Postnatal Sustentacular Cells as Chromaffin Progenitors and Tumor Cells of Origin in VHL-Related Paragangliomas

Postnatal Sustentacular Cells as Chromaffin Progenitors and Tumor Cells of Origin in VHL-Related Paragangliomas | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Postnatal Sustentacular Cells as Chromaffin Progenitors and Tumor Cells of Origin in VHL-Related Paragangliomas Petra Bullova, Peng Cui, Maria Arceo, Jiacheng Zhu, Wenyu Li, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6907400/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2025 Read the published version in npj Precision Oncology → Version 1 posted 10 You are reading this latest preprint version Abstract The cellular source of chromaffin cell regeneration after birth and its relationship to paraganglioma tumorigenesis remains incompletely defined. Here, we identify a postnatal population of SOX2/SOX10-expressing sustentacular glia-like cells in the organ of Zuckerkandl (OZ) and adrenal gland that give rise to chromaffin cells in vivo . These cells differ transcriptionally from embryonic chromaffin progenitors known as Schwann cell precursors and exhibit a unique progenitor signature. Genetic lineage tracing confirms their postnatal contribution to chromaffin cells, and SOX2 + PHOX2B + transitional cells were observed in both human and mouse OZ and adrenal tissues. Single-nuclei RNA-seq and inferCNA analysis of pheochromocytoma and paraganglioma (PPGL) revealed that while most sustentacular cells exhibit a stromal profile, a subset in VHL-mutated PPGLs harbor the hallmark 3p chromosomal loss shared with chief tumor cells, suggesting a clonal origin. In an additional PPGL, widespread SOX2 expression in PHOX2B + tumor cells supports this hypothesis. Finally, DLK1-NOTCH signaling was predicted as a central regulator of chromaffin–sustentacular communication, suggesting DLK1 fine-tunes chromaffin regeneration via NOTCH inhibition and may represent a therapeutic target in PPGL. Biological sciences/Cancer/Cancer stem cells Biological sciences/Cancer/Endocrine cancer Biological sciences/Cancer/Tumour heterogeneity SOX2 SOX10 chromaffin progenitor glia sustentacular cells adrenal medulla organ of Zuckerkandl PPGL paraganglioma neuroendocrine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The adrenal medulla is composed of neural crest-derived neuroendocrine chromaffin cells, which are responsible for catecholamine production, i.e. epinephrine and norepinephrine, mediating the body’s 'fight or flight' response. In addition to the adrenal medulla, chromaffin cells are also located in extra-adrenal sites, including the organ of Zuckerkandl (OZ) near the abdominal aorta and in small clusters along paravertebral sympathetic ganglia (paraganglia), producing predominantly norepinephrine 1 . Tumors (PPGL) arising in these tissues —pheochromocytomas (intradrenal) and paragangliomas (extradrenal) — often cause catecholamine overproduction, and can lead to severe hypertension and increased the risk of cardiovascular events 2 . PPGL have the highest heritability rates among all tumors, with 40% of patients carrying a susceptibility gene mutation in constitutional tissues 3 . Recent studies have demonstrated that embryonic chromaffin cells at the adrenal anlagen originate from neural crest-derived multipotent Schwann cell precursors (SCPs) 4 , 5 . SCPs migrate along visceral motor nerves to the developing adrenal anlagen, contributing to the majority of the adrenal chromaffin cell population embryonically 4 . During mouse embryogenesis, SCPs also give rise to paraganglia, including chromaffin cells in the OZ and some sympathetic neurons 6 . Notably, multipotent SCPs persist for a short period during embryonic development 4 , 5 , disappearing around mouse embryonic day 15, whereas the OZ reaches the largest size just before/after birth 7 and in humans, this peak occurs even later, around the third year of life before regressing 8 . Thus, the process by which chromaffin cells are generated and replenished postnataly is poorly understood. Putative chromaffin stem cells have been previously postulated 9 , 10 and also been recently reported by analyzing deep single cell sequencing of human postnatal adrenal glands 11 , 12 . In addition, chromaffin cell division might also repopulate chromaffin cells postnatally 13 . Here, we describe the existence of a distinct postnatal chromaffin progenitor population using in vivo lineage tracing, single-cell analysis, and immunofluorescence staining. We identified a progenitor population in the mouse adrenal medulla, organ of Zuckerkandl, human adrenal medulla, and PPGL with characteristics of sustentacular glia cells co-expressing SOX10 and SOX2. SOX2 is a well-known transcription factor that maintains multipotency and stemness by regulating genes associated with embryonic neural crest stem cells, embryonic multipotent crest derivatives (SCPs), and postnatal neural stem cells in neurogenic regions of the central nervous system 14 , 15 . While sustentacular glia cells have traditionally been seen as supportive cells in neuroendocrine tissues through paracrine signaling, our findings suggest they also serve as progenitor cells, contributing to chromaffin cell generation postnatally and supporting neuroendocrine tissue integrity through pathways involving NOTCH inhibitor DLK1 and WNT6 paracrine signaling. While the majority of sustentacular cells in PPGL are believed to act as non-neoplastic stromal components, we identified in two PPGL cases where a subset of SOX2 + sustentacular cells (8% and 15%) shared predicted copy number aberrations with neoplastic tumor cells. This suggest that, in rare instances, sustentacular cells may serve as the tumor cell of origin. These findings not only expand our understanding of postnatal chromaffin progenitors but also highlight the potential dynamic role of sustentacular cells in tumor biology. Investigating their contributions to the tumor microenvironment and progression could inform new diagnostic, prognostic, and therapeutic strategies. Results Postnatal SOX10 + cells are chromaffin progenitor cells in vivo To investigate whether SOX10 + glial cells contribute postnatally to the generation of chromaffin cells (TH + ) in the adrenal medulla, we performed fate tracing using the glia-specific inducible Cre-line Sox10 CreERT2+/− coupled to the R26R YFP+/− reporter. Genetic cell fate tracing was initiated postnatally in glial cells by tamoxifen (TAM)-induced recombination. TAM was administered orally to nursing females via gavage during the first 4 days after birth (P0–P4, Fig. 1 A). Analysis of adrenal medullas at postnatal days P5, P14, and P90 revealed that 4.25%, 14.17%, and 17.56% of YFP traced cells (Fig. 1 B), respectively, were positive for tyrosine hydroxyase (TH) (Fig. 1 D–G). However, the substantial increase in TH + chromaffin cells from P5 to P90 (a 3.7-fold increase, Fig. 1 C) could not be attributed entirely to postnatally derived SOX10 + cells. This suggests that perhaps neonatal TH + chromaffin cells (P0–P7) retain the capacity to proliferate, contributing to postnatal chromaffin cell expansion. To assess proliferation in the adrenal medulla, we administered EdU (5-ethynyl-2'-deoxyuridine), a thymidine analog that incorporates into newly synthesized DNA, 2 hours before euthanizing P5 and P14 mice. Adrenal glands were sectioned and stained for TH and EdU-labeled cells. At P5, we observed multiple EdU + TH + cells in the medullary region (Fig. 1 H), indicating that neonatal chromaffin cells retain the capacity for self-renewal. However, the proliferative capacity declined by P14 (Fig. 1 I). Taken together, our data demonstrate that TH + chromaffin cells can regenerate postnatally through two mechanisms: differentiation from SOX10 + progenitors and self-renewal during the neonatal stage. Postnatal SOX10 + cells in the adrenal medulla and the organ of Zuckerkandl (OZ) co-express the multipotency factor SOX2, resembling sustentacular cells. SOX2 co-expression has been observed alongside SOX10 in neural crest cells and embryonic multipotent crest derivatives, SCPs 16 – 19 . SCPs are the major source for the generation of embryonic chromaffin cells 4 , but not observed postnatally. To characterize chromaffin progenitors, we analyzed both postnatal and embryonic adrenal medulla and OZ for expression of SOX10, SOX2 and sympatho-adrenal lineage specification marker PHOX2B (Fig. 2 ). Immunofluorescence staining for SOX10 and SOX2 revealed that SOX10 + cells are positive for SOX2 + in the neonatal (P1) adrenal medulla (Fig. 2 A) and OZ (Fig. 2 B), persisting to adulthood (Suppl. Fig. S1 A), similarly as seen embryonically at day E17 (Fig. 2 C). However, hematoxylin and eosin (HE) staining revealed distinctive morphological features of SOX10 + SOX2 + cells in the postnatal medulla and OZ, distinguishing them from the embryonically SOX10 + SOX2 + glia/SCPs cells (E17). Postnatal SOX10 + SOX2 + cells have distinctive spindle-shaped nuclei and extended cellular processes that interface with the surrounding chromaffin cells, and resemble morphological features of sustentacular glia cells (Fig. 2 A–B). In contrast, embyonic SOX10 + SOX2 + cells were scattered among differentiating chromaffin cells, having round to oval nuclear shape and cell processes that were thin and difficult to discern (Fig. 2 C), indicating that these cells most likely represent satellite-glia and/or SCPs. Additionally, we observed a few SOX2 + PHOX2B + cells in the postnatal adrenal medulla and their occurrence was increased in the OZ at P1 (Fig. 2 A–B and Fig. 3 ), indicative of a transitional state between progenitors and committed sympatho-adrenal cells. PHOX2B expression indicates lineage commitment to neuroendocrine cell types and is expressed in chromaffin cells, sympathoblast and fully differentiated neurons. However, a SOX2 + PHOX2B + cell could represent a progenitor cell in transition, a cell that has not yet fully differentiated into chromaffin or sympathetic cells. Thus, we quantified SOX2 + PHOX2B + double-positive nuclei in the adrenal medulla (Fig. 3 A–B) and in the OZ (Fig. 3 C–D) at P1. Whereas in the adrenal medulla only a few SOX2 + PHOX2B + (0.6%) or SOX10 + SOX2 + PHOX2B + (5.2%) nuclei were detected, a larger proportion of SOX2 + PHOX2B + double-positive (13.5%) and triple-positive (6.5%) nuclei was observed in the OZ (Fig. 3 D–E). Notably, triple-positive cells exhibited markedly weaker PHOX2B expression compared to SOX2 + PHOX2B + double-positive cells that lacked SOX10, suggesting that SOX10 downregulation is required for full PHOX2B expression. The observation that a relatively large proportion (20.4%) of PHOX2B positive cells coexpressed SOX2 in the OZ, compared to only 1.1% in the adrenal gland, was unexpected (Fig. 3 E). We suspect that, at birth, when the OZ reaches its maximal size, sustentacular cells generate a large number of chromaffin cells, reflecting the OZ’s peak functional importance during the perinatal period. This may support critical physiological processes such as catecholamine production and stress response. Notably, SOX2 + PHOX2B + double-positive cells in both the OZ and adrenal gland expressed TH, consistent with noradrenergic fate restriction (Fig. 3 F–G and Suppl. Fig. S2 A–D). Next, we analyzed adrenal glands from children (n = 3), aged 20 weeks to 4 years, to investigate the co-expression of SOX10, SOX2, and PHOX2B and determine whether transitional cells, similar to those observed in mouse adrenal glands, are also present in human tissue (Fig. 4 , Suppl. Fig. S3). All SOX10 positive nuclei co-expressed SOX2, similar to adrenal sustentacular cells in mice (98.22% \(\:\pm\:\) 1.58% of the total SOX10 + cells were SOX2 + ). Among all DAPI positive cells counted, 1.6% \(\:\pm\:\) 1.59% were triple-positive for SOX10, SOX2, and PHOX2B, a percentage comparable to that observed in the mouse medulla at P1 (Fig. 4 C). These findings suggest that human adrenal chromaffin cells in children may also undergo postnatal regeneration, similar to what is observed in mice. However, the rarity of transitioning cells committing to differentiation aligns with previous findings, which describe the postnatal adrenal medulla as a nearly post-mitotic tissue with a stable and low proliferation rate. 13 , 20 . SOX2 + SOX10 + sustentacular cells form a distinct population at postnatal ages with a gene expression program different from embryonic glia/SCPs To investigate transcriptional differences between postnatal and embryonic glial cells, we profiled adrenal cell populations in mice across developmental stages. Postnatal (PN) adrenal samples ( n = 18; ages: P5, P14, P90, and aged) comprised 4,552 single cells, while embryonic (E17) medullary samples ( n = 7) included 2,549 single cells. All cells were analyzed using deep single-cell RNA sequencing with the SmartSeq2 protocol (Fig. 5 A, Suppl. Figure 4A–C). Quality-controlled data were subjected to unsupervised clustering, resulting in the identification of 7,101 adrenal cells and categorized into eight medullary clusters consisting of glia (6,11), chromaffin (0, 1, 2, and 5), embryonic neuroblasts (10) and embryonic TH-expressing cycling cells (8) (Fig. 5 A–B, Suppl. Fig. S4A–C). We performed in silico subsetting of adrenal medullary cell populations using previously established markers 11 . Clusters were annotated based on the expression of key marker genes; noradrenergic and adrenergic chromaffin markers ( Pnmt, Th, Dbh, Chga, Chgb, Epas1, Penk ), neuroblast markers ( Elavl4, Isl1, Gap43, Nefl, Prph ), glial markers ( Sox10, Plp1, Erbb3, Fabp7 ), and cycling markers ( Top2a, Mki67, Aspm, Bub1 ) (Suppl. Fig. S4D). Non-medullary populations, including cortical, mesenchymal, endothelial, and immune cell clusters, were also identified and annotated (Suppl. Fig. S4D). We found that the embryonic glia/SCP population at E17.5 (6-E_Glia/SCP) transcriptionally differed transcriptionally from the postnatal glia/sustentacular population (11-PN_Glia/sustentacular) (Fig. 5 C). Differentially expressed genes (DEGs) characterizing these populations were identified using specific DEG analysis methods (Wilcoxon Rank Sum test, Table S1 , detailed in Methods), revealing distinct transcriptional programs (Fig. 5 C–E). The embryonic glial/SCP and postnatal glial sustentacular cells shared the expression of canonical glial markers, including Sox10, S100b, Foxd3, Plp1, Erbb3 , and Fabp7 (Fig. 5 E, Suppl. Fig. S4D). Additionally, they co-expressed Sox2 , as previously demonstrated by immunofluorescence