MSCs enhance 4T1 mammosphere formation and tumorigenicity.

<p>For the mammosphere formation assay, 4T1 cells were cultured in regular medium or MSC-CM for 1 week. After typsinization, a single-cell suspension was confirmed by microscopic observation. The cells were then seeded at 2000 cells/100 µl/well into 96 well ultra-low-attachment plates and cultured in mammosphere-forming medium. The number of spheres formed was counted after 1 week incubation. (a) Number of spheres formed from 2000 cells pre-cultured with RPMI-1640 or MSC-CM and counted from five wells. Cells pre-cultured in MSC-CM formed mammospheres more efficiently than those cultured in RPMI-1640 medium (<i>p</i> = 0.03). Data are presented as the mean ± SD. To evaluate the <i>in vivo</i> tumorigenic potential of 4T1 cell, a total of either 5, 10 or 100 RFP-expressing 4T1 cells alone or premixed with 1×10⁵ GFP-expressing MSCs, were injected into the mammary fat pad. Tumor incidence was observed and imaged. (b) Representative image of tumorigenesis. The white and black arrows indicate the sites where 4T1 cells mixed with MSCs or 4T1 cells alone were injected, respectively. Tumor growth was recorded and is shown in (b). (c) Summary of tumor formation when analyzed using limiting dilution.