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Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

Cox RF, Jenkinson A, Pohl K, O'Brien FJ, Morgan MP - PLoS ONE (2012)

Bottom Line: It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells.Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media.The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

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Investigating the effect of increasing concentrations of β-glycerophosphate on 4T1 cell mineralization.Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC, 5 mM βG and 10 mM βG treated groups, beginning on days 14, 28 and 14 respectively. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, 5 mM βG and 10 mM βG treated groups on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay and normalized to protein. Increases in calcium were observed in the OC and 10 mM βG treated groups over time. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. βG = β-glycerophosphate.
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pone-0041679-g002: Investigating the effect of increasing concentrations of β-glycerophosphate on 4T1 cell mineralization.Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC, 5 mM βG and 10 mM βG treated groups, beginning on days 14, 28 and 14 respectively. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, 5 mM βG and 10 mM βG treated groups on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay and normalized to protein. Increases in calcium were observed in the OC and 10 mM βG treated groups over time. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. βG = β-glycerophosphate.

Mentions: 4T1 cells were grown in culture plates for 28 days in the presence of regular growth media (control), the osteogenic cocktail (OC; 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid), and increasing concentrations of β-glycerophosphate (βG; 2 mM, 5 mM and 10 mM). Positive staining for calcium was detected in the 10 mM βG group beginning on day 14 using alizarin red S (Figure 2A), with the intensity of the stain increasing over time up to day 28. Faint positive staining was detected in the 5 mM and 2 mM βG groups by day 28. These results were also confirmed using von Kossa staining, as shown by the day 28 representative images (Figure 2B). Positive staining for calcium phosphate (black/brown) was observed in the 5 mM and 10 mM βG group by this time point. A calcium assay was also used to quantify the results (Figure 2C). The greatest increase in calcium levels over time was observed in the OC group (P<0.001 vs. control on days 14, 21 and 28). In addition, by day 28 an 80-fold increase in calcium levels was detected in the 10 mM βG group, a 14-fold was observed in the 5 mM βG group and a 3.5-fold increase was detected in the 2 mM βG group, indicating a dose response. No changes in calcium normalized to protein were detected in the control group over time.


Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

Cox RF, Jenkinson A, Pohl K, O'Brien FJ, Morgan MP - PLoS ONE (2012)

Investigating the effect of increasing concentrations of β-glycerophosphate on 4T1 cell mineralization.Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC, 5 mM βG and 10 mM βG treated groups, beginning on days 14, 28 and 14 respectively. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, 5 mM βG and 10 mM βG treated groups on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay and normalized to protein. Increases in calcium were observed in the OC and 10 mM βG treated groups over time. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. βG = β-glycerophosphate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404045&req=5

pone-0041679-g002: Investigating the effect of increasing concentrations of β-glycerophosphate on 4T1 cell mineralization.Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC, 5 mM βG and 10 mM βG treated groups, beginning on days 14, 28 and 14 respectively. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, 5 mM βG and 10 mM βG treated groups on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay and normalized to protein. Increases in calcium were observed in the OC and 10 mM βG treated groups over time. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. βG = β-glycerophosphate.
Mentions: 4T1 cells were grown in culture plates for 28 days in the presence of regular growth media (control), the osteogenic cocktail (OC; 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid), and increasing concentrations of β-glycerophosphate (βG; 2 mM, 5 mM and 10 mM). Positive staining for calcium was detected in the 10 mM βG group beginning on day 14 using alizarin red S (Figure 2A), with the intensity of the stain increasing over time up to day 28. Faint positive staining was detected in the 5 mM and 2 mM βG groups by day 28. These results were also confirmed using von Kossa staining, as shown by the day 28 representative images (Figure 2B). Positive staining for calcium phosphate (black/brown) was observed in the 5 mM and 10 mM βG group by this time point. A calcium assay was also used to quantify the results (Figure 2C). The greatest increase in calcium levels over time was observed in the OC group (P<0.001 vs. control on days 14, 21 and 28). In addition, by day 28 an 80-fold increase in calcium levels was detected in the 10 mM βG group, a 14-fold was observed in the 5 mM βG group and a 3.5-fold increase was detected in the 2 mM βG group, indicating a dose response. No changes in calcium normalized to protein were detected in the control group over time.

Bottom Line: It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells.Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media.The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

Show MeSH
Related in: MedlinePlus