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First published online 16 January 2007
doi: 10.1242/jcs.03347
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Research Article |
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
* Author for correspondence (e-mail: max.bachem{at}uniklinik-ulm.de)
Accepted 15 November 2006
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Pancreatic adenocarcinoma, Tumor-host interaction, Matrix metalloproteinases, Pancreatic stellate cell, Mouse xenograft model
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Introduction |
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), and efficient therapies are currently unavailable (Landis et al., 1999; Smeenk et al., 2005). Further insights into the mechanisms causing the highly aggressive nature of this disease might reveal new prospects for therapy and diagnosis of pancreatic adenocarcinomas.
Compared with other epithelial neoplasms, pancreatic adenocarcinomas have an extremely rich and dense fibrotic stroma surrounding the malignant cells (Bachem et al., 2005; Seymour et al., 1994). We recently demonstrated that the desmoplastic reaction in pancreatic cancer is the result of the cross-talk between carcinoma cells and the pancreatic stellate cells (PSC) (Bachem et al., 2005). PSC are also involved in the development of pancreatic fibrosis following pancreatitis (Haber et al., 1999) and share a lot of similarities with hepatic stellate cells (Buchholz et al., 2005), which synthesize `fibrotic' matrix in diseased human liver (Bachem et al., 1998; Friedman, 1993). Both are activated from a quiescent state upon stimulation with TGF-
1 (Bachem et al., 1998). This process leads to the acquisition of a myofibroblast-like phenotype by the PSC, which has the following changes compared with its quiescent counterpart: (1) loss of retinyl droplets, (2) change in cellular size and morphology, (3) expression of iso-
smooth muscle actin, and (4) the ability to proliferate and (5) synthesize significant amounts of extracellular matrix proteins (Apte et al., 1998; Bachem et al., 1998).
Cancer cell-derived matrix metalloproteinase-2 (MMP-2) was recently shown to be associated with the development of the desmoplastic reaction and seems to promote tumor cell invasion in human pancreatic cancer (Ellenrieder et al., 2000). However, in most epithelial cancers the majority of MMP-2 is produced by stromal cells (Egeblad and Werb, 2002) and PSC also express the gelatinases MMP-2 and MMP-9 (Phillips et al., 2003). Mesenchymal cells are stimulated to produce MMP-1, MMP-2 and MMP-3 by basigin (BSG), a transmembrane glycoprotein enriched on several tumor cell types, which is identical to extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) (Biswas et al., 1995). Although BSG is a transmembrane protein, cancer cells do not stimulate MMPs through direct cell-cell contact with mesenchymal cells. Instead, the malignant cells stimulate MMPs in a paracrine fashion after BSG is shed from their cell surface via microvesicles (Sidhu et al., 2004) or by matrix-metalloproteinases (Haug et al., 2004).
It has been suggested that BSG plays an important role in tumor-mesenchymal cell interaction and in promoting tumor progression (Biswas et al., 1995; Caudroy et al., 2002; Kanekura et al., 2002; Sameshima et al., 2000; Taylor et al., 2002; Zucker et al., 2001). Experimental overexpression of BSG in MDA-MB-436 human mammary tumor cells, which express lower levels of BSG than most malignant cell lines, promotes invasive capacity (Caudroy et al., 2002) and anchorage-independent growth in vitro (Marieb et al., 2004). In vivo, one of the most marked responses to overexpression of BSG was the formation of fast-growing tumors in nude mice (Zucker et al., 2001).
Tumor-host communication is crucial for the progression of many human tumors and PSC are abundant in the desmoplasia, producing matrix-associated growth factors, as well as MMP-2, both of which have been associated with accelerated pancreatic cancer progression. The effect of PSC on pancreatic cancer progression, however, remains elusive.
To elucidate the role of cancer cell-PSC interaction with respect to progression of pancreatic cancer, we first investigated the source and the regulation of MMP-2 in the desmoplastic reaction. Moreover, we analyzed the influence of PSC on cancer cell invasion in the chorioallantoic membrane (CAM) assay and on tumorigenity in a xenograft model in nude mice.
We demonstrate that PSC are an important source of MMP-2 in pancreatic cancer and that the interaction of PSC and tumor cells stimulates MMP-2 in PSC and promotes invasiveness (CAM assay) as well as tumorigenity (nude mice xenograft model) of cancer cells. These findings suggest that the desmoplasia of pancreatic cancer promotes tumor progression.
