Delineating cellular heterogeneity of neuroblastoma for identifying therapeutically targetable vulnerabilities

Neuroblastoma is a heterogeneous pediatric neuroendocrine tumor and the most common extracranial solid malignancy in children. Although neuroblastoma etiology is elusive, it is thought to originate from the neural crest lineage, which includes multipotent Schwann cell precursors (SCPs) that can give rise to sympatho-adrenal cells, including chromaffin cells and sympathoblasts. Neuroblastoma has a wide range of clinical outcomes. Some tumors regress spontaneously without treatment, while others respond well to chemotherapy. Approximately half of neuroblastoma cases are highly aggressive, leading to refractory and relapsed disease with poor prognosis. High-risk neuroblastoma is often diagnosed with metastases, preferentially spreading to the bone marrow. Common genetic aberrations associated with the high-risk group include MYCN- amplification, segmental gain of chromosome arms 1q, 2p, 17q and deletion of chromosome arms 1p and 11q.

To study the cellular identity and clonal development of human neuroblastoma, we performed joint single-cell DNA and RNA sequencing and orthogonal validation by DNA-FISH with immunofluorescence staining in paper I. In addition to malignant adrenergic cells, we discovered aneuploid pre-malignant SCP-like cells in primary tumors. Clonal expansion was evident in both SCP-like and adrenergic subclones. Gain of chromosome 17 was a shared aberration of pre-malignant SCP-like cells across samples. Genetic analysis and phylogeny of tumor subclones suggest that migrating neural crest cells or multipotent SCPs, prone to aneuploidy, may represent putative tumor-initiating events in neuroblastoma. Abnormal SCP-like cells showed upregulated proliferation scores and downregulated antigen presentation via MHC molecule gene expression compared to non-malignant counterparts.

Modeling highly heterogeneous tumors like neuroblastoma is challenging. Since MYCN is a potent oncogenic driver in high-risk neuroblastoma, the TH-MYCN transgenic mouse model is widely used in preclinical studies. However, the extent to which TH-MYCN tumors model the disease has not been explored. In paper II, we comprehensively characterized the single-cell transcriptional landscape of TH-MYCN mouse tumors across various ages, both sexes and genotypes. Joint alignment analysis of tumor cells with normal fetal adrenal gland exhibited resemblance with embryonic chromaffin cells and primarily sympathoblasts. Chromaffin to sympathoblast transitions were observed in tumors, consistent with normal murine developmental trajectories. Comparative analysis with human MYCN-amplified neuroblastoma confirmed similarities in the adrenergic tumor cell compartment. Additionally, inferred ligand-receptor analysis revealed potential therapeutic targets in the NCAM and NOTCH signaling pathways.

Existing TH-MYCN cell lines lose the adrenergic identity of neuroblastoma and instead acquire a mesenchymal phenotype. To address this issue, we established novel ex vivo tumoroids that preserve PHOX2B expression and maintain the adrenergic cellular identity of the originating tumor. While ex vivo tumoroid cells demonstrated transcriptional resemblance with embryonic chromaffin cells and sympathoblasts, distinct adrenergic subclusters were enriched in culture. Ex vivo enriched gene expression profiles were associated with synaptic signaling, neuronal morphogenesis and metabolic processes. Some of the upregulated genes in the enriched subclusters were correlated with poor neuroblastoma survival.

The major cause of cancer-related death is bone marrow metastasis. To examine the cellular and transcriptional shifts associated with neuroblastoma bone marrow metastasis, we compared the single-cell transcriptomes of non-metastatic and metastatic bone marrow biopsies in paper III. Metastatic tumor cells presented an adrenergic phenotype and acquired a transcriptional signature associated with poor neuroblastoma prognosis. We detected an immunosuppressive microenvironment in the metastatic samples encompassing B cell depletion and enriched regulatory T cell activity. We further identified cytotoxic T cells and CD56bright NK cells with upregulated expression of inhibitory receptors in the bone marrow metastatic niche. By flow cytometry analysis, we confirmed the presence of tumor cells and compositional shifts in B cell and T cell populations in matched bone marrow biopsies. Moreover, metastatic samples with enriched macrophages and mature neutrophils contributed to interactions with disseminated tumor cells via NOTCH signaling and other immunoregulatory interactions.

Taken together, this thesis provides an overview of the tumor cell atlas of neuroblastoma and bone marrow metastatic niche remodeling, which may contribute to the identification of new therapeutic approaches. Furthermore, we highlight the translational potential of TH-MYCN in vivo and novel ex vivo models for the therapeutic testing of these new targets.

List of scientific papers

I. Olsen TK*, Otte J*, Mei S*, Embaie BT, Kameneva P, Cheng H, Gao T, Zachariadis V, Tsea I, Björklund Å, Kryukov E, Hou Z, Johansson A, Sundström E, Martinsson T, Fransson S, Stenman J, Fard SS, Johnsen JI, Kogner P, Adameyko I, Enge M, Kharchenko PV*, Baryawno N *. Joint single-cell genetic and transcriptomic analysis reveal pre- malignant SCP-like subclones in human neuroblastoma. Mol Cancer. 2024 Aug 31;23(1):180. https://doi.org/10.1186/s12943-024-02091-y

II. Embaie BT*, Sarkar H*, Alchahin AM, Otte J, Olsen TK, Tümmler C, Kameneva P, Artemov AV, Akkuratova N, Adameyko I, Stukenborg JB, Wickström M, Kogner P, Johnsen JI, Mei S, Kharchenko PV, Baryawno N. Comparative Single-Cell Transcriptomics of Human Neuroblastoma and Preclinical Models Reveals Conservation of an Adrenergic Cell State. Cancer Res. 2025 Mar 14;85(6):1015-1034. https://doi.org/10.1158/0008-5472.can-24-1507

III. Mei S*, Alchahin AM*, Embaie BT, Gavriliuc IM, Verhoeven BM, Zhao T, Li X, Jeffries NE, Pepich A, Sarkar H, Olsen TK, Wickström M, Stenman J, Reina-Bedoya O, Kharchenko PV, Saylor PJ, Johnsen JI, Sykes DB, Kogner P*, Baryawno N *. Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature. JCI Insight. 2024 Feb 15;9(6):e173337. https://doi.org/10.1172/jci.insight.173337

*Contributed equally.