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SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

Pasquier J, Abu-Kaoud N, Abdesselem H, Madani A, Hoarau-Véchot J, Thawadi HA, Vidal F, Couderc B, Favre G, Rafii A - BMC Cancer (2015)

Bottom Line: This interaction increases migration from primary sites as well as homing at distant sites.Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml.The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar. jep2026@qatar-med.cornell.edu.

ABSTRACT

Background: The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites.

Methods: Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases.

Results: We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion.

Conclusion: We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

No MeSH data available.


Related in: MedlinePlus

Differential effect of SDF-1alpha on MDA-MB231. a Adhesion assay testing the role of different concentration of SDF-1α. 50,000 eGFP MDA-MB231 were allowed to adhere for 1 h in presence or absence of SDF-1α (50, 100, 200 ng/ml). The maximum adhesion was observed at 200 ng/ml. b F-actin polymerisation in MDA-MB231. MDA-MB231 were grown on glass bottom slides with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling (red). More stress fiber and filipods can be seen (arrows) in MDA-MB231 treated with 50 or 100 ng/ml of SDF-1α. Scale bar 20 μm.c MDA-MB231 plasticity on Matrigel. MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel in presence or absence of SDF-1α (50, 100 or 200 ng/ml). Microscopic pictures of cellular networks after SDF-1α stimulation were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection (white) and network (black) are presented. The evaluation was made by counting on 10 different fields. The results are expressed as means with standard error. Interconnection and network number was increased when the cells are treated with 50 or 100 ng/ml of SDF-1α. d Wound closure assay. Migration ability of MDA-MB231 was tested after a scratch in presence of different concentration of SDF-1α (0, 50, 100 or 200 ng/ml). Only 50 and 100 ng/ml of SDF-1α enhanced MDA-MB231 motility. e Migration in agarose gel assay. MDA-MB231 cells were seeded in the central well. Media only was poured in the CTRL well as control and different concentration of SDF-1α were used in the right well for the 3 wells experiments (left panel) or simultaneity in the 5 wells experiments (right panel). Pictures were taken after 8 days and the distance travelled by the cells was calculated. The histograms present the results of 3 different experiments. f Cell cycle analysis. MDA-MB231 were treated with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. 50 and 100 ng/ml of SDF-1α increased the number of cells in phase S (green) and G2/M (purple) and decreased the one in G0/G1 (blue). The results presents in this figure are representative of three different experiments
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Fig3: Differential effect of SDF-1alpha on MDA-MB231. a Adhesion assay testing the role of different concentration of SDF-1α. 50,000 eGFP MDA-MB231 were allowed to adhere for 1 h in presence or absence of SDF-1α (50, 100, 200 ng/ml). The maximum adhesion was observed at 200 ng/ml. b F-actin polymerisation in MDA-MB231. MDA-MB231 were grown on glass bottom slides with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling (red). More stress fiber and filipods can be seen (arrows) in MDA-MB231 treated with 50 or 100 ng/ml of SDF-1α. Scale bar 20 μm.c MDA-MB231 plasticity on Matrigel. MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel in presence or absence of SDF-1α (50, 100 or 200 ng/ml). Microscopic pictures of cellular networks after SDF-1α stimulation were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection (white) and network (black) are presented. The evaluation was made by counting on 10 different fields. The results are expressed as means with standard error. Interconnection and network number was increased when the cells are treated with 50 or 100 ng/ml of SDF-1α. d Wound closure assay. Migration ability of MDA-MB231 was tested after a scratch in presence of different concentration of SDF-1α (0, 50, 100 or 200 ng/ml). Only 50 and 100 ng/ml of SDF-1α enhanced MDA-MB231 motility. e Migration in agarose gel assay. MDA-MB231 cells were seeded in the central well. Media only was poured in the CTRL well as control and different concentration of SDF-1α were used in the right well for the 3 wells experiments (left panel) or simultaneity in the 5 wells experiments (right panel). Pictures were taken after 8 days and the distance travelled by the cells was calculated. The histograms present the results of 3 different experiments. f Cell cycle analysis. MDA-MB231 were treated with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. 50 and 100 ng/ml of SDF-1α increased the number of cells in phase S (green) and G2/M (purple) and decreased the one in G0/G1 (blue). The results presents in this figure are representative of three different experiments

Mentions: We demonstrated that maximal adhesion was obtained at a SDF-1α concentration of 200 ng/ml (Fig. 3a). Confocal microscopy imaging of MDA-MB231 treated with SDF-1α revealed an increase of F-actin staining in the periphery of the cells at 50 and 100 ng/ml (Fig. 3b). Stress fibers and filopods formation required for the invasion of malignant cells into tissues, were observed only at a concentration of 50 and 100 ng/ml. We then evaluated the role of SDF-1α in cellular plasticity by quantifying network formation on matrigel after 24 h of culture (Fig. 3c). Matrigel assays allow rapid quantification of the relative invasive potential of metastatic cells [41]. In this assay non tumorigenic cells generally do not grow; while low metastatic tumor cells form large round colonies, while high metastatic cells form branching invasive colonies [42]. SDF-1α at 50 and 100 ng/ml increased the formation of intercellular connections while the 200 ng/ml treatment resulted in decrease branching. In a wound-healing migration assay, 50 and 100 ng/ml of SDF-1α induced maximal migration (Fig. 3d).Fig. 3


SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

Pasquier J, Abu-Kaoud N, Abdesselem H, Madani A, Hoarau-Véchot J, Thawadi HA, Vidal F, Couderc B, Favre G, Rafii A - BMC Cancer (2015)

Differential effect of SDF-1alpha on MDA-MB231. a Adhesion assay testing the role of different concentration of SDF-1α. 50,000 eGFP MDA-MB231 were allowed to adhere for 1 h in presence or absence of SDF-1α (50, 100, 200 ng/ml). The maximum adhesion was observed at 200 ng/ml. b F-actin polymerisation in MDA-MB231. MDA-MB231 were grown on glass bottom slides with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling (red). More stress fiber and filipods can be seen (arrows) in MDA-MB231 treated with 50 or 100 ng/ml of SDF-1α. Scale bar 20 μm.c MDA-MB231 plasticity on Matrigel. MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel in presence or absence of SDF-1α (50, 100 or 200 ng/ml). Microscopic pictures of cellular networks after SDF-1α stimulation were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection (white) and network (black) are presented. The evaluation was made by counting on 10 different fields. The results are expressed as means with standard error. Interconnection and network number was increased when the cells are treated with 50 or 100 ng/ml of SDF-1α. d Wound closure assay. Migration ability of MDA-MB231 was tested after a scratch in presence of different concentration of SDF-1α (0, 50, 100 or 200 ng/ml). Only 50 and 100 ng/ml of SDF-1α enhanced MDA-MB231 motility. e Migration in agarose gel assay. MDA-MB231 cells were seeded in the central well. Media only was poured in the CTRL well as control and different concentration of SDF-1α were used in the right well for the 3 wells experiments (left panel) or simultaneity in the 5 wells experiments (right panel). Pictures were taken after 8 days and the distance travelled by the cells was calculated. The histograms present the results of 3 different experiments. f Cell cycle analysis. MDA-MB231 were treated with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. 50 and 100 ng/ml of SDF-1α increased the number of cells in phase S (green) and G2/M (purple) and decreased the one in G0/G1 (blue). The results presents in this figure are representative of three different experiments
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Related In: Results  -  Collection

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Fig3: Differential effect of SDF-1alpha on MDA-MB231. a Adhesion assay testing the role of different concentration of SDF-1α. 50,000 eGFP MDA-MB231 were allowed to adhere for 1 h in presence or absence of SDF-1α (50, 100, 200 ng/ml). The maximum adhesion was observed at 200 ng/ml. b F-actin polymerisation in MDA-MB231. MDA-MB231 were grown on glass bottom slides with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling (red). More stress fiber and filipods can be seen (arrows) in MDA-MB231 treated with 50 or 100 ng/ml of SDF-1α. Scale bar 20 μm.c MDA-MB231 plasticity on Matrigel. MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel in presence or absence of SDF-1α (50, 100 or 200 ng/ml). Microscopic pictures of cellular networks after SDF-1α stimulation were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection (white) and network (black) are presented. The evaluation was made by counting on 10 different fields. The results are expressed as means with standard error. Interconnection and network number was increased when the cells are treated with 50 or 100 ng/ml of SDF-1α. d Wound closure assay. Migration ability of MDA-MB231 was tested after a scratch in presence of different concentration of SDF-1α (0, 50, 100 or 200 ng/ml). Only 50 and 100 ng/ml of SDF-1α enhanced MDA-MB231 motility. e Migration in agarose gel assay. MDA-MB231 cells were seeded in the central well. Media only was poured in the CTRL well as control and different concentration of SDF-1α were used in the right well for the 3 wells experiments (left panel) or simultaneity in the 5 wells experiments (right panel). Pictures were taken after 8 days and the distance travelled by the cells was calculated. The histograms present the results of 3 different experiments. f Cell cycle analysis. MDA-MB231 were treated with different concentration of SDF-1α (0, 50, 100 or 200 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. 50 and 100 ng/ml of SDF-1α increased the number of cells in phase S (green) and G2/M (purple) and decreased the one in G0/G1 (blue). The results presents in this figure are representative of three different experiments
Mentions: We demonstrated that maximal adhesion was obtained at a SDF-1α concentration of 200 ng/ml (Fig. 3a). Confocal microscopy imaging of MDA-MB231 treated with SDF-1α revealed an increase of F-actin staining in the periphery of the cells at 50 and 100 ng/ml (Fig. 3b). Stress fibers and filopods formation required for the invasion of malignant cells into tissues, were observed only at a concentration of 50 and 100 ng/ml. We then evaluated the role of SDF-1α in cellular plasticity by quantifying network formation on matrigel after 24 h of culture (Fig. 3c). Matrigel assays allow rapid quantification of the relative invasive potential of metastatic cells [41]. In this assay non tumorigenic cells generally do not grow; while low metastatic tumor cells form large round colonies, while high metastatic cells form branching invasive colonies [42]. SDF-1α at 50 and 100 ng/ml increased the formation of intercellular connections while the 200 ng/ml treatment resulted in decrease branching. In a wound-healing migration assay, 50 and 100 ng/ml of SDF-1α induced maximal migration (Fig. 3d).Fig. 3

Bottom Line: This interaction increases migration from primary sites as well as homing at distant sites.Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml.The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar. jep2026@qatar-med.cornell.edu.

ABSTRACT

Background: The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites.

Methods: Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases.

Results: We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion.

Conclusion: We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

No MeSH data available.


Related in: MedlinePlus