DataSheet_1_Architectural organization and molecular profiling of 3D cancer heterospheroids and their application in drug testing.docx

3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals. However, most solid tumors are composed of multiple cell types, including fibroblasts. In this study we developed multicellular tumor heterospheroids composed of cancer and fibroblasts cell lines. We developed heterospheroids by combining HT-29, MCF-7, PANC-1 or SW480 with 1BR.3.G fibroblasts, which we have previously reported support spheroid formation. We also tested fibroblast cell lines, MRC-5, GM00498 and HIF, but 1BR.3.G was found to best form heterospheroids with morphological similarity to in vivo tumor tissue. The architectural organization of heterospheroids was based on histological examination using immunohistochemistry. We found that HT-29 and MCF-7 cells developed spheroids with the cancer cells surrounding the fibroblasts, whereas PANC-1 cells interspersed with the fibroblasts and SW480 cells were surrounded by fibroblasts. The fibroblasts also expressed collagen-1 and FAP-α, and whole transcriptomic analysis (WTA) showed abundant ECM- and EMT-related expression in heterospheroids, thus reflecting a representative tumor-like microenvironment. The WTA showed that PANC-1 heterospheroids possess a strong EMT profile with abundant Vimentin and CDH2 expression. Drug testing was evaluated by measuring cytotoxicity of 5FU and cisplatin using cell viability and apoptosis assays. We found no major impact on the cytotoxicity when fibroblasts were added to the spheroids. We conclude that the cancer cell lines together with fibroblasts shape the architectural organization of heterospheroids to form tumor-like morphology, and we propose that the various 3D tumor structures can be used for drug testing directed against the cancer cells as well as the fibroblasts.

三维癌细胞培养为替代实验动物中的化合物测试提供了新的机遇。然而，大多数实体肿瘤由多种细胞类型组成，包括成纤维细胞。在本研究中，我们开发了一种由癌细胞和成纤维细胞系组成的细胞异质球体。我们通过将HT-29、MCF-7、PANC-1或SW480与1BR.3.G成纤维细胞相结合，后者我们此前报道支持球体形成，来构建异质球体。我们还测试了成纤维细胞系MRC-5、GM00498和HIF，但发现1BR.3.G能够与体内肿瘤组织形成形态相似的异质球体。异质球体的建筑组织基于组织学检查，采用免疫组化方法。我们发现，HT-29和MCF-7细胞形成了球体，其中癌细胞围绕着成纤维细胞，而PANC-1细胞则与成纤维细胞混合分布，SW480细胞则被成纤维细胞所包围。成纤维细胞还表达胶原蛋白-1和FAP-α，整体转录组分析（WTA）显示异质球体中富含ECM和EMT相关表达，从而反映了典型的肿瘤样微环境。WTA显示，PANC-1异质球体具有强烈的EMT特征，Vimentin和CDH2表达丰富。药物测试通过测量5FU和顺铂的细胞毒性以及细胞活力和凋亡实验来评估。我们发现当将成纤维细胞添加到球体中时，对细胞毒性没有产生显著影响。我们得出结论，癌细胞系与成纤维细胞共同塑造了异质球体的建筑组织，形成了肿瘤样形态，并提出各种三维肿瘤结构可用于针对癌细胞及成纤维细胞的药物测试。