Abstract RET is a receptor tyrosine kinase expressed in cells derived from the neural crest. RET activation and signalling is mediated by soluble ligands of the Glial Cell-Derived Neurotrophic Factor (GDNF) family. RET signalling plays critical roles during embryogenesis, mediating directional cell migration, proliferation, and survival. In addition to its normal developmental roles, oncogenic mutations or aberrant expression of RET are also linked to tumor spread and metastasis in multiple human tumor types. Specifically, activating mutations of RET have been identified in multiple-endocrine neoplasia type 2, while expression of wildtype RET is linked to increased local invasion in breast and pancreatic tumors. Previous studies of RET-mediated invasion and metastasis have used standard 2D culture methodologies, which may not be reflective of the in vivo microenvironment. Here, we have developed an in vitro 3D model of tumor growth, invasion, and metastasis to characterize the signals critical to these processes. Using SH-SY5Y cells, a neuroblastoma cell line that endogenously expresses RET, we assessed anchorage-independent growth and invasion into a surrounding collagen matrix in response to RET stimulation with GDNF. We have quantified the contributions of several RET downstream signalling pathways by individually blocking SRC, PI3K, FAK, STAT3, MEK, and integrin β-1 signals, for their effects on RET-mediated cell growth and invasion in 3D culture. Our data demonstrate that inhibiting either PI3K or MEK decreases the invasive potential of SH-SY5Y cells in response to RET activation, while inhibition of STAT3 had little effect. We have also shown that integrin β-1, FAK or SRC inhibition completely blocked invasion, consistent with an integrin dependent mode of invasion in SH-SY5Y cells. Additionally, we showed that FAK and SRC signalling were critical for SH-SY5Y survival in 3D microenvironments. While several RET-mediated signalling pathways have been implicated in cell motility and invasion in two-dimensional culture models, our findings suggest that specific signals may differ in their importance in a three-dimensional microenvironment. We are currently using our 3D models to further characterize the contributions of RET signalling to tumor cell invasiveness. These data may provide further insight into the role of RET expression in in vivo tumor spread. Citation Format: Sarah M. Maritan, Eric Y. Lian, Lois M. Mulligan. RET-mediated invasion in three-dimensional microenvironment models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5056.