ICAM-2 Limits Metastasis Figure 4. ICAM-2 expression inhibited the development of disseminated tumors but did not affect establishment or development of local subcutaneous tumors. A) ICAM-2 did not affect the growth of neuroblastoma cells as subcutaneous tumors in SCID mice, a measure of tumorigenic potential. B) ICAM-2 prevented the development of disseminated tumors, following intravenous tail vein injection of neuroblastoma cells, a measure of metastatic potential. C) Mice injected intravenously with SK-N-ASpIRES.ICAM-2 cells survived significantly longer (P,0.001) than mice receiving pIRESneo or pIRES.ICAM2DCD transfected cells. D) The failure of ICAM2DCD expression to suppress development of disseminated tumors was not due to cessation of expression of the transfected cDNA, since expression of ICAM-2DCD protein persisted until time of death. doi:10.1371/journal.pone.0003629.g004 (P,0.001) (Fig. 4C). These data showed that ICAM-2 abrogated the ability of SK-N-AS cells to develop disseminated neuroblas- toma tumors in SCID mice. In contrast to SK-N-ASpIRES.I- CAM2 cells, SK-N-ASpIRES.ICAM2DCD cells produced dis- seminated tumors (Fig. 3C) despite persistent expression of this truncated protein (Fig. 3D). We concluded that ICAM-2-mediated effects in this model depended on an intact intracellular domain. Together, in vitro and in vivo data suggest that the observed nonmetastatic phenotype could be due, at least in part, to the interaction of membrane-bound ICAM-2 with the cytoskeletal linker protein a-actinin. To address the possibility that the observed results were a consequence of artificial overexpression of ICAM-2, we used RNAi techniques to decrease the expression of ICAM-2 protein in SJNB-1A cells, the neuroblastoma cell line expressing the highest endogenous level of ICAM-2 expression (Fig. 1A) and investigated the phenotype of RNAi transfectants. At early passages, stably transfected SJNB-1A cells expressed ,10% of the level of ICAM-2, compared to control transfectants (Fig. 5A). Consistent with previous results, control siRNA-transfected SJNB-1A cells had a juxtamembrane distribution of actin fibers (high ICAM-2::juxtamembrane actin fibers) (Fig. 5B) whereas siRNA transfected SJNB-1A cells had transverse actin fibers (low ICAM-2::transverse fibers). In addition, mice injected intrave- nously with control transfected SJNB-1A cells (high ICAM-2) developed no detectable tumors, while 40% of mice (N = 10/ group) injected with ICAM-2 shRNA-transfected cells developed multiple tumors (Fig. 5C). While we would have expected 100% of the mice receiving ICAM-2 shRNA-transfected cells to develop tumors, we note that even in the presence of selection pressure in vitro, the suppression of ICAM-2 expression did not persist long-term (Fig. 5A, passage 15), and results in Fig. 5C are consistent with the transient downregulation of expression seen for ICAM-2 in SK-N-AS ICAM-2 shRNA cells. These results indicate that observations using neuroblastoma cells transfected to increase expression of ICAM-2 are unlikely to be due to artificial over-expression of this protein, since downregulation of endogenous ICAM-2 in SJNB-1A cells produced the expected phenotype: an inverse relationship between level of ICAM-2 expression with actin fiber redistribution and development of disseminated tumors in vivo was seen in both cDNA and RNAi transfected cell lines. PLoS ONE | www.plosone.org 6 November 2008 | Volume 3 | Issue 11 | e3629