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First published online 16 January 2007
doi: 10.1242/jcs.03347

Journal of Cell Science 120, 512-519 (2007)
Published by The Company of Biologists 2007
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Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay

Wilhelm Schneiderhan1,2, Fredy Diaz1, Martin Fundel1, Shaoxia Zhou1, Marco Siech3, Cornelia Hasel4, Peter Möller4, Jürgen E. Gschwend5, Thomas Seufferlein2, Thomas Gress2, Guido Adler2 and Max G. Bachem1,*

1 Department of Clinical Chemistry and Pathobiochemistry, University of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany
2 Department of Gastroenterology, University of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany
3 Department of Surgery, Hospital Aalen, Im Kälblesrain 1, 73430 Aalen, Germany
4 Department of Pathology, University of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany
5 Department of Urology, University of Ulm, Prittwitzstr. 43, 89075 Ulm, Germany


Figure 1
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  		Fig. 1. MMP expression in pancreatic cancer cell lines and mesenchymal cells. (A) Gelatin zymography (Zymo) of tumor cell and PSC supernatant reveals that MMP-2 is predominantly expressed in PSC. Supernatant was loaded adapted to the DNA level of the wells corresponding to the samples. (B) Gelatin zymography: the indicated volume of supernatant conditioned by MiaPaCa2, Panc1 and SW850 (TC SN) was incubated with PSC for 24 hours. Serum-free culture media was added to yield a total volume of 2000 µl. Supernatant of cancer cells stimulated the expression of MMP-2 in PSC. Supernatant was loaded adjusted to the DNA level of the wells corresponding to the samples. Results are representative of at least three independent experiments. (C) Densitometry of the zymography: each condition was determined in quandruplicate and values are expressed as means. Boxes represent the s.e.m. and error bars the s.d. The asterisk denotes a statistically significant difference (P<0.01) for MMP-2 in the supernatant of the group of PSC stimulated with 1000 µl cancer cell supernatant compared with control.

 

Figure 2
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  		Fig. 2. BSG expression in cultured tumor cell lines MiaPaCa2, SW850 and Panc1. (A) Immunofluorescence staining of BSG: the human pancreatic adenocarcinoma cell lines were immunostained for BSG. Immunoreactivity was detected by fluorescein isothiocyanate (FITC) (green). Control was performed by omission of the primary antibody. Initial magnification was 200x. (B) Western-blot analysis of RIPA lysates and supernatant of MiaPaCa2, SW850, Panc1 cells as well as PSC. A band of 40-50 kDa according to the molecular mass of HG BSG was detected in supernatants as well as lysates. The LG form of BSG appeared as a band of 30-35 kDa in tumor cell lysates. Highly glycosylated proteins do not focus if separated by SDS-PAGE and glycosylation differs among the different cell types. Supernatants were concentrated 10-fold before western blot analysis. Lysates and supernatants were loaded adjusted to their protein concentration or adjusted to the surface area of the confluent well. Control was performed by omission of the primary antibody. (C) BSG immunofluorescence staining (FITC, green) of cancer cells co-cultured with PSC. Compared with cancer cells, the intensity of the BSG fluorescence was much lower on PSC as the result of a lower density of BSG in the cell membrane of PSC. The large PSC with their irregular polygonal fibroblast-like shape can be distinguished from the small round cancer cells in the phase-contrast image. Nuclei were counterstained with propidium iodide (red). (D) Agarose gel electrophoresis demonstrating the qRT-PCR products of BSG with a predicted length of 120 bp that were reverse transcribed and amplified from RNA of cultured MiaPaCa2, SW850 and Panc1 carcinoma cell lines as well as PSC (left panel). Controls comprised substitution of water for either cDNA (no template) or reverse transcriptase (no RT). In addition, the results of qRT-PCR were expressed normalized using XS13 as a housekeeping gene (right panel). The diagram is representative for three independent experiments.

 

Figure 3
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  		Fig. 3. Effect of tumor cell supernatant-derived BSG on MMP expression in PSC. (A,B) As demonstrated by zymography (A) and western blot (B), BSG isolated from MiaPaCa2, Panc1 and SW850 stimulates MMP-2 and MMP-1-expression in PSC to the same extent as complete supernatant. Final concentration of purified BSG and BSG in supernatant were adjusted to the same concentration. Supernatant depleted of BSG by immunoprecipitation (ID supernatant) did not increase MMP levels compared with control.

 

Figure 4
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  		Fig. 4. Immunohistologic examination of BSG expression in human ductal adenocarcinomas of the pancreas. (A) Immunofluorescence micrographs demonstrating the distribution of BSG, cytokeratin, fibronectin and iso-{alpha} smooth muscle actin (FITC, green) expression in serial sections of a human carcinoma. The PSC marker iso-{alpha} smooth muscle actin is colocalized with the excessive deposition of fibronectin, a major constituent of the extracellular matrix of the tumor desmoplasia. BSG was only detected in cancer cells, which are identified by pancytokeratin staining, whereas the stromal cells of the desmoplasic reaction exhibit no immunoreactivity to BSG. Nuclei were counterstained with propidium iodide. Initial magnification was 100x. (B) Immunofluorescence micrograph of BSG (FITC, green) expression in an adenocarcinoma of the pancreas. The duct-like structures (#) with multilayered malignant cells exhibit a strong BSG immunoreactivity, whereas an adjacent normal duct structure (*) with regular epithelial cells shows no BSG expression. Nuclei were counterstained with propidium iodide. Initial magnification was 200x. (#, duct-like structure of an adenocarcinoma; *, regular pancreatic duct.) (C) Immunostaining of BSG and MMP-2 in sections of human pancreatic cancer using APAAP detection with Fast Red as a chromogen. BSG is expressed by malignant epithelial cells. MMP-2 could be detected in peritumoral stroma at the tumor-stroma interface adjacent to malignant epithelial cells with high BSG immunoractivity. Nuclei were counterstained with Mayer's hemalaun. Initial magnification was 200x.

 

Figure 5
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  		Fig. 5. Pancreatic stellate cells promote invasion and growth of pancreatic tumors in the CAM model and a xenograft model in nude mice. (A) If CAMs of chick embryos were inoculated with Panc1 cells alone, after 4 days no invasion of the membrane was observed by microscopic examination of hematoxylin-eosin-stained paraffin sections. In combination with human PSC, Panc1 cells formed significant tumors in all cases and markedly invaded the CAM. Initial magnification was 200x. (B) After subcutaneous injection of tumor cells into nude mice either alone on the right-side or in combination with PSC on the left-side, palpable tumors formed at the site of injection in all cases after 11 days. Tumor weight of explanted pancreatic tumors induced by subcutaneous injection of Panc1, MiaPaCa2 or SW850 either in the presence (+PSC) or absence (-PSC) of PSC in nude mice was determined at the end of the experiment. The squares represent the mean, the circles the single measured values, the boxes the standard error and the error bars the standard deviation observed for six animals. All groups with PSC were significantly larger than the respective group without PSC (*P<0.05).

 

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© The Company of Biologists Ltd 2007