9 Low-grade tumours (P n = 5; S n = 4) 9.5 High-grade tumours (P n = 7, S n = 
23.8 where P = proliferation assays, S = senescence assays. The ratio of proliferation:senescence was calculated for non-tumour, low grade tumour and high grade tumour primary cultures using the slope of proliferation learn more graphs and senescence values from Figure 2B. An increased ratio was observed in the stepwise progression from non-tumour to low grade tumour to high grade tumour categories. Alterations in putative progenitor cell subpopulations
correlate with aggressive tumours Since progenitor cells control the generation of new cells in a tissue, we questioned if alterations in progenitor populations could distinguish between aggressive and non-aggressive tumours. Several pieces of evidence suggested the presence of progenitors in primary cultures. Firstly, tumour and non-tumour cultures exhibited epithelial and myoepithelial co-differentiation (Figure 1). Secondly, they expressed the myoepithelial marker p63 (Figure 1C) which is also a progenitor marker [11]. Thirdly, selleck kinase inhibitor filter-grown cultures had basal electron-lucent, glycogen-rich cells (Figure 3a arrow) resembling those described as progenitor/stem cells in mammary duct basal GSK2245840 in vitro laminae [6]. Apically-located cells were attenuated and squamous-differentiated (Figure 3b , top arrow). Layering of dark filament-rich cells (Figure 3b arrows) with light glycogen-rich cells (Figure 3b arrowhead)
was observed in all cultures (Figure 3c). Figure 3 Ultrastructural identification of putative progenitor (-)-p-Bromotetramisole Oxalate cells in primary cultures. HMEC and tumour primary cultures
analyzed by TEM were observed to grow as multi-layers, with basally-located cells having plump morphologies (a, arrow) compared to the attenuated morphologies of apically-located cells. Filament-rich cells (b, arrows) were layered with glycogen-rich cells (b, arrowhead). A schematic representation of cellular organization is shown in (c). Flow cytometry was used to isolate putative progenitor populations from primary cultures and search for links with clinicopathological evidence of tumour progression. Non-tumour and tumour cultures were analyzed for expression of CALLA (myoepithelial) and EPCAM (epithelial) markers [4, 12]. All cultures had highest expression of CALLA and lowest expression of EPCAM single-positive cells, with double-negative (DN) populations exceeding double-positive (DP). Results were grouped according to clinicopathological factors of prognostic relevance, namely tumour grade and expression of ER and HER2 (Figure 4A). The DP population was significantly reduced in aggressive HG relative to LG tumour or non-tumour cultures (p < 0.05), while the CALLA population increased significantly. Both DN and EPCAM populations decreased slightly with increasing grade. Trends were similar in aggressive ER-negative tumour cultures, but not statistically significant.