Breast Cancer Cells Induce a Pro-inflammatory Response to Mitigate Immune Mediation in a 3D Culture Model

Abstract

Background: Growth factors and cytokines mediate complex interactions between cells within the breast tumour microenvironment. In advanced cancer, an excess of regulatory T (T_REG) lymphocytes and lack of natural killer (NK) cells in tumour-infiltrating lymphocyte populations may reflect a shift to pro-tumorigenic adaptive immune mechanisms. To facilitate targeted assessment of the interactions between tumour and immune cells ex vivo, three-dimensional (3D) culture systems are able to better recapitulate the in vivo microenvironment, recreating the anatomy of tumours.

Materials and methods: We used 3D breast tumour models to determine morphological alterations, and the levels of secreted transforming growth factor-β (TGFβ) and induced cytokines. 3D luminal phenotype models and basal phenotype models were generated by culturing NK cells and CD4⁺CD25⁺ T_REG cells with MCF-7 cells and MDA-MB-231 cells respectively, in growth factor-reduced Matrigel. TGFβ was qualitatively assessed by immunolocalisation and cytokine data from culture supernatant was acquired with a multiplex cytokine assay. Traditional statistical analysis and principal component analysis were employed to unravel the cytokine response.

Results and conclusion: We identified that an interleukin-6 (IL6)-chemokine axis associated with TGFβ is primarily responsible for differences detected between breast cancer models, with luminal and basal phenotype tumours responding differentially to immune mediation. Identified cytokines are implicated in facilitating tumour cell subversion of immune cell function to promote an invasive phenotype. Moreover, the disruption of the extracellular matrix and failure to form well-differentiated tumour masses/networks is indicative of enhanced malignancy. Tumour cells are implicated in promoting a pro-inflammatory microenvironment to attenuate NK cell function and in inducing a pro-tumorigenic profile that is facilitated by T_REG lymphocytes.

Keywords: 3D culture; Breast cancer; cytokines; natural killer cells; regulatory T-lymphocytes.