Extensive abnormal vesiculation patterns were identified in the peri-nuclear regions of tumour versus non-tumour cultures (Figure 2A, VNT versus VT). Multi-nucleation of tumour cells Forskolin in vivo was frequently observed, in parallel with compromised nuclear membranes (Figure 2A, NMNT versus NMT). Furthermore, tumour cell mitochondria were abnormal, elongated and occasionally fused (Figure 2A, MNT versus MT). Finally, non-tumour cells displayed a well-differentiated rough endoplasmic reticulum (RER) while that in tumour

cells was fragmented and dispersed (Figure 2A, RNT versus RT). Figure 2 Ultrastructural and functional differences distinguish non-tumour from tumour primary cultures. A. TEM analysis of non-tumour cells revealed modest numbers of cytoplasmic vesicles (V nt ), single nuclei, distinct nuclear double membranes (NM nt ), regular mitochondria (M nt ) and well-organized RER (R nt ). Tumour cells showed abnormal peri-nuclear vesicles (V t ), >1 nucleus per cell with thin nuclear membranes (NM t ), abnormal mitochondria (M t ) and disorganized RER (R t ). B. Proliferation was enhanced in HG tumour cultures relative to LG tumour cultures or non-tumour

cultures (left). Kinase Inhibitor Library mw Basal senescence, estimated by SA-β-galactosidase staining, was lower in tumour versus non-tumour cultures (right; p < 0.001). We next investigated if morphological differences were accompanied by cell fate differences (Figure 2B). Proliferation abilities were assessed by Cyquant assay on 4 non-tumour cultures and 12 tumour cultures either – 5 low grade (LG, grade 1-2) and 7 high grade (HG, grade 3). Values were calculated relative to a standard curve of fluorescence intensity versus known cell numbers (Additional file 2). A significant increase in proliferation was observed in high grade tumour cultures (HG; grade 3) relative to non-tumour

or low grade tumour cultures (LG; grades 1-2; Figure 2B, left). Since Cyquant proliferation assays quantify all cells rather than just actively-proliferating cells, we performed senescence-associated (SA) β-galactosidase assays [9] to estimate growth arrest (Figure 2B, right). Non-tumour cultures had two-fold higher SA-β-galactosidase staining than that in tumour cultures. This was independent of the grade of the originating tumour, and did not reflect an impaired capacity to senesce in response to exogenous stimulation (data not shown). As the balance between proliferation and senescence is more important than either parameter alone, we examined whether altered proliferation:senescence ratios in breast primary cultures could identify aggressive tumours. The proliferation:senescence relationship was estimated based on proliferation graph slopes and senescence values (Figure 2B). Our data revealed a stepwise increase in proliferation:senescence ratio from non-tumour through LG and finally HG tumours, correlating with a simple model of tumour progression (Table 1).