Identifying new drug targets to overcome therapy resistance in ER+ breast cancer is essential for enhancing patient survival and quality of life. Cancer-associated fibroblasts have been suggested as therapeutic targets for overcoming treatment resistance in ER+ breast cancer, given their ability to influence cancer cells via the secretion of signaling molecules, matrix proteins, and immune evasion mechanisms. Here, we establish a co-cultured-based high-throughput screen format to identify drug candidates that either inhibit the pro-tumoral effects of fibroblasts or overcome fibroblast-mediated treatment resistance.

This assay utilizes a co-culture format incorporating MCF-7 and BJhTERT cell lines to effectively model ER+ breast cancer cells and human immortalized fibroblasts, respectively. Both cell lines were transfected with lentiviruses to express fluorescent proteins—mCherry in MCF-7 cells and GFP in BJhTERT cells. The cells were cultured in DMEM and seeded at different densities and ratios in mono- and co-culture into 384-well plates using a Multidrop liquid dispenser. At 5, 16, and 24 hours post-seeding, some plates were fixed with 4% PFA and stained with Hoechst nuclear dye, while others were treated with a selective estrogen receptor modulator (SERM) or left untreated as controls. Twenty-four hours after treatment, the plates were fixed, imaged, and analyzed using an Operetta CLS high-content analysis system. The analyses were conducted by quantifying MCF-7 and BJhTERT cell numbers based on Hoechst, GFP, and mCherry signals.

Optimal cell densities and ratios in 384-well plates were identified as 500 MCF-7 cells and 1000 BJhTERT cells per well in both mono- and co-culture. Sixteen hours post-seeding was found to be the most suitable time to initiate treatment and observe fibroblast-driven growth effects. During this period, MCF-7 cells in monoculture exhibited a fold change of 1.3, indicating a 30% increase in cell numbers relative to the initial count. In co-culture with fibroblasts, the fold change was 1.9, reflecting a 90% increase and significantly greater cell growth than in monoculture, highlighting the growth-promoting effect of fibroblasts on MCF-7 cells. Tamoxifen administration at a concentration of 20 µM led to a significant reduction in MCF-7 cell numbers, with a 63% decrease in monoculture compared to 38% in co-culture. This indicates that the drug was more effective at inducing cell death in MCF-7 cells grown alone than in those co-cultured with fibroblasts, demonstrating the protective influence of fibroblasts.

Our co-culture-based assay format is well-suited to reveal the influence of fibroblasts on drug resistance in ER+ breast cancer cells. Furthermore, this model system will enable us to identify new fibroblast-related drug targets through high-throughput screening.