Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer

• Published in: Journal of immunological methods 2015-11, Vol.426, p.1-13
• Main Authors: Augustine, Tanya N., Dix-Peek, Thérèse, Duarte, Raquel, Candy, Geoffrey P.
• Format: Article
• Language: English
• Subjects: Biomarkers, Tumor - isolation & purification; Biomarkers, Tumor - metabolism; Breast cancer; Breast Neoplasms - immunology; Breast Neoplasms - pathology; Cell Communication - immunology; Cell Culture Techniques; Chemokine CCL4 - secretion; Endopeptidase K - pharmacology; Estrogen Receptor alpha - isolation & purification; Female; Flow Cytometry - methods; Humans; Killer Cells, Natural - immunology; Matrigel; MCF-7 Cells; Mucin-1 - isolation & purification; Natural Killer cells; Phenotype; Receptor, Epidermal Growth Factor - isolation & purification; Regulatory T cells; T-Lymphocytes, Regulatory - immunology; Three-dimensional culture; Tumor Microenvironment - immunology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2015.07.003

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell–cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines. •Developed a 3D culture system to study immune mediation of breast tumour progression•Natural Killer & regulatory T cells were cultured with tumour cell lines in Matrigel•3D model feasible for immunocytochemistry, RNA extraction & cytokine analysis•Tumour morphology, expression of biomarkers and CCL4 varied under immune mediation