SV40 large T antigen-transformed human primary normal and cancerous mammary epithelial cells are phenotypically similar but can be distinguished in 3D culture with selection medium.

Human normal mammary epithelial cells (NMECs) have 2 major in vitro growth restrictions, senescence and crisis. Cellular immortalization is considered a hallmark of malignancy. However, cancerous mammary epithelial cells (CMECs) that are thought to have passed growth barriers in vivo usually cannot be established long-term in vitro. Here we show that CMECs deprived of their natural environment and grown in conventional complete medium behave similar to NMECs, e.g., they stop producing telomerase and become senescent. Like NMECs, CMECs are rescued by SV40 large T (LT) from senescence but not from crisis. The telomere length of both LT-transformed NMEC (N-LT) and CMEC (C-LT) cells first shortens but later partially recovers after telomerase activation. Both cell types upregulate ErbB2 expression, acquire genetic changes, remain long-term dependent on LT and ErbB2 and are nontumorigenic. Despite these similarities, N-LT and C-LT cells cultured in selection medium show different growth characteristics in 3D culture and in vivo tumorigenesis. Thus, CMECs are under a comparable in vitro selective pressure in conventional monolayer culture as NMECs despite their in vivo malignancy. This data demonstrate that most primary breast cancer cells are still unable to overcome the in vitro growth restrictions and suggest that the relationship of in vitro immortalization and in vivo carcinogenesis should be re-evaluated.