Mitogenic and progenitor programs in single pilocytic astrocytoma cells.

Zachary J. Reitman*, Brenton R. Paolella*, Guillaume Bergthold, Kristine Pelton, Sarah Becker, Robert Jones, Claire E. Sinai, Hayley Malkin, Ying Huang, Leslie Grimmet, Zachary T. Herbert, Yu Sun, Jessica L. Weatherbee, John Alberta, John F. Daley, Orit Rozenblatt-Rosen, Alexandra L. Condurat, Kenin Qian, Prasidda Khadka, Rosalind A. Segal, Daphne Haas-Kogan, Mariella G. Filbin, Mario L. Suva, Aviv Regev, Charles Stiles, Mark W. Kieran, Liliana Goumnerova, Keith L. Ligon, Alex K. Shalek, Pratiti Bandopadhayay†#, Rameen Beroukhim†#

Contact: rameen_beroukhim@dfci.harvard.edu, pratiti_bandopadhayay@dfci.harvard.edu

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) program that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.

Workflow. Samples are collected at the time of surgery, freshly dissociated to single-cell suspensions and sorted based on A2B5 glial progenitor markers into 96-well plates. Full-length scRNA-seq is performed following the Smart-Seq2 protocol.  A custom spike-in BRAF oligonucleotide is incorporated into the cDNA library preparation to maximize KIAA1549-BRAF fusion detection in scRNA-seq data and using a custom qPCR assay.