ICAM-2 Limits Metastasis Figure 5. RNAi techniques indicated that the observed ICAM-2-induced phenotype was unlikely to be due to artificial overexpression of this protein. A) SJNB-1A cells express a relatively high endogenous level of ICAM-2 (Fig. 1A). The expression of ICAM-2 was inhibited in a population of SJNB-1A cells transfected to stably express ICAM-2 shRNA. This inhibition was transient even in the presence of selection pressure by G418 in vitro. B) Consistent with the proposed hypothesis, the high endogenous level of ICAM-2 expression in SJNB-1A cells is co- incident with a juxtamembrane distribution of actin fibers (left panel); and when ICAM-2 expression is decreased, actin fibers are transverse (right panel). C) Inhibition of ICAM-2 expression increased the development of disseminated neuroblastoma tumors in SCID mice injected intravenously with SJNB-1A ICAM-2 shRNA cells. doi:10.1371/journal.pone.0003629.g005 In primary neuroblastoma tumors, as well as in cell lines, ICAM-2 expression and metastatic potential are inversely related We hypothesized that if the above observations reflected the biology of primary tumors, ICAM-2 would be expressed by primary neuroblastoma cells with limited potential for develop- ment of progressive metastatic disease. This hypothesis was investigated by immunostaining for ICAM-2 of a neuroblastoma tissue microarray (TMA) obtained from the Children’s Oncology Group. The TMA contained 90 independent patient samples, of which 89 samples gave interpretable results. Photomicrographs of representative tumor specimens demon- strating the range of staining intensity of primary tumor samples are shown in Fig. 6. The left panels of this Figure show immunoperoxidase staining with a control rabbit polyclonal antiserum and are appropriately negative. Of the right panels, the top panel shows a weak (1+) but unequivocal level of ICAM-2 expression by 100% of normal lymphocytes infiltrating human tonsilar tissue (Expression Score = 100%61+ = 100). Lymphocytes are one of the few types of normal cells that express ICAM-2 protein, and the photomicrograph reflects the known low-level expression that has been reported for the few normal cells that do express ICAM-2 [19]. The three lower right-hand panels demonstrate ICAM-2 staining in representative stage 4, stage 2, and stage 4S neuroblastoma tumors. The stage 4 tumor is a high-risk, poorly differentiated, Schwannian stroma-poor tumor comprised of immature neoplastic cells. Patients with tumors of this stage and histology have a poor prognosis with virtually certain development of metastatic disease. Consistent with results obtained using neuroblastoma cells lines, ICAM-2 expression (Expression Score = 0) and metastatic potential are inversely related. The stage 2 tumor is a differentiating neuroblastoma, Schwannian stroma-poor, comprised of some immature neoplastic cells (,10% of tumor cells) as well as a sizeable population of neoplastic cells that exhibit histologic features of ganglionic differentiation (,90% of tumor cells). The immature neuroblastoma cells are negative for ICAM-2, but all tumor cells exhibiting histologic features of ganglionic differentiation express high levels (3+) of ICAM-2. The overall score for this tumor specimen is 90%63+ = 270. The third example is a stage 4S tumor, which represents a special subtype of neuroblastoma with favorable prognosis [34]. It is defined as PLoS ONE | www.plosone.org 7 November 2008 | Volume 3 | Issue 11 | e3629