(PQ1) Identifying and targeting human glioblastoma migrating in the peritumoral niche

胶质母细胞瘤是最常见和最致命的原发脑肿瘤，通过以下方式广泛传播到整个大脑 劫持正常神经干细胞使用的细胞迁移途径。移行性胶质母细胞瘤持续存在于 肿瘤切除后周围脑(癌前野)，最终复发并致患者死亡。 胶质母细胞瘤的迁移是这种毁灭性疾病的标志，然而还没有人分离和分析 移行性胶质母细胞瘤的单细胞转录组与正常(成熟和胎儿)或瘤周的比较 星形胶质细胞。我们认为移行性胶质母细胞瘤和瘤周星形胶质细胞使用胎儿星形胶质细胞。 促进胶质母细胞瘤迁移的基因，这些基因在患者内部和患者之间是一致的， 而这些基因的抑制将阻止胶质母细胞瘤的迁移。解构和靶向胶质母细胞瘤 在人类瘤周星形胶质细胞微核内迁移，并为患者提供个性化治疗建议， 我们开发了三种利用原始人体组织的创新方法。目标1：确定 移行性胶质母细胞瘤在多大程度上由人胎儿星形胶质细胞基因表达决定，不同 来自正常和胶质母细胞瘤的星形胶质细胞。单细胞分离、rna-seq及转录组分析 匹配的人脑胶质母细胞瘤、瘤周和正常脑将用于鉴定脑细胞亚型和 瘤周脑内移行性胶质母细胞瘤。移行性胶质母细胞瘤基因标记与 胎儿星形胶质细胞基因和样本内和样本之间的一致性将进一步得到验证。目标2：测试 瘤周星形胶质细胞促进胶质母细胞瘤通过胎儿星形胶质细胞迁移的程度 基因表达。从新鲜手术中分离的匹配的人脑胶质母细胞瘤和瘤周星形胶质细胞 通过我们新的免疫泛素分离技术，标本将进行RNA-SEQ和培养。我们 疑似肿瘤周围星形胶质细胞和正常胎儿星形胶质细胞在转录和功能上的相似性。 在使用原生人类进行的跨井迁移试验中，候选通路将被促进或抑制 胶质母细胞瘤。目的3：检测是否可以通过敲除蛋白来抑制胶质母细胞瘤的迁移 移行性胶质母细胞瘤中的胎儿星形胶质细胞基因或瘤周星形胶质细胞。候选基因 在移行性胶质母细胞瘤(AIM 1)和瘤周星形胶质细胞(AIM 2)中发现的基因具有治疗前景，以及 将首先在体外和体外使用原发的人类胶质母细胞瘤进行验证。在这些方面显示出希望的目标 移行性胶质母细胞瘤或瘤周星形胶质细胞将接受人脑胶质母细胞瘤的验证研究- 小鼠颅内异种移植模型的建立。对照和初级标本将清晰地成像以 证实胶质母细胞瘤与瘤周星形胶质细胞的动态相互作用。这个项目有直接的翻译 作为靶向胶质母细胞瘤迁移的可能性将把胶质母细胞瘤限制在局部疾病，改善对 通过手术切除和放射治疗来减少恶性进展。

Glioblastoma, the most common and deadly primary brain tumor, disseminates widely throughout the brain by hijacking the cell migration pathways used by normal neural stem cells. Migrating glioblastoma persist in the surrounding brain (pre-malignant field) after tumor resection, ultimately recurring and killing the patient. Glioblastoma migration is the hallmark of this devastating disease, yet no one has isolated and analyzed the single cell transcriptome of migrating glioblastoma as compared to normal (mature and fetal) or peritumoral astrocytes. We propose that migrating glioblastoma and peritumoral astrocytes employ fetal astrocyte genes to promote glioblastoma migration, that these genes are consistent within and across patients, and that inhibition of these genes will halt glioblastoma migration. To deconstruct and target glioblastoma migrating within the human peritumoral astrocyte microniche and suggest personalized therapies for patients, we have developed three innovative methods that leverage primary human tissue. Aim 1: To determine the extent to which migrating glioblastoma are defined by human fetal astrocyte gene expression, distinct from normal and glioblastoma astrocytes. Single cell isolation, RNA-seq, and transcriptome analysis of matched human glioblastoma, peritumoral, and normal brain will be used to identify brain cellular subtypes and migrating glioblastoma within the peritumoral brain. Migrating glioblastoma genetic markers that overlap with fetal astrocyte genes and are consistent within and across samples will be further validated. Aim 2: To test the extent to which peritumoral astrocytes facilitate glioblastoma migration through fetal astrocyte gene expression. Matched human glioblastoma and peritumoral astrocytes, isolated from fresh surgical specimens through our novel immunopanning separation technique, will undergo RNA-seq and culture. We suspect transcriptional and functional similarities between peritumoral astrocytes and normal fetal astrocytes. Candidate pathways will be promoted or inhibited in transwell migration assays using primary human glioblastoma. Aim 3: To test whether glioblastoma migration can be inhibited through knockdown of either fetal astrocyte genes in migrating glioblastoma or peritumoral astrocytes. Candidate genes identified in migrating glioblastoma (Aim 1) and peritumoral astrocytes (Aim 2) hold therapeutic promise, and will first be validated using primary human glioblastoma in vitro and ex vivo. Targets showing promise in these validation studies of either migrating glioblastoma or peritumoral astrocytes will undergo human glioblastoma- in-mouse intracranial xenograft modeling. Control and primary specimens will be imaged with CLARITY to confirm the dynamic glioblastoma-peritumoral astrocyte interactions. This project has direct translational potential as targeting glioblastoma migration will confine glioblastoma to a local disease, improving response to surgical resection and radiation by decreasing malignant progression.