title: Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.
abstract: 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.
authors.affiliation: School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa. Electronic address: tanya.augustine@wits.ac.za.
authors.forename: Tanya N
authors.initials: TN
authors.lastname: Augustine
authors.affiliation: School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa.
authors.forename: Thérèse
authors.initials: T
authors.lastname: Dix-Peek
authors.affiliation: School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa.
authors.forename: Raquel
authors.initials: R
authors.lastname: Duarte
authors.affiliation: School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa.
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Candy
mesh_terms: D014408:Biomarkers, Tumor / Q000302:isolation & purification / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology* / Q000473:pathology*; D002450:Cell Communication / Q000276:immunology*; D018929:Cell Culture Techniques; D054407:Chemokine CCL4 / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D066246:ErbB Receptors / Q000302:isolation & purification; D047628:Estrogen Receptor alpha / Q000302:isolation & purification; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D061986:MCF-7 Cells; D018396:Mucin-1 / Q000302:isolation & purification; D010641:Phenotype; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D059016:Tumor Microenvironment / Q000276:immunology
chemical_list: D014408:Biomarkers, Tumor; C066507:CCL4 protein, human; D054407:Chemokine CCL4; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D018396:Mucin-1; D066246:ErbB Receptors; D019286:Endopeptidase K
keywords: Breast cancer; Matrigel; Natural Killer cells; Regulatory T cells; Three-dimensional culture