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Results |
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), we elucidated the role of PSC as a source of MMPs in the desmoplasia of human adenocarcinomas of the pancreas. Expression of MMPs by PSC was compared with MMP expression by tumor cells. Gelatinase activity was hardly detectable by zymography in supernatants of tumor cells but was very strong in PSC supernatants (Fig. 1A). A prominent band of approximately 65 kDa corresponding to proMMP-2 was detected in PSC supernatants and Panc1 supernatant (Fig. 1A). If supernatant of SW850 and MiaPaCa2 was concentrated 10-fold, minimal amounts of MMP-2 could be detected here as well (data not shown).
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). When adjusted to the total protein concentration, PSC exhibited an expression of cell-associated BSG that was comparable to cancer cells, yet the concentration of soluble BSG shed by PSC into the supernatant was considerably lower (Fig. 2B). When samples were loaded adjusted to the surface area of the cells, the expression level of cell-associated BSG in PSC was much lower than in cancer cells, indicating a lower density of BSG in the membrane of PSC (Fig. 2B). The lower concentration of BSG in the membrane of PSC was also demonstrated by immunofluorescence microscopy of stellate cells co-cultured with cancer cells (Fig. 2C). The PSC can be distinguished from the small round cancer cells in the phase-contrast image by their larger size and their irregular polygonal shape (Fig. 2C). BSG expression by tumor cells was also demonstrated to be higher than that of PSC on the RNA level by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The PCR product with a predicted length of 120 bases was amplified with BSG-specific primers on a light-cycler platform and XS13 was used as a housekeeping gene (Fig. 2D, right panel). The negative controls comprised a no-template control and omission of the reverse transcriptase (Fig. 2D, left panel). To evaluate the relative importance of BSG in paracrine stimulation of MMP synthesis by PSC compared with the other components of the cancer cell-conditioned medium, we compared the ability of purified BSG, complete tumor cell supernatant and BSG-immunodepleted tumor cell supernatant to stimulate MMP expression when added to PSC. Purified BSG exhibited the same effect on MMP-2 concentration in PSC supernatant as cancer cell-conditioned medium that had the same final BSG concentration (Fig. 3A). In addition, the effect of cancer cell-conditioned medium was completely abolished by immunodepletion of BSG from the cancer cell-conditioned medium (Fig. 3A). The gelatin zymography data were verified by western blot analysis (Fig. 3B). In addition, western-blot analysis revealed a strong increase in MMP-1 concentration in PSC supernatant, occurring in parallel to the effect of BSG on MMP-2 (Fig. 3B).
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In vivo BSG is expressed in human pancreatic cancer mainly by tumor cells, whereas MMP-2 is enriched in peritumoral stroma
We examined the localization of BSG and MMPs in relation to cancer cells and PSC respectively by immunofluorescence microscopy of serial sections of 10 human pancreatic adenocarcinomas (Fig. 4). The cancer cells, identified by pancytokeratin staining, are surrounded by an extensive peritumoral fibrosis, which was identified by its strong reactivity with antibodies to the matrix proteins fibronectin (Fig. 4A) and collagen type I (data not shown). To demonstrate that PSC are located in the peritumoral stroma, immunostaining with the PSC marker iso- smooth muscle actin was performed. Iso- smooth muscle actin (Fig. 4A) was colocalized with the proteins of the extracellular matrix of the peritumoral fibrosis. BSG immunostaining revealed an intense BSG expression predominantly co-localized with carcinoma cells (Fig. 4A-C) but not with normal monolayer epithelial cells of pancreatic duct structures (Fig. 4B). In accordance with the in vitro results, immunoreactivity to MMP-2 was mainly detected in the stroma of pancreatic cancer, and particularly at the tumor-stroma interface (Fig. 4C).
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To assess the influence of PSC on cancer cell tumorigenicity, xenograft tumors were induced by subcutaneous injection of Panc1, MiaPaCa2 or SW850 cells in the presence of PSC on the left-side of six nude mice. As a control, tumor cells were injected on the right-side in the absence of PSC. After 11 days of incubation, palpable tumors formed in all mice at the site of injection. PSC alone were not tumorigenic in nude mice (data not shown).
