Hi-C profiling of cancer spheroids identifies 3D-growth-specific chromatin interactions in breast cancer endocrine resistance [RNA-Seq]

Organoids or spheroids have emerged as a physiologically relevant in vitro preclinical model to study patient-specific diseases. A recent study used spheroids of MCF10 cells to model breast cancer progression and identified targetable alterations more similar to those in vivo. Thus, it is practical and essential to explore and characterize the spheroids of the commonly used human breast cancer (BC) cells. This study conducted Hi-C analyses in three-dimensional (3D) spheroids of MCF10A, MCF7 and MCF7TR cells and compared TADs and looping genes with those in 2D monolayers. We found that chromatin domains and looping genes' strength have drastically changed during the 3D culture growth, although we identified very similar numbers of TADs and looping genes. We further identified novel 3D growth-specific looping genes within Hippo relevant pathways, of which two genes showed potential prognostic values in measuring the outcome of the endocrine treatment. We finally confirmed a few selected Hippo relevant pathways genes with enhanced looping in breast cancer patient tissues' organoid. Hence, our work has provided significant insights into our understanding of 3D-growth-specific chromatin architecture in tamoxifen-resistant breast cancer. Overall design: RNA-seq experiment of 3 cell types in 2D monolayers or 3D spheroids, 3 biological replicates for each condition.

类器官（Organoids）与球体（spheroids）已成为研究患者特异性疾病的生理相关体外临床前模型。近期一项研究利用MCF10细胞球体模拟乳腺癌进展，并鉴定出与体内状态更为相似的可靶向分子异常。因此，对常用人类乳腺癌（Breast Cancer, BC）细胞球体进行探索与特征解析，兼具实用性与必要性。本研究对MCF10A、MCF7及MCF7TR细胞的三维（3D）球体开展Hi-C分析，并将拓扑关联域（Topologically Associating Domains, TADs）与染色质环互作基因的特征与二维（2D）单层培养细胞进行对比。我们发现，尽管鉴定得到的TAD数量与染色质环互作基因数量相近，但三维培养过程中染色质结构域及染色质环互作基因的互作强度发生了显著变化。此外，我们在Hippo信号通路中鉴定出新型的3D生长特异性染色质环互作基因，其中两个基因在评估内分泌治疗预后方面具有潜在应用价值。最后，我们在乳腺癌患者组织类器官中验证了部分经筛选的、互作强度增强的Hippo通路相关基因。综上，本研究为理解他莫昔芬耐药乳腺癌中3D生长特异性染色质结构提供了重要见解。实验整体设计：对3种细胞类型分别开展二维单层培养与三维球体培养的RNA测序（RNA-seq）实验，每组设置3次生物学重复。