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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02467

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Tumour necrosis factor confers an invasive, transformed phenotype on mammary epithelial cells

¹ Department of Cell Physiology and Metabolism, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland
² Institute of Physiological Chemistry and Pathobiochemistry, Münster University Hospital, Münster 48149, Germany

View larger version (120K): [in a new window]   Fig. 1. Effect of proinflammatory cytokines on the behaviour of 2A4 mammary epithelial cells grown in collagen gels. The cells were suspended in a gelling collagen solution. After a 10 minute incubation at 37°C to allow collagen gelation, defined medium with or without the indicated cytokines was added above the gels and renewed after 3 days. (A) 2A4 cells grown in a collagen gel for 6 days under control conditions form compact ball-like colonies. (B) 2A4 cells treated for 6 days with IL-1ß (2 ng ml-1) form loose cell clusters. (C) 2A4 cells treated for 6 days with TNF- (10 ng ml-1) do not form cohesive colonies but scatter throughout the collagen matrix, mostly as individual cells. Scale bars, 50 µm. (D) Quantification of the effects of IL-6, IL-1, IL-1ß and TNF- was carried out after 6 days of treatment by counting the number of apparently single cells in five randomly selected fields per experimental condition in each of three independent experiments. Data were expressed as mean number of cells ± s.e.m. and statistical significance was determined using the Student's unpaired t test. P=0.025 for values of IL-6 compared with controls; P<0.0005 for values of IL-1, IL-1ß and TNF- compared with controls, as well as for values of TNF- compared with IL-6, IL-1, IL-1ß.

View larger version (80K): [in a new window]   Fig. 2. TNF- induces 3D and 2D scattering of 2A4 mammary epithelial cells. (A) 2A4 cells grown for 9 days in a collagen gel in defined medium have formed compact ball-like structures. (B) 2A4 cells were grown for 3 days in a collagen gel to allow the formation of spheroidal cell clusters, at which time 10 ng ml-1 TNF- was added for an additional 6 days. TNF- treatment has resulted in the dissociation of preformed colonies and the dispersal of individual cells within the collagen matrix. (C) 2A4 cells seeded in plastic wells (2D culture) in DMEM-F12 with ITS+ Premix and grown for 4 days have formed discrete islands of closely apposed cells. (D) 2A4 cells were grown in DMEM-F12 with ITS+ Premix for 2 days to allow formation of small compact colonies before addition of TNF- (10 ng ml-1) for an additional 48 hours. TNF- has induced a pronounced 2D scattering of 2A4 cells. (E) 2A4 cells maintained for 5 days in the presence of TNF- are fully scattered. (F) Parallel cultures were treated 48 hours with TNF- to induce cell dispersion, at which time the medium was changed and the cells were incubated for an additional 72 hours without TNF-. The cells have re-established close intercellular contacts and formed compact epithelial-like colonies. (A-F) Scale bars, 100 µm. (G) Time course and dose dependence of TNF--induced 3D scattering. 2A4 cells were grown in collagen gels for 3 days and subsequently incubated for an additional 6 days with or without increasing concentrations of TNF-. Quantification was carried out by counting the number of apparently single cells per photographic field after 3 days and 6 days of treatment. Data represent the mean number of single cells ± s.e.m. P<0.05 for values of 1 ng ml-1 TNF- compared with controls (3 days); P<0.01 for values of 1 ng ml-1 TNF- compared with controls (6 days); P<0.005 for values of 3 ng ml-1 TNF- compared with controls (3 days). (H) Time course and dose dependence of TNF--induced 2D scattering. 2A4 cells were grown for 2 days in gelatine-coated wells and subsequently incubated with or without increasing concentrations of TNF-. Scattering was quantified by counting cells that were completely isolated or showed only a minimal area of contact with their neighbours. Data represent the mean number of isolated cells ± s.e.m. in nine randomly selected photographic fields. P<0.05 for values of 3 ng ml-1 TNF- compared with controls (24 hours); P<0.0025 for values of 10 ng ml-1 TNF- compared with controls (24 hours); P<0.025 for values of 1 ng ml-1 TNF- compared with controls (48 hours); P<0.0005 for values of 3 ng ml-1 TNF- compared with controls (48 hours). Scale bars, 100 µm.