Tumors grew progressively up to a final weight of 40±27 mg (mean ± s.d.) if induced with Panc1, up to 33±17 mg with MiaPaCa2, and up to 60±12 mg with SW850, respectively (Fig. 5B). PSC significantly increased the weight of Panc1 tumors by 3.3-fold to 131±66 mg, the weight of MiaPaCa2 tumors by 3.7-fold to 123±35 mg, and the weight of SW850 tumors by 2.1-fold to 126±46 mg (Fig. 5B).
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Discussion |
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). The phenomenon of tumor-associated desmoplasia is most frequently described in squamous cell carcinomas and bilo-pancreatic carcinomas (Kunz-Schughart et al., 2002). We recently demonstrated that the characteristic dense fibrous peritumoral stroma of pancreatic adenocarcinoma forms as a result of the interaction between tumor cells and PSC (Bachem et al., 2005). However, it is not known whether the desmoplastic reaction promotes, inhibits or has no effect on tumor progression.
It has recently been demonstrated that MMP-2 is associated with development of the desmoplasia of pancreatic carcinoma (Ellenrieder et al., 2000). MMPs have long been associated with metastasis, poor prognosis and the malignant phenotype of tumors (Egeblad and Werb, 2002) and they are now reported to be involved in the invasion of pancreatic cancer as well (Ellenrieder et al., 2000). In recent years it has become clear that transformed cells secrete MMPs, and yet a major fraction of MMPs is produced by fibroblasts in the tumor stroma (Egeblad and Werb, 2002; Noel et al., 1994). Our current results show that PSC constitutively express high levels of MMP-2 compared with the pancreatic tumor cell lines MiaPaCa2, SW850 and Panc1. Immunohistology confirmed that MMP-2 immunoreactivity is found on mesenchymal cells adjacent to cancer cells colocalized with iso--smooth muscle actin. Our results demonstrate for the first time that the stromal PSC also significantly contribute to MMP-2 secretion in pancreatic cancer in vivo and in vitro. Moreover, our results suggest that secretion of MMP-2 by PSC can exceed that of cancer cells by far. Although the exact sources for MMPs in pancreatic cancer need to be identified in further detailed studies, these result indicate that PSC are a major source and further emphasize the role of the host reaction in pancreatic cancer progression.
We observed that the MMP-2 synthesis of PSC was even further increased in the presence of tumor cell-conditioned medium, suggesting an interaction of tumor cells and PSC by soluble factors. Here we demonstrate by RT-PCR, western blot and immunofluorescence staining that BSG, a type-2 membrane glycoprotein that induces MMPs in stromal cells (Biswas et al., 1995), is expressed and released by MiaPaCa2, SW850 and Panc1. We and others have demonstrated that the soluble form of BSG is a result of shedding of the membrane-associated form (Haug et al., 2004; Sidhu et al., 2004). BSG expression by pancreatic adenocarcinomas was also shown by immunohistology of surgically resected human tumors in vivo. BSG has been demonstrated to be enriched in many human malignancies including lymphomas, gliomas, breast cancer, ovarian cancer and certain forms of skin cancer (Davidson et al., 2003; Sameshima et al., 2000; Taylor et al., 2002; Thorns et al., 2002) and the expression of BSG correleates with malignant potential and poor survival (Dalberg et al., 2000; Davidson et al., 2003; Marionnet et al., 2003).
The molecular mechanisms by which BSG promotes the malignant potential of tumors are poorly understood but its property to stimulate MMPs in mesenchymal cells has gained considerable attention (Kanekura et al., 2002; Li et al., 2001; Taylor et al., 2002). In our study, BSG purified from tumor cell supernatant stimulated MMP-1 and MMP-2 synthesis in PSC to the same extent as tumor cell supernatant. These findings demonstrate that BSG is a soluble factor released by cancer cells that plays a significant role in the interaction of cancer cells and PSC resulting in the stimulation of MMP-2. BSG seems to be a major factor for MMP stimulation compared with other cancer cell-derived soluble factors. This assumption is supported by our observation that the effect of conditioned medium was almost completely abolished after BSG immunodepletion.
Although we are only beginning to understand how mesenchymal cells and MMPs support tumor growth (Liotta and Kohn, 2001; Zucker et al., 2000), it has been reported that fibroblasts from MMP knockout mice have lost their ability to support tumor growth (Itoh et al., 1999). This emphasizes the pivotal role of mesenchymal cell-derived MMP secretion for tumor progression.
