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

(PQ1) 识别和靶向在瘤周微环境中迁移的人类胶质母细胞瘤

• 批准号: 9883759
• 负责人: Melanie Hayden Gephart
• 金额: $ 35.97万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883759
• 关键词: Adult; Affect; Astrocytes; Biological Assay; Brain; Candidate Disease Gene; Cell Line; Cell Migration Pathway; Cell Separation; Cells; Coculture Techniques; Code; Data; Development; Diagnostic radiologic examination; Disease; Drowning; Excision; Galectin 1; Gene Expression; Gene Expression Profile; Genes; Genetic Markers; Genetic Transcription; Glioblastoma; Grouping; Histologic; Human; Image; Immunohistochemistry; In Vitro; Individual; Lead; Malignant - descriptor; Malignant neoplasm of brain; Maps; Methods; Microscopic; Microtomy; Migration Assay; Modeling; Mus; Neurosurgeon; Operative Surgical Procedures; Pathway interactions; Patients; Peptides; Positioning Attribute; Pre-Clinical Model; Primary Brain Neoplasms; Primary Cell Cultures; Process; Radiation; Rodent; Sampling; Scientist; Serum-Free Culture Media; Slice; Specimen; Techniques; Testing; Therapeutic; Time; Tissue imaging; Validation; Xenograft Model; Xenograft procedure; brain cell; cell motility; cell type; fetal; fetus cell; gene interaction; genetic signature; human disease; human tissue; improved; innovation; interest; knock-down; migration; multidisciplinary; neoplastic cell; nerve stem cell; new therapeutic target; novel; personalized medicine; premalignant; response; scaffold; single cell analysis; single-cell RNA sequencing; transcriptome; transcriptome sequencing; tumor; validation studies; wound healing

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.

胶质母细胞瘤是最常见和致命的原发性脑肿瘤，通过以下方式在整个大脑中广泛传播： 劫持正常神经干细胞使用的细胞迁移途径。迁移性胶质母细胞瘤持续存在 肿瘤切除后大脑周围（恶变前区域），最终复发并导致患者死亡。 胶质母细胞瘤迁移是这种毁灭性疾病的标志，但没有人分离和分析其 迁移性胶质母细胞瘤的单细胞转录组与正常（成熟和胎儿）或瘤周相比 星形胶质细胞。我们建议迁移性胶质母细胞瘤和瘤周星形胶质细胞利用胎儿星形胶质细胞 促进胶质母细胞瘤迁移的基因，这些基因在患者体内和患者之间是一致的， 抑制这些基因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生态位内迁移并建议为患者提供个性化治疗， 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 迁移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来定义，不同 来自正常和胶质母细胞瘤星形胶质细胞。单细胞分离、RNA-seq 和转录组分析 匹配的人类胶质母细胞瘤、瘤周和正常大脑将用于识别脑细胞亚型和 瘤周脑内迁移的胶质母细胞瘤。迁移的胶质母细胞瘤遗传标记与 胎儿星形胶质细胞基因在样本内和样本间的一致性将得到进一步验证。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。从新鲜手术中分离出匹配的人胶质母细胞瘤和瘤周星形胶质细胞 通过我们新颖的免疫淘选分离技术获得的样本将进行 RNA 测序和培养。我们 怀疑瘤周星形胶质细胞和正常胎儿星形胶质细胞之间的转录和功能相似性。 在使用原代人的 Transwell 迁移测定中，候选途径将被促进或抑制 胶质母细胞瘤。目标 3：测试是否可以通过敲低胶质母细胞瘤的迁移来抑制 迁移性胶质母细胞瘤或瘤周星形胶质细胞中的胎儿星形胶质细胞基因。候选基因 在迁移性胶质母细胞瘤（目标 1）和瘤周星形胶质细胞（目标 2）中鉴定的具有治疗前景，并且 首先将使用原代人胶质母细胞瘤进行体外和离体验证。在这些方面显示出希望的目标 迁移性胶质母细胞瘤或瘤周星形胶质细胞的验证研究将进行人类胶质母细胞瘤- 小鼠颅内异种移植模型。对照样本和初级样本将进行清晰成像 确认动态胶质母细胞瘤-瘤周星形胶质细胞相互作用。该项目有直接翻译 靶向胶质母细胞瘤迁移的潜力将把胶质母细胞瘤限制为局部疾病，从而改善对胶质母细胞瘤的反应 手术切除和放射治疗可减少恶性进展。