View larger version (31K): [in a new window]   Fig. 3. TNF- stimulates invasion of collagen gels. 2A4 cells were plated on top of a collagen gel, allowed to form a monolayer and subsequently incubated with or without different concentrations of TNF-. 5 days later, the cultures were photographed at a focal plane below the surface to visualize cells that have penetrated the gel. In untreated cultures (A), only rare cells migrate from the surface monolayer into the underlying collagen matrix. By contrast, in cultures treated with TNF- (3 ng ml-1) (B), there is extensive invasion of the collagen gel. Scale bars, 100 µm. (C) Invasion was quantified by counting the number of all cellular structures (i.e. apparently single cells and cell cords) present in a photographic field at a focal plane beneath the surface monolayer. Five randomly selected fields per condition were analysed in each of three separate experiments. *P<0.025 (compared with control values); **P<0.0005 (compared with control values).

View larger version (56K): [in a new window]   Fig. 4. TNF- stimulates anchorage-dependent and -independent growth of 2A4 cells. (A,B) 35 mm tissue culture dishes were seeded with 2A4 cells at a density of 2x105 cells per dish in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was renewed and the cultures were left untreated or were treated with 10 ng ml-1 TNF-. 5 days later, control cells have formed only small colonies (A), whereas TNF--treated cells have formed a nearly confluent monolayer (B). Scale bars, 200 µm. (C) Dose dependence of TNF--induced cell proliferation. 2A4 cells were seeded in 16 mm wells at 1x104 cells per well in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was removed, fresh medium was added, and the cultures were incubated with or without various concentrations of TNF-. Medium and treatments were renewed every 2-3 days. Cells in triplicate wells were harvested by trypsinization after 9 days and counted with a haemocytometer. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. *P<0.0005 (compared with control values). (D) Time course of TNF--induced cell proliferation. 2A4 cells were seeded in 16 mm wells at a concentration of 1x104 cells per well in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was changed and the cells were incubated for 4 days, 8 days, 12 days and 16 days with or without 10 ng ml-1 TNF-. Cells in triplicate wells were harvested and counted at the indicated time points. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. *P<0.0005 (compared to controls at 4 days). (E,F) Assay of anchorage-independent growth. 2A4 cells were suspended at 3x104 cells ml-1 in agarose gels and grown for 14 days in the absence (E) or presence (F) of TNF-. Scale bar, 200 µm. (G) Quantification of colony formation. Three randomly selected fields per experimental condition were photographed, and the number of colonies greater than 40 µm was determined in each photographic field. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. *P<0.025; **P<0.001 (compared with control values).

View larger version (114K): [in a new window]   Fig. 5. TNF- disrupts contact-mediated inhibition of cell proliferation. 2A4 cells were seeded at saturation density within a glass cylinder placed in the centre of a 22 mm well. After 24 hours, the cylinder was removed and the monolayer was incubated with or without TNF-. (A) When left untreated for 9 days, the initial cobblestone-like monolayer of 2A4 cells remain strictly contact inhibited. (B) Over the same time period, 2A4 cells treated with 5 ng ml-1 TNF- have formed thick ridges overlying the initial monolayer. (C) Thin section perpendicular to the plane of a control culture showing that the cells grow as a single layer. (D) Thin section perpendicular to the plane of a culture treated with 3 ng ml-1 TNF-. The cells pile up forming multiple cell layers. Scale bars, 200 µm (A,B); 10 µm (C,D).

View larger version (137K): [in a new window]   Fig. 6. TNF--induced cell scattering is mediated by TNF-R1. (A,B) 2A4 cells were seeded in plastic wells in DMEM-F12 medium supplemented with ITS+ Premix and grown for 24 hours. The cultures were then incubated for 2 hours with 40 µg ml-1 hamster monoclonal antibodies to either mouse TNF-R1 or TNF-R2 before addition of 10 ng ml-1 TNF-. Pictures were taken 72 hours later. TNF--induced scattering is blocked by the anti-TNF-R1 antibody (A) but not by the anti-TNF-R2 antibody (B). (C,D) 2A4 cells were suspended in a collagen gel in defined medium. Goat irrelevant IgG (C) or goat anti-TNF-R1 agonist antibody (50 µg ml-1) (D) were added both to the gel at the time of cell embedding and to the medium above the gel. The anti-TNF-R1 agonist antibody has induced 3D scattering of 2A4 cells (D). Scale bars, 10 µm.

