α2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro and Enhances Its Metastatic Potential In Vivo

Background: Cell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process. Methodology/Principal Findings: ST3Gal III overexpressing pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28 were generated. They showed an increase of the tumour associated antigen sialyl-Lewis x. The transfectants ’ E-selectin binding capacity was proportional to cell surface sialyl-Lewis x levels. Cellular migration positively correlated with ST3Gal III and sialyl-Lewis x levels. Moreover, intrasplenic injection of the ST3Gal III transfected cells into athymic nude mice showed a decrease in survival and higher metastasis formation when compared to the mock cells. Conclusion: In summary, the overexpression of ST3Gal III in these pancreatic adenocarcinoma cell lines underlines the rol

α2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro and Enhances Its Metastatic Potential In Vivo

Background: Cell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process. Methodology/Principal Findings: ST3Gal III overexpressing pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28 were generated. They showed an increase of the tumour associated antigen sialyl-Lewisx. The transfectants’ E-selectin binding capacity was proportional to cell surface sialyl-Lewisx levels. Cellular migration positively correlated with ST3Gal III and sialyl-Lewisx levels. Moreover, intrasplenic injection of the ST3Gal III transfected cells into athymic nude mice showed a decrease in survival and higher metastasis formation when compared to the mock cells. Conclusion: In summary, the overexpression of ST3Gal III in these pancreatic adenocarcinoma cell lines underlines the role of this enzyme and its product in key steps of tumour progression such as adhesion, migration and metastasis formatio

Flow cytometry profiles of the cell surface glycan structures of the pancreatic adenocarcionoma cells.

<p><b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g002" target="_blank">Figure 2A</a></b>. Capan-1 (continous dot outline: ………), CP (spaced dot outline: . . . . . ), C31 (bold outline:_______), C32 (plain outline:______). <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g002" target="_blank">Figure 2B</a></b>. MDAPanc-28 (continous dot outline: ………), MP (spaced dot outline: . . . . . ), M34 (bold outline:_______), M33 (plain outline:______). Experiments were performed for triplicate. Representative cytometry histograms are shown. Anti-Le^x MAb binds to Galβ1,4[Fucα1,3]GlcNAc-; anti-SLe^x MAb binds to NeuAcα2-3Galβ1,4[Fucα1,3]GlcNAc-; anti-H2 MAb binds to [Fucα1,2]Galβ1,4GlcNAc-; anti-Le^y MAb binds to [Fucα1,2]Galβ1,4[Fucα1,3]GlcNAc-; SNA lectin (<i>Sambucus nigra</i> agglutinin) binds to NeuAcα2–6Galβ- structures.</p

Binding assay to rh-E-selectin.

<p>Capan-1 variant cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g003" target="_blank"><b>Figure 3A</b></a>) and MDAPanc-28 variant cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g003" target="_blank"><b>Figure 3B</b></a>), previously incubated with PBS-1% BSA (light bars) or anti-SLe^x MAb (dark bars), were added to 96-well microplates coated with rh E-selectin or PBS-1% BSA (negative control). Adherent cells were estimated with a MTT-based colorimetric assay. Results are expressed as the Specific binding to E-selectin (O.D. 570 nm of cells bounded to E-selectin – O.D. 570 nm of cells bonded to PBS-1% BSA) <i>versus</i> cells previously incubated or not with anti-SLe^x MAb. Data represents the mean ± SD of 3 separate experiments, each in five replicates (n = 15). * Significantly different (<i>P<0.001</i>).</p

Kaplan-Meier plots of estimated survival after injection of MP (MDAPanc-28 mock cells) and M34 (MDAPanc-28 ST3Gal III transfected cells).

<p>Cells (7×10⁶) were intrasplenically injected in nude mice on day 1 of the experiment. Mice were daily examined and sacrificed when they looked sick. The differences between groups were assessed by the long-rank test (<i>P</i> = 0.019; n = 7–8/group).</p

ST3Gal III expression normalized to β-actin of the pancreatic adenocarcionoma cells.

<p>MDAPanc-28 parental cells, MP: MDAPanc-28 mock cells, M34 and M33: MDAPanc-28 cells transfected with the ST3Gal III gene. Capan-1<b>:</b> parental cells, CP: Capan-1 mock cells, C31 and C32: Capan-1 cells transfected with the ST3Gal III gene. Data represents the mean ± SD of 3 separate experiments, each in six replicates (n = 18). * Significantly different (<i>P<0.001</i>).</p

E-selectin induction on Primary Cultured Hepatic Sinusoidalendothelium (HSE) cells (Figure 4A).

<p>HSE cells were incubated with  =  anti-murine CD62 E (E-selectin) MAb or  =  isotype-matched control antibody. Parental Capan-1 cells were labelled with calcein and added to HSE control cells, TNF-α stimulated HSE cells, LPS stimulated HSE cells or IL-1β stimulated HSE cells. Results are expressed as the % Specific adhesion to HSE cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone.0012524-VidalVanaclocha2" target="_blank">[78]</a>. Data represents the mean ± SD of 3 separate experiments, each in three replicates (n = 9). * Significantly different (<i>P<0.001</i>) when comparing anti-E-selectin incubated HSEC to control cells. # Significantly different (<i>P<0.001</i>) when comparing treatments. <b>Tumour cell adhesion assay to Primary Cultured HSE cells (</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g004" target="_blank"><b>Figure 4B</b></a><b>).</b> Capan-1 variant cells were labelled with Calcein and added to IL-1β stimulated HSE cells or (<b>−</b>) not stimulated HSE control cells. >  =  anti-murine CD62 E (E-selectin) MAb was added to IL-1β HSE cells or <b>-</b> HSE cells before tumour cell addition. * Significantly different (<i>P<0.001</i>) when comparing Capan-1, CP, and C31 cells. # Significantly different (<i>P<0.001</i>) when comparing each clone (Capan-1, CP and C31) adhesion for the different HSE cell treatments.</p

Cell migration assay.

<p>Capan-1 variant cells (Capan-1,CP and C31) <i>(</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g005" target="_blank"><i>Figure 5A</i></a><i>)</i> and MDAPanc-28 variant cells (MDAPanc-28, MP and M34) <i>(</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012524#pone-0012524-g005" target="_blank"><i>Figure 5B</i></a><i>)</i> were seeded onto 8 µm-pores-Type I-Collagen coated inserts, placed on top of wells containing DMEM-1% FBS and incubated at 37°C (6 h for Capan-1 model and 18 h for MDAPanc-28 model). Non-migrated cells were eliminated and migrated cells fixed, stained and counted. Results are expressed as migrated cells per well. Data represents the mean ± SD of the values obtained in 3 separate experiments, (n = 9). * Significantly different (<i>P<0.001</i>).</p