Abstract 1802: Fibroblast-driven tamoxifen resistance in ER+ breast cancer: Experimental validation and development of a high-throughput co-culture screening assay.

Abstract New strategies are needed to overcome tamoxifen resistance in ER+ breast cancer. While the contribution of the tumor microenvironment to drug resistance remains incompletely understood, fibroblasts have emerged as potential therapeutic targets, due to their ability to modulate tumor behavior and drug sensitivity via paracrine signaling, extracellular matrix remodeling, and immune regulation. This study aimed to validate the protective role of fibroblasts against tamoxifen and to establish a co-culture assay suitable for high-throughput drug screening. We first performed functional assays to assess fibroblast-mediated protection using human ER+ breast cancer (MCF-7) and human fibroblast (BJhTERT) cell lines. Flow cytometry experiments with the CD140 marker distinguished BJhTERT (CD140+) from MCF-7 (CD140-) in co-culture and revealed that tamoxifen induced close to 90% cell death in MCF-7 monocultures measured by a viability dye 7-AAD (7-aminoactinomycin D), whereas only 25% of MCF-7 cells died under co-culture conditions with BJhTERT. In parallel, qPCR analysis confirmed that tamoxifen downregulated proliferative (CCND1) and estrogen response (ESR1, PGR, TFF1, GREB1) genes. Building on these findings, we developed a high-throughput co-culture assay. MCF-7 and BJhTERT cells were transduced with mCherry and GFP lentiviral reporters, respectively, and seeded into 384-well plates at optimized densities (500 and 1,000 cells/well). Cells were treated with tamoxifen or DMSO, fixed at defined time points, and mCherry, GFP, and Hoechst nuclear stain signals were analyzed using high-content imaging. Sixteen hours post-seeding was chosen as the optimal time for treatment initiation. Over that time, MCF-7 monocultures showed a fold change of 1.3 (30% increase) in cell numbers relative to the initial count, whereas MCF-7 in co-culture showed a fold change of 1.9 (90% increase), indicating a significantly greater growth-promoting effect of fibroblasts. Tamoxifen at a concentration of 20 µM reduced MCF-7 cell numbers by 63% in monoculture versus 38% in co-culture, confirming the protective stromal effect. In conclusion, our experiments validate fibroblast-ER+ breast cancer crosstalk as a mechanism of tamoxifen resistance. Furthermore, the development of a co-culture-based high-throughput assay, that models fibroblast-mediated tamoxifen resistance, provides a platform for novel screening strategies aimed at targeting the tumor-stroma interactions in ER+ breast cancer. Citation Format: Elisabet Rodríguez-Tomàs, Francesco Massai, Arne Östman. Fibroblast-driven tamoxifen resistance in ER+ breast cancer: Experimental validation and development of a high-throughput co-culture screening assay abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1802.