Galectin-3 in Pancreatic Cancer Figure 6. Inconsistent effect of Gal-3 knockdown and overexpression on cell migration as measured by Boyden-chamber assay. Transiently Gal-3 silenced cell lines (A), stably Gal-3 silenced S2-007 cell clones (B) or stably Gal-3 overexpressing PaTu 8988t cell clones (C) were cultured in Boyden-chamber inserts for 2 or 4 h. Cells migrating through the pores of the inserts along a fetal calf serum gradient were fixed, stained methylene blue and counted using light microscopy. Numbers of migrated cells transiently transfected with different Gal-3 specific siRNA (siGal-3 1 and 2) were normalized to control siRNA transfected cells (NC). Migrated stable Gal-3 knockdown cells (shGal-3 1, 2 and 3) and stable Gal-3 overexpressing cells (pGal-3 1, 2 and 3) were normalized to the mean of control vector transfected stable cell clones (shC 1, 2, 3 and pC 1, 2) respectively. Data includes a minimum of three independent experiments. * Indicates p,0.05 as compared with control siRNA transfected cells (double-sided unpaired t-test). doi:10.1371/journal.pone.0020859.g006 To this end, xenograft tumors were induced in nude mice by subcutaneous injection of S2-007 cells with stable Gal-3 knockdown or control vector-transfected cells, respectively. No significant difference in tumor volumes between Gal-3 knockdown and control tumors was observed (Fig. 7C). Histological exami- nation of the tumors also did not reveal any systematic differences in differentiation, local invasiveness or microvessel density between the different groups (data not shown). In order to confirm persistent reduction of Gal-3 levels in the knockdown tumors, mRNA was prepared from bulk tumor tissue and analyzed by qRT PCR. As expected, Gal-3 mRNA levels were significantly lower in the tumors derived from the S2-007 knockdown clones than in those derived from the control vector- transfected clones (Fig. 7D). Discussion Deregulation of Gal-3 expression has been described in several human cancers, although the prognostic significance of the observed changes remains a matter of controversy [37,38]. Pancreatic ductal adenocarcinoma is among those human tumors in which a significant overexpression of Gal-3 both on mRNA as well as on the protein level is well established [27,30]. Our own results from microarray analyses of microdissected pancreatic tumor tissues indicated that Gal-3 is expressed by the tumor cells themselves rather than by the stromal cells which typically make up the bulk of the tumor [28]. In the current study, we confirm overexpression of Gal-3 mRNA in the tumor tissues by qRT-PCR and demonstrate that strong Gal-3 expression is retained in the majority of pancreatic cancer cell lines in vitro. As is the case with data on the prognostic role in cancer, reports on putative cellular functions of Gal-3 in cancer cells are in part inconclusive or even contradictory. While Honjo et al. report loss of (serum-independent) proliferative capacity and abrogation of anchorage-independent growth in breast cancer cells following silencing of Gal-3 expression [26], Matarrese et al. did not observe any difference in cell growth or proliferation upon modulation of Gal-3 expression, but describe enhanced resistance to apoptosis following overexpression of Gal-3 [36]. Likewise, the same group that observed the growth inhibitory effects of Gal-3 silencing in PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20859