Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, accounting for 15% of all childhood cancer deaths. Despite recent improvements in outcome for children with early stage disease, the majority of children with high-risk neuroblastoma survive less than 5 years. Nearly all newly diagnosed patients achieve apparent remission, but patients with high-risk disease relapse and die from metastatic progression within 2-5 years. Our long-range goal is to develop therapies that prevent metastatic relapse. We recently made the novel observation that intercellular adhesion molecule-2 (ICAM-2) prevents development of disseminated tumors in a murine model of metastatic neuroblastoma. Interestingly, ICAM-2 suppressed metastatic but not tumorigenic potential in preclinical models, supporting a novel mechanism of regulating metastatic disease. Aside from this observation, little is known about the function of ICAM-2 in tumor cells. Importantly, and consistent with data generated using neuroblastoma cell lines, we also showed that primary human neuroblastoma cells expressing ICAM-2 are those recognized clinically to have limited metastatic potential. Because the actin cytoskeletal network is a primary regulator of cell motility and metastatic potential, we hypothesized that ICAM-2 affected the phenotype of neuroblastoma cells by interacting with the actin cytoskeletal linker protein α-actinin. We used in silico modeling to examine the likelihood that the cytoplasmic domain of ICAM-2 binds directly to α-actinin. We then used site-directed mutagenesis to express variants of ICAM-2 with mutated α-actinin binding domains, and compared the impact of ICAM-2 wild type (WT) and each variant on neuroblastoma cell motility. We used dual immunofluorescence and co-immunoprecipitation approaches to demonstrate co-localization of ICAM-2 WT and α-actinin in situ; and we evaluated ICAM-2 WT and variants for their ability to produce disseminated tumors in in vivo models. In vitro and in vivo characteristics of cells expressing ICAM-2 variants with modified α-actinin binding domains differed from cells expressing ICAM-2 WT and also from cells that express no detectable ICAM-2. Unlike the WT protein, ICAM-2 variants did not completely suppress development of disseminated neuroblastoma tumors in vivo. We concluded that the interaction of ICAM-2 with α-actinin was critical to conferring the ICAM-2-mediated non-metastatic phenotype in NB cells. This work was supported by the University of Alabama at Birmingham-Comprehensive Cancer Center-New Faculty Development Award (P30 CA013148). Citation Format: Joseph M. Feduska, Stephen G. Aller, Stuart Cramer, Robert C.A.M. van Waardenburg, Karina J. Yoon. The cytoplasmic domain of ICAM-2 interacts with α-actinin to confer a non-metastatic phenotype in neuroblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 118. doi:10.1158/1538-7445.AM2014-118