Abstract 1678: Single-cell landscape of the TH-MYCN neuroblastoma mouse model

Abstract

Abstract Background and aims: Neuroblastoma (NB) is the most common and deadly malignancy of infancy. It is an embryonal neuroendocrine tumor arising from the neural crest lineage. Amplification of MYCN is a frequent genetic abnormality, found in approximately 20% of NB tumors, and represents a critical stratifying prognostic marker. Given that MYCN plays a key role in NB tumorigenesis and aggressiveness, the TH-MYCN transgenic mouse model is widely used in NB research. This model is characterized by the overexpression of MYCN in neuroectodermal cells, driven by tyrosine hydroxylase (TH) promoter. This overexpression gives rise to tumors exclusively in the sympathoadrenal system, reflecting human NB features. We aimed to explore the transcriptional landscape of TH-MYCN tumors by single-cell RNA sequencing (scRNA-seq). Methods: Tumors from three homozygous and two hemizygous TH-MYCN mice were dissociated into single cells, profiled by scRNA-seq (Chromium 3’ v2 kit, 10x Genomics), sequenced on the NextSeq platform (Illumina). A total of 41666 single cells were analyzed using pagoda2 and conos. TH-MYCN tumor single-cell transciptomes were further aligned with previously published transcriptomes from the adrenal gland of three mice at embryonic day (E)13.5. To evaluate malignant tumor cells, copy number variants (CNV) were analyzed using inferCNV. Immunohistochemistry was used to validate the bioinformatically predicted cell types. Results: scRNA-seq of tumors from TH-MYCN mice revealed 16 major cell populations, spanning stromal, immune and tumor compartments. MYCN+ tumor cells predominantly resemble sympathetic neuroblasts (Isl1, Stmn1, Nefl, Nefm), while MYCN+ chromaffin-like tumor cells (Pnmt, Cdkn1c, Th, Dbh) were rare. Immunofluorescence stainings confirmed that tumors predominantly consist of proliferating sympathetic neuroblasts. Tumor cell nests were surrounded by supportive stromal and immune cells. The adrenergic tumor cell composition observed in TH-MYCN tumors is highly reminiscent of human NB. Interestingly, we discovered a cluster of immature adrenergic tumor cells aligning with normal fetal cells transitioning from bridge to chromaffin state (characterized by Sox11, Atrx, Xist, Ptprs, Tcf4). Distinct genomic aberrations, particularly unique chromosomal deletions, were found in the immature adrenergic tumor cells relative to their sympathoblast and chromaffin tumor cell counterparts. Conclusions: Examining the cellular composition of TH-MYCN tumors at a transcriptional level gives a detailed view of tumor cell heterogeneity at an unprecedented resolution. This comprehensive atlas of TH-MYCN tumors provides a resource for unmasking novel therapeutic targets. Citation Format: Bethel Tesfai Embaie, Adele Alchahin, Thale Kristin Olsen, Hirak Sarkar, Shenglin Mei, Ninib Baryawno. Single-cell landscape of the TH-MYCN neuroblastoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1678.