staining (Fig. 2 ). However, the embryonic glia/SCP population significantly upregulated Wwtr1 , Lmo4 , and Moxd1 (Fig. 5 C–E), genes known to play critical roles in maintaining cell stemness and proliferation 21 – 23 . Notably, Moxd1 ( Monooxygenase DBH Like 1 ) has been previously characterized as highly enriched in embryonic SCPs 24 , 25 (Fig. 5 E). In contrast, postnatal glia-sustentacular cells showed significant upregulation of Wnt6 , Notch1 , Hey2 , Sfrp5/1 , and Tgfb2 (Fig. 5 E, Table S1 ). These genes suggest involvement in processes such as cell signaling, fate determination, and maintenance of progenitor-like characteristics 26 – 32 . Gene ontology (GO) enrichment analysis of the specific DEG list confirmed the enrichment of WNT, BMP, and NOTCH signaling pathways (Fig. 5 F, Table S2 ). These findings are consistent with a recent study 12 , which also identified a distinct transcriptional identity for postnatal SOX2 + sustentacular cells, differentiating them from embryonic Schwann cell precursors (SCPs). Together, these data emphasize the unique molecular signature of postnatal sustentacular cells and support their role as a specialized glial progenitor population in the adrenal medulla. Sustentacular and chromaffin cell-cell communication reveals NOTCH1 and WNT signaling in regeneration and paracrine support. To explore how the identified signaling pathways interact within the adrenal tissue, we performed cell-cell communication analysis using CellChat-tool in different adrenal age groups in mice. CellChat enables systematic analysis of cell-cell communication from single-cell transcriptomics data by quantifying the signaling communication probability between two cell groups, incorporating the core interactions between ligands and receptors 33 . We observed that the NOTCH signaling pathway was predominantly active in postnatal chromaffin cells (Cluster 1 NOR and Cluster 2 ADR) with receptors mainly on sustentacular cells (Cluster 11), (Suppl. Fig. S4E–G). Analysis of individual ligand-receptor pairs predicted that, across all postnatal ages (P5, P14, P90, and aged), the inhibitory NOTCH ligand Dlk1 was primarily expressed and sent by chromaffin cells and received by the Notch1 receptor expressed in sustentacular cells (Fig. 5 G–H, Suppl. Fig. S4H). Similarly this communication was also observed in embryonic glia, chromaffin and neuroblast populations at E17 (Suppl. Fig. S4I). Dlk1 is a known inhibitor of the NOTCH signaling pathway, acting by preventing activation of the NOTCH receptor 34 , 35 . DLK1 is also expressed in sympathoadrenal and chromaffin cells downstream of PHOX2B 36 , supporting its role in lineage-specific regulatory programs. Recent studies have demonstrated that inhibition of the NOTCH signalling pathway leads to an increased number of Th + cells in both sympathetic ganglia and the adrenal gland, whereas activation of the pathway has the opposite effect 37 . Thus, it is plausible that DLK1 expression in postnatal chromaffin cells fine-tunes the regenerative capacity of sustentacular cells during chromaffin differentiation. Notably, DLK1 expression was similarly observed in human neuroendocrine PPGL tumor cells (Suppl. Fig. S5C), suggesting that DLK1 expression in tumor cells might also impact tumor cell differentiation through the tumor microenvironment as observed recently in neuroblastoma 38 . Furthermore, analysis of all NOTCH ligand-receptor communications in chromaffin and glial populations across developmental stages revealed the expression of NOTCH activating ligand Jag2 in chromaffin cells (Fig. 5 I). NOTCH target genes Hes1 , Hey2 and Heyl were expressed highly in sustentacular cells (Fig. 5 J). This suggests a dual role for NOTCH signaling, involving both inhibitory and activating interactions, which may finetune the regenerative capacity of sustentacular cells (Fig. 5 K). In addition to the identified NOTCH-mediated communication between sustentacular cells and chromaffin cells, WNT signaling emerged as another highly enriched pathway in sustentacular cells, as highlighted by a top-regulated GO term (Fig. 5 F). Among the most differentially expressed genes in sustentacular cells were Wnt6 and Sfrp1/5 (Fig. 5 C–E, Suppl. Fig. S5J). CellChat analysis of WNT signaling pathways predicts ligand-receptor communication between WNT6 from sustentacular cells, and the receptors Fzd3/5 and Lrp5/6 expressed in chromaffin cells during postnatal stages (Fig. 5 L, Suppl. Fig. S5J). Interestingly, the expression of Sfrp1/5 in sustentacular cells suggests a potential dual regulatory role for WNT signaling (Fig. 5 M), as these proteins can inhibit WNT receptor activation, balancing inhibitory and activating interactions. Additionally, chromaffin cells highly expressed the WNT co-receptor Lgr5 (Fig. 5 M, Suppl. Fig S4J), which, although not directly reported to interact with WNT6, is known to enhance WNT signaling by stabilizing WNT receptors (e.g., Frizzled receptors) on the cell surface through interactions with R-spondins (RSPOs). The co-expression of Fzd3 and Lgr5 in chromaffin cells (Fig. 5 M) supports the model of paracrine WNT signaling from sustentacular cell-derived WNT6 to chromaffin cells. This finding underscores the critical role of sustentacular cells in supporting chromaffin cell survival under specific physiological conditions. Deep single-nucleus RNAseq analysis of human PPGL shows SOX2 and SOX10 expression in neoplastic cells Sustentacular cells are commonly observed in PPGL, though their prevalence and characteristics vary depending on the tumor type and its microenvironment. Traditionally considered non-neoplastic glial-like cells, sustentacular cells form a supportive framework around chromaffin cells in both the normal paraganglia and PPGL. However, our genetic tracing revealed that sustentacular glial cells can function as chromaffin progenitor cells postnatally (Fig. 1 ). To investigate whether they could also serve as the tumor cell of origin, rather than being confined to structural and supportive roles within the tumor microenvironment, we performed deep single-cell sequencing on PPGL ( n = 9) together with cancer-specific copy number aberration (CNA) inference across both neuroendocrine and sustentacular glial cells. We sequenced 3,418 single nuclei from 9 patients and profiled 2,586 cells passing quality control (Fig. 6 A, Suppl. Fig. S5A, Methods). After assigning each cell to a cell type (Fig. 6 B, Suppl. Fig. S5B–C), we focused on the neuroendocrine compartment to see whether we could find expression of SOX2 or SOX10 in neoplastic cells, and the impact of these genes’ expression. To separate neoplastic from non-neoplastic neuroendocrine cells, copy number aberrations (CNA) were inferred in all neuroendocrine cells, as well as in Schwann cells, the latter to explore whether we can find sustentacular-like neoplastic cells in PPGL. In total 1893/1921 (99%) of all neuroendocrine cells were classified as neoplastic (Fig. S5D), and the inferred patterns of chromosomal aberrations largely confirmed genomic data. All four tumors with genetically confirmed Von Hippel-Lindau ( VHL) mutations (samples 195, 262, 198 and 267) were found to have a chromosome 3p loss, consistent with a second hit in VHL -mutated malignancies 39 (Fig. 6 C). Further validating our approach, the sample 266, with a known NF1 mutation, showed the corresponding 17q deletion (Fig. 6 C). Similarly, out of the tumors with known chromosome 1p status, inferred copy numbers aligned with known genetics in all cases (Fig. 6 C, Table S3). None of the sustentacular cells (SC) sequenced shared any CNA with the neuroendocrine tumor cells and thus were classified as non-neoplastic (Fig. 6 D, 6 F–G) In some cases (sample 199 Fig. 6 D) only a few sustentacular cells were sequenced. Thus, we additionally performed anti-SDHB immunofluorescence staining on sample 199 ( SDHB mutant) to validate its neoplastic status (Fig. 6 E). PPGL harboring germline SDHB mutations are known to display loss of SDHB immunoreactivity 40 and thus SDHB immuno-staining provides a valuable tool to investigate the neoplastic status of sustentacular cells. Neuroendocrine TH + PHOX2B + tumor cells were organized in “zellballen” and confirmed loss of SDHB expression, however surrounding SOX10 + sustentacular cells were positive for SDHB, indicating being normal and confirming results derived by inferred CNA (Fig. 6 E). While no sustentacular cells were classified as neoplastic, a subset of neoplastic neuroendocrine cells expressed either SOX10 (sample 198: 6.25%) or SOX2 (sample 264: 12.1%). Neoplastic cells expressing either SOX2 (sample 264, Fig. 6 G) or SOX10 ( sample 198 Fig. 6 F ) shared CNA with other cells from the corresponding tumors, and the quantitative metrics used to determine neoplastic-CNA signal and correlation (see Methods) - were comparable across neuroendocrine cells regardless of SOX2 / SOX10 positivity (Suppl. Fig. S5D–E). The SOX10 positive cells were found across multiple tumors, with low numbers in each. In contrast to SOX10, SOX2 positive neoplastic cells were only found to any significant degree in one tumor, sample 264, where 40/331 (12%) of neoplastic cells had at least one read from SOX2 (Fig. 6 G). Combined immunofluorescence staining for SOX2 and PHOX2B revealed that, in this sample, the majority of neoplastic chief cells (PHOX2B + ) were SOX2 positive (Fig. 6 H), indicating that our inability to show SOX2 mRNA expression in the majority of neuroendocrine cells was an effect of technical dropouts rather than biological heterogeneity within the tumor. Indeed, no genes were significantly differentially expressed between cells with and without identified SOX2 mRNA in this sample. To assess the impact of SOX2 expression in neoplastic cells, we repeatedly performed differential gene expression analysis between this tumor and each of the remaining eight tumors separately, searching for genes that were recurrently over/underexpressed (log2FC > = 1, p < 0.05, t-test). 42 genes were significantly overexpressed in sample 264 in all eight pairwise comparisons – notably ASCL1 , HOXA9 and TUBB3 – suggesting a link between malignant SOX2 expression and a stem-like, neuronal-like phenotype (Fig. S5F, Table S4). Achaete-scute homolog 1 (ASCL1), a proneural transcription factor, plays a central role in neurogenesis 41 – 43 . Furthermore, ASCL1 activates PHOX2A, which subsequently induces the expression of genes essential for catecholamine biosynthesis 44 . It is plausible that a similar ASCL1-mediated program operates postnatally, with sustentacular progenitor cells contributing to the chromaffin lineage. In PPGL, sustained ASCL1 expression may promote tumor cell plasticity and maintain an undifferentiated state. Schwann cells share 3p deletions with neuroendocrine cells in VHL -mutated tumors Although we could not find any evidence of neoplastic sustentacular cells in our data, the discovery of SOX2 expression in neuroendocrine neoplastic cells indicated that these cells might exist, but that the low cell numbers sequenced by Smartseq2 and heavy skewing towards neuroendocrine cells in our dataset prevented their discovery. Thus, we re-analyzed a larger, droplet-based snRNAseq dataset of PPGL samples 45 , providing both more samples and higher numbers of cells per sample. In five samples with > = 100 Schwann-cell-like cells (SCLCs), as defined by the authors, we re-inferred CNA in SCLCs and neuroendocrine cells, using as reference (Methods) both stromal cells from the same patient as well as, to avoid false positive copy number events due to Schwann-cell-specific transcriptional programs, SCLCs from two normal adrenal medulla samples in the same dataset. SCLCs were classified as malignant if they showed deletions or amplifications consistent with any of the copy number events found in the neuroendocrine cells. Through this approach, in two samples (E024, E042) we were able to find subsets of SCLCs (16/199, 8% and 18/122, 15%) with inferred 3p deletions, consistent with the neuroendocrine cells, but none of the additional copy number events found (Fig. 7 A–B). Both samples exhibited germline VHL mutations, suggesting that these SCLCs could represent sustentacular glial cells that have undergone a second-hit deletion through the loss of 3p, encompassing the remaining wild-type VHL allele. For Von Hippel-Lindau disease-related PPGL, the inactivation or loss of both alleles of the VHL gene, as predicted by the Knudson two hit theory, is required. Thus, in both of these cases (E024, E042), the biallelic inactivation of VHL as an initiating tumor driving event resulted from the combination of germline VHL mutation and subsequent chromosomal 3p loss in SCLCs. Analyzing differentially expressed genes between SCLCs with or without 3p deletions, we found that 20 genes were consistently upregulated in non-neoplastic SCLCs in both samples. Notably, only four of these ( CNTN4, BHLHE40 , RAF1 and CAPN7 ) are located on chromosome 3p, indicating that the inferred 3p deletion is not an artifact of transcriptional downregulation. In “pre-neoplastic” SCLCs, only one gene per sample was significantly upregulated – HNRNPA2B1 (E024) and NRXN1 (E042) (Table S5). Notably, the latter was found to be expressed in both neuroendocrine and Schwann cells in our own PPGL data (Fig. 5 B). Discussion Previously, single-nuclei transcriptomes of human adult adrenal glands identified a putative progenitor population with intermediate markers of chromaffin cells, supported with the directional transition from progenitor cells to chromaffin cells predicted by RNA velocity 11 . In this study, we provide in vivo evidence for the existence of such chromaffin progenitor populations postnatally, using in vivo lineage tracing, single-cell RNA sequencing, and immunofluorescence in mouse and human adrenal glands. Our findings establish SOX2 + SOX10 + cells as a distinct progenitor population capable of giving rise to chromaffin cells after birth. Our single-cell transcriptomic analysis of embryonic and postnatal glial cells revealed that chromaffin regeneration by SOX2 + SOX10 + cells postnatally represents a source distinct from their embryonic origin, the SCPs. While SCPs serve as the primary source of embryonic chromaffin cells during development 4 , their absence postnatally underscores the critical role of an alternative progenitor source in maintaining a growing tissue. Our study provides insights into the role of adrenal SOX2 + SOX10 + glial cells, characterized as sustentacular cells, as postnatal chromaffin progenitors. The