Characterization of Estrogen Receptor Mutant Breast Cancer Three-Dimensional Cell Cultures

Background: Breast cancer is one of the leading types of cancer among women globally. Many primary breast cancers are estrogen receptor-positive (ER+) and responsive to anti-estrogenic therapies; however, after long-term treatment, these tumors can mutate the estrogen receptor to survive. These mutations make the tumors more triple-negative-like and, therefore, more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D) monolayer cell culture, which does not translate effectively in animal models and humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with actual tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they first must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Three different MCF-7 ER+ mutant cells (D538G, E380Q, and Y537S) were seeded at a density of 3000 cells per well in a low-attachment, round-bottomed 96-well plate. After seven days of culture, spheroids were imaged. Spheroids were measured, and size differences were quantified compared to the control, wild-type, ER+ MCF-7 cell line. RNA was extracted from spheroids, quantified, and reverse transcribed to make cDNA. cDNA was used to perform qRT-PCR to determine baseline gene expression differences between ER+ control MCF-7s and ER+ mutant MCF-7 cell lines. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways in the mutant lines. These 3D cultures also vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why these mutants tend to behave in a more triple-negative manner in humans. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture to identify future therapies – cells behave differently in different culturing contexts, 3D being more consistent with actual tumor behavior. Therefore, characterizing ER+ mutants before beginning drug treatment studies is crucial to understanding how compounds affect cancer cells and identifying differences in different ER+ mutants to know better how to treat them in the future.