A Single-cell Atlas of Schwannoma Across Genetic Backgrounds and Anatomic Locations

Background: Schwannomas are nerve sheath tumors arising at cranial and peripheral nerves, either sporadically or in patients with a schwannomatosis-predisposition syndrome. There is limited understanding of the transcriptional heterogeneity of schwannomas across genetic backgrounds and anatomic locations. Methods: Here, we prospectively profile by single-cell full-length transcriptomics tumors from 22 patients with NF2-related schwannomatosis, non-NF2-related schwannomatosis, and sporadic schwannomas, resected from cranial and peripheral nerves. We profiled 11,373 cells, including neoplastic cells, fibroblasts, T cells, endothelial cells, myeloid cells and pericytes. Results: We characterize the intra-tumoral genetic and transcriptional heterogeneity of schwannoma, identifying six distinct transcriptional metaprograms, with gene signatures related to stress, myelin production, antigen presentation, interferon signaling, glycolysis and extracellular matrix. We demonstrate the robustness of our findings with analysis of an independent cohort. Conclusion: Overall, our atlas describes the spectrum of gene expression across schwannoma entities at the single-cell level and will serve as important resource for the community. Adult patients with radiographic evidence of schwannoma provided preoperative informed consent to take part in the study in all cases after the Institutional Review Board Protocols DF/HCC 10- 417 and DF/HCC 15-370B. Among these patients were patients that had received a diagnosis of NF2-related schwannomatosis or non-NF2-related schwannomatosis based on current clinical diagnostic criteria. Patients were males and females. Sample processing and single-cell RNA-seq data generation: Fresh tumors were collected directly from the operating room at the time of surgery and presence of schwannoma was confirmed by frozen section. Tumors were mechanically and enzymatically dissociated using a papain-based tumor dissociation kit (Miltenyi Biotec). Tumor cells were blocked in 1% bovine serum albumin in Hanks buffered saline solution (BSA / HBSS). Tumors were first stained first with CD45- Vioblue direct antibody conjugate (clone REA747, Miltenyi Biotec) for 30 min at 4deg C. Cells were washed with cold PBS, and then re- suspended in 1mL of BSA / HBSS containing 1 mM calcein AM (Life Technologies) and 0.33 mM TO-PRO-3 iodide (Life Technologies) to co-stain for 30 min before sorting. CD45 negative cells were stained with calcein AM (Life Technologies) for viability and CD24-APC (human antibody, clone REA832, Miltenyi Biotec) and CD44-VioBlue (human antibody, clone REA690, Miltenyi Biotec) to sort subpopulations of viable non-immune cells. Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488 nm (calcein AM, 530/30 filter), 640nm (TO-PRO-3 or CD24-APC, 670/14 filter), and 405 nm (CD45-VioBlue or CD44-VioBlue, 450/50 filter) lasers. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein AM positive and TO-PRO-3 negative. We sorted individual, viable, immune, and non-immune single cells into 96- well plates containing TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen on dry ice immediately after sorting and stored at -80deg C prior to whole transcriptome amplification, library preparation and sequencing. Smart-seq2 whole transcriptome amplification, library construction, and sequencing were performed.

背景：神经鞘瘤（Schwannomas）是起源于颅神经与外周神经的神经鞘肿瘤，可散发出现，或发生于携带神经鞘瘤易感综合征的患者群体中。目前学界对不同遗传背景与解剖位置下神经鞘瘤的转录异质性认知仍较为有限。

方法：本研究通过单细胞全长转录组学技术，前瞻性表征分析了22例患者的肿瘤样本，这些患者分别患有NF2相关神经鞘瘤病、非NF2相关神经鞘瘤病以及散发性神经鞘瘤，肿瘤均切除自颅神经与外周神经。本次研究共分析11373个细胞，涵盖肿瘤细胞、成纤维细胞、T细胞、内皮细胞、髓系细胞与周细胞。

结果：本研究刻画了神经鞘瘤的瘤内遗传与转录异质性，鉴定出6种不同的转录元程序，其相关基因特征分别对应应激、髓鞘生成、抗原呈递、干扰素信号通路、糖酵解与细胞外基质。我们通过对独立队列的分析验证了研究发现的稳健性。

结论：总体而言，本研究的转录组图谱在单细胞水平上描绘了不同类型神经鞘瘤的基因表达谱，将为相关领域研究者提供重要的研究资源。

所有经影像学确诊为神经鞘瘤的成年患者，均在术前签署了知情同意书，本研究的开展符合机构审查委员会（Institutional Review Board）的DF/HCC 10-417与DF/HCC 15-370B方案。本研究纳入的患者根据现行临床诊断标准，被分为NF2相关神经鞘瘤病、非NF2相关神经鞘瘤病两组，同时涵盖男性与女性患者。

样本处理与单细胞RNA测序数据生成：手术切除的新鲜肿瘤样本直接从手术室获取，并通过冰冻切片确认存在神经鞘瘤组织。采用基于木瓜蛋白酶的肿瘤解离试剂盒（Miltenyi Biotec），通过机械联合酶解的方法对肿瘤组织进行解离。将肿瘤细胞重悬于含1%牛血清白蛋白的汉克斯平衡盐溶液（BSA/HBSS）中进行封闭。首先使用CD45-VioBlue直接抗体偶联物（克隆REA747，Miltenyi Biotec）在4℃下染色30分钟。随后用预冷PBS洗涤细胞，并重悬于1mL含1mM钙黄绿素AM（Life Technologies）与0.33mM TO-PRO-3碘化物（Life Technologies）的BSA/HBSS溶液中，共染色30分钟以标记活细胞与死细胞，随后进行分选。分选出CD45阴性的细胞后，使用钙黄绿素AM标记活细胞，同时结合CD24-APC（人源抗体，克隆REA832，Miltenyi Biotec）与CD44-VioBlue（人源抗体，克隆REA690，Miltenyi Biotec），对非免疫活细胞亚群进行分选。分选使用FACSAria Fusion定制分选系统（Becton Dickinson），配置488nm（钙黄绿素AM，530/30滤光片）、640nm（TO-PRO-3或CD24-APC，670/14滤光片）以及405nm（CD45-VioBlue或CD44-VioBlue，450/50滤光片）激光器。采用标准严格的前向散射高度与面积阈值以区分双细胞聚集体（doublet），仅对单个细胞设门分析。活的单个细胞被定义为钙黄绿素AM阳性且TO-PRO-3阴性。将单个活的免疫与非免疫细胞分选至含有1%β-巯基乙醇的TCL缓冲液（QIAGEN）的96孔板中。分选完成后立即将板置于干冰上冷冻，并在-80℃下保存，直至进行全转录组扩增、文库制备与测序。后续采用Smart-seq2技术完成全转录组扩增、文库构建与测序。