Figure 2
Increased migration of breast cancer cells due to the changes in a paracrine signaling and gene expression in MSC-CM-exposed SKBR3. A) Paracrine signaling in tumor and stromal cell cocultures. AT-MSCs were directly cocultured with EGFP-SKBR3 for 2 days and the cytokine levels were evaluated by human Bio-PlexTM 27-plex Cytokine Assay. Direct coculture of the tumor and stromal cells resulted in induction of IL-5, IL-7, IL-10, GM-CSF, IFN-γ and MIP-1a (‡). EGFP-SKBR3 or AT-MSCs alone did not produce detectable levels of these cytokines. Levels of cytokines IL-4, IL-9, eotaxin, IP-10, MCP-1 were significantly higher in comparison to the theoretically calculated additive value of EGFP-SKBR3 and AT-MSCs alone. Values were calculated as means of two independent experiments performed in duplicates, *p < 0.05, **p < 0.01. B) Expression analysis demonstrated the induction of VEGFR2 and c-Kit receptor expression in MSC-CM exposed EGFP-SKBR3 cells for 6 days. The expression of EGFR1, VEGFA, SCF and c-Met was detected in EGFP-SKBR3 cells. Induced expression of VEGFR2 and c-Kit was detected in MSC-CM cultured EGFP-SKBR3. Representative outcome is shown; the experiments were repeated at least three times with different MSCs isolates and similar outcome. C) EGFP-SKBR3 cells exhibited increased migration in the presence of MSC-CM as evaluated by live-cell imaging in a scratch wound assay. Confluent monolayers of EGFP-SKBR3 cells were wounded and the migration in the presence of MSC-CM or standard culture medium was observed for 72 hrs. Quantitative evaluation of a relative wound density demonstrated the capability of the secreted soluble factors by AT-MSCs to significantly increase the migration of tumor cells. Data are expressed as means of three independent measurements each run in quadruplicates ± SD. D) Increased migration of EGFP-SKBR3 in MSCs-CM could be significantly inhibited by 200 nM Sunitinib (VEGFR2, PDGFRβ and c-Kit inhibitor); and not by 150 nM Pazopanib (multi-target kinase inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFR, FGFR, c-Kit and c-Fms) or 250 nM Sorafenib (VEGFR-2, Raf-1 and B-Raf inhibitor).