Sphere formation reverses the metastatic and cancer stem cell phenotype of the murine mammary tumour 4T1, independently of the putative cancer stem cell marker Sca-1

Breast cancer stem cells (BCSCs) initiate and sustain breast cancers, and several putative markers have been proposed to prospectively isolate BCSC from the non-cancer stem cell population. The candidate BCSC marker Sca-1 is a GPI-linked membrane protein expressed on activated lymphocytes, hematopoietic stem cells and mammary stem cells. Sca-1+ cells were purified from the murine mammary tumour cell line 4T1. However, this did not enrich for a stem-like, tumour initiating or metastatic cell population in vitro or in vivo. Sphere formation, which induced high levels of Sca-1, reduced BCSC gene expression with near complete loss of spontaneous metastasis from sphere-derived tumours. This was associated with decreased expression of TGFB2 and reduced activation of the TGFβ signalling pathway in spheres. Both TGFB2 expression in vitro and spontaneous metastasis in vivo could be restored upon re-differentiation of sphere cells by exposure to serum, and this occurred with retention of the majority of Sca-1 expression. We conclude that while putative BCSC, including spheres, can have high Sca-1 expression, Sca-1 itself is not a marker of BCSC in established 4T1 tumours or the cell line.