Fig 2: ICAM-2 variants significantly delayed but did not completely inhibit development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Mice received intravenous injections of SK-N-AS cells expressing no detectable ICAM-2 (neo, N = 10), ICAM-2 WT (N = 10), or one of four hypo-glycosylated forms of ICAM-2 (N = 5/group). Kaplan-Meier survival plots were analyzed by log-rank (Mantel-Cox) test using GraphPad Prism 5 software (Version 5.02). Mice receiving cells expressing hypo-glycosylated ICAM-2 survived longer than mice receiving cells expressing no detectable ICAM-2, but not as long as mice receiving cells expressing ICAM-2 WT.

Fig 3: Morphology of ICAM-2 variants derived from SK-N-AS neuroblastoma cells. Cells plated at low density (~20% confluence) in CoStar tissue culture flasks were cultured at 37°C at 5% CO2 in humidified chambers, and allowed to proliferate to ~70% confluence. SK-N-AS cells transfected to express WT or gsv ICAM-2 have nascent neuronal morphologies. In comparison to SK-N-ASpIRES.ICAM-2 transfectants (WT), gsv transfectants were morphologically more pleomorphic and discohesive. All transfectants grew as attached monolayers. When detached and sized using a Coulter Counter, no differences in cell size were observed.

Fig 4: ICAM-2 WT and glycosylation variants inhibit NB cell migration in scratch assays. Expression of WT and gsv ICAM-2 inhibited SK-N-AS cell motility in vitro. A) Scratch assays to evaluate cell motility were performed using standard methods, and images acquired at 24 hour intervals. At each time point, the distance remaining across the scratch was calculated in pixels. Only wells in which initial scratches were of similar width were included in analyses. B) Analysis by two-way ANOVA followed by Bonferroni post test showed that at 72 hours post scratch, all variants except gsv4,5 migrated more slowly than transfectants that expressed no detectable ICAM-2 (neo) (P < 0.05* [gsv1-6] and P < 0.001*** [gsv1,4,5 and gsv1,2,5]). At 72 hours post scratch, all gsv transfectants migrated more rapidly than transfectants expressing ICAM-2 WT (P<0.01 – P<0.001, P values not indicated on graph).

Fig 5: Schematic structure of ICAM-2 and ICAM-2 variants. Schematic representation of the structures of ICAM-2 and hypo-glycosylated ICAM-2 glycosylation site variants (gsv). ICAM-2 wild type (WT) contains six glycosylated amino acids in its extracellular domain (ED), at residues 47, 82, 105, 153, 176 and 187. The ED also contains two immunoglobulin-like domains (designated IgLD-1 and -2). Short transmembrane (TD) and cytoplasmic (CD) domains are also present. The N-terminus of the nascent protein contains a 21-amino acid signal peptide (sp) that is not present in the 254 amino acid mature protein. Variants were constructed by substituting alanines (A) for asparagines (N) at the indicated amino acid residues. For example, gsv1,4,5 contains alanines at mutation sites 1, 4, and 5 (amino acids 47, 153, and 176); therefore, gsv1,4,5 is not able to be glycosylated at these three sites. Variant gsv1-6 has alanines at all six glycosylation sites, and is non-glycosylated when expressed in SK-N-AS cell transfectants.