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. Overall design: 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.