Project Summary The microenvironment plays a unique role in the development of melanoma, which arises in the skin. The skin displays externally visible signs of aging, such as decreased elasticity and a loss of collagen integrity. Much work has focused recently on how changes in collagen can affect tumor metastasis, both biomechanically and biochemically. Data from several groups indicate that the cross-talk between stromal fibroblasts and transformed melanocytes is important for invasion, melanoma growth, and even therapy resistance. Our proteomics analyses indicate that factors used to crosslink collagen, such as HAPLN1 are secreted by young, but not aged dermal fibroblasts, and Wnt5A is secreted by aged, but not young fibroblasts. The interplay between these molecules may contribute to age-related changes in collagen integrity. We hypothesize that changes regulated by age-related alterations in the extracellular matrix (ECM) initiate or promote a pro- metastatic program, and impact therapy resistance. We will query the contribution of aged skin biochemical and architectural contributions in governing melanoma’s metastatic progression as well as resistance to targeted therapy. We propose an alternate theory of matrix stiffness that hypothesizes that increasing stiffness will have a non-linear effect on metastatic progression, and therapy resistance. We will use pathophysiologically relevant in vitro 3D stromal systems (3D skin reconstructions), animal models of melanoma, a novel simultaneous multi-channel immunofluorescent analysis (SMIA) of formalin-fixed and paraffin embedded (FFPE) human tissue cohorts in combination with a customized new software written for the bulk analysis and acquisition of SMIA-generated images, single molecule RNA imaging coupled with high-throughput single cell imaging and sequencing, all of which will feed back into an increasingly intricate mathematical modeling for improved understanding and better patient personalized (i.e., metastatic and efficacy of drug treatment) prediction capabilities. We expect our data will reveal a synergistic picture of the mechanochemical interactions between the aged microenvironment and singular tumor cells that the individual approaches could not have deciphered. This team project involves experts in the biology of melanoma metastasis, Wnt signaling and aging (Weeraratna), single cell RNA systems biology (Raj), tumor-stromal interactions and digital imaging quantitative analyses (Cukierman) and computational and mathematical predictive modeling (Shenoy).