Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

精金质颅咽管瘤（ACP）是临床上具有挑战性的肿瘤，其中大多数在CTNNB1中具有激活性突变。它们在组织学上很复杂，显示出囊性和固体成分，后者由不同形态的细胞类型组成（例如，β-catenin聚集簇细胞和上皮呈弥散性上皮细胞），周围是与免疫细胞发生的小胶质细胞反应。在这里，我们对18个ACP样品进行了RNA测序，并将这些数据与现有的ACP转录组数据集整合在一起。迄今为止，尚无研究检查肿瘤不同细胞区室中基因表达的模式。为了实现这一目标，我们将激光捕获显微切割技术与计算机分析技术相结合，以揭示与上皮肿瘤细胞（集群和上皮性上皮细胞），神经胶质组织或免疫浸润相关的基因组。我们使用来自两个ACP小鼠模型的这些人类ACP分子标记和RNA-Seq数据来揭示细胞簇在分子上与釉质结相似，釉质结是控制正常牙齿形态发生的关键信号传导中心。支持这一发现，我们表明人类簇细胞表达高水平的分泌因子的FGF，TGFB和BMP家族的几个成员，通过针对磷酸化蛋白pERK1 / 2，pSMAD3和pSMAD1 / 5的免疫染色证明了向邻近细胞的信号传递在人类和小鼠的ACP中均为/ 9。我们揭示了用曲美替尼（一种临床批准的MEK抑制剂）抑制MAPK / ERK途径，可导致人和小鼠ACP外植体培养物中增殖减少和细胞凋亡增加。最后，我们分析了神经胶质反应性组织中的突出分子特征，以表征炎症微环境并揭示了人类ACP中炎性小体的激活。我们通过对来自ACP患者的实体瘤和囊性液中的免疫细胞标记物进行免疫染色，细胞因子ELISA和蛋白质组分析来验证这些结果。我们的数据支持一种新的分子范式，用于将ACP肿瘤发生理解为自然牙齿发育的异常模拟，并通过揭示人类ACP中MAPK / ERK的激活和炎性体途径开启了新的治疗机会。