One of the most remarkable properties of PSC was their ability to support tumor progression, as demonstrated by the CAM- and xenograft assay. PSC substantially promote the growth and invasion of Panc1 cells through the CAM of chick embryos and support tumorigenity of tumor xenografts in nude mice. These results demonstrate that the interaction of cancer cells with PSC propagates the invasion of pancreatic cancer in vivo. In breast cancer and prostatic cancer, the role of the stromal microevironment, which consists primarily of fibroblasts, is also being increasingly appreciated (De Wever and Mareel, 2002; Liotta and Kohn, 2001).
Here we demonstrate that PSC represent a significant source of MMP-2 in pancreatic cancer, which is known to promote tumor progression. Moreover, cancer cells release BSG to stimulate MMP-2 in PSC, indicating that cross-talk of cancer cells and mesenchymal cells might further increase MMP-2 production in the desmoplasia. Finally, we show by in vivo models that the interaction of human cancer cells with PSC promotes invasiveness and tumorigenity of malignant epithelial cells of the pancreas. In conclusion, our data suggest that the tumor-stroma interaction in the desmoplastic reaction of pancreatic cancer promotes tumor progression.
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Materials and Methods |
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). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1:1 vol./vol.) containing 10% fetal calf serum (FCS) and used from passage 3-8. To study stimulated MMP synthesis, BSG or tumor cell-conditioned media were added to 3-5x104 PSC/cm2 grown in six-well plates in the absence of FCS. Cultures were stopped 24 hours later. To obtain carcinoma cell-conditioned media, 10 ml DMEM was conditioned by incubation with confluent tumor cell lines (MiaPaCa2, Panc1 and SW850) in 75 cm2 culture flasks for 48 hours.
Purification of BSG
Panc1-, SW850- or MiaPaCa-conditioned media were dialyzed against 20 mM Tris, pH 8.0 at 4°C overnight and applied to an equilibrated anion-exchange column (BioCAD Workstation, Applied Biosystems, Foster City, CA). Bound proteins were eluted with a linear salt gradient. The BSG-containing fraction was added to an immunoaffinity matrix for 12 hours at 4°C, which was prepared by coupling a monoclonal anti-human BSG antibody (Serotec, Oxford, UK) to protein-G-agarose using ImmunoPure plus an Immunoglobulin G (IgG) Orientation Kit (Pierce, Rockford, IL). The immunoaffinity matrix was washed with 20 mM Tris, pH 8.0, several times before BSG was eluted with elution buffer (Pierce, Rockford, IL). The eluted protein was neutralized with 1 M Tris, pH 9.5, and analyzed for purity by SDS-PAGE and silver staining.
BSG immunoassay
To quantify BSG, Nunc-maxisorb 96-well plates (Nunc, Roskilde, Denmark) were coated with 50 µl/well polyclonal rabbit anti-mouse antibody (DAKO, Glostrup, Denmark) diluted 1:220 in coating buffer (50 mM NaHCO3, pH 9.1) at 4°C overnight. Subsequently, 100 µl/well monoclonal anti-CD147 antibody (R&D Systems, Minneapolis, MN) diluted 1:2000 in coating buffer was allowed to bind for 4 hours at room temperature. Unspecific binding sites were blocked by incubation with assay buffer [50 mM Tris, 150 mM NaCl, 0.5% (w/v) albumin, 0.5% (w/v) NaN3, pH 7.7] for 2 hours at room temperature before incubation with 100 µl/well of sample or standard at 4°C overnight. Following incubation of 100 µl/well biotinylated polyclonal anti-CD147 antibody (R&D Systems) diluted 1:1000 in assay buffer for 4 hours, streptavidin europium (PerkinElmer/Wallac, Turku, Finland) was incubated diluted 1:500 in assay buffer for 1 hour. Finally, time-resolved fluorescence was measured on a Victor multilabelcounter (PerkinElmer/Wallac) after enhancement solution was added. Each incubation step was followed by a washing step with 150 mM NaCl, 50 mM Tris, 0.5% (v/v) Tween, pH 7.4. A dilution series of conditioned tumor cell supernatant was used as a standard and results were expressed as arbitrary units. All samples were determined in duplicate.
Immunodepletion of conditioned media
Monoclonal anti-BSG IgG (150 µl) coupled to protein-G-agarose was incubated with 2 ml conditioned media for 2 hours at 4°C. After centrifugation, immunodepletion of the supernatant was confirmed by western blotting.
