The largest single cell study to date of the childhood cancer, neuroblastoma, has answered important questions about the genesis of the disease.
The researchers from the Wellcome Sanger Institute, Great Ormond Street Hospital (GOSH) and the Princess Máxima Center for Pediatric Oncology, discovered that all neuroblastomas arise from a single type of embryonic cell called sympathoblasts.
The study, published today (5 February 2021) in Science Advances, sought to understand why neuroblastomas range in severity, with some easy to treat and others having relatively low five-year survival rates. The fact that all neuroblastomas arise from sympathoblasts makes them an attractive drug target, because these cells exist only in the tumour after the child is born.
It begins in the abdomen, usually in the adrenal glands – hormone-producing glands above the kidneys. Neuroblastoma is remarkable in that its severity can vary greatly between individuals. In some children the cancer will disappear without treatment, whereas in others the cancer is relentless. The five-year survival rate for neuroblastoma is one of the lowest of all childhood cancers.
This varied outlook prompted the researchers to ask whether the range of severity could be caused by neuroblastomas arising from different cell types at different stages of the child’s development in the womb. This was made possible by the advent of single cell mRNA sequencing, a high-resolution technology that can identify different cell types present in a tissue according to the genes expressed by individual cells.
In this study, gene expression of 19,723 cancer cells was analysed and compared to a reference of 57,972 developmental adrenal cells in the hope of identifying the cell types from which neuroblastomas arise and to find novel treatment targets.
Dr Jan Molenaar, a senior author of the study from the Princess Máxima Center for Pediatric Oncology in the Netherlands, said: “What is most striking about our findings is that despite the great diversity of clinical behaviour of neuroblastoma, there is an overarching neuroblastoma cell type that is found in all patients. The identification of sympathoblasts as the root of all neuroblastoma is an important step towards understanding how the disease develops and, hopefully, how we can treat it.”
Currently, many cancer treatments cause serious side effects for the patient. But in recent years, technological advances have sped up drug development by allowing researchers to identify differences between the biological processes, such as the expression of a particular gene, within healthy human cells and those within cancerous ones. These differences can be exploited to attack cancer cells without affecting the patient’s healthy cells.
The presence of sympathoblasts, a developmental cell type not normally found in children after they are born, makes it a promising drug target for the treatment of neuroblastoma.
Dr Karin Straathof, a senior author of the study from Great Ormond Street Hospital, said: “Neuroblastoma is an unusual cancer in that some tumours resolve without intervention, yet the disease still has one of the lowest five-year survival rates of any childhood cancer.
This study fills important gaps in our knowledge of what neuroblastoma cells are and revealed novel treatment targets. My hope is that new, less intrusive therapies can be developed by targeting sympathoblasts, a developmental cell type that exists only in neuroblastoma tumours after a child is born.”
As well as facilitating the discovery of sympathoblasts as the root of neuroblastoma, the single-cell reference map of the developmental adrenal gland will also contribute to the Human Cell Atlas project**. The project aims to create comprehensive reference maps of all types of human cells – the fundamental units of life – as a basis for understanding human health and diagnosing, monitoring, and treating disease.
Dr Sam Behjati, a senior author of the study from the Wellcome Sanger Institute and Cambridge University Hospitals, said: “Our study shows the power of looking at individual childhood cancer cells in revealing not just one, but a plethora of novel treatment ideas. This raises the exciting prospect that a single cell atlas of all types of paediatric tumours may transform our understanding of childhood cancer.”
Neuroblastoma, the most common extracranial solid malignancy among children under 15 years of age, accounts for 8%–10% of pediatric tumors [1, 2]. In general, neuroblastoma is thought to originate from undifferentiated neural crest cells (NCC), which can become any of several different cell types, depending on the location within the embryo .
