Unmasking epithelial-mesenchymal transition in a breast cancer primary culture: a study report

Luigi Minafra,Valentina Bravatà,Massimo Viola,Cristina Messa,Rossana Norata,Cecilia Gelfi,Carmelo Lupo

doi:10.1186/1756-0500-5-343

Abstract

BackgroundImmortalized cancer cell lines are now well-established procedures in biomedicine for a more complete understanding of cellular processes in cancer. However, they are more useful in preparation of fresh tumour tissue, in order to obtain cancer cells with highly preserved individual tumour properties. In the present study we report an analytical investigation on a breast cancer primary cell culture isolated from a surgical specimen obtained from a patient with an infiltrating ductal carcinoma. The objective of the research was to reveal unrecognized aspects of neoplastic cells, typical of the tumour from where the cells were derived, but masked in fixed tissue sections, in order to better predict the aggressive potentiality of the tumour.FindingsUsing a combination of mechanical and enzymatic treatment, the tumour tissue was dissociated immediately after surgical removal. The primary cells were isolated by differential cell centrifugation and grown in selective media. Immunocytochemistry and quantitative RT-PCR analysis were performed to detect the presence of specific biomarkers at protein and transcript level.The isolated primary breast cancer cells displayed phenotypic behaviour, characteristic of malignant cells and expression of several mesenchymal markers, revealing a strong signature for the epithelial-to-mesenchymal transition associated to a stellate morphology with a number of cellular protrusions and the attitude to overgrow as multilayered overlapping cellular foci.ConclusionsOur data are a further meaningful indication that primary cell cultures represent a powerful system that could be applied to those cases deserving a deeper investigation at molecular level in order to design individualized anticancer therapies in the future.

Highlights

Immortalized cancer cell lines are well-established procedures in biomedicine for a more complete understanding of cellular processes in cancer
The medium composition was the following: DMEM with antibiotics supplemented with 10 mM Hepes (Invitrogen) and 1 mg/ml bovine serum albumin (BSA), 10 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 5 ng/ml epidermal growth factor (EGF), 5 μg/ml apo-transferrin
Isolation and in vitro expansion of primary cells from the breast tumour specimen Primary tumour cells were isolated from breast tissue of a patient with an infiltrating ductal carcinoma (IDC) and with the following histologic features: moderately differentiated, G2, pT2, pN1a, MIB-1 (KI-67) proliferation index 21%, Estrogen receptor (ER)+/Progesterone Receptor (PR)+, HER2

Summary

Introduction

Immortalized cancer cell lines are well-established procedures in biomedicine for a more complete understanding of cellular processes in cancer They are more useful in preparation of fresh tumour tissue, in order to obtain cancer cells with highly preserved individual tumour properties. Histological and molecular classification of breast cancer have revealed at least four major subgroups: 1) basal-like: estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), HER2negative (HER2-); 2) luminal A: estrogen receptor-positive (ER+, low grade); 3) luminal B: ER+, high grade; 4) HER2positive (HER2+) [1]. All these subgroups generally correspond to different patients’ prognoses and responsiveness to therapy. These parameters appear still insufficient to give a reliable prevision of the clinical outcome of the individual patient

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