Targeting epithelial plasticity in pancreatic cancer

由细胞可塑性调节的动态细胞命运决定对包括治疗施加压力在内的改变的环境线索的反应是肿瘤适应的关键。这种可塑性的细胞行为促使肿瘤的发生、肿瘤细胞从原发部位剥离和转移。此外，可塑性导致肿瘤的异质性以及对治疗的抵抗力。我们最近建立了一个新的PDAC类器官形态发生系统，它允许我们在肿瘤类器官形态发生过程中以动态的方式捕捉肿瘤的可塑性和异质性。有趣的是，来自不同分子簇的PDAC细胞可以重复产生复杂的三维结构，从而推断出分子亚型。在这个项目中，我们将利用有机物形态发生实验来确定驱动PDAC表型异质性的分子程序。在WP 1中，我们将通过转录图谱分析分支有机化合物表型的整个光谱，获得对有机化合物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剖面图来剖析有机体内的异质性。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械的标记调节因子对PDAC有机类化合物的生长过程进行量化和动态建模来阐明胰腺癌类有机化合物发展的分子动力学和机制。此外，在WP 3中，我们将量化分支形态发生过程中涉及的应变和应力，方法是使用UV激光在有机化合物相邻的基质中进行局部切割，从而导致松弛，从而使我们能够确定结构内的张力。最后，在WP4中，我们将通过调节有机体内和有机体内表型异质性的驱动因素的扰动来确定细胞可塑性的功能作用。这将通过使用临床前和临床药物在表型映射的PDAC有机化合物中的药物筛选来实现，以识别改变药物的异质性。限制表型异质性的药物将在PDAC患者衍生的有机化合物(PDO)的大小组中进行测试，并与一线多元化疗相结合。总体而言，我们提出了一种创新的跨学科策略来识别和干扰驱动细胞可塑性和异质性的分子机制。最终结果将是一个合理的策略，以了解驱动有机类物质异质性和可塑性的分子和生物物理机制，以及在不同PDAC亚型衍生的有机类物质形态发生的特定发育阶段干预药理表型多样性。这两种现象都被认为是化疗耐药和转移转移的关键调节因素，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.