NCCs may partially differentiate into neuroblastoma or ganglioneuroblastoma, which are malignant, or may differentiate into benign ganglioneuroma . The primary site of neuroblastoma is typically the adrenal medulla or tissues that originate from the sympathetic nervous system .
lthough low- and intermediate-risk patients generally have a favorable outcome, high-risk patients show a high mortality rate and fewer than 50% of patients have long-term survival despite recent intensified treatment [5, 6].
Several genome-wide analysis studies have been reported which aimed to improve prognosis and develop a new therapeutic strategy for high-risk neuroblastoma [2, 7, 8]. Segmental chromosomal aberration is common in high-risk neuroblastoma, such as amplification of the MYCN oncogene  deletions of chromosomes 1p, 3p, 4p, and 11q, and gains of chromosomes 1q, 2p, and 17q [10–12]. These chromosomal aberrations greatly influence the clinical course .
In contrast, recent high-throughput genome-wide studies have revealed that there were few recurrent somatic alterations in high-risk neuroblastoma, except MYCN  ALK [14–17] ATRX [18, 19] and TERT . These genetic alterations do not account for the entire genetic mechanism leading to high-risk neuroblastoma. Thus, effective targeted therapies for intractable neuroblastoma remain limited.
Based on the super-enhancer landscape of neuroblastoma cell lines, there are two neuroblastoma subtypes: Noradrenergic (ADRN)-type and Mesenchymal (MES)-type, showing distinct expression patterns in core regulatory circuitry (CRC)-related genes [21, 22]. However, expression profiling of high-risk neuroblastoma is not yet fully understood. In the present study, we classified 30 cases of International Neuroblastoma Staging System (INSS)  Stage 4 neuroblastomas, based on expression profiles of whole transcriptome sequencing (WTS). Then we conducted a combined analysis with data on mutations and copy number alteration (CNA) to explore a new therapeutic strategy for high-risk neuroblastoma.
In the present study, we classified INSS Stage 4 neuroblastoma into two subtypes, MES- and ADRN-clusters, based on WTS expression profiles. These two clusters were validated with independent large cohort and consistent with the previous classification of the super-enhancer landscape of neuroblastoma cell lines [21, 22]. A small set of core transcription factors forms an interconnected auto-regulatory loop, CRC, which is often driven by super-enhancers . ADRN-type neuroblastoma, showing sympathetic noradrenergic identity, was characterized by CRC modules formed by several transcription factors, such as HAND2, PHOX2B, and GATA3, leading to high expression of these genes.
CRC modules, including AP-1 transcription factors, defined the MES- subtype [21, 22]. Because the formation of these CRCs is associated with differentiation, the pathway analysis results between MES- and ADRN-clusters might provide information about the origin of neuroblastomas. In the present study, enrichment of secretion- and vesicle-related pathways in the MES-cluster represented the features of chromaffin cells, composing about 80% of the adrenal medulla.
In contrast, neuron- and axon-related pathways were enriched in the ADRN-cluster, which indicated sympathetic nervous system features. Furthermore, primary lesions in MES-cluster cases were limited to the adrenal gland or the retroperitoneum, whereas ADRN-cluster cases occurred in tissues originating from the sympathetic nervous system.
These results were consistent with previous reports showing that most chromaffin cells originated from SCPs rather than from sympathoblasts, although both SCPs and sympathoblasts are NCC-derived . Thus, there was a strong correlation between differentiation and classification of super-enhancer or expression profiles in neuroblastoma.
Primary neuroblastoma is a mixture of both MES- and ADRN-type cells, with a balance toward the ADRN type, in most samples [21, 22]. Because of intratumor heterogeneity and the usage of bulk biopsy samples, the classification of expression profiles in the present study might indicate the main cell type component in each neuroblastoma sample.
Therefore, we need to be careful to say that our classification, MES- and ADRN-clusters, is directly associated with the origin of neuroblastoma. There are two possible reasons for the intratumor heterogeneity of neuroblastoma (containing both MES- and ADRN-type cells). First, critical alterations leading to neuroblastoma might occur in NCCs before differentiating into SCPs.
In this case, mutated NCCs might differentiate into both MES- and ADRN-types. Second, critical alterations may occur in a later development stage, when NCC derivatives have been already determined to become either MES- or ADRN-type cells. In this case, either of the committed MES- or ADRN-type cells might interconvert to the other type.
In fact, ADRN cell lines transitioned toward MES-type profiles upon chemotherapy [21, 22]. To elucidate the relevance of intratumor heterogeneity and differentiation, further analyses including single-cell analysis might be required.
Cases in the ADRN-cluster were classified into MYCN- and ATRX-clusters in the present study. MYCN-cluster cases showed high expression in ALDH18A1 (encoding P5CS) and PYCR1, important enzymes in converting glutamate to proline. In Burkitt lymphoma, MYC suppressed POX/PRODH expression and increased P5CS and PYCR1, leading to reprogramming of proline and glutamine metabolism .
This tumor metabolic reprogramming contributes to tumor cell proliferation despite tumor-specific proline vulnerability as a compensatory mechanism . In invasive breast carcinoma and kidney cancers, extensive proline production is necessary to maintain tumorigenic growth because tumor cell proliferation depends on proline . As a result, high expression of PYCR1 was induced in these cancer cells. Similar to these cancers, neuroblastoma especially in MYCN-cluster might possess a tumor-specific proline vulnerability, which could be used as a target metabolic pathway for treatment.
On the other hand, SLC18A2 encoding VMAT2 was expressed significantly in ATRX-cluster cases and its inhibition with GZ-793A (VMAT2 inhibitor) represented significant attenuation of cell proliferation. VMAT2 is a membrane protein that transports monoamines from the cytosol into synaptic vesicles. Production of monoamines such as dopamine and noradrenaline is a hallmark of neuroblastoma.
These monoamines are degraded by enzymes, COMT and monoamine oxidase (MAO), and are finally excreted as monoamine metabolites homovanillic acid and vanillylmandelic acid [42, 43] Excess free dopamine in the cytosol undergoes oxidation  producing ROS .
Since this generation of ROS induces cytotoxicity and neurodegeneration, COMT, MAO, and VMAT2 play an important role in preventing dopamine oxidation in dopaminergic neurons . Therefore, Tolcapone, a potent COMT inhibitor to treat Parkinson’s disease, was reported in neuroblastoma cell lines to induce oxidative stress leading to caspase-3-mediated apoptosis and to inhibit tumor proliferation .
The efficacy of Tolcapone in neuroblastoma cell lines was also demonstrated in the present study and showed synergy effect with VMAT2 inhibitor in MYCN-cluster representative IMR-32 cell line. However, this synergistic effect was not observed when treated with higher Tolcapone concentration (20μM), suggesting that MYCN-cluster phenotype might use additional pathways for their proliferation and survival because Tolcapone and VMAT2 inhibitor target the same monoamine metabolism pathway.
Furthermore, a report from the Children’s Oncology Group showed correlation of metaiodobenzylguanidine (MIBG) avidity with high VMAT2 expression in neuroblastoma cases without MYCN amplification . Thus, the combination of VMAT inhibitor with COMT/MAO inhibitor (MYCN-cluster) or MIBG radiation therapy (ATRX-cluster) might be a potential therapeutic strategy for treating neuroblastoma cases.
In the present study, 30 cases with INSS Stage 4 neuroblastoma was classified into MES- and ADRN-clusters based on expression profiles. These two clusters showed association with the differentiation process and the origin of neuroblastoma (Fig 6). Further classification of the ADRN-cluster identified MYCN- and ATRX-clusters, characterized by genetic alterations and metabolism with potential therapeutic strategy.
Treatment of high-risk neuroblastoma has already been intensified to the maximum limit without exceeding toxicity levels harmful to the patients. Thus, targeting metabolic reprogramming distinct in each cluster might be helpful for the development of a new therapeutic strategy for high-risk neuroblastoma.