identification of SOX2 + PHOX2B + transitional cells in both human and mouse adrenal medulla, as well as in extra-adrenal chromaffin tissues such as the OZ, further supports the notion of a dynamic and continuous process of chromaffin differentiation during the perinatal and postnatal periods. Single-cell transcriptomic analysis enabled us to quantify the signaling communication of core interactions between ligands and receptors across cell populations. We found that maintaining neuroendocrine tissue integrity involves dynamic signaling interactions of the DLK1-NOTCH and WNT pathways. A recently published study 12 also describes sustentacular cells as chromaffin stem cells and highlights the role of paracrine signaling through WNT secretion, further supporting our findings. Furthermore, we characterized the role of SOX2 + SOX10 + sustentacular cells in their contribution to PPGL tumorigenesis, uncovering instances where sustentacular cells may serve as tumor cells of origin. We could identify two PPGL samples where subsets of glial cells (8% and 15%) had inferred 3p deletions shared with the neuroendocrine tumor cells, inferred 3p deletions, which were shared with the neuroendocrine tumor cells, suggesting a common parental cell origin. Both samples harbored germline VHL mutations. This supports the notion that a second-hit deletion through the loss of 3p, encompassing the remaining wild-type VHL allele, led to VHL inactivation—a prerequisite for VHL loss-driven tumorigenesis. No additional copy number alterations were shared with the neuroendocrine tumor cells in either case, indicating that 3p loss was an early or initiating event in tumorigenesis, preceding other genetic alterations associated with tumor progression. Thus, we termed these sustentacular cells harboring the 3p deletion as pre-neoplastic. A recent study using CNV predictions also identified rare aneuploid glial cells, termed malignant SCP-like cells, in a related neuroendocrine tumor, neuroblastoma 46 . However, it is possible that this population instead resembles sustentacular glia-like cell, which are also a component of the tumor microenvironment in low-risk neuroblastomas. In one case of PPGL, the majority of neoplastic neuroendocrine PHOX2B + tumor cells coexpressed SOX2. ASCL1, a proneural transcription factor, was found significantly upregulated in this tumor compared to all other samples with SOX2-negative tumor cells. This establishes a link between SOX2 expression and a stem-like phenotype. The regulatory transcription factor Achaete-scute homolog 1 (ASCL1) plays a central role in neurogenesis 41 – 44 . Similarly, ASCL1 might also act as a pioneering factor postnatally, mediating a program in sustentacular progenitor cells that contributes to the chromaffin fate. Notably, the expression of SOX2 in PPGL has previously been reported 47 , supporting a stem-like or progenitor-associated phenotype. Sustained ASCL1 expression in these pheochromocytoma cells may thus reflect an inability of these tumor cells to fully differentiate, keeping them in a plastic and immature state. In PPGL, the tumor-initiating event is well understood in the context of germline predisposition, but the cell of origin for oncogenic transformation leading to PPGL remains unclear. One of the intriguing aspects of PPGL is their unusually high prevalence of germline predisposition compared to most other adult cancers, despite being a rare cancer type. This has been suggested to be linked to specific developmental windows that permit oncogenic initiation 48 . While the majority of PPGL are thought to arise from either embryonic or postnatal chromaffin tissue, our findings suggest that tumorigenesis can also be initiated in chromaffin progenitor cells, specifically sustentacular cells. This raises the possibility that developmental timing and cellular plasticity play critical roles in determining susceptibility to oncogenic transformation. Finally, identifying SOX2 + SOX10 + sustentacular cells as postnatal chromaffin progenitors may have significant clinical implications. Understanding the molecular mechanisms governing their differentiation and plasticity could unveil novel diagnostic and prognostic markers for PPGL, enabling earlier and more precise disease stratification. The presence of SOX2 + PHOX2B + transitional cells in both adrenal and extra-adrenal chromaffin tissues underscores a dynamic regulatory network, potentially driven by key signaling pathways such as NOTCH inhibition by DLK1 and paracrine WNT signaling, which could serve as therapeutic targets. In this context, the identification of DLK1 expression in chromaffin cells and PPGL’s is particularly intriguing, as DLK1 has recently been described as an immunotherapeutic target in neuroblastoma, with DLK1 silencing promoting differentiation 38 . Materials and Methods Animals Mouse experimental procedures were permitted by the Stockholm North committee for animal experiments. R26R YFP and Sox10 CreERT2 mice have been described 49 . C57BL/6 mice were kept in rooms with controlled 12-h light/dark cycles, temperature, and humidity with food and water provided. Animal care were in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). Tamoxifen-induced lineage tracing, EdU administration and tissue preparation are described in supplementary materials. Human tissue specimens and tissue preparation Tumor tissue samples (PPGL n = 9) were collected from individuals operated and diagnosed at the Karolinska University Hospital, Stockholm, Sweden, and previously characterized for mutations in PPGL susceptibility genes 50 . Post-mortem human adrenal glands were obtained from the NIH Neurobiobank (University of Maryland, Baltimore, MD) as previously described 11 . Tissue preparation are described in supplementary materials. Immunofluorescence, Hematoxylin and eosin (H&E) staining and Statistical analyses Staining protocoll and antibodies are described in supplentarial materials. In short, tissue sections were deparaffinized in xylene (HistoLab Products AB, #02070) and rehydrated using a series of ethanol (Solveco) and distilled water washes. Antigen retrieval was performed in citrate target retrieval solution (Agilent, #S1699) for 20 min at 96–100°C. The slides were cooled down, washed, and hydrophobic barrier was created using ImmEdge Pen (Vector laboratories, #H-4000). Alternatively, mouse cryosections were left to dry at room temperature for 15 min and washed 3× in PBS. Statistical analyses of immunofluorescence images are detailed in supplentarial materials. Ethical considerations Collection and analyses of human samples (PPGL) are covered by the ethical approval numbers 01-136 local ethical committee KI forskningsetikkommitté Nord) and 2020–04226 (Swedish Ethical Review Authority). All samples were obtained following an informed patient consent. Post-mortem human adrenal glands for staining’s were obtained from the NIH Neurobiobank (University of Maryland, MD) under the same ethical permit from Stockholm Regional Ethical Review Board and the Karolinska University Hospital Research Ethics Committee (KI 2007/069 and KI 2001/136). Ethical permits for animal studies were approved by the appropriate local and national authorities (Jordbruksverket, Sweden). Mouse tissue single-cell sequencing, data pre-processing and quality control Single-cell suspension preparation for single-cell RNA sequencing is described in supplementary materials and further processed by the Eukaryotic Single Cell Genomics (ESCG) facility at SciLifeLab. Single-cell raw sequencing data was pre-processed as described in our previous work 11 . The quality control of mice individual cells included removing cells expressing more than 10000 or fewer than 200 genes, removing cells whose total expression was due to mitochondrial genes in more than 10%, and removing from the expression matrix genes that are expressed in fewer than 3 cells. Mouse single-cell data clustering and cell type identification are further described in supplementary materials. Specific differential expressed genes (DEGs) and GO term pathway analysis To identify specific DEGs in mouse embryonic and postnatal glia cells, we conducted differential expression analysis by comparing each cellular cluster against another cluster in a pairwise manner. This process was repeated iteratively for all possible cluster pairs and described in supplementary materials including gene-ontology enrichment analysis and cellulare interaction networks. Human single-nuclei sequencing, pre-processing and quality control Human samples were processed using a single-nuclei sequencing protocol, while mouse samples underwent a single-cell sequencing protocol. The protocols for processing both human and mouse samples were described in our previous work 11 . Human single-nuclei data pre-processing, quality control, clustering and cell type assignment are described in supplementary materials. CNA inference Copy number aberration (CNA) inference and neoplastic cell definitions are described in supplentarial materials. In short, CNA inference was performed similarly to 51 . Since not all samples had enough stromal cells to be used as reference for CNA inference, a pooled reference of fibroblasts, endothelial cells adrenocortical cells, deriving from multiple patients was created. To avoid creating batch effects, from each patient with at least 15 fibroblasts, endothelial or adrenocortical cells, 15 cells from that cell type were randomly sampled. This created a pooled reference comprising 30 fibroblasts, 45 endothelial cells and 30 adrenocortical cells, which was used for all CNA inference. Data and code availability Raw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: https://github.com/susanneschlisio/Bullova-et-al . The raw sequencing data of human and mouse samples will be publicly accessible at the date of publication. Declarations Acknowledgments Raw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: https://github.com/susanneschlisio/Bullova-et-al . The raw sequencing data of both human and mouse samples will be publicly accessible as the date of publication. The single-cell transcriptome data was generated at the Eukaryotic Single-cell Genomics facility at Science for Life Laboratory in Stockholm, Sweden. The computations and data handling for this project were performed on resources provided by the the National Academic Infrastructure for Supercomputing in Sweden (NAISS) at sens2018122, NAISS 2024/5-221, NAISS 2024/6-129, and NAISS 2024/6-332. We thank the NIH NeuroBioBank providing human adrenal glands. We thank Oscar Bedoya Reina for his computational support with the smartseq2 RNAseq analysis. Author contributions: Conceptualization: P.B., J.Z., M.M. and S.S. Methodology: P.B., P.C., M.A., J.Z.,W.L., V.P., M.P., K.S., M.E.K, C.S. and M.M. Formal analysis: P.B., P.C., M.A., J.Z., W.L., V.P., M.P., K.S., C.S., M.M. and S.S. Resources: C.L. C.J. Writing—original draft: M.M., P.B., J.Z., and S.S. Writing—review and editing: P.B., P.C., M.A., J.Z., W.L., K.S., C.S. M.E.K., C.L., C.J., M.M S.S. Visualization: P.C. J.Z., Supervision: M.M. and S.S. Project administration: S.S. Funding acquisition: S.S. was funded by the Swedish Research Council, Swedish Childhood Cancer Fund, the Swedish Cancer Society, ParaDiff Foundation, ERC Synergy grant (KILL-OR-DIFFERENTIAT) and Radiumhemmets Forskningsfonder. P.B. was funded by the Swedish Cancer Society. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Raw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: https://github.com/susanneschlisio/Bullova-et-al . 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Supplementary Files BullovasupplementarymaterialsJune2025.pdf BullovaSupplementaryTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2025 Read the published version in npj Precision Oncology → Version 1 posted Editorial decision: Revision requested 30 Jul, 2025 Reviews received at journal 30 Jul, 2025 Reviews received at journal 28 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 06 Jul, 2025 Submission checks completed at journal 18 Jun, 2025 First submitted to journal 16 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6907400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":481995692,"identity":"83ddef47-6ed6-4d1d-8b4c-389b32d48f7b","order_by":0,"name":"Petra Bullova","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Bullova","suffix":""},{"id":481995693,"identity":"4c2fb9d9-b699-4809-bbd0-45c8c5033cd1","order_by":1,"name":"Peng Cui","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Cui","suffix":""},{"id":481995694,"identity":"ca1b79ee-6823-490b-a553-0dc43e4e6410","order_by":2,"name":"Maria Arceo","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Arceo","suffix":""},{"id":481995695,"identity":"7258c288-d7ba-4638-b6e8-bb86c34d62e9","order_by":3,"name":"Jiacheng Zhu","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiacheng","middleName":"","lastName":"Zhu","suffix":""},{"id":481995696,"identity":"fbbb0a01-7fd6-4618-9072-113dbb49e4a4","order_by":4,"name":"Wenyu Li","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenyu","middleName":"","lastName":"Li","suffix":""},{"id":481995697,"identity":"c7fee93b-495c-42eb-9c4e-6526f670bed8","order_by":5,"name":"Monika Plescher","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"","lastName":"Plescher","suffix":""},{"id":481995698,"identity":"6e0b05e9-9995-4b16-90df-9c2b0fb6894e","order_by":6,"name":"Valentin Poltorachenko","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Valentin","middleName":"","lastName":"Poltorachenko","suffix":""},{"id":481995699,"identity":"8419cd01-6265-4571-93ca-58f543d6a4f1","order_by":7,"name":"Katerina Stripling","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Katerina","middleName":"","lastName":"Stripling","suffix":""},{"id":481995704,"identity":"5e50cb4a-4d33-4213-a131-545712c5a8d1","order_by":8,"name":"Christian Santangeli","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Santangeli","suffix":""},{"id":481995707,"identity":"08f2af0f-ca75-4674-b400-c357a31c920b","order_by":9,"name":"Maria Eleni Kastriti","email":"","orcid":"","institution":"Medical University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Eleni","lastName":"Kastriti","suffix":""},{"id":481995710,"identity":"6c21dd3b-0bae-4d23-8719-d23167d33ef0","order_by":10,"name":"Catharina Larsson","email":"","orcid":"","institution":"Karolinska Institutet, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Catharina","middleName":"","lastName":"Larsson","suffix":""},{"id":481995711,"identity":"1d85b944-5c73-4322-b2a8-dcd68f0af51a","order_by":11,"name":"C. 