-D gels (GEL). Stiffer 3-D gels (XPC) were prepared from the GEL via plastic compression and genipin crosslinking. The differential physicochemical material properties of our models were characterized via AFM, SEM, and Raman spectroscopy. A375 and VMM-1 cells lines were cultured on-top of the GEL, XPC, and TCTP surfaces. Cellular proliferation was measured with the AlamarBlue assay on day 1, 3, 5, and 7. Additionally, A375 and VMM-1 cells were fixed and stained after 48h to visualize cellular morphology and actin stress fibers as well as to quantify cellular spreading. Further, cryostat sections of the substrates were stained in order to visualize the distribution of biomarkers, pFAK and integrin αvβ3, that are involved in focal adhesion (FA) formation. Lastly, an 84-gene array RT-qPCR analysis and western blot were performed in order to assess the differential expression of genes and proteins involved in cellular adhesion and ECM-remodeling. Results and Conclusions: The AlamarBlue assay indicated significantly (p<0.05) increased cell proliferation with increasing substrate moduli. Fluorescence microscopy indicated that cells grown on the stiffer substrates displayed greater actin stress fiber distribution as well as significantly increased cell spreading. Further, the size and distribution of pFAK and integrin αvβ3 suggested the presence of mature and stable FA on the stiffer substrates and temporary focal complexes on the soft gel. The western blot confirmed the expression of metastatic markers within our models, which importantly were found to increase in expression with increasing substrate moduli. The 84-gene array RT-qPCR analysis displayed significant differential gene expression patterns for the cells grown on GEL, XPC, and TCTP. These results confirm that our models are capable of inducing differential melanoma phenotypes. This further highlights the influence of ECM physicochemical properties on metastatic progression, as well as it also demonstrates the need for reproducible and tunable 3-D tumor models for melanoma research and drug screening applications.