Macrophage Mediation of Stemness in Hormone-Dependent Breast Cancer Cells under Combined Tamoxifen and Cannabidiol Treatment

Abstract

Late tumour recurrence and drug resistance remain obstacles in the treatment of hormone- dependent breast cancers. A possible limiting factor to the efficacy of standard therapeutic agents, such as Tamoxifen, is the propensity of breast tumour cells to convert between stem- like and differentiated states. This plasticity is influenced by macrophages in the tumour microenvironment, which can polarise into either anti-tumorigenic (M1) or pro-tumorigenic (M2) phenotypes, with evidence to suggest breast tumour progression is reliant on the interactions of tumour stem cells and M2 macrophages. It has been postulated that Cannabidiol (CBD) can mitigate these pro-tumorigenic interactions by inhibiting M2 macrophage polarisation. However, CBD is often administered with standard therapeutic agents, and the effects of combination treatment on macrophage polarisation and breast cancer stemness remains unclear. Thus, the present study set out to investigate the effects of Tamoxifen and CBD co-treatment on macrophage mediation of stemness in breast tumour cells in vitro. In brief, stem-like (CD44+CD133+) and non-stem-like (CD44-CD133-) breast cancer cells were magnetically isolated from an MCF7 cell line parent population. After culture in a Modified Essential-8 medium (ME-8), cells were phenotypically assessed for the expression of stemness markers CD44, CD133 and CD24 using flow cytometry. A co-culture system of MCF7 cells (parent, stem-like or non-stem-like) and RAW 264.7 macrophages was established and treated with combined Tamoxifen and CBD; the concentration, duration and administration model of which was optimised. The phagocytic capacity and nitrite production of macrophages was evaluated as measures of macrophage function. Corresponding alterations in genes associated with the cytoskeleton and autophagy, as indirect measures of epithelial-to-mesenchymal transition (EMT), were assessed in MCF7 cells. Results indicated that Tamoxifen and CBD co-treatment promoted M0 RAW 264.7 macrophage polarisation towards an M1-like phenotype, enhancing their capacity for phagocytosis and nitric oxide synthesis. Moreover, M0 RAW 264.7 macrophages and Tamoxifen and CBD co-treatment altered the expression of β-actin, vimentin and beclin-1 in MCF7 cells whereby gene expression was enhanced in parent and non-stem-like MCF7 cells, and reduced in stem-like MCF7 cells thus reflecting changes in their cytoskeletal dynamics and autophagic processes. The findings of this study affirm a potential role for combined Tamoxifen and CBD in anti- tumour immunity that warrants further exploration.