View larger version (77K): [in a new window]   Fig. 7. TNF--induced 3D scattering and invasion of collagen gels require metalloproteinase activity. (A,B) 2A4 cells were grown in collagen gels in defined medium for 3 days and subsequently incubated for an additional 6 days with 10 ng ml-1 TNF- together with either the synthetic metalloproteinase inhibitor BB94 (1 µM) (B) or the inactive isomer BB1268 (3 µM) (A). Scale bars, 100 µm. (C) Quantification of the effect of increasing concentrations of BB94. 3D scattering is inhibited in a dose-dependent manner by BB94 (black columns) but is not affected by the inactive isomer BB1268 (open column). Quantification of 3D scattering was carried out after 6 days of treatment. Data represent the mean number of single cells per photographic field ± s.e.m. from at least three independent experiments. *P<0.01 compared with cultures incubated with TNF- alone. (D) Inhibition of collagen-gel invasion by BB94. 2A4 cells were plated onto a collagen gel and subsequently incubated with 10 ng ml-1 TNF- in the presence of either BB94 or the inactive isomer BB1268. 5 days later, invasion was quantified by counting the number of all cellular structures (i.e. apparently single cells and cell cords) present in a photographic field at a focal plane beneath the surface monolayer. *P<0.05 compared with cultures incubated with TNF- alone; **P<0.0005 compared with cultures incubated with TNF- alone. (E) By gelatine zymography, proteolytic activity corresponding to the molecular weight of MMP-9 is not detectable in conditioned media from untreated 2A4 cells but is clearly present in conditioned media from TNF--treated cells. Conditioned media from murine J3B1A cells were used as a migration standard for MMP-9. The results illustrated were obtained with conditioned media collected from three independent experiments.

View larger version (65K): [in a new window]   Fig. 8. TNF--induced 3D scattering is dependent on 2ß1 integrin. (A-C) TNF- increases the adhesion of 2A4 cells to collagen-coated dishes. 2A4 cells were harvested from confluent cultures following a 48-hour preincubation in the absence (A) or presence (B) of 10 ng ml-1 TNF- in defined medium and were seeded into collagen- or gelatine-coated bacteriological dishes. After a 60 minute incubation at 37°C, nonadhering cells were washed away and adhered cells were fixed and photographed using an inverted photomicroscope. Nonspecific adhesion to dishes that had not been coated with collagen was negligible. Scale bars, 100 µm. (C) Quantification of cell adhesion. Five randomly selected fields were photographed in each dish and the number of attached cells per field was counted on positive prints. Results represent the mean number of cells per field ± s.e.m. from three independent experiments. Mean values were compared using Student's unpaired t test. *P<0.001 versus control values; **not significantly different from control values. (D,E) TNF- increases cell surface expression of the 2 integrin subunit. Cells were trypsinized and stained with monoclonal antibody against the mouse 2 subunit followed by secondary FITC-conjugated antibody, and were analysed by flow cytometry. (D) The plots show 2A4 cells incubated with monoclonal antibody against the 2 integrin subunit (grey profile) or secondary antibody alone (open profile) after 48 hours without (top) or with (bottom) treatment with 10 ng ml-1 TNF-. The flow-cytometric plots shown are representative of five independent experiments. (E) Cells were treated with TNF- and incubated with monoclonal antibody against 2-integrin subunit as described in D. Mean fluorescence intensities ± s.e.m. from five independent experiments were normalized to secondary antibody fluorescence. Mean values were compared using Student's unpaired t test. P<0.005 for values of TNF- versus control. (F-H) Blocking antibodies to ß1 integrins abrogate TNF--induced 3D scattering in a concentration-dependent manner. Cells were grown in collagen gels in defined medium for 3 days and were subsequently incubated for an additional 6 days with 10 ng ml-1 TNF- in the absence (F) or presence (G) of function-blocking antibody against ß1 integrin (10 µg ml-1). (H) Dose-response analysis of the effect of increasing concentrations of anti-ß1-integrin antibody. An isotype-matched irrelevant (anti-TNP) antibody was used as a control (open column). Quantification of 3D scattering was carried out after 6 days of treatment. Data represent the mean number of single cells per photographic field ± s.e.m. from at least three independent experiments. *P<0.025 compared with cultures incubated with TNF- alone; **P<0.0005 compared with cultures incubated with TNF- alone. (I,L) The 2ß1-integrin antagonist rhodocetin abolishes TNF--induced 3D scattering. (I) Cells were grown in collagen gels in defined medium for 3 days and subsequently incubated for an additional 6 days with 10 ng ml-1 TNF- in the presence of 200 nM rhodocetin. (L) Dose-response analysis of the effect of increasing concentrations of rhodocetin. *P<0.0125 for 50 nM rhodocetin compared with cultures incubated with TNF- alone. (F,G,I) Scale bars, 100 µm.

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