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The \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;R26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eYFP/+\u003c/em\u003e\u003c/sup\u003e pups induced with Tam by breast-feeding during these 4 days. After 1, 10, or 86 days, the adrenal glands harvested at P5, P14, or P90 for sectioning, staining, and analysis. \u003cstrong\u003e(B)\u003c/strong\u003e Bar plot for medullary (TH\u003csup\u003e+\u003c/sup\u003e) cells number (P5, 676.50 ± 65.50, \u003cem\u003en\u003c/em\u003e = 2; P14, 1067.00 ± 75.14, \u003cem\u003en\u003c/em\u003e = 4; P90, 2542.00 ± 128.90, \u003cem\u003en\u003c/em\u003e = 6. \u003cem\u003eP\u003c/em\u003e value from Kruskal-Wallis test is 0.0004. \u003cem\u003eP\u003c/em\u003e values from Dunn’s multiple comparisons test for P5 \u003cem\u003evs.\u003c/em\u003e P14 is \u0026gt; 0.9999; P5 \u003cem\u003evs.\u003c/em\u003e P90 is 0.0197; P14 \u003cem\u003evs.\u003c/em\u003e P90 is 0.0951). The number in each bar indicates sample number (n). Data are presented as mean ± s.e.m. *, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05. \u003cstrong\u003e(C)\u003c/strong\u003e Percent stacked bar plot for the ratios of YFP and TH double-positive (YFP\u003csup\u003e+\u003c/sup\u003e TH\u003csup\u003e+\u003c/sup\u003e) cells in total YFP positive (YFP\u003csup\u003e+\u003c/sup\u003e) cells (P5, 4.25% ± 4.25%, \u003cem\u003en\u003c/em\u003e = 2; P14, 14.17% ± 3.40%, \u003cem\u003en\u003c/em\u003e = 4; P90, 17.56% ± 2.56%, \u003cem\u003en\u003c/em\u003e = 6. \u003cem\u003eP\u003c/em\u003e value from Kruskal-Wallis test is 0.0827. \u003cem\u003eP\u003c/em\u003e values from Dunn’s multiple comparisons test for P5 \u003cem\u003evs.\u003c/em\u003e P14 is 0.6006; P5 \u003cem\u003evs.\u003c/em\u003e P90 is 0.0944; P14 \u003cem\u003evs.\u003c/em\u003e P90 is 0.9482). The numbers on the top in each bar indicates averaged total YFP positive (YFP\u003csup\u003e+\u003c/sup\u003e) cells number at each timepoint. \u003cstrong\u003e(D–F)\u003c/strong\u003e Immunofluorescence staining for the adrenal glands harvested at P5, P14, and P90 after Tam-milk fed. Full arrowheads indicate YFP and TH double-positive (YFP\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e) cells. Scale bars in the adrenal gland images and insets are 100 μm and 20 μm, respectively. \u003cstrong\u003e(G)\u003c/strong\u003e H\u0026amp;E and immunofluorescent staining for the same window in (F). Empty arrowheads indicate YFP and SOX10 double-positive (YFP\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e) cells showing lineage tracing efficiency. Full arrowheads indicate YFP and TH double-positive (YFP\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e) cells. Scale bars are 20 μm. \u003cstrong\u003e(H and I)\u003c/strong\u003e Immunofluorescent staining for the adrenal glands harvested at P5, and P14 after Tam-milk fed. Empty arrows indicate EdU and TH double-positive (EdU\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e) cells. Scale bars in the adrenal gland images and insets are 100 μm and 20 μm. \u003cem\u003eAbbreviations: \u003c/em\u003eP, postnatal day; Tam, tamoxifen; d, days; TH, tyrosine hydroxylase; YFP, yellow fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-Ethynyl-2’-deoxyuridine; \u003cem\u003en\u003c/em\u003e, sample number; \u003cem\u003evs.\u003c/em\u003e, versus.\u003c/p\u003e","description":"","filename":"Figures21.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/54275f78ac9d0b9b977935ab.png"},{"id":86260689,"identity":"92d8c484-c068-4499-8b14-617f8158c455","added_by":"auto","created_at":"2025-07-08 14:27:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2890170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePostnatal SOX2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eSOX10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-expressing cells in the adrenal medulla and organ of Zuckerkandl (OZ) resemble sustentacular glia cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Immunofluorescence and H\u0026amp;E staining of P1 adrenal gland. Arrows indicate glial cells co-expressing SOX2 and SOX10. Scale bars in the adrenal glands and insets are 100μm and 20μm, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence and H\u0026amp;E staining of P1 Organ of Zuckerkandl (OZ). Arrows indicate glial cells co-expressing SOX2 and SOX10. Scale bars in the Organ of Zuckerkandl and insets are 100μm and 20μm, respectively. \u003cstrong\u003e(C)\u003c/strong\u003e Immunofluorescence and H\u0026amp;E staining of E17 adrenal gland. Arrows indicate glial cells co-expressing SOX2 and SOX10. Scale bars in the adrenal glands and insets are 100μm and 20μm, respectively.\u003c/p\u003e","description":"","filename":"Figures22.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/f82574ddc43e1c373a79a444.png"},{"id":86259364,"identity":"fc529ba6-edb6-4987-b9f3-c8434bd9d82f","added_by":"auto","created_at":"2025-07-08 14:11:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2553231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOX2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ePHOX2B\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e double-positive cells in the postnatal adrenal medulla and OZ express tyrosine hydroxylase, indicating a transitional state toward sympatho-adrenal fate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative image showing immunofluorescence staining for SOX2, PHOX2B and SOX10 in P1 adrenal gland. Empty arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells, full arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive glial cells, and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive glial cells. Scale bars in the adrenal glands and insets are 100 μm and 20 μm, respectively.\u0026nbsp; \u003cstrong\u003e(B)\u003c/strong\u003e Percent-stacked bar plot for the ratios of SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells (0.6% ± 0.2%), SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive cells (3.5% ± 0.5%)\u0026nbsp; and SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive cells (5.2% ± 0.7%) in total cell number in adrenal medulla. Kruskal-Wallis test; adjusted \u003cem\u003eP\u003c/em\u003e value from Dunn’s multiple comparisons test for SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e vs SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e is 0.0216, and for SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e vs SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e is 0.0003; \u003cem\u003en\u003c/em\u003e = 8. Data are presented as mean ± s.e.m. *, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05; ***, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.001. \u003cstrong\u003e(C)\u003c/strong\u003e Representative image showing immunofluorescence staining for SOX2, PHOX2B and SOX10 in mouse P1 organ of Zuckerkandl. Empty arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells, full arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive glial cells, and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive glial cells. Scale bars in the adrenal glands and insets are 100 μm and 20 μm, respectively.\u0026nbsp; \u003cstrong\u003e(D)\u003c/strong\u003e Percent-stacked bar plot for the ratios of SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells (13.5% ± 1.3%), SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive cells (7.6% ± 0.9%) and SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive cells (6.5% ± 1.2%) in total cell number in organ of Zuckerkandl. Kruskal-Wallis test; adjusted P value from Dunn’s multiple comparisons test for SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e vs SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e is 0.0148; \u003cem\u003en\u003c/em\u003e = 6. Data are presented as mean ± s.e.m. *, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05. \u003cstrong\u003e(E)\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003ePercent-stacked bar plot for the ratios of SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells in total number of PHOX2B-positive cells in adrenal gland (1.1% ± 0.5%; \u003cem\u003en\u003c/em\u003e = 8) vs organ of Zuckerkandl (20.4% ± 1.4%; \u003cem\u003en\u003c/em\u003e = 6). Mann-Whitney t-test; \u003cem\u003eP\u003c/em\u003e value 0.0007. Data are presented as mean ± s.e.m. ***, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.001. \u003cstrong\u003e(F)\u003c/strong\u003e Representative image showing immunofluorescence staining for SOX2, PHOX2B and TH in mouse P1 adrenal gland. Full arrows indicate SOX2\u003csup\u003e \u003c/sup\u003epositive cells and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e triple-positive cells.\u0026nbsp; Scale bars in the adrenal glands and insets are 100 μm and 20 μm, respectively.\u0026nbsp; \u003cstrong\u003e(G)\u003c/strong\u003e Representative image showing immunofluorescence staining for SOX2, PHOX2B and TH in mouse P1 organ of Zuckerkandl. Full arrows indicate SOX2\u003csup\u003e \u003c/sup\u003epositive cells and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e triple-positive cells. Scale bars in the organ of Zuckerkandl and insets are 100 μm and 20 μm, respectively.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figures23.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/bd12c3814ce7cb8d87ac45e6.png"},{"id":86259371,"identity":"6fc769e6-8dc9-420e-bb75-1bb3fdf9dcc0","added_by":"auto","created_at":"2025-07-08 14:11:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2371255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOX2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ePHOX2B\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e transitional cells are present in human adrenal medulla.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative image showing H\u0026amp;E and immunofluorescence staining for SOX2, PHOX2B and SOX10 in 4-year-old human adrenal gland. The box shows one of 5 counting medullary regions in this sample. Full arrows indicate PHOX2B positive cells, full arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive sustentacular glial cells, and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive glial cells. Scale bars in the adrenal gland and insets are 100μm and 20μm, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Representative image showing H\u0026amp;E and immunofluorescence staining for SOX2, PHOX2B and SOX10 in 20-week-old human adrenal gland. The box shows one of 5 counting medullary regions in this sample. Full arrows indicate PHOX2B positive cells, full arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive sustentacular glial cells, and full triangles indicate SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive glial cells. Scale bars in the adrenal gland and insets are 100 μm and 20 μm, respectively. \u003cstrong\u003e(C)\u003c/strong\u003e Percent-stacked bar plot for the ratios of PHOX2B positive cells (56.89% ± 0.51%, indicated by full arrow), SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e double-positive cells (9.90% ± 1.76%, indicated by full arrowhead) and SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e triple-positive cells (1.60% ± 0.92%, indicated by full triangle) in cell number within all counting medullary regions (3 samples with 5 counting medullary regions each). Kruskal-Wallis test; adjusted \u003cem\u003eP\u003c/em\u003e value from Dunn’s multiple comparisons test for PHOX2B\u003csup\u003e+\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e is 0.5391, for PHOX2B\u003csup\u003e+\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e is 0.0219, and for SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e is 0.5391; \u003cem\u003en\u003c/em\u003e = 3. Data are presented as mean ± s.e.m. *, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures24.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/7bd0e1a8f0882ecab918a050.png"},{"id":86259370,"identity":"86aa9b49-f32f-4f55-9907-386d79697a95","added_by":"auto","created_at":"2025-07-08 14:11:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1785747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePostnatal sustentacular cells form a distinct population with a gene expression program different from that of embryonic SCPs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e UMAP plot showing cellular clusters and their annotations derived from scRNA-Seq data of mouse adrenal tissue. \u003cstrong\u003e(B)\u003c/strong\u003e UMAP plot of adrenal medullary cells, color-coded by gross age groups. \u003cstrong\u003e(C) \u003c/strong\u003eVolcano plot displaying specific differential expressed genes (DEGs) comparing embryonic (green) and postnatal (blue) glia cells. Genes of interest are labeled with their respective symbols. \u003cstrong\u003e(D)\u003c/strong\u003e Dot plot showing the expression of specific DEGs, as labeled in panel (C), in embryonic and postnatal glia cells. \u003cstrong\u003e(E) \u003c/strong\u003eUMAP plot illustrating the expression of selected genes representing shared glia markers (upper panel), embryonic glia-specific genes (middle panel), and postnatal glia-specific genes (lower panel). The UMAP plot in the top left corner highlights the two glial cell subsets from panel (A). \u003cstrong\u003e(F)\u003c/strong\u003e Bubble plot presenting the top 25 “signaling” related GO term enriched in the specific DEGs of postnatal glia cell. \u003cstrong\u003e(G)\u003c/strong\u003e Network diagram showing inferred DLK1-NOTCH1 ligand-receptor interactions among cell types in P90 mouse cells. \u003cstrong\u003e(H)\u003c/strong\u003eNetwork diagram showing inferred DLK1-NOTCH1 ligand-receptor interactions among cell types in aged mouse cells. \u003cstrong\u003e(I) \u003c/strong\u003eDot plot illustrating ligand-receptor communication pairs related to the NOTCH (upper panel) and WNT (lower panel) signaling pathways between glia cells and chromaffin cells across all five age groups. \u003cstrong\u003e(J)\u003c/strong\u003e Dot plot illustrating expression patterns of NOTCH signaling pathway-related genes across identified medullary cell populations. \u003cstrong\u003e(K)\u003c/strong\u003e Model for chromaffin cell regeneration from sustentacular cells via NOTCH inhibition. \u003cstrong\u003e(L)\u003c/strong\u003eDot plot illustrating ligand-receptor communication pairs related to the WNT signaling pathways between glia cells and chromaffin cells across all five age groups. (\u003cstrong\u003eM\u003c/strong\u003e) Dot plot illustrating expression patterns of WNT signaling pathway-related genes across identified medullary cell populations.