PO.ET03.02 · 实验与分子治疗
成纤维细胞驱动的ER+乳腺癌tamoxifen耐药：实验验证与高通量共培养筛选试验的开发
Fibroblast-driven tamoxifen resistance in ER+ breast cancer: Experimental validation and development of a high-throughput co-culture screening assay
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
需要新策略来克服ER+乳腺癌的tamoxifen耐药。虽然肿瘤微环境对耐药的贡献仍未被充分理解，但成纤维细胞已成为潜在的治疗靶点，因为它们能够通过旁分泌信号传导、细胞外基质重塑和免疫调节来调控肿瘤行为和药物敏感性。本研究旨在验证成纤维细胞对tamoxifen的保护作用，并建立适用于高通量药物筛选的共培养试验。
我们首先进行功能实验，使用人ER+乳腺癌（MCF-7）和人成纤维细胞（BJhTERT）细胞系评估成纤维细胞介导的保护作用。使用CD140标志物的流式细胞术实验在共培养中区分了BJhTERT（CD140+）与MCF-7（CD140-），并揭示tamoxifen在MCF-7单培养中诱导接近90%的细胞死亡（用活力染料7-AAD（7-氨基放线菌素D）测定），而在与BJhTERT共培养条件下只有25%的MCF-7细胞死亡。同时，qPCR分析证实tamoxifen下调了增殖相关（CCND1）和雌激素应答（ESR1、PGR、TFF1、GREB1）基因。
基于这些发现，我们开发了一种高通量共培养试验。MCF-7和BJhTERT细胞分别用mCherry和GFP慢病毒报告基因转导，并以优化密度（每孔500和1,000个细胞）接种至384孔板。细胞用tamoxifen或DMSO处理，在设定时间点固定，并使用高内涵成像分析mCherry、GFP和Hoechst核染色信号。选择接种后16小时作为开始处理的最佳时间。在此期间，MCF-7单培养相对于初始计数显示出1.3的倍数变化（增加30%），而共培养中的MCF-7显示出1.9的倍数变化（增加90%），表明成纤维细胞具有显著更强的促生长作用。浓度为20 µM的tamoxifen在单培养中使MCF-7细胞数量减少63%，而在共培养中减少38%，证实了间质的保护作用。
总之，我们的实验验证了成纤维细胞-ER+乳腺癌串扰是tamoxifen耐药的一种机制。此外，基于共培养、模拟成纤维细胞介导tamoxifen耐药的高通量试验的开发，为旨在靶向ER+乳腺癌中肿瘤-间质相互作用的新型筛选策略提供了平台。
查看英文原文 English 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.
利益披露 Disclosure
E. Rodríguez-Tomàs, None..
F. Massai, None..
A. Östman, None.