tumorsphere formation and alters expression of breast cancer stem cell-associated surface

8 In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and 9 in basal epithelial cell populations including gland-reconstituting mammary stem cells 10 (MaSC), with largely undefined functions. Here, we studied how vimentin deficiency affects 11 mouse mammary gland development. Our results demonstrate that in adult vimentin 12 knockout mice (Vim-/-) mammary ductal outgrowth is delayed. The adult Vim-/glands are 13 characterised by dilated ducts, an imbalance in the proportion of basal to luminal mammary 14 epithelial cells and a reduction in cells expressing Slug (Snai2), an established MaSC 15 regulator. All of these features are indicative of reduced progenitor cell activity. Accordingly, 16 isolated Vim-/mammary epithelial cells display reduced capacity to form mammospheres, 17 and altered organoid structure, compared to wt counterparts, when plated in a 3D matrix in 18 vitro. Importantly, altered basal epithelial cell number translates into defects in Vim-/19 mammary gland regeneration in vivo in cleared fat pad transplantation studies. 20 Furthermore, we show that vimentin contributes to stem-like cell properties in triple 21 negative MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces 22 tumorsphere formation and alters expression of breast cancer stem cell-associated surface 23 markers. Together, our findings identify vimentin as a positive regulator of stemness in the 24 developing mouse mammary gland and in breast cancer cells. 25 26