Short-term primary culture of epithelial cells derived from human breast tumours

Abstract

Summary As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cellenriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17p-hydroxysteroid dehydrogenase type 1. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.

Production of VEGF and expression of the VEGF receptors Flt-1 and KDR in primary cultures of epithelial and stromal cells derived from breast tumours

“…Because of the nature of our cell separation prcedure combined with the high sensitivity of the PCR technique, it was possible that we may have been amplifying Flt-1 and KDR message from occult endothelial cells rather than epithelial cells or fibroblasts. However, we have previously characterized these cultures by immunocytochemistry and flow cytometry (Speirs et al, 1998). More importantly, using RT-PCR for the endothelial marker factor VIII, we were unable to detect a positive signal in breast cancer cell cultures, confirming the absence of stray endothelial cells in our epithelial and stromal populations.…”

“…Individual epithelial and stromal preparations were isolated using a differential centrifugation method followed by culture in selective media (Speirs et al, 1996b) and were previously fully characterized by immunostaining, flow cytometry, gene expression and enzyme assay (Speirs et al, 1998). Clinicopathological details of the tumours from which the cultures were established are presented in Table 1.…”

In vivo and in vitro expression of steroid-converting enzymes in human breast tumours: associations with interleukin-6

“…However, we also observed the gene in breast primary cultures. These cultures have been fully characterized using a number of cellular and genetic markers and under the culture conditions described are unlikely to contain adipocytes (Speirs et al, 1996b(Speirs et al, , 1998a. Furthermore, the observation of 17-HSD type III expression in human pituitary adenomas (Green et al, 1999) and in a human colonic carcinoma cell line (English et al, 1997) lends further support for an active role of 17-HSD type III in steroid metabolism.…”

“…Tumours were digested overnight in 0.1% collagenase III (Life Technologies; Speirs et al, 1996a). The tissue digest was separated into epithelial or stromal fractions by differential centrifugation followed by culture in selective medium as previously described (Speirs et al, 1996b(Speirs et al, , 1998a. Cell were lysed in situ by the addition of Trizol directly to the culture dishes and the resulting RNA reverse transcribed into cDNA, which was amplified by PCR as outlined above.…”

Estrogen receptor β acts as a dominant regulator of estrogen signaling

“…The concentrations of genistein required for activation of ERs and in particular ERb are well within the range of what can be measured in the circulation of individuals on a diet rich in iso¯avonoids. The presence of ERb protein and mRNA in normal human breast tissue as well as in tumor samples suggests a putative role for ERb in the development of breast cancer (Dotzlaw et al, 1999;Leygue et al, 1998;Sasano et al, 1999;Speirs et al, 1999). Measurement of ERa has long been used as a diagnostic tool in the clinical evaluation of breast tumors.…”