Spatial characterization of the BT-20 triple negative breast cancer spheroidal model

Abstract

Breast cancer is a commonly diagnosed cancer in women, with increasing diagnosis and mortality rates. The triple-negative sub-type of the disease is characterised by lack of hormone-receptor overexpression, exhibition of poorly characterised molecular aberrations, and treatment failure. Chemotherapy is the current mainstay; however, it fails to slow tumor progression, and this is partially attributed to the lack of characterisation of biological features that drive treatment failure. There is, therefore, a need to characterise the biological features of triple-negative breast cancer, in order to develop effective therapies against the disease. Researchers widely use monolayer cell culture in pre-clinical screening of anticancer drugs. However, the development of effective anti-cancer drugs is hampered by limitations inherent to these culture systems, as they insufficiently mimic the physiological characteristics of tumours in vivo. Spheroids have been suggested as a bridge to the gap between monolayers and animal models, as they combine the flexibility and cost-effectiveness of cell culture with the spatial and molecular attributes of tissue. This study aimed to grow and characterise a spheroid model of triple-negative breast cancer with regards to growth, morphology, and drug sensitivity. The hanging drop and liquid overlay techniques were compared to select a method for growing spheroids. Spheroid growth was assessed using phase contrast microscopy and the bicinchoninic acid assay. Viability was assessed using the fluorescein diacetate (FDA)/propidium iodide (PI) assay. Haematoxylin and eosin staining were used for morphological evaluation. An iridium complex was used to investigate the induction of hypoxia. The sulphorodamine B and acid phosphatase assays, FDA/PI staining and phase contrast microscopy were used to assess the 72-h cytotoxicity effects of doxorubicin in monolayers and spheroids. Immunostaining and optical clearing were used to visualise the spatial distribution of the Ki-67 antigen and cadherins in spheroids.