Ardila Diana Catalina, Aggarwal Vaishali, Singh Manjulata, Chattopadhyay Ansuman, Chaparala Srilakshmi, Sant Shilpa
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Health Sciences Library System, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Cancers (Basel). 2021 Mar 20;13(6):1429. doi: 10.3390/cancers13061429.
Collective cell migration is a key feature of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) among many other cancers, yet the microenvironmental factors and underlying mechanisms that trigger collective migration remain poorly understood. Here, we investigated two microenvironmental factors, tumor-intrinsic hypoxia and tumor-secreted factors (secretome), as triggers of collective migration using three-dimensional (3D) discrete-sized microtumor models that recapitulate hallmarks of DCIS-IDC transition. Interestingly, the two factors induced two distinct modes of collective migration: directional and radial migration in the 3D microtumors generated from the same breast cancer cell line model, T47D. Without external stimulus, large (600 µm) T47D microtumors exhibited tumor-intrinsic hypoxia and directional migration, while small (150 µm), non-hypoxic microtumors exhibited radial migration only when exposed to the secretome of large microtumors. To investigate the mechanisms underlying hypoxia- and secretome-induced directional vs. radial migration modes, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory microtumors compared with non-hypoxic, non-migratory small microtumors as controls. We propose unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration. Gene Set Enrichment Analysis (GSEA) and protein-protein interaction (PPI) network analysis revealed enrichment and potential interaction between hypoxia, EMT, and migration gene signatures for the hypoxia-induced directional migration. In contrast, hypoxia and EMT were not enriched in the secretome-induced radial migration, suggesting that complete EMT may not be required for radial migration. Survival analysis identified unique genes associated with low survival rate and poor prognosis in TCGA-breast invasive carcinoma dataset from our tumor-intrinsic hypoxia gene signature (CXCR4, FOXO3, LDH, NDRG1), hypoxia-induced EMT gene signature (EFEMP2, MGP), and directional migration gene signature (MAP3K3, PI3K3R3). NOS3 was common between hypoxia and migration gene signature. Survival analysis from secretome-induced radial migration identified ATM, KCNMA1 (hypoxia gene signature), and KLF4, IFITM1, EFNA1, TGFBR1 (migration gene signature) to be associated with poor survival rate. In conclusion, our unique 3D cultures with controlled microenvironments respond to different microenvironmental factors, tumor-intrinsic hypoxia, and secretome by adopting distinct collective migration modes and their gene expression analysis highlights the phenotypic heterogeneity and plasticity of epithelial cancer cells.
集体细胞迁移是导管原位癌（DCIS）向浸润性导管癌（IDC）转变的关键特征，在许多其他癌症中也是如此，然而触发集体迁移的微环境因素和潜在机制仍知之甚少。在这里，我们使用三维（3D）离散大小的微肿瘤模型研究了两种微环境因素，即肿瘤内在缺氧和肿瘤分泌因子（分泌组），作为集体迁移的触发因素，该模型概括了DCIS-ID C转变的特征。有趣的是，这两种因素诱导了两种不同的集体迁移模式：在由同一乳腺癌细胞系模型T47D产生的3D微肿瘤中定向迁移和径向迁移。在没有外部刺激的情况下，大（600 µm）的T47D微肿瘤表现出肿瘤内在缺氧和定向迁移，而小（150 µm）的非缺氧微肿瘤仅在暴露于大微肿瘤的分泌组时才表现出径向迁移。为了研究缺氧和分泌组诱导的定向与径向迁移模式的潜在机制，我们对缺氧和分泌组诱导的迁移微肿瘤与作为对照的非缺氧、非迁移的小微肿瘤进行了差异基因表达分析。我们提出了与肿瘤内在缺氧、缺氧诱导的上皮-间质转化（EMT）以及缺氧诱导的定向迁移和分泌组诱导的径向迁移相关的独特基因特征集。基因集富集分析（GSEA）和蛋白质-蛋白质相互作用（PPI）网络分析揭示了缺氧诱导的定向迁移中缺氧、EMT和迁移基因特征之间的富集和潜在相互作用。相比之下，缺氧和EMT在分泌组诱导的径向迁移中未富集，这表明径向迁移可能不需要完全的EMT。生存分析从我们的肿瘤内在缺氧基因特征（CXCR4、FOXO3、LDH、NDRG1）、缺氧诱导的EMT基因特征（EFEMP2、MGP）和定向迁移基因特征（MAP3K3、PI3K3R3）中确定了与TCGA乳腺癌浸润癌数据集中低生存率和不良预后相关的独特基因。NOS3在缺氧和迁移基因特征之间是共同的。分泌组诱导的径向迁移的生存分析确定ATM、KCNMA1（缺氧基因特征）以及KLFⱨ、IFITM1、EFNA1、TGFBR1（迁移基因特征）与低生存率相关。总之，我们具有可控微环境的独特三维培养物通过采用不同的集体迁移模式对不同的微环境因素、肿瘤内在缺氧和分泌组做出反应，并且它们的基因表达分析突出了上皮癌细胞的表型异质性和可塑性。