\u003c/p\u003e","description":"","filename":"Figures25.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/bcd93704a56f26a9bbd0e2af.png"},{"id":86259376,"identity":"28e93e42-f264-4716-8e9b-ad7b1825c8aa","added_by":"auto","created_at":"2025-07-08 14:11:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3521346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing with infer-CNA analysis of human PPGL tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e UMAP of 2,586 single nuclei colored by assigned cell type.\u003cstrong\u003e (B)\u003c/strong\u003e Accuracy of cell type assignment.\u003cstrong\u003e \u003c/strong\u003eDot plot showing expression of selected marker genes by cell type.\u003cstrong\u003e (C)\u003c/strong\u003e Inferred CNAs match genomic data.\u003cstrong\u003e \u003c/strong\u003eFor each sample (rows), inferred copy number profiles (red=amplification, blue=deletion) averaged across all malignant cells per sample. Columns are chromosomal positions, averaged into five bins per chromosome. \u003cstrong\u003e(D)\u003c/strong\u003e CNA plot of PPGL #199. Each row is a cell, each column a gene, arranged by chromosomal position. Cells are arranged by SOX2-status (SOX2-pos if at least one SOX2 read). Ref, reference; SC, Schwann cells (sustentacular); pos, positive; neg, negative. \u003cstrong\u003e(E) \u003c/strong\u003eSDHB, TH and SOX10 immunofluorescence and H\u0026amp;E staining of PPGL #199 as indicated. Arrows indicate sustentacular cells co-expressing SDHB and SOX10, scale bars in the tumor and insets are 200μm and 20μm, respectively.\u003cstrong\u003e (F)\u003c/strong\u003e CNA plot of PPGL #198. Each row is a cell, each column a gene, arranged by chromosomal position. Neuroendocrine cells are arranged by SOX10 expression (SOX10-pos if at least one SOX10 read). At the top, inferred CNA profiles from all Schwann cells in this sample. \u003cstrong\u003e(G)\u003c/strong\u003e CNA plot of PPGL #264. Each row is a cell, each column a gene, arranged by chromosomal position. Cells are arranged by SOX2-status (SOX2-pos if at least one SOX2 read).\u003cstrong\u003e (H)\u003c/strong\u003e SOX2, PHOX2B and SOX10 immunofluorescence staining in PPGL #264. Empty arrowheads indicate SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive tumor cells, full arrowheads indicate SOX10\u003csup\u003e+\u003c/sup\u003eSOX2\u003csup\u003e+\u003c/sup\u003e sustentacular glial cells, and full arrow indicate PHOX2B single positive tumor cells. Scale bars in the tumor and insets are 200μm and 20μm, respectively.\u003c/p\u003e","description":"","filename":"Figures26.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/c898d899146716473b450b48.png"},{"id":86259374,"identity":"3bc2b04d-f0ed-4fda-929f-3f6536349b13","added_by":"auto","created_at":"2025-07-08 14:11:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4315327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInferring CNA from VHL related PPGL reveals pre-neoplastic sustentacular cells sharing 3p deletions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e CNA plot of PPGL #E042 and #E042. Each row is a cell, each column a gene, arranged by chromosomal position. Glia cells (SCLCs) are defined as pre-neoplastic (pre-neo) if they have an inferred 3p deletion (highlighted with red circle). The reference cells consist of stromal cells from the same patient and SCLCs from normal adrenal medulla. All SCLCs are shown, while 500 neuroendocrine and 100 reference cells were randomly sampled for illustration purposes. Glia cells are refererred as schwann cell like cells (SCLCs) from previously defined Zethoven et al. dataset \u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figures27.png","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/300d8aec679b7e9267706c15.png"},{"id":93956042,"identity":"e383c9e0-aed9-4225-a7b4-a46d92a3d8af","added_by":"auto","created_at":"2025-10-20 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20270711,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/14a83620-3cf5-41de-92ee-97121f1320e2.pdf"},{"id":86259380,"identity":"2183fc63-f3ad-4ed8-bb68-7f933785835e","added_by":"auto","created_at":"2025-07-08 14:11:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23479716,"visible":true,"origin":"","legend":"","description":"","filename":"BullovasupplementarymaterialsJune2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/64c704c22c0fe6572a825583.pdf"},{"id":86259705,"identity":"59b7e96b-42d1-4c69-8746-0b67003a47c0","added_by":"auto","created_at":"2025-07-08 14:19:51","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1549490,"visible":true,"origin":"","legend":"","description":"","filename":"BullovaSupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6907400/v1/cd57bae37fddf0770456b783.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Postnatal Sustentacular Cells as Chromaffin Progenitors and Tumor Cells of Origin in VHL-Related Paragangliomas","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe adrenal medulla is composed of neural crest-derived neuroendocrine chromaffin cells, which are responsible for catecholamine production, i.e. epinephrine and norepinephrine, mediating the body\u0026rsquo;s 'fight or flight' response. In addition to the adrenal medulla, chromaffin cells are also located in extra-adrenal sites, including the organ of Zuckerkandl (OZ) near the abdominal aorta and in small clusters along paravertebral sympathetic ganglia (paraganglia), producing predominantly norepinephrine \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Tumors (PPGL) arising in these tissues \u0026mdash;pheochromocytomas (intradrenal) and paragangliomas (extradrenal) \u0026mdash; often cause catecholamine overproduction, and can lead to severe hypertension and increased the risk of cardiovascular events \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. PPGL have the highest heritability rates among all tumors, with 40% of patients carrying a susceptibility gene mutation in constitutional tissues \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent studies have demonstrated that embryonic chromaffin cells at the adrenal anlagen originate from neural crest-derived multipotent Schwann cell precursors (SCPs) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. SCPs migrate along visceral motor nerves to the developing adrenal anlagen, contributing to the majority of the adrenal chromaffin cell population embryonically \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. During mouse embryogenesis, SCPs also give rise to paraganglia, including chromaffin cells in the OZ and some sympathetic neurons \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, multipotent SCPs persist for a short period during embryonic development \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, disappearing around mouse embryonic day 15, whereas the OZ reaches the largest size just before/after birth \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and in humans, this peak occurs even later, around the third year of life before regressing \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus, the process by which chromaffin cells are generated and replenished postnataly is poorly understood. Putative chromaffin stem cells have been previously postulated \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and also been recently reported by analyzing deep single cell sequencing of human postnatal adrenal glands \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In addition, chromaffin cell division might also repopulate chromaffin cells postnatally \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we describe the existence of a distinct postnatal chromaffin progenitor population using \u003cem\u003ein vivo\u003c/em\u003e lineage tracing, single-cell analysis, and immunofluorescence staining. We identified a progenitor population in the mouse adrenal medulla, organ of Zuckerkandl, human adrenal medulla, and PPGL with characteristics of sustentacular glia cells co-expressing SOX10 and SOX2. SOX2 is a well-known transcription factor that maintains multipotency and stemness by regulating genes associated with embryonic neural crest stem cells, embryonic multipotent crest derivatives (SCPs), and postnatal neural stem cells in neurogenic regions of the central nervous system \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. While sustentacular glia cells have traditionally been seen as supportive cells in neuroendocrine tissues through paracrine signaling, our findings suggest they also serve as progenitor cells, contributing to chromaffin cell generation postnatally and supporting neuroendocrine tissue integrity through pathways involving NOTCH inhibitor DLK1 and WNT6 paracrine signaling.\u003c/p\u003e\u003cp\u003eWhile the majority of sustentacular cells in PPGL are believed to act as non-neoplastic stromal components, we identified in two PPGL cases where a subset of \u003cem\u003eSOX2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e sustentacular cells (8% and 15%) shared predicted copy number aberrations with neoplastic tumor cells. This suggest that, in rare instances, sustentacular cells may serve as the tumor cell of origin. These findings not only expand our understanding of postnatal chromaffin progenitors but also highlight the potential dynamic role of sustentacular cells in tumor biology. Investigating their contributions to the tumor microenvironment and progression could inform new diagnostic, prognostic, and therapeutic strategies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePostnatal SOX10\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ecells are chromaffin progenitor cells\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether SOX10\u003csup\u003e+\u003c/sup\u003e glial cells contribute postnatally to the generation of chromaffin cells (TH\u003csup\u003e+\u003c/sup\u003e) in the adrenal medulla, we performed fate tracing using the glia-specific inducible Cre-line \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e coupled to the \u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eYFP+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e reporter. Genetic cell fate tracing was initiated postnatally in glial cells by tamoxifen (TAM)-induced recombination. TAM was administered orally to nursing females via gavage during the first 4 days after birth (P0\u0026ndash;P4, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of adrenal medullas at postnatal days P5, P14, and P90 revealed that 4.25%, 14.17%, and 17.56% of YFP traced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), respectively, were positive for tyrosine hydroxyase (TH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;G). However, the substantial increase in TH\u003csup\u003e+\u003c/sup\u003e chromaffin cells from P5 to P90 (a 3.7-fold increase, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) could not be attributed entirely to postnatally derived SOX10\u003csup\u003e+\u003c/sup\u003e cells. This suggests that perhaps neonatal TH\u003csup\u003e+\u003c/sup\u003e chromaffin cells (P0\u0026ndash;P7) retain the capacity to proliferate, contributing to postnatal chromaffin cell expansion. To assess proliferation in the adrenal medulla, we administered EdU (5-ethynyl-2'-deoxyuridine), a thymidine analog that incorporates into newly synthesized DNA, 2 hours before euthanizing P5 and P14 mice. Adrenal glands were sectioned and stained for TH and EdU-labeled cells. At P5, we observed multiple EdU\u003csup\u003e+\u003c/sup\u003eTH\u003csup\u003e+\u003c/sup\u003e cells in the medullary region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), indicating that neonatal chromaffin cells retain the capacity for self-renewal. However, the proliferative capacity declined by P14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Taken together, our data demonstrate that TH\u003csup\u003e+\u003c/sup\u003e chromaffin cells can regenerate postnatally through two mechanisms: differentiation from SOX10\u003csup\u003e+\u003c/sup\u003e progenitors and self-renewal during the neonatal stage.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePostnatal SOX10\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ecells in the adrenal medulla and the organ of Zuckerkandl (OZ) co-express the multipotency factor SOX2, resembling sustentacular cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSOX2 co-expression has been observed alongside SOX10 in neural crest cells and embryonic multipotent crest derivatives, SCPs \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. SCPs are the major source for the generation of embryonic chromaffin cells \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, but not observed postnatally. To characterize chromaffin progenitors, we analyzed both postnatal and embryonic adrenal medulla and OZ for expression of SOX10, SOX2 and sympatho-adrenal lineage specification marker PHOX2B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Immunofluorescence staining for SOX10 and SOX2 revealed that SOX10\u003csup\u003e+\u003c/sup\u003e cells are positive for SOX2\u003csup\u003e+\u003c/sup\u003e in the neonatal (P1) adrenal medulla (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and OZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), persisting to adulthood (Suppl. