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Differential mechanical response of mesenchymal stem cells and fibroblasts to tumor-secreted soluble factors.

McGrail DJ, Ghosh D, Quach ND, Dawson MR - PLoS ONE (2012)

Bottom Line: In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion.Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1.These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells--including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)--are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.

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Effect of TCM on Cell Migration.Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.
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pone-0033248-g007: Effect of TCM on Cell Migration.Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.

Mentions: Previous studies documented increased MSC migration toward chemotactic factors after 18–24 hours [23], [24], [35]. Our multiple particle tracking studies revealed dramatic changes in intracellular mechanics within 1 hour. To determine if these mechanical changes were correlated with increased mobility, the migration of control and treated cells toward CM or TCM was measured hourly for 3 hours using transwell inserts with 3 µm (Fig. 7A) or 8 µm (Fig. 7B) pores. Without chemotactic factors present, few MSCs and 3T3 fibroblasts migrated through 3 µm pores; however, when TCM was added, there was a significant increase in MSC migration within 3 hours (p<0.01, Fig. 7A). MSC migration through 8 µm pores was almost always significantly greater than 3T3 fibroblast migration (Fig. 7B–C)). MSCs also responded more rapidly to TCM, with increased MSC migration within 2 hours (p<0.001) and increased 3T3 fibroblast migration within 3 hours (p<0.01). To determine if mechanical changes exhibited by MSCs in the first hour were indicative of a more migratory phenotype, we pre-treated both cell types for one hour with TCM before seeding in 8 µm transwell inserts. Pre-treatment with TCM increased MSC and 3T3 fibroblast migration toward CM and TCM, though more significantly toward TCM (Fig. 7C). Statistical analysis also revealed synergistic effects of TCM pre-treatment and migration toward TCM for both cell types, suggesting that pre-treatment not only increases motility but also causes a preferential migration towards chemotactic gradients.


Differential mechanical response of mesenchymal stem cells and fibroblasts to tumor-secreted soluble factors.

McGrail DJ, Ghosh D, Quach ND, Dawson MR - PLoS ONE (2012)

Effect of TCM on Cell Migration.Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306382&req=5

pone-0033248-g007: Effect of TCM on Cell Migration.Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.
Mentions: Previous studies documented increased MSC migration toward chemotactic factors after 18–24 hours [23], [24], [35]. Our multiple particle tracking studies revealed dramatic changes in intracellular mechanics within 1 hour. To determine if these mechanical changes were correlated with increased mobility, the migration of control and treated cells toward CM or TCM was measured hourly for 3 hours using transwell inserts with 3 µm (Fig. 7A) or 8 µm (Fig. 7B) pores. Without chemotactic factors present, few MSCs and 3T3 fibroblasts migrated through 3 µm pores; however, when TCM was added, there was a significant increase in MSC migration within 3 hours (p<0.01, Fig. 7A). MSC migration through 8 µm pores was almost always significantly greater than 3T3 fibroblast migration (Fig. 7B–C)). MSCs also responded more rapidly to TCM, with increased MSC migration within 2 hours (p<0.001) and increased 3T3 fibroblast migration within 3 hours (p<0.01). To determine if mechanical changes exhibited by MSCs in the first hour were indicative of a more migratory phenotype, we pre-treated both cell types for one hour with TCM before seeding in 8 µm transwell inserts. Pre-treatment with TCM increased MSC and 3T3 fibroblast migration toward CM and TCM, though more significantly toward TCM (Fig. 7C). Statistical analysis also revealed synergistic effects of TCM pre-treatment and migration toward TCM for both cell types, suggesting that pre-treatment not only increases motility but also causes a preferential migration towards chemotactic gradients.

Bottom Line: In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion.Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1.These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells--including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)--are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.

Show MeSH
Related in: MedlinePlus