Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Abstract

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.

Figure 1. Gal-4 mRNA expression in pancreatic cancer cell lines.

Gal-4 mRNA expression of normal human pancreatic duct epithelial-like cell line (hTERT-HPNE) and 9 different human pancreatic cancer cell lines was analyzed by quantitative real-time PCR and depicted as the relative amount of Gal-4 transcripts (± SEM) compared to the expression of the endogenous reference gene GAPDH. (*** p≤0.001 versus all other cell lines, using one way ANOVA with post Dunnett two sided t tests).

Figure 2. Gal-4 and Gal-4 binding sites in PaTu-S and PaTu-T cells.

Detection of endogenous Gal-4, and Gal-4 ligands, in PaTu-S and PaTu-T cells by flow cytometry. A histogram of one representative experiment is depicted for each condition of least two independent experiments. A) Dot plots of Gal-4 staining of permeabilized PaTu-S and PaTu-T cells. Gal-4 was detected at 4°C with anti-hGal-4 Abs in fixed permeabilized cells. Secondary Abs staining without anti-hGal-4 Abs was used as background autofluorescence control. B) Presence of endogenous bound Gal-4 to the surface of PaTu-S and PaTu-T after washing the cells with 500 mM lactose prior to Gal-4 staining. The presence of Gal-4 was established by FACS analysis using anti-hGal-4 Abs at 4°C. Endogenous Gal-4 bound to the surface is shown by a black line. C) The presence of Gal-4 binding sites on PaTu-S and PaTu-T cells was determined after washing the cells with 500 mM lactose prior to Gal-4 staining. The binding of externally added recombinant (rec) hGal-4 (5 µg/ml, black line) was investigated. Binding of rec hGal-4 to the surface could be inhibited by adding lactose (dark field). Background staining with secondary Abs is depicted as light grey fields in B and C.

Figure 3. Immunocytochemical localization of Gal-4 in PaTu-S cells.

Photographs of representative ICC analysis of the cellular localization of Gal-4 in PaTu-S cells. Gal-4 was detected using Alexa-labeled anti-Gal-4 Abs (green), Actin was stained using Phalloidin (red) and nucleus staining obtained using HOESCHS (blue); the third panel shows the merging of the different stainings. Bar = 25 µm.

Figure 4. Gal-4 protein levels in PaTu-S and PaTu-T cells, and localization of Gal-4 in PaTu-T/Gal-4.

A) Proteins from whole-cell extracts (75 ug total protein) and culture medium (4 days culture, 25 ul) of PaTu-S (P-S), PaTu-T (P-T), PaTu-T/Gal-4 (P-T/Gal-4) and PaTu-T/mock (P-T/M) were separated by SDS-PAGE. After transfer of the proteins to a nitrocellulose membrane, the blots were stained using goat anti-hGal-4 for detection of Gal-4, and mouse anti-tubulin as control for the presence of intracellular protein. B) Photographs of representative ICC analysis of the cellular localization of Gal-4 in PaTu-T/Gal-4 and PaTu-T/mock cells. Gal-4 was detected using Alexa-labeled anti-Gal-4 Abs (green), Actin was stained using Phalloidin (red) and nucleus staining obtained using HOESCHS (blue); the third panel shows the merging of the different stainings. Bar = 25 µm.

Figure 5. In vitro cell migration of PaTu-T cells.

A scratch (wound healing) assay was performed with PaTu-T, PaTu-T/Gal-4 and PaTu-T/mock cells. PaTu-T/mock and PaTu-T/Gal-4 cells were seeded on a 24 well plate and scratched on the surface with a 200-µl pipette tip. Relative values were set at 100% of the gap width at the time of the scratch. A) Representative photographs at time points 0, 6, 19 and 24 hours after the wound (scratch) for all conditions are depicted. B) Histogram representation of data analyzed from photographs taken at 0 h; 6 h, 19 h and 24 h after the scratch. Measurements were done in duplicate in 3 separate experiments, and data are depicted as average gap width ± SEM. (* p≤0.05 and ** p≤0.01, using one way ANOVA Tukey t tests).

Figure 6. Metastasis assay of zebrafish casper embryos transplanted with PaTu-S and PaTu-T cells.

A) Schematic depiction of the time schedule of the transplantation experiments. Embryos were injected at the yolk sack at 32–38 h developmental stage. siRNA was introduced twice at day -4 and day -1, respectively. At 1–6 hours post fecundation (hpf) the embryos were evaluated for viability and fitness. Scoring of metastasis formation was performed at day 1, 2 and 3 by detection of the localization of CMDiI (red) fluorescent cells. B) Representative photographs of a casper embryo at one cell developmental stage, and an embryo injected with fluorescent red cells. At day 0 cells are present only at the yolk sac of the embryo and at day 3 cells had migrated from the yolk sac throughout the embryo, including the caudal vein, hart and liver. C) Metastasis assay of zebrafish casper embryos transplanted with PaTu-T/Gal-4 and PaTu-T/mock cells. The number of embryos presenting metastasis is shown per day and the total percentage of embryos with metastasis formation after 3 days is depicted as a histogram. The data are derived from 4 separate experiments, and are depicted as the average metastasis formation ± SEM. D) Gal-4 mRNA levels of PaTu-S/Gal-4 KD (KD G4) and mock siRNA treated (KD M) PaTu-S cells were determined by quantitative RT-PCR as a control for the efficiency of the siRNA treatment. E) Histogram showing the total percentage of embryos, transplanted with fluorescent PaTu-S/mock-KD and PaTu-S/Gal-4-KD cells, with metastasis formation after 1–3 days. Data are derived from 3 separate experiments, and are depicted as average metastasis formation ± SEM. Significance of the data is determined by paired sample T-tests (* p≤0.05, ** p≤0.01 and, *** p≤0.001).