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), similarly as seen embryonically at day E17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, hematoxylin and eosin (HE) staining revealed distinctive morphological features of SOX10\u003csup\u003e+\u003c/sup\u003e SOX2\u003csup\u003e+\u003c/sup\u003e cells in the postnatal medulla and OZ, distinguishing them from the embryonically SOX10\u003csup\u003e+\u003c/sup\u003eSOX2\u003csup\u003e+\u003c/sup\u003e glia/SCPs cells (E17). Postnatal SOX10\u003csup\u003e+\u003c/sup\u003eSOX2\u003csup\u003e+\u003c/sup\u003e cells have distinctive spindle-shaped nuclei and extended cellular processes that interface with the surrounding chromaffin cells, and resemble morphological features of sustentacular glia cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;B). In contrast, embyonic SOX10\u003csup\u003e+\u003c/sup\u003eSOX2\u003csup\u003e+\u003c/sup\u003e cells were scattered among differentiating chromaffin cells, having round to oval nuclear shape and cell processes that were thin and difficult to discern (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that these cells most likely represent satellite-glia and/or SCPs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, we observed a few SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e cells in the postnatal adrenal medulla and their occurrence was increased in the OZ at P1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;B and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicative of a transitional state between progenitors and committed sympatho-adrenal cells. PHOX2B expression indicates lineage commitment to neuroendocrine cell types and is expressed in chromaffin cells, sympathoblast and fully differentiated neurons. However, a SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e cell could represent a progenitor cell in transition, a cell that has not yet fully differentiated into chromaffin or sympathetic cells. Thus, we quantified SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive nuclei in the adrenal medulla (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B) and in the OZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;D) at P1. Whereas in the adrenal medulla only a few SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e (0.6%) or SOX10\u003csup\u003e+\u003c/sup\u003eSOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e (5.2%) nuclei were detected, a larger proportion of SOX2\u0026thinsp;+\u0026thinsp;PHOX2B\u0026thinsp;+\u0026thinsp;double-positive (13.5%) and triple-positive (6.5%) nuclei was observed in the OZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;E). Notably, triple-positive cells exhibited markedly weaker PHOX2B expression compared to SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells that lacked SOX10, suggesting that SOX10 downregulation is required for full PHOX2B expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe observation that a relatively large proportion (20.4%) of PHOX2B positive cells coexpressed SOX2 in the OZ, compared to only 1.1% in the adrenal gland, was unexpected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We suspect that, at birth, when the OZ reaches its maximal size, sustentacular cells generate a large number of chromaffin cells, reflecting the OZ\u0026rsquo;s peak functional importance during the perinatal period. This may support critical physiological processes such as catecholamine production and stress response. Notably, SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e double-positive cells in both the OZ and adrenal gland expressed TH, consistent with noradrenergic fate restriction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;G and Suppl. Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e\u003cp\u003eNext, we analyzed adrenal glands from children (n\u0026thinsp;=\u0026thinsp;3), aged 20 weeks to 4 years, to investigate the co-expression of SOX10, SOX2, and PHOX2B and determine whether transitional cells, similar to those observed in mouse adrenal glands, are also present in human tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Suppl. Fig. S3). All SOX10 positive nuclei co-expressed SOX2, similar to adrenal sustentacular cells in mice (98.22% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.58% of the total SOX10\u003csup\u003e+\u003c/sup\u003e cells were SOX2\u003csup\u003e+\u003c/sup\u003e). Among all DAPI positive cells counted, 1.6% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.59% were triple-positive for SOX10, SOX2, and PHOX2B, a percentage comparable to that observed in the mouse medulla at P1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that human adrenal chromaffin cells in children may also undergo postnatal regeneration, similar to what is observed in mice. However, the rarity of transitioning cells committing to differentiation aligns with previous findings, which describe the postnatal adrenal medulla as a nearly post-mitotic tissue with a stable and low proliferation rate. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSOX2\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eSOX10\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003esustentacular cells form a distinct population at postnatal ages with a gene expression program different from embryonic glia/SCPs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate transcriptional differences between postnatal and embryonic glial cells, we profiled adrenal cell populations in mice across developmental stages. Postnatal (PN) adrenal samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;18; ages: P5, P14, P90, and aged) comprised 4,552 single cells, while embryonic (E17) medullary samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) included 2,549 single cells. All cells were analyzed using deep single-cell RNA sequencing with the SmartSeq2 protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Suppl. Figure\u0026nbsp;4A\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuality-controlled data were subjected to unsupervised clustering, resulting in the identification of 7,101 adrenal cells and categorized into eight medullary clusters consisting of glia (6,11), chromaffin (0, 1, 2, and 5), embryonic neuroblasts (10) and embryonic TH-expressing cycling cells (8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B, Suppl. Fig. S4A\u0026ndash;C). We performed in silico subsetting of adrenal medullary cell populations using previously established markers \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Clusters were annotated based on the expression of key marker genes; noradrenergic and adrenergic chromaffin markers (\u003cem\u003ePnmt, Th, Dbh, Chga, Chgb, Epas1, Penk\u003c/em\u003e), neuroblast markers (\u003cem\u003eElavl4, Isl1, Gap43, Nefl, Prph\u003c/em\u003e), glial markers (\u003cem\u003eSox10, Plp1, Erbb3, Fabp7\u003c/em\u003e), and cycling markers (\u003cem\u003eTop2a, Mki67, Aspm, Bub1\u003c/em\u003e) (Suppl. Fig. S4D). Non-medullary populations, including cortical, mesenchymal, endothelial, and immune cell clusters, were also identified and annotated (Suppl. Fig. S4D).\u003c/p\u003e\u003cp\u003eWe found that the embryonic glia/SCP population at E17.5 (6-E_Glia/SCP) transcriptionally differed transcriptionally from the postnatal glia/sustentacular population (11-PN_Glia/sustentacular) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Differentially expressed genes (DEGs) characterizing these populations were identified using specific DEG analysis methods (Wilcoxon Rank Sum test, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, detailed in Methods), revealing distinct transcriptional programs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E).\u003c/p\u003e\u003cp\u003eThe embryonic glial/SCP and postnatal glial sustentacular cells shared the expression of canonical glial markers, including \u003cem\u003eSox10, S100b, Foxd3, Plp1, Erbb3\u003c/em\u003e, and \u003cem\u003eFabp7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Suppl. Fig. S4D). Additionally, they co-expressed \u003cem\u003eSox2\u003c/em\u003e, as previously demonstrated by immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the embryonic glia/SCP population significantly upregulated \u003cem\u003eWwtr1\u003c/em\u003e, \u003cem\u003eLmo4\u003c/em\u003e, and \u003cem\u003eMoxd1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E), genes known to play critical roles in maintaining cell stemness and proliferation \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eMoxd1\u003c/em\u003e (\u003cem\u003eMonooxygenase DBH Like 1\u003c/em\u003e) has been previously characterized as highly enriched in embryonic SCPs \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eIn contrast, postnatal glia-sustentacular cells showed significant upregulation of \u003cem\u003eWnt6\u003c/em\u003e, \u003cem\u003eNotch1\u003c/em\u003e, \u003cem\u003eHey2\u003c/em\u003e, \u003cem\u003eSfrp5/1\u003c/em\u003e, and \u003cem\u003eTgfb2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These genes suggest involvement in processes such as cell signaling, fate determination, and maintenance of progenitor-like characteristics \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Gene ontology (GO) enrichment analysis of the specific DEG list confirmed the enrichment of WNT, BMP, and NOTCH signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These findings are consistent with a recent study \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which also identified a distinct transcriptional identity for postnatal SOX2\u0026thinsp;+\u0026thinsp;sustentacular cells, differentiating them from embryonic Schwann cell precursors (SCPs). Together, these data emphasize the unique molecular signature of postnatal sustentacular cells and support their role as a specialized glial progenitor population in the adrenal medulla.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSustentacular and chromaffin cell-cell communication reveals NOTCH1 and WNT signaling in regeneration and paracrine support.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore how the identified signaling pathways interact within the adrenal tissue, we performed cell-cell communication analysis using CellChat-tool in different adrenal age groups in mice. CellChat enables systematic analysis of cell-cell communication from single-cell transcriptomics data by quantifying the signaling communication probability between two cell groups, incorporating the core interactions between ligands and receptors \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe observed that the NOTCH signaling pathway was predominantly active in postnatal chromaffin cells (Cluster 1 NOR and Cluster 2 ADR) with receptors mainly on sustentacular cells (Cluster 11), (Suppl. Fig. S4E\u0026ndash;G). Analysis of individual ligand-receptor pairs predicted that, across all postnatal ages (P5, P14, P90, and aged), the inhibitory NOTCH ligand Dlk1 was primarily expressed and sent by chromaffin cells and received by the Notch1 receptor expressed in sustentacular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;H, Suppl. Fig. S4H). Similarly this communication was also observed in embryonic glia, chromaffin and neuroblast populations at E17 (Suppl. Fig. S4I). Dlk1 is a known inhibitor of the NOTCH signaling pathway, acting by preventing activation of the NOTCH receptor \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. DLK1 is also expressed in sympathoadrenal and chromaffin cells downstream of PHOX2B \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, supporting its role in lineage-specific regulatory programs. Recent studies have demonstrated that inhibition of the NOTCH signalling pathway leads to an increased number of Th\u003csup\u003e+\u003c/sup\u003e cells in both sympathetic ganglia and the adrenal gland, whereas activation of the pathway has the opposite effect \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, it is plausible that DLK1 expression in postnatal chromaffin cells fine-tunes the regenerative capacity of sustentacular cells during chromaffin differentiation. Notably, DLK1 expression was similarly observed in human neuroendocrine PPGL tumor cells (Suppl. Fig. S5C), suggesting that DLK1 expression in tumor cells might also impact tumor cell differentiation through the tumor microenvironment as observed recently in neuroblastoma \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, analysis of all NOTCH ligand-receptor communications in chromaffin and glial populations across developmental stages revealed the expression of NOTCH activating ligand \u003cem\u003eJag2\u003c/em\u003e in chromaffin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). NOTCH target genes \u003cem\u003eHes1\u003c/em\u003e, \u003cem\u003eHey2\u003c/em\u003e and \u003cem\u003eHeyl\u003c/em\u003e were expressed highly in sustentacular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). This suggests a dual role for NOTCH signaling, involving both inhibitory and activating interactions, which may finetune the regenerative capacity of sustentacular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eIn addition to the identified NOTCH-mediated communication between sustentacular cells and chromaffin cells, WNT signaling emerged as another highly enriched pathway in sustentacular cells, as highlighted by a top-regulated GO term (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Among the most differentially expressed genes in sustentacular cells were \u003cem\u003eWnt6\u003c/em\u003e and \u003cem\u003eSfrp1/5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E, Suppl. Fig. S5J). CellChat analysis of WNT signaling pathways predicts ligand-receptor communication between \u003cem\u003eWNT6\u003c/em\u003e from sustentacular cells, and the receptors \u003cem\u003eFzd3/5\u003c/em\u003e and \u003cem\u003eLrp5/6\u003c/em\u003e expressed in chromaffin cells during postnatal stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, Suppl. Fig. S5J). Interestingly, the expression of \u003cem\u003eSfrp1/5\u003c/em\u003e in sustentacular cells suggests a potential dual regulatory role for WNT signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM), as these proteins can inhibit WNT receptor activation, balancing inhibitory and activating interactions.\u003c/p\u003e\u003cp\u003eAdditionally, chromaffin cells highly expressed the WNT co-receptor \u003cem\u003eLgr5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM, Suppl. Fig S4J), which, although not directly reported to interact with WNT6, is known to enhance WNT signaling by stabilizing WNT receptors (e.g., Frizzled receptors) on the cell surface through interactions with R-spondins (RSPOs). The co-expression of \u003cem\u003eFzd3\u003c/em\u003e and \u003cem\u003eLgr5\u003c/em\u003e in chromaffin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM) supports the model of paracrine WNT signaling from sustentacular cell-derived WNT6 to chromaffin cells. This finding underscores the critical role of sustentacular cells in supporting chromaffin cell survival under specific physiological conditions.