A single cell atlas of human glioma under therapy

Most clinical trials for glioblastoma (GBM) enroll patients at recurrence. However, most pre-clinical animal models are of primary disease. Most studies have used tissues from primary tumors. Knowledge of GBM cellular composition at recurrence is limited. We profiled 80 human IDH-wild-type GBM specimens via single-nucleus RNA sequencing, 40 primary tumors and 40 patient-matched recurrent tumors. Select cohorts were also profiled via single-cell assay for transposase-accessible chromatin, single-cell digital-spatial, and single-cell spatial-transcriptomic assays. All patients were treated only with standard-of-care therapy: temozolomide, radiation and surgical resection. The cohort had a 1.2 male/female ratio, ages 35–76. We found a significant increase at recurrence in cells with the Verhaak mesenchymal phenotype and fewer proneural cells. Although there was an increase in the percentage of cycling cells overall, the fraction of cycling proneural cells decreased while the fraction of cycling mesenchymal cells increased. These changes were concomitant with a significant increase in the percentage of tumor-infiltrating monocytic-lineage cells derived from the periphery. Taken together, these findings support a proneural-to-mesenchymal shift at recurrence due to an increase in the birth rate of mesenchymal cells, supported by an expansion of myeloid-derived cells from peripheral blood. Although the percentages of glioblastoma-infiltrating T-cells are generally low (~1% on average in our data), we found a statistically significant increase in T-cell abundance at recurrence. We identified a cohort of T-cell outliers that demonstrate 2-to-8-fold increases in T-cells over average levels. We validated that T-cells in these tumors have extravasated beyond the perivascular space and into the cellular tumor via immunohistochemistry and spatial profiling. We present our ongoing analysis of the paracrine signals, upstream transcription-factor expression, and cis-regulatory grammars utilized in immune-outlier GBMs. We describe how these programs change under therapy. These studies shed light on the effect of standard therapy in shaping GBM composition at recurrence. We performed bulk RNA sequencing (RNA-seq), single-nuclei RNA sequencing (snRNA-seq), single-cell assay for transposase-accessible chromatin using sequencing(scATAC-seq), and spatial transcriptomics and proteomics of specimens from untreated human gliomas. We integrated the public single cell GBM data and our data, which showed that proneural and mesenchymal cells resided on a single axis. We investigated the cell composition and showed that mesenchymal cells increased in recurrence compared with the primary GBM. We identified therapeutic targets gene via intergration of spatial data and cell-cell interction analysis from snRNA-seq daata. Lastly, we performed an in vitro screen of drug combinations that target genes identified from our single-cell analysisof GBM. We profiled spatial proteomics (SP) for six formalin-fixed paraffin embedded (FFPE) slides from three recurrent outlier-cases and their matched primary cases by using the Nanostring GeoMx platform. We next performed spatial transcriptomics (ST) on 10 slides from five patient-matched primary-recurrent pairs. ------------------------------------------------------------------------------- Authors state that the raw data is "not available due to privacy concerns".目前绝大多数胶质母细胞瘤（glioblastoma, GBM）临床试验均纳入复发期患者，然而多数临床前动物模型针对的是原发性疾病，且多数研究使用的组织样本均来自原发性肿瘤。目前学界对复发期胶质母细胞瘤的细胞组成认知仍十分有限。本研究通过单细胞核RNA测序（single-nucleus RNA sequencing）对80例人类异柠檬酸脱氢酶（IDH）野生型胶质母细胞瘤样本进行了分型，其中包括40例原发性肿瘤与40例患者匹配的复发肿瘤。部分队列还通过单细胞转座酶可及性染色质测序（single-cell assay for transposase-accessible chromatin using sequencing, scATAC-seq）、单细胞数字空间检测及单细胞空间转录组检测进行了分型。所有受试者仅接受了标准治疗方案：替莫唑胺、放疗及手术切除。本队列的男女比例为1.2:1，患者年龄跨度为35~76岁。我们发现，复发期样本中具有Verhaak间质表型的细胞比例显著升高，而前神经元细胞占比则有所降低。尽管整体增殖细胞比例有所上升，但增殖性前神经元细胞的占比却出现下降，而增殖性间质细胞的占比则显著升高。上述变化与外周来源的肿瘤浸润单核细胞系细胞占比的显著升高相伴出现。综上，上述研究结果支持复发期胶质母细胞瘤存在从前神经元表型向间质表型的转变，这一转变源于间质细胞生成率的提升，同时伴有外周血髓系来源细胞的扩增。尽管胶质母细胞瘤浸润性T细胞的占比普遍较低（本研究数据中平均占比约为1%），但我们发现复发期T细胞的丰度存在统计学意义上的显著升高。我们还鉴定出一类T细胞异常亚群，其T细胞占比较平均水平升高2~8倍。通过免疫组化与空间分型检测，我们验证了这些肿瘤中的T细胞已穿过血管周围间隙，浸润至肿瘤细胞实质中。本研究目前正在分析免疫异常型胶质母细胞瘤中所涉及的旁分泌信号、上游转录因子表达及顺式调控语法。我们阐述了上述调控程序在治疗压力下的变化情况。本研究揭示了标准治疗对复发期胶质母细胞瘤细胞组成的塑造作用。我们对未接受治疗的人类神经胶质瘤样本进行了批量RNA测序（bulk RNA-seq）、单细胞核RNA测序（snRNA-seq）、单细胞转座酶可及性染色质测序（scATAC-seq）以及空间转录组与蛋白质组检测。我们整合了公开的胶质母细胞瘤单细胞数据与本研究产生的数据，结果显示前神经元细胞与间质细胞处于同一分化轴上。我们对细胞组成进行了分析，结果证实相较于原发性胶质母细胞瘤，复发期样本中的间质细胞占比有所升高。我们通过整合空间数据与来自单细胞核RNA测序的细胞间相互作用分析，鉴定出了治疗靶点基因。最后，我们针对通过单细胞胶质母细胞瘤分析筛选得到的靶点基因开展了体外药物联合筛选实验。我们使用Nanostring GeoMx平台，对来自3例复发异常亚组患者及其匹配的原发性患者的6张福尔马林固定石蜡包埋（FFPE）切片进行了空间蛋白质组（spatial proteomics, SP）分型。随后，我们对5例患者匹配的原发-复发配对样本的10张切片开展了空间转录组（spatial transcriptomics, ST）检测。作者声明，原始数据因隐私保护问题无法公开获取。