ICAM-2 Limits Metastasis Figure 3. The effect of ICAM-2 on actin distribution and cell motility depends on an intact intracellular domain. A) SK-N-AS cells were transfected to express a truncated form of ICAM-2 with a deleted cytoplasmic domain (ICAM2DCD). Expression of full length ICAM-2 and ICAM2DCD was similar in respective transfected cell lines. B) ICAM-2, a-actinin, and actin co-precipitated only in transfectants expressing full length ICAM-2. C) ICAM2DCD had no effect on actin fiber distribution. (Compare with Fig. 2A). D) ICAM2DCD did not affect migration of SK-N-AS cells in scratch assays. Pixel width of scratches for pIRESneo and pIRES.ICAM2DCD transfectants at 0 hours and 27 hours were similar. No gap remained for either cell line 45 hours after scratch. doi:10.1371/journal.pone.0003629.g003 ICAM-2 did not affect growth of neuroblastoma cells as subcutaneous xenografts, but did inhibit development of disseminated neuroblastoma tumors in a mouse model of metastatic neuroblastoma Because cell adhesion molecules and the actin cytoskeleton have been reported to regulate tumorigenic potential, we injected SK- N-ASpIRESneo and SK-N-ASpIRES.ICAM2 cells subcutaneous- ly into SCID mice (5 mice per group, bilateral flank injections) and observed the mice for tumor development. Both cell types produced subcutaneous tumors within the same time frame (Fig. 4A). Subcutaneous local tumor growth was rapid and mice were euthanized for humane reasons before metastases were observed. Since both ICAM-2 and control transfectants readily formed subcutaneous tumors, we concluded that ICAM-2 was unlikely to be acting as a tumor suppressor. However, data from in vitro experiments showed that ICAM-2 expression was coincident with actin fiber redistribution and inhibition of cell motility. Since cell motility and the consequent migratory and invasive potential are essential to the metastatic process, we used a preclinical model of metastatic neuroblastoma to investigate whether ICAM-2 expression affected the development of disseminated tumors in SCID mice. In this model of metastatic neuroblastoma there is no primary tumor per se; but tumor cells injected intravenously must migrate through the endothelial cell monolayer of vasculature and invade stromal tissue to seed metastatic tumor foci and facilitate tumor progression. Mice injected intravenously with SK-N-ASpIRESneo cells (or other neuroblastoma cell lines) develop tumors at multiple anatomic locations and require euthanasia within 2–3 months (Fig. 4B, left panels) due to progressive disease [33]. In contrast, mice receiving SK-N-ASpIRES.ICAM2 cells intravenously devel- oped no detectable tumors for .12 months and survived significantly longer than mice receiving SK-N-ASpIRESneo cells PLoS ONE | www.plosone.org 5 November 2008 | Volume 3 | Issue 11 | e3629