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDeep single-nucleus RNAseq analysis of human PPGL shows SOX2 and SOX10 expression in neoplastic cells\u003c/h2\u003e\u003cp\u003eSustentacular cells are commonly observed in PPGL, though their prevalence and characteristics vary depending on the tumor type and its microenvironment. Traditionally considered non-neoplastic glial-like cells, sustentacular cells form a supportive framework around chromaffin cells in both the normal paraganglia and PPGL. However, our genetic tracing revealed that sustentacular glial cells can function as chromaffin progenitor cells postnatally (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To investigate whether they could also serve as the tumor cell of origin, rather than being confined to structural and supportive roles within the tumor microenvironment, we performed deep single-cell sequencing on PPGL (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) together with cancer-specific copy number aberration (CNA) inference across both neuroendocrine and sustentacular glial cells. We sequenced 3,418 single nuclei from 9 patients and profiled 2,586 cells passing quality control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Suppl. Fig. S5A, Methods). After assigning each cell to a cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Suppl. Fig. S5B\u0026ndash;C), we focused on the neuroendocrine compartment to see whether we could find expression of \u003cem\u003eSOX2\u003c/em\u003e or \u003cem\u003eSOX10\u003c/em\u003e in neoplastic cells, and the impact of these genes\u0026rsquo; expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo separate neoplastic from non-neoplastic neuroendocrine cells, copy number aberrations (CNA) were inferred in all neuroendocrine cells, as well as in Schwann cells, the latter to explore whether we can find sustentacular-like neoplastic cells in PPGL. In total 1893/1921 (99%) of all neuroendocrine cells were classified as neoplastic (Fig. S5D), and the inferred patterns of chromosomal aberrations largely confirmed genomic data. All four tumors with genetically confirmed Von Hippel-Lindau (\u003cem\u003eVHL)\u003c/em\u003e mutations (samples 195, 262, 198 and 267) were found to have a chromosome 3p loss, consistent with a second hit in \u003cem\u003eVHL\u003c/em\u003e-mutated malignancies \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Further validating our approach, the sample 266, with a known \u003cem\u003eNF1\u003c/em\u003e mutation, showed the corresponding 17q deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Similarly, out of the tumors with known chromosome 1p status, inferred copy numbers aligned with known genetics in all cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Table S3).\u003c/p\u003e\u003cp\u003eNone of the sustentacular cells (SC) sequenced shared any CNA with the neuroendocrine tumor cells and thus were classified as non-neoplastic (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u0026ndash;G) In some cases (sample 199 Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) only a few sustentacular cells were sequenced. Thus, we additionally performed anti-SDHB immunofluorescence staining on sample 199 (\u003cem\u003eSDHB\u003c/em\u003e mutant) to validate its neoplastic status (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). PPGL harboring germline \u003cem\u003eSDHB\u003c/em\u003e mutations are known to display loss of SDHB immunoreactivity \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and thus SDHB immuno-staining provides a valuable tool to investigate the neoplastic status of sustentacular cells. Neuroendocrine TH\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e tumor cells were organized in \u0026ldquo;zellballen\u0026rdquo; and confirmed loss of SDHB expression, however surrounding SOX10\u003csup\u003e+\u003c/sup\u003e sustentacular cells were positive for SDHB, indicating being normal and confirming results derived by inferred CNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eWhile no sustentacular cells were classified as neoplastic, a subset of neoplastic neuroendocrine cells expressed either \u003cem\u003eSOX10\u003c/em\u003e (sample 198: 6.25%) or \u003cem\u003eSOX2\u003c/em\u003e (sample 264: 12.1%). Neoplastic cells expressing either \u003cem\u003eSOX2\u003c/em\u003e (sample 264, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) or \u003cem\u003eSOX10 (\u003c/em\u003esample 198 Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cem\u003e)\u003c/em\u003e shared CNA with other cells from the corresponding tumors, and the quantitative metrics used to determine neoplastic-CNA signal and correlation (see Methods) - were comparable across neuroendocrine cells regardless of \u003cem\u003eSOX2\u003c/em\u003e/\u003cem\u003eSOX10\u003c/em\u003e positivity (Suppl. Fig. S5D\u0026ndash;E).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eSOX10\u003c/em\u003e positive cells were found across multiple tumors, with low numbers in each. In contrast to \u003cem\u003eSOX10, SOX2\u003c/em\u003e positive neoplastic cells were only found to any significant degree in one tumor, sample 264, where 40/331 (12%) of neoplastic cells had at least one read from \u003cem\u003eSOX2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Combined immunofluorescence staining for SOX2 and PHOX2B revealed that, in this sample, the majority of neoplastic chief cells (PHOX2B\u003csup\u003e+\u003c/sup\u003e) were SOX2 positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), indicating that our inability to show \u003cem\u003eSOX2\u003c/em\u003e mRNA expression in the majority of neuroendocrine cells was an effect of technical dropouts rather than biological heterogeneity within the tumor. Indeed, no genes were significantly differentially expressed between cells with and without identified \u003cem\u003eSOX2\u003c/em\u003e mRNA in this sample.\u003c/p\u003e\u003cp\u003eTo assess the impact of \u003cem\u003eSOX2\u003c/em\u003e expression in neoplastic cells, we repeatedly performed differential gene expression analysis between this tumor and each of the remaining eight tumors separately, searching for genes that were recurrently over/underexpressed (log2FC\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, t-test). 42 genes were significantly overexpressed in sample 264 in all eight pairwise comparisons \u0026ndash; notably \u003cem\u003eASCL1\u003c/em\u003e, \u003cem\u003eHOXA9\u003c/em\u003e and \u003cem\u003eTUBB3\u003c/em\u003e \u0026ndash; suggesting a link between malignant \u003cem\u003eSOX2\u003c/em\u003e expression and a stem-like, neuronal-like phenotype (Fig. S5F, Table S4). Achaete-scute homolog 1 (ASCL1), a proneural transcription factor, plays a central role in neurogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Furthermore, ASCL1 activates PHOX2A, which subsequently induces the expression of genes essential for catecholamine biosynthesis \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. It is plausible that a similar ASCL1-mediated program operates postnatally, with sustentacular progenitor cells contributing to the chromaffin lineage. In PPGL, sustained ASCL1 expression may promote tumor cell plasticity and maintain an undifferentiated state.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSchwann cells share 3p deletions with neuroendocrine cells in\u003c/b\u003e \u003cb\u003eVHL\u003c/b\u003e\u003cb\u003e-mutated tumors\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough we could not find any evidence of neoplastic sustentacular cells in our data, the discovery of \u003cem\u003eSOX2\u003c/em\u003e expression in neuroendocrine neoplastic cells indicated that these cells might exist, but that the low cell numbers sequenced by Smartseq2 and heavy skewing towards neuroendocrine cells in our dataset prevented their discovery. Thus, we re-analyzed a larger, droplet-based snRNAseq dataset of PPGL samples \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, providing both more samples and higher numbers of cells per sample.\u003c/p\u003e\u003cp\u003eIn five samples with \u0026gt;\u0026thinsp;=\u0026thinsp;100 Schwann-cell-like cells (SCLCs), as defined by the authors, we re-inferred CNA in SCLCs and neuroendocrine cells, using as reference (Methods) both stromal cells from the same patient as well as, to avoid false positive copy number events due to Schwann-cell-specific transcriptional programs, SCLCs from two normal adrenal medulla samples in the same dataset. SCLCs were classified as malignant if they showed deletions or amplifications consistent with any of the copy number events found in the neuroendocrine cells.\u003c/p\u003e\u003cp\u003eThrough this approach, in two samples (E024, E042) we were able to find subsets of SCLCs (16/199, 8% and 18/122, 15%) with inferred 3p deletions, consistent with the neuroendocrine cells, but none of the additional copy number events found (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B). Both samples exhibited germline \u003cem\u003eVHL\u003c/em\u003e mutations, suggesting that these SCLCs could represent sustentacular glial cells that have undergone a second-hit deletion through the loss of 3p, encompassing the remaining wild-type \u003cem\u003eVHL\u003c/em\u003e allele. For Von Hippel-Lindau disease-related PPGL, the inactivation or loss of both alleles of the \u003cem\u003eVHL\u003c/em\u003e gene, as predicted by the Knudson two hit theory, is required. Thus, in both of these cases (E024, E042), the biallelic inactivation of \u003cem\u003eVHL\u003c/em\u003e as an initiating tumor driving event resulted from the combination of germline \u003cem\u003eVHL\u003c/em\u003e mutation and subsequent chromosomal 3p loss in SCLCs. Analyzing differentially expressed genes between SCLCs with or without 3p deletions, we found that 20 genes were consistently upregulated in non-neoplastic SCLCs in both samples. Notably, only four of these (\u003cem\u003eCNTN4, BHLHE40\u003c/em\u003e, \u003cem\u003eRAF1\u003c/em\u003e and \u003cem\u003eCAPN7\u003c/em\u003e) are located on chromosome 3p, indicating that the inferred 3p deletion is not an artifact of transcriptional downregulation. In \u0026ldquo;pre-neoplastic\u0026rdquo; SCLCs, only one gene per sample was significantly upregulated \u0026ndash; \u003cem\u003eHNRNPA2B1\u003c/em\u003e (E024) and \u003cem\u003eNRXN1\u003c/em\u003e (E042) (Table S5). Notably, the latter was found to be expressed in both neuroendocrine and Schwann cells in our own PPGL data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreviously, single-nuclei transcriptomes of human adult adrenal glands identified a putative progenitor population with intermediate markers of chromaffin cells, supported with the directional transition from progenitor cells to chromaffin cells predicted by RNA velocity \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In this study, we provide \u003cem\u003ein vivo\u003c/em\u003e evidence for the existence of such chromaffin progenitor populations postnatally, using \u003cem\u003ein vivo\u003c/em\u003e lineage tracing, single-cell RNA sequencing, and immunofluorescence in mouse and human adrenal glands. Our findings establish SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e cells as a distinct progenitor population capable of giving rise to chromaffin cells after birth.\u003c/p\u003e\u003cp\u003eOur single-cell transcriptomic analysis of embryonic and postnatal glial cells revealed that chromaffin regeneration by SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e cells postnatally represents a source distinct from their embryonic origin, the SCPs. While SCPs serve as the primary source of embryonic chromaffin cells during development \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, their absence postnatally underscores the critical role of an alternative progenitor source in maintaining a growing tissue. Our study provides insights into the role of adrenal SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e glial cells, characterized as sustentacular cells, as postnatal chromaffin progenitors. The identification of SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e transitional cells in both human and mouse adrenal medulla, as well as in extra-adrenal chromaffin tissues such as the OZ, further supports the notion of a dynamic and continuous process of chromaffin differentiation during the perinatal and postnatal periods.\u003c/p\u003e\u003cp\u003eSingle-cell transcriptomic analysis enabled us to quantify the signaling communication of core interactions between ligands and receptors across cell populations. We found that maintaining neuroendocrine tissue integrity involves dynamic signaling interactions of the DLK1-NOTCH and WNT pathways. A recently published study \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e also describes sustentacular cells as chromaffin stem cells and highlights the role of paracrine signaling through WNT secretion, further supporting our findings.\u003c/p\u003e\u003cp\u003eFurthermore, we characterized the role of SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e sustentacular cells in their contribution to PPGL tumorigenesis, uncovering instances where sustentacular cells may serve as tumor cells of origin. We could identify two PPGL samples where subsets of glial cells (8% and 15%) had inferred 3p deletions shared with the neuroendocrine tumor cells, inferred 3p deletions, which were shared with the neuroendocrine tumor cells, suggesting a common parental cell origin. Both samples harbored germline \u003cem\u003eVHL\u003c/em\u003e mutations. This supports the notion that a second-hit deletion through the loss of 3p, encompassing the remaining wild-type \u003cem\u003eVHL\u003c/em\u003e allele, led to \u003cem\u003eVHL\u003c/em\u003e inactivation\u0026mdash;a prerequisite for \u003cem\u003eVHL\u003c/em\u003e loss-driven tumorigenesis. No additional copy number alterations were shared with the neuroendocrine tumor cells in either case, indicating that 3p loss was an early or initiating event in tumorigenesis, preceding other genetic alterations associated with tumor progression. Thus, we termed these sustentacular cells harboring the 3p deletion as pre-neoplastic. A recent study using CNV predictions also identified rare aneuploid glial cells, termed malignant SCP-like cells, in a related neuroendocrine tumor, neuroblastoma \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, it is possible that this population instead resembles sustentacular glia-like cell, which are also a component of the tumor microenvironment in low-risk neuroblastomas.\u003c/p\u003e\u003cp\u003eIn one case of PPGL, the majority of neoplastic neuroendocrine PHOX2B\u003csup\u003e+\u003c/sup\u003e tumor cells coexpressed SOX2. ASCL1, a proneural transcription factor, was found significantly upregulated in this tumor compared to all other samples with SOX2-negative tumor cells. This establishes a link between SOX2 expression and a stem-like phenotype. The regulatory transcription factor Achaete-scute homolog 1 (ASCL1) plays a central role in neurogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Similarly, ASCL1 might also act as a pioneering factor postnatally, mediating a program in sustentacular progenitor cells that contributes to the chromaffin fate. Notably, the expression of SOX2 in PPGL has previously been reported \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, supporting a stem-like or progenitor-associated phenotype. Sustained ASCL1 expression in these pheochromocytoma cells may thus reflect an inability of these tumor cells to fully differentiate, keeping them in a plastic and immature state.\u003c/p\u003e\u003cp\u003eIn PPGL, the tumor-initiating event is well understood in the context of germline predisposition, but the cell of origin for oncogenic transformation leading to PPGL remains unclear. One of the intriguing aspects of PPGL is their unusually high prevalence of germline predisposition compared to most other adult cancers, despite being a rare cancer type. This has been suggested to be linked to specific developmental windows that permit oncogenic initiation \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile the majority of PPGL are thought to arise from either embryonic or postnatal chromaffin tissue, our findings suggest that tumorigenesis can also be initiated in chromaffin progenitor cells, specifically sustentacular cells. This raises the possibility that developmental timing and cellular plasticity play critical roles in determining susceptibility to oncogenic transformation.\u003c/p\u003e\u003cp\u003eFinally, identifying SOX2\u003csup\u003e+\u003c/sup\u003eSOX10\u003csup\u003e+\u003c/sup\u003e sustentacular cells as postnatal chromaffin progenitors may have significant clinical implications. Understanding the molecular mechanisms governing their differentiation and plasticity could unveil novel diagnostic and prognostic markers for PPGL, enabling earlier and more precise disease stratification. The presence of SOX2\u003csup\u003e+\u003c/sup\u003ePHOX2B\u003csup\u003e+\u003c/sup\u003e transitional cells in both adrenal and extra-adrenal chromaffin tissues underscores a dynamic regulatory network, potentially driven by key signaling pathways such as NOTCH inhibition by DLK1 and paracrine WNT signaling, which could serve as therapeutic targets. In this context, the identification of DLK1 expression in chromaffin cells and PPGL\u0026rsquo;s is particularly intriguing, as DLK1 has recently been described as an immunotherapeutic target in neuroblastoma, with DLK1 silencing promoting differentiation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e Mouse experimental procedures were permitted by the Stockholm North committee for animal experiments. \u003cem\u003eR26R\u003c/em\u003e\u003csup\u003e\u003cem\u003eYFP\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSox10\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e mice have been described \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. C57BL/6 mice were kept in rooms with controlled 12-h light/dark cycles, temperature, and humidity with food and water provided. Animal care were in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). Tamoxifen-induced lineage tracing, EdU administration and tissue preparation are described in supplementary materials.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHuman tissue specimens and tissue preparation\u003c/h3\u003e\n\u003cp\u003eTumor tissue samples (PPGL \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) were collected from individuals operated and diagnosed at the Karolinska University Hospital, Stockholm, Sweden, and previously characterized for mutations in PPGL susceptibility genes \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Post-mortem human adrenal glands were obtained from the NIH Neurobiobank (University of Maryland, Baltimore, MD) as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Tissue preparation are described in supplementary materials.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence, Hematoxylin and eosin (H\u0026amp;E) staining and Statistical analyses\u003c/h2\u003e\u003cp\u003eStaining protocoll and antibodies are described in supplentarial materials. In short, tissue sections were deparaffinized in xylene (HistoLab Products AB, #02070) and rehydrated using a series of ethanol (Solveco) and distilled water washes. Antigen retrieval was performed in citrate target retrieval solution (Agilent, #S1699) for 20 min at 96\u0026ndash;100\u0026deg;C. The slides were cooled down, washed, and hydrophobic barrier was created using ImmEdge Pen (Vector laboratories, #H-4000). Alternatively, mouse cryosections were left to dry at room temperature for 15 min and washed 3\u0026times; in PBS. Statistical analyses of immunofluorescence images are detailed in supplentarial materials.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003eCollection and analyses of human samples (PPGL) are covered by the ethical approval numbers 01-136 local ethical committee KI forskningsetikkommitt\u0026eacute; Nord) and 2020\u0026ndash;04226 (Swedish Ethical Review Authority). All samples were obtained following an informed patient consent. Post-mortem human adrenal glands for staining\u0026rsquo;s were obtained from the NIH Neurobiobank (University of Maryland, MD) under the same ethical permit from Stockholm Regional Ethical Review Board and the Karolinska University Hospital Research Ethics Committee (KI 2007/069 and KI 2001/136). Ethical permits for animal studies were approved by the appropriate local and national authorities (Jordbruksverket, Sweden).\u003c/p\u003e\n\u003ch3\u003eMouse tissue single-cell sequencing, data pre-processing and quality control\u003c/h3\u003e\n\u003cp\u003eSingle-cell suspension preparation for single-cell RNA sequencing is described in supplementary materials and further processed by the Eukaryotic Single Cell Genomics (ESCG) facility at SciLifeLab. Single-cell raw sequencing data was pre-processed as described in our previous work \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The quality control of mice individual cells included removing cells expressing more than 10000 or fewer than 200 genes, removing cells whose total expression was due to mitochondrial genes in more than 10%, and removing from the expression matrix genes that are expressed in fewer than 3 cells. Mouse single-cell data clustering and cell type identification are further described in supplementary materials.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSpecific differential expressed genes (DEGs) and GO term pathway analysis\u003c/h2\u003e\u003cp\u003eTo identify specific DEGs in mouse embryonic and postnatal glia cells, we conducted differential expression analysis by comparing each cellular cluster against another cluster in a pairwise manner. This process was repeated iteratively for all possible cluster pairs and described in supplementary materials including gene-ontology enrichment analysis and cellulare interaction networks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHuman single-nuclei sequencing, pre-processing and quality control\u003c/h2\u003e\u003cp\u003eHuman samples were processed using a single-nuclei sequencing protocol, while mouse samples underwent a single-cell sequencing protocol. The protocols for processing both human and mouse samples were described in our previous work \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Human single-nuclei data pre-processing, quality control, clustering and cell type assignment are described in supplementary materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCNA inference\u003c/h2\u003e\u003cp\u003eCopy number aberration (CNA) inference and neoplastic cell definitions are described in supplentarial materials. In short, CNA inference was performed similarly to \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Since not all samples had enough stromal cells to be used as reference for CNA inference, a pooled reference of fibroblasts, endothelial cells adrenocortical cells, deriving from multiple patients was created. To avoid creating batch effects, from each patient with at least 15 fibroblasts, endothelial or adrenocortical cells, 15 cells from that cell type were randomly sampled. This created a pooled reference comprising 30 fibroblasts, 45 endothelial cells and 30 adrenocortical cells, which was used for all CNA inference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eData and code availability\u003c/h2\u003e\u003cp\u003eRaw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/susanneschlisio/Bullova-et-al\u003c/span\u003e\u003cspan address=\"https://github.com/susanneschlisio/Bullova-et-al\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The raw sequencing data of human and mouse samples will be publicly accessible at the date of publication.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: https://github.com/susanneschlisio/Bullova-et-al . The raw sequencing data of both human and mouse samples will be publicly accessible as the date of publication. The single-cell transcriptome data was generated at the Eukaryotic Single-cell Genomics facility at Science for Life Laboratory in Stockholm, Sweden. The computations and data handling for this project were performed on resources provided by the the National Academic Infrastructure for Supercomputing in Sweden (NAISS) at sens2018122, NAISS 2024/5-221, NAISS 2024/6-129, and NAISS 2024/6-332. We thank the NIH NeuroBioBank providing human adrenal glands. We thank Oscar Bedoya Reina for his computational support with the smartseq2 RNAseq analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conceptualization: P.B., J.Z., M.M. and S.S. Methodology: P.B., P.C., M.A., J.Z.,W.L., V.P., M.P., K.S., M.E.K, C.S. and M.M. Formal analysis: P.B., P.C., M.A., J.Z., W.L., V.P., M.P., K.S., C.S., M.M. and S.S. Resources: C.L. C.J. Writing\u0026mdash;original draft: M.M., P.B., J.Z., and S.S. Writing\u0026mdash;review and editing: P.B., P.C., M.A., J.Z., W.L., K.S., C.S. M.E.K., C.L., C.J., M.M S.S. Visualization: P.C. J.Z., Supervision: M.M. and S.S. Project administration: S.S. Funding acquisition: S.S. was funded by the Swedish Research Council, Swedish Childhood Cancer Fund, the Swedish Cancer Society, ParaDiff Foundation, ERC Synergy grant (KILL-OR-DIFFERENTIAT) and Radiumhemmets Forskningsfonder. P.B. was funded by the Swedish Cancer Society.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Raw single-cell/nuclei sequencing data have been submitted to XXX Database with accession number XXX. The analysis pipeline has been uploaded to GitHub and public available at: https://github.com/susanneschlisio/Bullova-et-al . The raw sequencing data of both human and mouse samples will be publicly accessible as the date of publication.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKastriti, M.E., Kameneva, P. \u0026amp; Adameyko, I. Stem cells, evolutionary aspects and pathology of the adrenal medulla: A new developmental paradigm. \u003cem\u003eMol Cell Endocrinol\u003c/em\u003e 518, 110998 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLenders, J.W., Eisenhofer, G., Mannelli, M. \u0026amp; Pacak, K. Phaeochromocytoma. \u003cem\u003eLancet\u003c/em\u003e 366, 665\u0026ndash;675 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrona, J., Taieb, D. \u0026amp; Pacak, K. 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Here, we identify a postnatal population of SOX2/SOX10-expressing sustentacular glia-like cells in the organ of Zuckerkandl (OZ) and adrenal gland that give rise to chromaffin cells \u003cem\u003ein vivo\u003c/em\u003e. These cells differ transcriptionally from embryonic chromaffin progenitors known as Schwann cell precursors and exhibit a unique progenitor signature. Genetic lineage tracing confirms their postnatal contribution to chromaffin cells, and SOX2\u0026thinsp;+\u0026thinsp;PHOX2B\u0026thinsp;+\u0026thinsp;transitional cells were observed in both human and mouse OZ and adrenal tissues. Single-nuclei RNA-seq and inferCNA analysis of pheochromocytoma and paraganglioma (PPGL) revealed that while most sustentacular cells exhibit a stromal profile, a subset in VHL-mutated PPGLs harbor the hallmark 3p chromosomal loss shared with chief tumor cells, suggesting a clonal origin. In an additional PPGL, widespread SOX2 expression in PHOX2B\u0026thinsp;+\u0026thinsp;tumor cells supports this hypothesis. Finally, DLK1-NOTCH signaling was predicted as a central regulator of chromaffin\u0026ndash;sustentacular communication, suggesting DLK1 fine-tunes chromaffin regeneration via NOTCH inhibition and may represent a therapeutic target in PPGL.\u003c/p\u003e","manuscriptTitle":"Postnatal Sustentacular Cells as Chromaffin Progenitors and Tumor Cells of Origin in VHL-Related Paragangliomas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-08 14:11:47","doi":"10.21203/rs.3.rs-6907400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-30T12:57:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-30T09:54:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T17:08:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209821959503757891882590971634936713101","date":"2025-07-09T07:39:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326774097613265592544150149869682963035","date":"2025-07-07T13:35:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211096512607912259626607889721324471815","date":"2025-07-07T06:21:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T05:46:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-06T11:57:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-18T05:37:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2025-06-16T16:15:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86d81b88-4aa0-4b5e-807b-15e1939c7223","owner":[],"postedDate":"July 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51176447,"name":"Biological sciences/Cancer/Cancer stem cells"},{"id":51176448,"name":"Biological sciences/Cancer/Endocrine cancer"},{"id":51176449,"name":"Biological sciences/Cancer/Tumour heterogeneity"}],"tags":[],"updatedAt":"2025-10-20T16:03:31+00:00","versionOfRecord":{"articleIdentity":"rs-6907400","link":"https://doi.org/10.1038/s41698-025-01145-8","journal":{"identity":"npj-precision-oncology","isVorOnly":false,"title":"npj Precision Oncology"},"publishedOn":"2025-10-15 15:58:07","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-07-08 14:11:47","video":"","vorDoi":"10.1038/s41698-025-01145-8","vorDoiUrl":"https://doi.org/10.1038/s41698-025-01145-8","workflowStages":[]},"version":"v1","identity":"rs-6907400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6907400","identity":"rs-6907400","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}