Targeting epithelial plasticity in pancreatic cancer

由细胞可塑性调节的动态细胞命运决定响应于改变的环境线索，包括治疗施加的压力，对于肿瘤适应至关重要。这种可塑性细胞行为驱动肿瘤起始、肿瘤细胞从原发部位分层和转移性生长。此外，可塑性有助于肿瘤异质性以及对治疗的抵抗。我们最近产生了一种新的PDAC类器官形态发生系统，使我们能够在肿瘤类器官形态发生过程中以动态方式捕获肿瘤可塑性和异质性。有趣的是，来自不同分子簇的PDAC细胞可重复地产生复杂的三维结构，从而可以推断分子亚型。在这个项目中，我们将利用类器官形态发生测定来鉴定驱动PDAC表型异质性的分子程序。在WP 1中，我们将通过对整个分支类器官表型谱的转录谱分析来获得对类器官间异质性的独特见解。同时，我们将使用具有空间分辨率的单细胞谱分析来剖析类器官内的异质性。在WP 2中，我们的目标是通过使用活细胞成像结合细胞骨架机制的标记调节剂对PDAC类器官的生长过程进行定量和动态建模，阐明胰腺癌类器官发育中的分子动力学和机制。此外，在WP 3中，我们将通过使用UV激光在类器官相邻基质中应用局部切割来量化分支形态发生期间涉及的应变和应力，从而导致松弛，这反过来又使我们能够确定结构内的张力。最后，在WP 4中，我们将通过扰动调节类器官间和类器官内表型异质性的驱动程序来确定细胞可塑性的功能作用。这将通过在表型映射的PDAC类器官中使用临床前和临床药物进行药物筛选来实现，以鉴定改变异质性的药物。限制表型异质性的药物将在一大组PDAC患者源性类器官（PDO）中与一线综合化疗联合进行测试。总的来说，我们提出了一个创新的和跨学科的战略，以确定和扰动分子机制驱动细胞的可塑性和异质性。最终的结果将是一个合理的策略，以了解驱动类器官异质性和可塑性的分子和生物物理机制，以及在来自不同PDAC亚型的类器官形态发生的定义发育阶段干预神经元表型多样性。这两种现象被认为是化疗耐药性和转移性播散的关键调节因子，特别是在PDAC中。

Dynamic cell fate decisions regulated by cellular plasticity in response to altered environmental cues including treatment-imposed pressure are critical for tumors to adapt. Such plastic cellular behaviors drive tumor initiation, delamination of tumor cells from the primary site and metastatic outgrowth. Furthermore, plasticity contributes to tumor heterogeneity as well as resistance to therapy. We have recently generated a novel PDAC organoid morphogenesis system which allows us to capture tumor plasticity and heterogeneity in a dynamic fashion during tumor organoid morphogenesis. Interestingly, PDAC cells from distinct molecular clusters reproducibly generate complex three-dimensional structures that allow to infer the molecular subtype. In this project, we will exploit the organoid morphogenesis assay to identify the molecular programs driving phenotypic heterogeneity in PDAC. In WP 1, we will acquire unique insight into inter-organoid heterogeneity by transcriptional profiling of the entire spectrum of branched organoid phenotypes. At the same time, we will dissect intra-organoid heterogeneity using single cell profiling with spatial resolution. In WP 2, we aim to elucidate the molecular dynamics and mechanics in pancreatic cancer organoid development by quantification and dynamic modelling of the growth process of PDAC organoids using live cell imaging in combination with labeled regulators of the cytoskeletal machinery. In addition, in WP 3 we will quantify involved strains and stresses during branching morphogenesis by applying localized cuts in the organoid-adjacent matrix using UV lasers resulting in relaxation which allows us in turn to determine the tension within the structures. Lastly, in WP 4, we will determine the functional role of cellular plasticity by perturbation of drivers regulating inter- and intra-organoid phenotypic heterogeneity. This will be achieved by a drug screen using pre-clinical and clinical drugs in phenotypically mapped PDAC organoids to identify heterogeneity altering drugs. Drugs that limit phenotypic heterogeneity will be tested in a large panel of PDAC patient-derived organoids (PDOs) in combination with first-line poly-chemotherapy. Overall, we propose an innovative and interdisciplinary strategy to identify and perturbate molecular mechanisms driving cellular plasticity and heterogeneity. The final outcome will be a rational strategy to understand the molecular and biophysical mechanism driving organoid heterogeneity and plasticity as well as to intervene pharmacologically phenotypic diversity at defined developmental phases of organoids morphogenesis derived from distinct PDAC subtypes. Both phenomena are considered key regulators of resistance to chemotherapy and metastatic dissemination, particularly in PDAC.