Molecular triggers for neuropathy and neural invasion in genetically engineered mouse models of pancreatic ductal adenocarcinoma

神经侵袭(NI)是一个独立的预后因素，也是PDAC局部复发和顽固性癌痛的主要驱动力之一，但其发病机制尚不清楚。在第一个资助期，我们的目标是在PDAC的创新模型中模拟NI，以了解其潜在的机制。我们筛选了250多个等位基因组合的表型，发现了新的类似人类的、真正具有神经侵袭性的PDAC GEMM。在批量和空间转录组分析中，我们发现神经肽Y(NPY)在癌细胞中过表达，并且胰腺星状细胞的“炎性”亚型(上调IL6)与NI有关。在基因筛查中，我们发现了NI与O-糖基化相关基因Galnt2和St3Gal6的失活突变之间的新联系。最后，我们产生了一种新的“神经有机体”结构，它将癌症有机体与神经脊细胞相结合，以诱导癌症神经支配。在这个项目中，我们将以这些发现为基础，通过追求以下目标来更深入地研究PDAC中NI的基础：1)在细胞水平上了解NPY介导的神经侵袭在PDAC中的机制；2)深入研究IL6信号在PDAC中NI中的影响；3)了解O-糖基化如何与PDAC中的神经侵袭联系在一起；以及4)产生和筛选PDAC中神经侵袭和神经可塑性的新模型。在WP1中，我们将研究NPY在PDAC中介导NI的机制。我们将从我们的NPY过度表达的小鼠突变体中分离癌细胞，并在我们的3D培养模型中测试它们的生物学特性、它们的磷蛋白质组以及与神经元和雪旺细胞的相互作用。在WP2中，我们将研究神经侵袭性PDAC模型中的神经元来源的IL6和星状细胞如何增加NI。我们将分析具有构成IL6信号的小鼠(KPC；L-gp130)以及来自该模型的背根节神经元和癌细胞的共培养中的NI。我们将把小鼠癌细胞与有或没有NI的小鼠的星状细胞共同植入，并随后分析NI。在WP3中，我们将分析O-糖基化是如何与NI联系起来的。我们将比较Galnt2和St3Gal6沉默的PDAC细胞的“糖糖”，以确定差异糖基化蛋白，并分析针对这些蛋白质编辑的PDAC细胞的体外和体内神经侵袭能力。在最后的WP4中，我们将探索PDAC中的NI的新模型。在这里，我们将原位植入小鼠PDAC有机物与小鼠神经脊细胞的混合物，并分析神经支配和医院感染。总而言之，这些研究将为NI的发病机制提供新的见解，并指出新的、潜在的可操作靶点。

Neural invasion (NI) is an independent prognostic factor and one of the main drivers of local recurrence and intractable cancer pain in PDAC, but its pathomechanism is barely understood. In the first funding period, we aimed modelling NI in innovative models of PDAC for understanding its underlying mechanisms. We screened phenotypes of over 250 allele combinations and discovered novel human-like, truly neuro-invasive GEMM of PDAC. In bulk and spatial transcriptome analyses, we identified overexpression of neuropeptide Y (Npy) in cancer cells, and the "inflammatory" subtype of pancreatic stellate cells (that upregulate Il6) to be associated with NI. In genetic screens, we found a novel link between NI and inactivating mutations in O-gylcosylation-associated genes Galnt2 and St3Gal6. Finally, we generated a novel "innervated organoid" setup, which combines cancer organoids with neural crest cells for inducing cancer innervation. In this project, we will build upon these findings to delve deeper into the underpinnings of NI by pursuing the following aims: 1) Understand the mechanism behind NPY-mediated neural invasion in PDAC at the cellular level; 2) Delve into the impact of IL6 signaling during NI in PDAC; 3) Understand how O-glycosylation is linked to neural invasion in PDAC; and 4) Generate and screen for novel models of neural invasion and neuroplasticity in PDAC. In WP1, we will study the mechanisms of Npy-mediated NI in PDAC. We will isolate the cancer cells from our Npy-overexpressing mouse mutants and will test their biological traits, their phosphoproteome and interactions with neurons and Schwann cells in our 3D culture models. In WP2, we will investigate how neuron-derived IL6, and stellate cells of neuro-invasive PDAC models augment NI. We will analyze NI in mice with constitutive IL6 signalling (KPC;L-gp130) and in co-cultures of DRG neurons and cancer cells from this model. We will co-implant murine cancer cells with stellate cells of mice with/without NI and subsequently analyze NI. In WP3, we will analyze how O-glycosylation is linked to NI. We will compare the "glycome" of Galnt2- and St3Gal6-silenced PDAC cells for identifying the differentially glycosylated proteins, and analyze the in vitro and in vivo neuro-invasiveness of PDAC cells that have been edited for these proteins. In the final WP4, we will explore novel models of NI in PDAC. Here, we will orthotopically implant murine PDAC organoids in mixture with murine neural crest cells and analyze innervation and NI. Collectively, these investigations will provide a novel insight into the pathomechanisms of NI and point out novel, potentially actionable targets.