Biophysical Interrogation of Signals that Drive GBM Invasion

阻碍多形性胶质母细胞瘤(GBM)成功治疗的一个主要问题是高度 侵袭性细胞扩散到脑实质。这些细胞逃避手术切除，并经常扩散。 远端在脑实质内。在单个GBM内存在多个在空间上不同的异型群体， 导致疾病的遗传异质性，并导致复杂的内在和外在细胞 入侵机制。一个标志性突变--表皮生长因子受体(EGFR)的扩增 在60%的病例中，最常见的是以异质性的方式发生，并经常与 外显子2-7的缺失，产生了一个结构性活性突变体EGFRvIII。虽然重要的焦点一直是 根据其激酶活性，相对较少的人知道EGFRvIII是否有能力通过 与黏附受体的相互作用。我们的初步数据支持EGFRvIII在它干扰的情况下扮演双重角色 并通过外在信号征募未转化的对应者 减少混合种群的粘附性。根据我们的发现，我们假设粘合剂的这种差异 活性是由于与EGFRvIII相关的差异信号，而这种受体传达了这种表型 通过细胞因子的产生转化为未转化的对应物(Inda，gene&Dev，2010；Zanca，gene&Dev， 2017)协同入侵薄壁组织。有了这个假设，我们将使用附着力测量 剖析细胞内在EGFR介导的侵袭机制的技术；鉴于 对于肿瘤，我们还将结合新开发的粘连分选技术和高通量测序 识别细胞外在机制和后续干预目标的技术。以下几行 将进行的实验包括：1)实施生物物理分析和信号通路分析 询问EGFRvIII的细胞内在活性如何导致不稳定的黏附和侵袭性表型；2) EGFRvIII细胞外源性、分泌体介导的wtEGFR培养的生化和功能分析 细胞黏附表型；3)黏附分类群体的表达和表观遗传学分析 为了定义一个迁移组特征，它在暴露于EGFRvIII分泌组后在wtEGFR细胞中的稳定性，以及 “受过教育的”wtEGFR将表观遗传特征传播到幼稚的wtEGFR细胞的能力。

One major issue confounding successful treatment of glioblastoma multiforme (GBM) is the presence of highly invasive cells disseminating into the brain parenchyma. These cells evade surgical resection and often spread distally in brain parenchyma. Multiple and spatially distinct heterotypic populations exist within a single GBM, giving rise to the disease’s genetic heterogeneity and leading to complex cell intrinsic and extrinsic mechanisms of invasion. Amplification of the epidermal growth factor receptor (EGFR), a hallmark mutation present in 60% of cases, most often occurs in a heterogeneous manner and is frequently associated with deletion of exons 2-7, creating a constitutively active mutant, EGFRvIII. While significant focus has been placed on its kinase activity, comparatively little is known about EGFRvIII’s ability to enhance migration via interaction with adhesion receptors. Our preliminary data supports a dual role for EGFRvIII where it interferes with intrinsic adhesion receptors and also recruits non-transformed counterparts via extrinsic signaling to reduce adhesion of a mixed population. Based on our findings, we hypothesize that this difference in adhesive activity is due to differential signaling associated with EGFRvIII, and that this receptor conveys this phenotype to non-transformed counterparts through cytokine production (Inda, Genes & Dev, 2010; Zanca, Genes & Dev, 2017) to cooperatively invade parenchyma. With this hypothesis, we will use adhesion measurement technologies to dissect cell intrinsic EGFR-mediated invasion mechanisms; given the heterogeneity within tumors, we will also combine newly developed adhesion sorting technologies with high throughput sequencing technologies to identify cell extrinsic mechanisms and targets for subsequent intervention. The following lines of experimentation will be carried out: 1) implementation of biophysical assays and signaling pathway analyses to interrogate how cell intrinsic activity of EGFRvIII leads to labile adhesion and an invasive phenotype; 2) biochemical and functional analysis of the EGFRvIII cell extrinsic, secretome-mediated education of wtEGFR cell adhesive phenotype; 3) expression and epigenetic analyses on adhesion-sorted populations will be used to define a migratome signature, its stability in wtEGFR cells after exposure to the EGFRvIII secretome, and the ability of “educated” wtEGFR to propagate that epigenetic signature to naïve wtEGFR cells.