1. Volume 128, Issue 4, Pages 907-921 (April 2005)
Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells 
Max G. Bachem, Marion Schünemann, Marco Ramadani, Marco Siech, Hans Beger, Andreas Buck, Shaoxia Zhou, Alexandra Schmid- Kotsas, Guido Adler 
Gastroenterology 
Volume 128, Issue 4, Pages (April 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Fluorescence micrographs showing the immunoreactivity of α-SMA, desmin, collagen type I, collagen type III, and fibronectin in tissue sections of 2 patients with pancreas adenocarcinoma. An intense immunofluorescence reaction to collagen type I (E and F), collagen type III (G and H), and fibronectin (I and J) was observed around dilatated ducts and in fibrotic septa between areas representing carcinoma. Ducts were always negative, and carcinoma cells were negative or at most weakly stained for extracellular matrix proteins. High numbers of α-SMA-positive cells (A and B) and desmin-positive cells (C and D) were present within fibrotic areas. (A, C, E, G, and I) patient 1; (B, D, F, H, and J) patient 2. (A and B) α-SMA; (C and D) desmin; (E and F) collagen type I; (G and H) collagen type III; (I and J) fibronectin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Fluorescence micrographs showing the immunoreactivity of vimentin, desmin, α-SMA, and collagen type III in cultured human PSCs. Cells were obtained by the outgrowth method from fibrotic tissue of patients with ductal adenocarcinomas (A, C, E, and G) and patients with chronic pancreatitis (B, D, F, and H). Several days after seeding of 1- to 3-mm3 tissue blocks, stellate and spindle-shaped cells began to grow out. After reaching confluency (2 to 3 weeks later), cells were passaged and seeded with a density of 0.5 × 104 cells per square centimeter on glass coverslips. Cells were cultured in the presence of 10% fetal calf serum. Five days after seeding, cultures were stopped, and indirect immunofluorescence stainings using anti-vimentin (A and B), anti-desmin (C and D), anti-α-SMA (E and F), and anti-collagen type III (G and H) were performed. Cells originating from pancreas adenocarcinomas (A, C, E, and G) and cells originating from chronic pancreatitis (B, D, F, and H) expressed vimentin (100%), desmin (20%–50%), α-SMA (>95%), and collagen type III (100%). Cell morphology was mostly (>70%) stellate-like, but some cells were also spindle shaped. There was no difference in the percentage of spindle-shaped cells between cultures outgrowing from carcinomas and cultures outgrowing from fibrotic tissue of chronic pancreatitis (original magnification, 200×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Effect of pancreas carcinoma cell line supernatants on proliferation of cultured PSCs. PSCs were isolated by the outgrowth method by using the tissue of patients with pancreas adenocarcinomas. After reaching confluence, cells were trypsinized and subcultured in the presence of 10% fetal calf serum. Two days after passage, fetal calf serum was reduced to 0.1%, and 50–600 μL/mL pancreas carcinoma cell line supernatants (conditioned as described in the Materials and Methods section) were added to the PSC culture (cells of the third to fifth passage). (A) Seventy-two hours after stimulation, cultures were stopped, and PSC proliferation was measured by fluorometric DNA quantification. (B) Six hours after stimulation, BrdU (final concentration, 5 × 10−5 mol/L) was added for another 18 hours. Thereafter, cultures were stopped, and BrdU incorporation was determined as described in the Materials and Methods section. (C) Carcinoma cell supernatants were preincubated for 1 hour with PDGF-neutralizing antibodies [anti-PDGF(AA) and anti-PDGF(BB) or a control antibody (goat immunoglobulin G), each 1 μg/mL] before the conditioned media were added to cultured PSCs. The nonspecific immunoglobulin did not reduce the stimulatory effects of the carcinoma cell supernatants. The results are expressed as fraction of control (cells grown in DMEM with 0.1% fetal calf serum) and are the mean ± SD of 3 experiments (each condition performed in triplicate wells). *Statistically significant difference (P < .05) compared with control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Fluorescence micrographs showing the immunoreactivity of fibronectin, collagen type I, and collagen type III in cultured human PSCs stimulated with conditioned media of MiaPaCa2 cells and Panc1 cells. Cultured human PSCs (fourth passage) grown on glass coverslips in the presence of 0.1% FCS were stimulated for 48 hours with 500 μL/mL MiaPaCa2- and Panc1-conditioned media. After 48 hours, cultures were acetone-fixed and immunostained for fibronectin (A, D, and G), collagen type I (B, E, and H), and collagen type III (C, F, and I). Staining and immunofluorescence microscopy was performed (as described in the Materials and Methods section). To compare staining intensities, all micrographs were taken by using the same exposure time (original magnification, 400×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Stimulation of collagen type I and c-fibronectin synthesis in cultured human PSCs by carcinoma cell supernatants. (A and C) Two days after passage, PSCs were stimulated for 24 hours with 200, 400, and 600 μL/mL MiaPaCa2- and Panc1-conditioned media, respectively. PSCs were cultured in DMEM/Ham’s F12 with 0.1% fetal calf serum, ascorbic acid (100 μg/mL), and β-aminopropionitrile (100 μg/mL). After 24 hours, cultures were stopped, and collagen type I (A) and c-fibronectin (B) were measured in PSC supernatants