Purified malignant mammary epithelial cells maintain hormone responsiveness in culture

Abstract

Currently, the therapy for breast cancer is determined by immunohistochemical staining of the primary tumour for oestrogen receptor alpha (ERa). However, a proportion of ERa-positive patients fail to respond to tamoxifen and a proportion of ERa-negative patients show response. Here, we describe a novel procedure for the purification of malignant breast epithelial cells in an attempt to identify these patients at an early stage. Using this procedure, we are able to purify malignant cells to 490% purity as determined by immunohistochemical staining, cytology and fluorescent in situ hybridisation (FISH). While the malignant cells can be maintained in culture they do not proliferate in contrast to purified breast epithelial cells from reduction mammoplasties. Moreover, ERa and progesterone receptor (PR) expression is maintained in malignant cells, whereas normal epithelial cells rapidly lose ERa and PR. Functional studies were performed on the separated malignant cells in terms of their response to oestradiol and tamoxifen. Four out of the seven ERa-positive tumours showed a significant reduction in cell numbers after tamoxifen treatment compared to oestradiol, ERa negative tumours failed to show a response. We conclude that (a) it is possible to purify and maintain breast cancer cells for a sufficient period to permit functional studies and (b) ERa is retained in culture facilitating the use of these cells in studies of the mechanism of endocrine response and resistance in vitro.

FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer

“…Another anti-FoxM1 therapeutic strategy that originated from findings in cancer cell lines was that the transcription activity of FoxM1 can be inhibited by the tumour suppressor p14ARF (p19ARF in mice), through a region mapped to residues 26-46 of the protein (43). In addition, administration of a cell-penetrating ARF [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] peptide to mice diminished FoxM1 function in vivo, causing selective apoptosis and reduced proliferation and angiogenesis in hepatocellular carcinomas (HCC) (44). Our data point to the intriguing possibility that the concomitant targeting of FoxM1 and HER2 may provide additional levels of cell death, delay or circumvent the development of resistance, whilst retaining tumour-selectivity.…”

“…The two filtrates were refiltered with progressively smaller filter sizes (50-28 mm). The tumour epithelial cells were immunoaffinity purified using super-paramagnetic, polystyrene beads (Dynal Ltd., New Ferry, Wirral, UK) coated with a mouse IgG1 monoclonal antibody (MAb Ber-EP4) specific for two (34 and 39 kDa) glycopolypeptide membrane antigens (32). Cells were then cytospun for 5 min, air dried and stored at -20˚C.…”

“…As with the immunohistochemical stained samples, HER2 status of the purified epithelial cells from primary tumours were determined by fluorescent in situ hybridisation (FISH). Validation of the purified tumour cells was performed by staining with hematoxylin/eosin (H&E) or cytokeratins 8 and 18 (MAb CAM 5.2, Becton-Dickinson, Oxford, UK) (32).…”

A Novel HSV-1 Virus, JS1/34.5−/47−, Purges Contaminating Breast Cancer Cells From Bone Marrow

“…Based on these results, it was decided to use JS1/34. tumor cells were purified from fresh breast tumors using a purification protocol developed in our laboratories (27). Because the number of purified primary tumor cells obtained from fresh breast tumor specimens are limiting, primary tumor cells purified from five specimens were pooled and infected with JS1/34.5 À /47 À /GFP at MOI 0.1 and infectivity assessed by examining GFP expression using fluorescence microscopy.…”

“…Primary breast tumor cells were purified according to the method of Kothari et al (27). Briefly, fresh breast tumor tissue was cut up and digested at 37jC with type IA collagenase (1 mg/mL) in RPMI 1640 supplemented with 5% FCS and 2 mmol/L L-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 50 units/mL polymixin B, and 2.5 mg/mL amphotericin B for 2 to 5 hours.…”