SDS-PAGE, silver staining and western blot
Basigin western blot was performed as previously described (Haug et al., 2004). Briefly, cells were lysed in a modified radioimmunoprecipitation assay (RIPA) buffer containing proteinase inhibitors and resolved by SDS-PAGE using an 8% gel. Supernatants were concentrated ten times using centrifugal filter devices (Millipore, Billerica, MA) with a 10 kDa cut-off before they were loaded adjusted to the DNA content of the corresponding wells. DNA was determined according to Labarca and Paigen (Labarca and Paigen, 1980) by fluorophotometry using Hoechst 33258 dye.
Gels were either stained using GelCode SilverSNAP kit (Pierce) or immunoblotted with antibodies against BSG (Serotec), MMP-1 (Chemicon, Temecula, CA) and MMP-2 (R&D Systems) and visualized by chemiluminescence.
Zymography
Supernatant adjusted to the DNA content of the corresponding well mixed with sample buffer (0.98 M Tris, pH 6.8, 1% SDS, 4% glycerol, 0.006% bromphenol blue) was separated on a 7% polyacrylamide gel containing 1 mg/ml gelatin. Thereafter, gels were soaked in 2.5% Triton X-100 for 1 hour, followed by incubation in developing buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.2% Brij-35) at 37°C for 16 hours.
Immunohistology and cytology
For detection of BSG, iso- smooth muscle actin, fibronectin, collagen type I and pancytokeratin cells seeded on glass coverslips and cryosections of human pancreatic adenocarcinomas or tumors induced in nude-mice were fixed in ice-cold acetone. After fixation, unspecific binding sites were blocked by incubation with TNB (0.1 M Tris, 0.15 M NaCl, 0.5% bovine serum albumin, pH 7.5). Subsequently, the specimens were incubated with anti-MMP-2 (R&D Systems), anti-BSG (clone MEM-M6/1; Serotec), anti-iso- smooth muscle actin (DAKO), anti-pancytokeratin (clone MNF-116; DAKO), anti-collagen type I (Chemicon) or anti-fibronectin (Behring, Marburg, Germany) antibodies diluted in TNB. Immunoreactivity was visualized using tyramide signal amplification (NEN Life Science Products, Boston, MA) and a streptavidin-conjugated fluorochrome or alkaline phosphatase-anti-alkaline phosphatase (APAAP) staining using FastRed (DAKO) as substrate. Nuclei were counterstained with propidium iodide, Hoechst 33258 or Mayer's hemalaun.
RNA-isolation and PCR
RNA was extracted from MiaPaCa, Panc1 and SW850 by phenol/chloroform following the method described by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). mRNA (1 µg) was used to synthesize complementary DNA using 200 U SuperScript reverse-transcriptase (Invitrogen, Carlsbad, CA, USA) and 0.5 µg oligo(dT) primer. PCR amplification was performed on a light-cycler platform using the Roche Light-Cycler Fast-Start SYBR-Green DNA Kit (Roche, Mannheim, Germany) according to the instructions of the manufacturer. The sequence of the sense primer was 5'-CCGAGGACGTCCTGGAT-3' and 5'-CGGGCCACCTGCCTCA-3' for the antisense primer. Negative controls comprised replacement of either cDNA or reverse transcriptase with water. XS13 was used as a housekeeping gene. The PCR product was analyzed by etidium-bromide staining on a 1.2% agarose gel. The predicted size of the PCR product was 120 bp.
CAM assay
The CAM assay was performed as previously described (Kunzi-Rapp et al., 2001). Briefly, 0.5x106 Panc1 cells were seeded either alone or in combination with 0.5x106 PSC on the CAM of fertilized chicken eggs. Following four days of incubation at 37°C and 60% relative humidity, the tumor cells with the surrounding CAM were sampled, fixed in 4% paraformaldehyde, paraffin-embedded and stained with hematoxylin-eosin for histologic examination.
Animals and tumors
Xenografts of all carcinoma cell lines were initiated by subcutaneous injection of 2x106 tumor cells into six nude mice on the right-side alone or in combination with 1.8x106 PSC on the left-side. After 11 days the mice were sacrificed and the tumors were dissected, weighed and prepared for further histological examination. All experiments were performed in accordance with institutional guidelines for the care and use of experimental animals.
Statistical analysis
Multivariate analysis of variance followed by Fischer LSD post-hoc analysis was performed using Statistica® 6.1 for Windows® to test for significant statistical differences between groups.
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Acknowledgments |
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References |
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