by immunoassay as described in the Materials and Methods section. DNA was measured in the cell layer. Collagen type I and c-fibronectin concentrations were related to the DNA concentration and expressed as means ± SD of 3 independent experiments; each condition was performed in triplicate culture wells. *Statistically significant difference (P < .05) compared with control. The inserts (B and D) show Northern blot hybridizations of collagen I (α1) (B, upper insert), fibronectin (D, upper insert), and 18S ribosomal RNA (B and D, lower insert). To obtain RNA, PSCs were cultured in 75-cm2 flasks in DMEM/Ham’s F12 with 0.1% fetal calf serum. Twelve hours after stimulation (5 mL of carcinoma cell supernatant/10 mL of medium), RNA was isolated, and Northern blot hybridizations were performed as described in the Materials and Methods section. Lane 1, 0.1% fetal calf serum (control); lane 2, 5 mL of MiaPaCa2-supernatant/10 mL of medium; lane 3, 5 mL of Panc1 supernatant/10 mL of medium.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Inhibition of stimulated c-fibronectin synthesis by neutralizing antibodies to TGF-β1, FGF-2, and PDGF. Carcinoma cell line supernatants were preincubated for 1 hour with neutralizing antibodies to PDGF(AA) and PDGF(BB) (each 1 μg/mL), FGF-2 (1 μg/mL), and TGF-β1 (10 μg/mL) before the supernatants (300 μL/mL) were added to cultured PSCs growing in DMEM/Ham’s F12 with 0.1% fetal calf serum, ascorbic acid (100 μg/mL), and β-aminopropionitrile (100 μg/mL). After 24 hours, cultures were stopped, and c-fibronectin (B) was measured in PSC supernatants by immunoassay as described in the Materials and Methods section. DNA was measured in the cell layer. Concentrations of c-fibronectin were related to the DNA concentration and are expressed as means ± SD of 3 independent experiments; each condition was performed in triplicate culture wells. Preincubation of the carcinoma cell supernatants with nonspecific immunoglobulins (goat immunoglobulin G for PDGF and rabbit immunoglobulin G for TGF-β and FGF-2) did not reduce the stimulatory effects. *Statistically significant difference (P < .05) compared with the stimulation with 300 μL/mL carcinoma cell supernatant without specific antibody preincubation.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Growth rate of subcutaneous tumors induced in nude mice. A total of 2 × 106 Panc1 cells (A, group IIc), 2 × 106 MiaPaCa2 cells (B, group IIa), or 2 × 106 SW850 cells (C, group IIe) were injected alone and together with 2 × 106 PSCs subcutaneously on to nu/nu mice. The tumor size was determined with a calliper rule, and the tumor volume was calculated by using the formula (a × b × c)/2, where a and b are the shorter and longer diameter of the tumors, respectively, and c is the thickness. Animals were killed after 11 days, and tumors were excised and immediately frozen in liquid nitrogen until cutting to obtain sections for immunostainings.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
Immunohistochemistry of α-SMA and desmin and immunofluorescence of collagen type I, collagen type III, and fibronectin in tissue sections of subcutaneous tumors induced in nude mice. A total of 6 × 106 MiaPaCa2 cells were injected alone (A, C, E, G, and I) and together with PSCs (B, D, F, H, and J) subcutaneously on to nu/nu mice. Animals were killed after 21 days. Tumors were excised and immediately frozen in liquid nitrogen until sectioning and immunostaining for α-SMA (A and B), desmin (C and D), collagen type I (E and F), collagen type III (G and H), and fibronectin (I and J). High numbers of α-SMA-positive and desmin-positive cells were found in the tumors arising from the combined injection of carcinoma cells and PSCs (B and D). Furthermore, immunofluorescence stainings of collagen I, collagen III, and fibronectin showed that the desmoplastic reaction was more intense in the tumors developing after injection of carcinoma cells together with PSCs (F, H, and J) compared with tumors arising from single application of MiaPaCa2 cells (E, G, and I).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 9
Immunofluorescence of collagen type I and immunohistochemistry of cytokeratin in tissue sections of subcutaneous tumors induced in nude mice. A total of 2 × 106 Panc1 cells (A–D) and 2 × 106 SW850 cells (E–H) were injected alone (A, C, E, and G) and together with PSCs (B, D, F, and H) subcutaneously on to nu/nu mice. Animals were killed after 11 days, and tumors were excised and immediately frozen in liquid nitrogen until sectioning and immunostaining for collagen type I (A, B, E, and F) and cytokeratin (C, D, G, and H). Immunofluorescence stainings of collagen I showed that the desmoplastic reaction was more intense in the tumors developing after injection of carcinoma cells together with PSCs (B and F) compared with tumors arising from single application of Panc1 and SW850 cells (A and E). Carcinoma cells of tumors growing after subcutaneous injection of Panc1 cells and SW850 cells showed intense cytokeratin stainings (C, D, G, and H). In Panc1 tumors (C), cancer cells were dispersed throughout the sections, whereas in the presence of stellate cells (D), the carcinoma cell lines grew in dense colonies surrounded by extracellular matrix. Morphometric analysis showed that in Panc1 tumors, compared with Panc1-PSC tumors, the carcinoma cell density was equal, but in SW850-PSC tumors, carcinoma cell density was significantly higher compared with that in SW850 tumors.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions