Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor 2 (HER2). The lack of dependence on estrogen by TNBC cells makes anti-estrogen chemotherapy ineffective. Compounding this, within solid tumors, differential blood supply creates an oxygen and nutrient gradient, providing cells close to the vasculature with a more hospitable environment, while those in the core are deprived. In the search for treatments that may display efficacy against such tumours, it is necessary to make use of in vitro systems that accurately depict the clinical setting. Traditional two-dimensional (2D) culturing fails to replicate this environment, however, they are commonly used when assessing biological activity of new chemical entities. Three-dimensional (3D) cultures, in the form of spheroids, should enact a similar gradient which includes the proliferative outer layer, a quiescent inner zone and a necrotic center. The aim of the study is to compare the growth characteristics of BT-20 triple-negative breast carcinoma cells in a traditional 2D culture to a 3D model established by the Department of Physiology, University of Pretoria.
BT-20 spheroids (40 000 cells/well) were grown using traditional culturing and the liquid overlay method for monolayer (2D) and spheroid (3D) cultures, respectively. Spheroid volume was assessed using light microscopy, while viability was visualized by live-dead staining. Metabolic capacity was determined using the resazurin cleavage assay. Protein content was determined using the bicinchoninic acid assay. Cytotoxicity of doxorubicin was determined in monolayers by sulforhodamine B staining after 72 h. Monolayer cultures and spheroids (day 4) were exposed to the IC25, IC50 and IC75 of doxorubicin for 72 h, after which protein content and acid phosphatase (APH) activity were determined using spectrophotometry, cellular kinetics by flow cytometry, and p53 expression detected by Western blot analysis.
BT-20 spheroids displayed structural integrity and viability over the growth period, with decreasing size and increasing numbers of membrane compromised cells (suggestive of necrosis) at Day 4. No necrosis was observed at Days 7 or 10. Due to spheroids compaction and lack of resorufin formation, metabolic activity could not be assessed accurately, highlighting the density of the spheroid as a potential contributor to reduced drug susceptibility. Neither spheroid protein content nor APH activity changed throughout the culturing period, while the monolayer cultures presented with higher values. Doxorubicin displayed an IC25, IC50 and IC75 of 1.4 _M, 3.6 _M and 11.75 _M respectively in monolayer cultures. Spheroid size, protein content and APH activity was affected only at the IC75, accompanied by an increase in the percentage of sub-G1-phase cells linked to a reduction in G1-phase cells. Lower doxorubicin concentrations resulted in increased spheroid size, protein content and APH activity. Expression of p53 was non-significantly increased after exposure to the IC25 of doxorubicin in both models, however, expression was lower in spheroids than in monolayers. Non-significant alterations to cell cycle kinetics was evident, with decreased G0/G1-phase cells, increased G2/M-phase cells and increased p53 expression, which suggest that a late cell cycle blockade was induced. In addition, the non-significant lower expression of p53 in treated spheroids suggests that the 3D-conformation exhibited reduced chemosensitivity to doxorubicin.
Cultured 3D spheroids presented with higher resistance to doxorubicin compared to monolayer cultures. Given the nature of in vivo tumours, a 3D model as platform for drug screening may present as a more representative model during drug development studies.