Galectin-3 in Pancreatic Cancer Figure 7. Gal-3 knockdown decreases anchorage independent cell growth, but has no effect on tumor growth in a xenograft mouse model. (A) Gal-3 was transiently silenced in pancreatic cancer cell lines using two independent siRNA sequences (siGal-3 1 and 2). Knockdown and control cells were seeded in soft agar and colony formation assessed after 7 days. Results were normalized to control siRNA transfected cells (NC). Data includes a minimum of three independent experiments. * Indicates p,0.05 as compared with control siRNA transfected cells (double-sided unpaired t- test). (B) Western blot analysis of nuclear (n) and cytoplasmatic (c) protein fractions derived from pancreatic cancer cell lines. Gal-3 is shown in the upper lane. b-actin staining was used as loading control. Staining for the nuclear factor ORC2 was used to assess the purity of the subcellular fractions. (C) Xenograft tumors were induced in nude mice by subcutaneous injection of two stable Gal-3 knockdown S2-007 cell clones (shGal-3 2 and 3) and two nonsilencing control shRNA transfected S2-007 clones (shC 2 and 3). Six mice per experimental group were analyzed. Box-and-whisker plots illustrate tumor volumes on day 22 post injection. Whiskers denote 1.56interquartile range (IQR). Values outside this range are shown as filled triangles. (D) Gal-3 mRNA levels in bulk tissue of xenograft tumors where determined by qRT-PCR. RPLP0 was used as the reference gene. doi:10.1371/journal.pone.0020859.g007 breast cancer cells described strong anti-tumor effects of Gal-3 knockdown in prostate cancer cells, including reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in nude mouse xenografts [24]. In contrast, Califice et al. did not observe any effect of Gal-3 expression on proliferation of prostate cancer cells in any constellation, and reported that Matrigel invasion, anchorage- independent growth and nude mouse xenograft formation was promoted only by cytoplasmic Gal-3 expression, while nuclear localization of Gal-3 had the opposite effect [37]. A common theme in many of these studies is the fact that often very few or only a single cell line was analyzed, and that some of the effects were only observed under very specific experimental conditions. We therefore decided to conduct a comprehensive study on the putative role of Gal-3 in pancreatic cancer cells by analyzing in parallel the effect of transient silencing of Gal-3 in a panel of five well-established cell lines with high endogenous Gal-3 expression, as well as study the long-term effects of modulation of Gal-3 expression by stable knockdown in S2-007 cells (high endogenous Gal-3 expression) or stable overexpression in PaTu 8988t cells (negligible endogenous Gal-3 expression), respectively. Our results demonstrate that transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth in individual pan- creatic cancer cell lines. These isolated effects were reminiscent of some of the results described in other in vitro cancer cell systems. However, although the five cell lines expressed endogeneous Gal-3 at very comparable levels, both on the mRNA as well as on the protein level, functional effects remained limited to individual cell lines and failed to show a consistent pattern across the spectrum of analyzed cell lines. Subcellular localization of the protein did not vary systematically and did not provide an explanation for the differences in susceptibility to Gal-3 knockdown, contrary to the suggestion by Califice et al. [37]. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpres- sion approaches in vitro. Most importantly, the attenuation of anchorage-independent growth of S2-007 cells after transient Gal-3 knockdown did not translate into significant differences in growth characteristics of stable knockdown and control clones in nude mouse xenograft tumors in vivo. While this manuscript was in preparation, a study by Kobayashi et al. was published that examined the effects of transient Gal-3 silencing in Panc-1, AsPC-1 and BxPc-3 pancreatic cancer cell References lines [39]. Similar to our results, the authors observed no influence on proliferation of the cells, but contrary to our results describe an inhibitory effect of Gal-3 knockdown on cell migration and invasion. While the observed effects were consistent across the three cell lines, only one siRNA sequence was used in the study, and no in vivo experiments were performed. It is interesting to note that in our experiments, we observed significant inhibition of cell migration in S2-007 and MIA PaCa-2 cells with one of the two siRNAs used (siGal-3 1); however, this effect was not observed for siGal-3 2, and was altogether absent in the other cell lines (Fig. 6). From the data presented by Kobayashi et al., it can not be ruled out that the observed effects were either specific to the three cell lines used and/or were attributable to off-target effects of the single siRNA used in the study. Our results do not rule out paracrine and/or systemic effects of the expression of (secreted) Gal-3 by pancreatic cancer cells. Jiang et al. have recently suggested that Gal-3 plays a role in the interaction between SW1990 pancreatic cancer cells and special- ized stromal cells of the pancreas termed pancreatic stellate cells (PSC) [40]. In another very recent study, Senapati et al. described that binding of exogenous Gal-3 to MUC4 on the cell surface of CD18/HPAF pancreatic cancer cells enhanced the attachment of the cancer cells to HUVEC endothelial cells in in vitro assays, speculating that MUC4/Gal-3 interaction may facilitate extrav- asation and metastasis of tumor cells in vivo [41]. However, the clinical significance of these observations remains unclear, especially in view of the fact that no Gal-3 secretion by CD18/ HPAF cells was detected. Moreover, in a large study with 104 pancreatic cancer cases, Shimamura et al. reported a statistically highly significant correlation of high expression of Gal-3 in the primary tumors with reduced metastatic potential and increased post-operative survival [42]. In summary, experimental data on the putative pathophysiological role(s) of Gal-3 in cancer remain controversial, and a clear picture of the function of Gal-3 in the onset and progression of cancer in general as well as pancreatic cancer in particular is not yet emerging. Author Contributions Conceived and designed the experiments: MB TMG. Performed the experiments: AH AG. Analyzed the data: AH MB AG. Contributed reagents/materials/analysis tools: YC. Wrote the paper: AH. First authors: AH AG. Senior authors: TMG MB. 1. 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