expressed ICAM-2 and that 65% of these (24 of 37) expressed ICAM-2 at relatively high levels (a score of .150 of a possible 300). Interestingly, all 89 samples scored either #90 or $160, with no intermediate results. Consistent with the preclinical data from neuroblastoma cell lines, ICAM-2 expression was associated with favorable histologic features (exact 95% confidence interval, 62.6% to 95.3%) such as ganglionic differentiation or low-risk stage [34,35]. Preclinical and TMA data support the hypothesis that ICAM-2 is associated with a nonmetastatic phenotype in primary neuroblastoma cells or cell lines. Consistent with our hypothesis, we observed the highest levels of ICAM-2 expression in neuroblastoma tumor cells associated with the most favorable predicted clinical outcome. Namely, tumor cells exhibiting histologic evidence of ganglionic differentiation were the predominant subset of ICAM-2-positive cells. Discussion The novel findings of this study are that neuroblastoma cells express ICAM-2 and that this expression is associated with juxtamembrane distribution of actin fibers, decreased cell motility in vitro, and suppressed development of disseminated tumors in a preclinical model of metastatic neuroblastoma. These functions for ICAM-2 were independent of immune stimulation, since the tumorigenic potential of ICAM-2-expressing neuroblastoma cells as xenografts in SCID mice was unaffected and cell phenotype was altered in vitro as well as in vivo. Results with primary tumor specimens also reflected the inverse relationship between ICAM-2 expression and predicted low-risk for progressive metastatic disease, as was seen with the preclinical model. Our data are in agreement with published literature in that the novel function reported here for ICAM-2 is similar to that of the related cell adhesion molecule CEACAM1 which binds directly to actin to inhibit tumorigenesis [36], but without the requirement for a cytoplasmic linker protein. ICAM-2 and CEACAM1 are also similar in that their association with the actin cytoskeleton and their suppression of tumor growth or metastasis (for CEAMCAM1 or ICAM-2, respectively) depend on an intact intracellular domain [37 and data herein]. Multiple other functions have been reported for ICAM-2 in different cell types. For example, gastric carcinoma tumor cells transfected to express ICAM-2 induce an immune response sufficient to produce a significant anti-tumor effect in immune competent mice [22]. Potentially, the dual functions of stimulating an immune response and inhibiting tumor cell motility could contribute to a more favorable clinical outcome. ICAM-2 has also been reported to increase the motility of endothelial cells and leukocytes, and also to activate AKT leading to inhibition of apoptosis [24–27]. Interestingly, ICAM-2 expression facilitated rather than inhibited endothelial cell motility. The increase in endothelial cell motility was directly related to an increased phosphorylation of Rac, a small guanosine triphosphatase (GTPase) and known regulator of actin organization, cell motility, and angiogensis [27]. ICAM-2 also facilitated leukocyte migration, albeit in a stimulus-specific manner [24]. Also in contrast to published studies with osteosarcoma and breast cancer cells [10,11], we detected no involvement of the linker protein ezrin in the phenotype that ICAM-2 conferred on neuroblastoma cells. The as yet unexplained differences in function of ICAM-2- interacting proteins in different cell types are, nonetheless, relatively common observations. Disease progression in uveal malignant melanoma [38] is associated with a relatively high level of expression of the ERM cytoskeletal linker protein ezrin. Conversely, disease progression is associated with a relatively low level of expression of ICAM-2 Limits Metastasis this protein in serous ovarian carcinoma [39]. Similarly, the linker protein a-actinin-4 decreases the tumorigenic potential of neuro- blastoma cells [7] but is associated with a progressive phenotype in breast and colorectal cancer [4,40]. We speculate that varying levels of expression of ICAM-2- interacting proteins by specific cell types or the protein composition of extracellular matrices or of adjacent cells in different tissues may modulate the state of activation (conforma- tion) and/or the function of ICAM-2. In tumor cells in particular, constitutive activation of proteins up- or down-stream of the bidirectional signals mediated by CAMs might also affect ICAM-2 function and cell phenotype. Quite likely, while some of the protein interactions of ICAM-2 and the pathways or functions affected by these interactions are known (e.g., binding of ICAM-2 to CD11a/CD18 and CD11b/CD18 integrins facilitates adhesion of human leukocytes to endothelial cells [20]; and ICAM-2 induces phosphorylation of Rac and modulates motility of endothelial cells [27]), others will undoubtedly be identified. However, despite the multiple proteins and pathways known to be affected by cell adhesion molecules in general and ICAM-2 in particular, complete membrane-to actin linkages that regulate CAM function in tumor cells have been difficult to identify. Several laboratories, however, have described the involvement of specific linker proteins and documented the impact of these linker proteins on the migratory and metastatic potential of tumor cells. In leukemic cells, for example, membrane-bound tetraspanin proteins EWI-2 and EWI-F bind to cytoskeletal linker ezrin- radixin-moesin (ERM) proteins to regulate cell migration [12]. This binding links cell membranes (EWI-1 and EWI-F proteins) with cytoplasmic actin cytoskeletal proteins (ERM proteins), but simultaneous association of these complexes with actin fibers has not yet been reported. Also of note, individual linker proteins appear to function differently in cells from different tumor types. In neuroblastoma cells, we detected an association of ICAM-2 with a-actinin, and data with competitive peptides and truncated proteins suggest that this association plays a role in the regulation of the observed ICAM-2-mediated phenotype. Potentially, in addition to limiting the interaction of ICAM-2 with a-actinin, truncation of the intracellular domain of ICAM-2 may also have prevented the association of membrane-bound ICAM-2 with other as-yet-uniden- tified intracellular proteins. However, the hypothesis that the novel function for ICAM-2 described here depends at least partly on the association of membrane-bound ICAM-2 with a-actinin and actin is consistent with published reports documenting the role of a-actinin as a determinant of metastatic potential [4,5,40,41] and the observation that a-actinin associates with an unknown membrane protein to decrease the tumorigenic or metastatic potential of neuroblastoma cells [7]. Our data indicate that ICAM-2 may be such a membrane protein. Our results are also consistent with the correlations between the juxtamembrane localization of actin fibers and decreased motility or decreased metastatic potential that have been reported for mouse embryonic fibroblasts [42] and for primary oral squamous cell carcinoma tumors [43], respectively. Finally, the finding that ICAM-2 expression inhibited tumor cell motility in scratch and modified Boyden chamber invasion assays in vitro merits further discussion. With respect to metastasis, in vitro cell motility assays may indicate how likely a tumor cell is 1) to detach from a primary tumor (and enter systemic circulation); or 2) to migrate through the vascular endothelial layer at an anatomic site distant from the primary tumor; or, 3) once through the endothelium, to invade the normal tissue stroma to facilitate development of tumor at secondary sites. We suggest that the component of the metastatic process most likely affected by ICAM-2 is the invasion of normal stroma. This PLoS ONE | www.plosone.org 10 November 2008 | Volume 3 | Issue 11 | e3629