Medium was replaced for standard culture medium to incubate overnight. Next day, SKBR3 cells were mixed with CFDA SE labeled selleck chemicals llc AT MSCs in a ratio 2,1 and plated onto 6 well plate for direct co culture. Doxorubicin at final concentration 50 ng ml was added to the respective wells one day later and cells were treated for 48 hrs. Apoptotic cells were stained with Phycoerythrin labeled Annexin V, dead cells were detected with DAPI viability dye. Cells were analyzed using BD CantoII cytometer equipped with FACSDiva program. FCS Express software was used for the evaluation. Statistical analysis Studies involving comparison between the two groups were analyzed by an unpaired Students t test in GraphPad Prism software. The value of p 0. 05 was considered statistically significant.
Results AT MSCs stimulate an EMT and mammosphere formation in the breast cancer cells SKBR3 Previously we have described that AT MSCs secrete a plethora of chemokines and growth factors which might affect the tumor cell behavior. When SKBR3 cells were maintained in MSC CM morphological changes in the majority of tumor cells could be observed. Very similar effect could be observed in the EGFP SKBR cells directly cocultured with the AT MSCs for 6 days. Cells shifted from the epithelial like cobble stone morphology to the spindle like fibroblastoid ap pearance. EGFP SKBR3 cells acquired mesenchymal like phenotype that resembled an epithelial to mesenchymal transition with scattered colony appearance and increased adherence. Up regulation of the EMT associated markers in MSC CM exposed EGFP SKBR3 cells was confirmed.
MSC CM treated tumor cells exhibited sig nificantly higher expression of EMT regulators TWIST, Snail1, Snail2, related genes SMA and fibroblast activating protein in compari son to unaffected EGFP SKBR3 cells. The EMT process was previously linked to contribute to increased stemness and an upregulation of Oct and Nanog was also de tected in MSC CM exposed EGFP SKBR3. Paracrine factors secreted by AT MSCs also substantially supported SKBR3 mammosphere formation. We hypothesized that it was due to stimulation of signa ling pathways downstream of receptor tyrosine kinases by MSCs secretome. Indeed, the pharmacological inhibition of phosphatidylinositol 3 kinase with specific in hibitor LY294002 or p38 mitogen activated protein kinase with inhibitor SB203580 prevented mammosphere formation in MSC CM.
selleck kinase inhibitor The viability of SKBR3 in MSC CM and standard culture con ditions was decreased to the same extent by these inhibi tors. Paracrine signaling and migration of SKBR3 cells is influenced by AT MSCs In order to further characterize the intercellular cross talk, we analyzed a cytokine secretion pattern in the SKBR3 MSCs cocultures. Detectable levels of IL 5, IL 7, IL 10, GM CSF, IFN and MIP 1a could be measured in the medium from the cocultured cells. These chemokines were below detectable level in the SKBR3 or MSC CM medium.