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: 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.

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: WT and gsv ICAM-2 suppressed anchorage-independent growth in vitro. All four glycosylation site ICAM-2 variants suppressed anchorage-independent growth. Soft agar assays were performed using standard methods. Number of colonies of >20 cells were visualized 14-21 days after plating and results analyzed using a 2-tailed t test and GraphPad Prism software. P = 0.0124*. P < 0.0001***. Not shown in this Figure are control experiments demonstrating that the growth in soft agar of parental SK-N-AS cells was equivalent to control SK-N-ASpIRESneo transfectants.