Gene expression dynamics of branching morphogenesis in pancreatic cancer organoids

The transcriptional dynamics occurring at the self-organizing glandular epithelial during branching morphogenesis are pivotal for deciphering various processes ranging from normal tissue growth to cancer formation. For the past decade matrigel© has been widely used as extracellular matrix for the propagation of organoids, here we report that only in the presence of a higher than 70% collagen matrix symmetry breaking can be observed. Comparing the transcriptomic profile of collagen and matrigel grown-organoids we identified that matrigel organoids are more basal-like, have a higher EMT signature (GSEA) compared to the more classical collagen grown organoids. In this study we used single primary murine pancreatic ductal adenocarcinoma cells (PDAC) cells embedded in floating collagen gels and adapted the media supplementation, thus allowing organoids to self-organize into highly complex branched structures, replicating the in vivo tumour architecture. During organoid development we identified four distinct morphogenetic phases, each characterized by a unique pattern of cell proliferation, invasion, matrix deformation and protein expression. In our search for novel molecular drivers of branching morphogenesis we compared the transcriptome of two distinct phases, an early development (day 7) vs a late mature time point (day 13). We compared these two distinct developmental time points in using PCA, GSEA and respective heat maps of major processes illustrating the overexpression of genes associated with: proliferation, ECM interaction, signalling by Rho GTPases and EMT in day-7 organoids and genes associated with ion channel transport at day-13 organoids. An important aspect raised during our investigation was the effect of different genotypes (Ptf1aCre/+; KrasG12D/+, Pdx1Cre/+; KrasG12D/+; TP53fl/fl) on the branch formation of organoids. To this end we generated organoids derived from KC and KPC tumours, which interestingly gave rise to strikingly similar branching organoids. Although the morphology was not significantly altered the transcriptional profiling showed higher proliferation (Myc targets, E2F targets), EMT score for the KPC organoids. We propose that the spatiotemporal synchronized processes of cell proliferation, matrix remodeling, contraction and ion channel flux are key-events of the different morphogenic phases and lead to the formation of these complex structures. Importantly, these dynamic processes are accompanied by strong transcriptional profile changes, with the organoids undergoing de-differentiation during branch elongation and later on activating an epithelial gene expression program upon maturation. Taken together, these results illustrate the ability of tumour cells to self-organize in multicellular complex structures and provide with a novel system to study branching morphogenesis and tumour biology in vitro. Bulk gene expression data from collagen and matrigel grown KC PDAC organoids. Early time points (day 7) vs late time points (day 13) mature organoids & KC vs KCP branching PDAC organoids.

分支形态发生过程中，自组织腺体上皮细胞内发生的转录动态变化，对于解析从正常组织生长到肿瘤发生的诸多生物学过程至关重要。十余年来，基质胶（matrigel）一直被广泛用作类器官（organoids）培养的胞外基质（extracellular matrix）；本研究发现，仅当胶原基质占比高于70%时，方可观察到对称破缺现象。通过对比胶原基质与基质胶培养类器官的转录组特征，本研究发现，相较于经典的胶原基质培养类器官，基质胶培养类器官更偏向基底样表型，且上皮间质转化（EMT）特征评分更高（基于基因集富集分析GSEA）。本研究采用悬浮于胶原凝胶中的原代小鼠胰腺导管腺癌（PDAC）细胞，并优化培养基补充配方，使类器官能够自组织形成高度复杂的分支结构，复刻体内肿瘤的组织结构。在类器官发育过程中，我们明确了四个独立的形态发生阶段，每个阶段均具有独特的细胞增殖、侵袭、基质重塑与蛋白质表达模式。为探寻分支形态发生的新型分子驱动因子，我们对比了两个关键发育阶段的转录组：早期发育阶段（第7天）与晚期成熟阶段（第13天）。本研究通过主成分分析（PCA）、基因集富集分析（GSEA）以及核心生物学过程的热图，对比了这两个发育时间点的转录组特征：第7天类器官中高表达的基因主要与细胞增殖、胞外基质相互作用、Rho GTP酶（Rho GTPases）信号通路及上皮间质转化（EMT）相关，而第13天类器官中高表达的基因则主要与离子通道转运相关。本研究还探讨了不同基因型（Ptf1aCre/+; KrasG12D/+, Pdx1Cre/+; KrasG12D/+; TP53fl/fl）对类器官分支形成的影响。为此我们分别构建了源自KC与KPC肿瘤的类器官，有趣的是二者均形成了形态极为相似的分支类器官。尽管二者的形态无显著差异，但转录组分析显示，KPC类器官的增殖相关特征（Myc靶点、E2F靶点）及上皮间质转化（EMT）评分更高。我们提出，细胞增殖、基质重塑、收缩及离子通道通量的时空同步过程，是不同形态发生阶段的核心事件，并最终促成复杂分支结构的形成。重要的是，这些动态过程伴随显著的转录组特征改变：类器官在分支延伸阶段发生去分化，而在成熟阶段则激活上皮基因表达程序。综上，本研究结果证实肿瘤细胞能够自组织形成多细胞复杂结构，并为体外研究分支形态发生与肿瘤生物学提供了全新的实验体系。本数据集包含胶原基质与基质胶培养的KC型PDAC类器官的批量基因表达数据，涵盖早期发育阶段（第7天）与晚期成熟阶段（第13天）的类器官，以及KC与KPC型分支PDAC类器官的转录组数据。