opinion is based primarily on results from immunostaining of the TMA, and from scratch and Boyden chamber assays. Firstly, ICAM-2 was expressed predominantly by primary neuroblastoma cells with some degree of ganglionic differentiation, and therefore predicted low metastatic potential. The notable exception to this was the 3+ staining of undifferentiated neuroblasts of some 4S tumors. Patients with 4S tumors frequently have liver, dermal, and/or limited (,10%) bone marrow, as well as adrenal, involvement at diagnosis, i.e., secondary tumors are present at diagnosis. However, unlike stage 4 tumors, 80% of patients with 4S disease do not develop progressive disease at metastatic sites. Rather, 4S tumors usually regress spontaneously without treat- ment, suggesting a limited invasive potential. Secondly, scratch assays performed in the presence of Matrigel, in which extracellular matrix proteins can influence cell function, showed readily distinguishable differences between control and ICAM-2 transfectants. However, when these assays were performed in the absence of Matrigel (data not shown), no differences in cell motility were observed. Thirdly, results from standard Boyden chamber invasion assays, in which cells invade a layer of Matrigel to migrate toward a chemoattractant, corroborated results of scratch assays. The presence of extracellular matrix proteins in vitro influenced ICAM-2-mediated cell function. Fourthly, Stage 4 neuroblastomas are progressively invasive tumors with a generally poor outcome; while most Stage 4S tumors are self-limiting variants of neuroblastoma despite transient involvement of skin, liver, or bone marrow. The data are consistent with the hypothesis that ICAM-2 limits the invasive potential of neuroblastoma cells. We suggest further that the significance of ICAM-2 expression can be compared with that of MYCN amplification. If MYCN is amplified, it is regarded as a prognostic indicator for poor outcome. If MYCN is not amplified, prognosis may still be poor— normal MYCN copy number is uninformative [28]. Similarly, ICAM-2 expression is associated with a more favorable prognosis. However, lack of ICAM-2 expression is not necessarily associated with poor prognosis; it is simply uninformative. In summary, the observed ICAM-2/a-actinin/actin interaction appears to represent a complete membrane-to-actin linkage that contributes to regulation of the motility (possibly, the invasive potential) and therefore the metastatic phenotype of neuroblasto- ma cells. Our long-range goal is to determine the specific protein domains essential to this regulation and to investigate the possibility that small molecule agonists can be developed to mimic the effects of ICAM-2 expression in neuroblastoma cells. Materials and Methods Immunoprecipitations and Immunoblots Immunoprecipitations of ICAM-2, a-actinin, ezrin, and actin done with antibodies IC2/2, MAB1682 (Chemicon International, Temecula, CA), C-15 (Santa Cruz Biotechnologies), and 4968 (Cell Signaling Technology), respectively. Immunoblots for ICAM-2, a-actinin, ezrin, actin, AKT, and phospho-AKT were done with AF244 (R&D Systems, Minneapolis, MN) and C-20 (Santa Cruz Biotechnologies, Beverly, MA), CBL231 (Research Diagnostics, Inc., Flanders, NJ), C-20 and C-15 (Santa Cruz Biotechnologies), 4968, 9272, and 4051 (Cell Signaling Technol- ogy), respectively. AKT and phospho-AKT standards were from Cell Signaling Technology. Isotype-matched control antibodies were from Jackson Immunolabs, Inc. (West Grove, PA). Cell Motility Assays Modified Boyden chamber invasion assays were done by standard methods [44]. Briefly, cells were overlaid on ‘‘solidified’’ ICAM-2 Limits Metastasis Matrigel (upper chamber), with a chemoattractant (lower chamber) comprised of 10% fetal bovine serum in Dulbecco’s Minimum Essential Medium supplemented with fibronectin (20 mg/mL) Cells that migrated through (invaded) the Matrigel layer and to the distal side of a membrane containing 8 micron pores were recorded as photomicrographs using standard inverted microscopy, and quantitated using an AlphaEase FC software (Alpha Innotech, San Leandro, CA). Results were expressed as the mean percentage of dark pixels/total pixels in 106 fields+S.D. from a minimum of five fields per experiment, of six replicate experiments. ‘‘Scratch’’ or ‘‘wound healing’’ assays were also done by standard methods. In brief, cell were plated in chamber well slides and cultured in Dulbecco’s Minimum Essential Medium supple- mented with 10% fetal bovine serum until confluent. A 200 microliter pipet tip was used to ‘‘wound’’ the confluent monolayers, which were immediately overlaid with 25% Matrigel. As soon as the Matrigel solidifed, photomicrographs were taken using an inverted microcroscope. Images were recorded at 0, 15, 24, and 48 hours after scratch. Plasmids Containing ICAM-2 cDNA The cDNA of ICAM-2 was isolated from human umbilical vein cell RNA (Clonetics, San Diego, CA). Amplification was performed at 95uC for 1 min, then 30 cycles of 94uC for 30 sec, 55uC for 30 sec, 72uC for 1 min, and 72uC for 7 min. Primers for full length ICAM-2 were: forward (59GCTTCCGCTGCCTGGATTCT39) and reverse (59AAGTCCAGGTGTTGTATTC39), and for trun- cated ICAM-2: forward (59ACGCAGGATCCGCCAGA- GATGTCCTCTTTCG) and reverse (TTGGATCCTGAT- GAAGCAGAGCAGGACAGATG). cDNAs inserted into pIRESneo2 (Clontech, San Jose, CA) were transfected with FuGene6 (Roche Diagnostics, Indianapolis, IN), and transfectants selected with G418. The St. Jude Hartwell Center for Bioinfor- matics sequenced all plasmids. RNAi-mediated decrease in endogenous ICAM-2 expression The siRNA or shRNA sequence used to decrease expression of endogenous ICAM-2 (CAAGAAGCTGGCGGTTGA) was de- signed using Invivogen siRNA Wizard and Dharmacon siDesign Center. siRNA sequences for transient transfections were synthe- sized in the St. Jude Hartwell Center for Technology. shRNA for stable transfections were done using psiRNA-hH1-ICAM2 (Invivogen). In this plasmid, shRNA expression is regulated by the H1 promoter, and neomycin resistance gene is regulated by expression of the SV40 promoter. Transfections were done with FuGene6 (Roche Applied Science, Indianapolis, IN). Stably transfected cells were selected using G418, and the entire population of G418-resistant cells was used for in vivo experiments. Control transfections were done with transfection reagent only or with scrambled RNAi and transfection reagent. Specificity of RNAi was assessed by probing immunoblots of RNAi and control transfectants for a minimum of three additional, irrelevant proteins. Competitive peptides that inhibit ICAM-2/a-actinin or a- actinin/actin binding Peptides comprised of amino acids that mediate binding of ICAM-2 to a-actinin or of a-actinin to actin, or scrambled control peptides were linked to the antennapedia 16-mer that facilitates intracellular peptide accumulation. The antennapedia 16-mer (RQIKIWFQNRRMKWKK [45] was coupled to the 59-end of PLoS ONE | www.plosone.org 11 November 2008 | Volume 3 | Issue 11 | e3629