2022 08 27 505530v1 Full

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.27.505530; this version posted August 28, 2022.
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Cell Atlas at Single-Nuclei Resolution of the Adult Human Adrenal Gland and
Adrenocortical Adenomas
Barbara Altieri1,8, A. Kerim Secener2,3,4,8, Somesh Sai2,3,4, Cornelius Fischer2,3, Silviu Sbiera1,
Panagiota Arampatzi5, Sabine Herterich6, Laura-Sophie Landwehr1, Sarah N. Vitcetz2,3, Caroline
Braeuning2, Martin Fassnacht1,6,9*, Cristina L. Ronchi1,7,9*, Sascha Sauer2,3,5,9*
1
Division of Endocrinology and Diabetes, Department of Internal Medicine I, University
Hospital, University of Würzburg, Würzburg (Germany).

2
Max Delbrück Center for Molecular Medicine, Berlin (Germany).
3
Berlin Institute of Health, Berlin (Germany).
4
Institute of Biochemistry, Department of Biology, Chemistry and Pharmacy, Free University
Berlin (Germany).
5
Core Unit SysMed, University of Würzburg, Würzburg (Germany).
6
Central Laboratory University Hospital Würzburg, Würzburg (Germany).
7
Institute of Metabolism and System Research, University of Birmingham, Birmingham (United
Kingdom).
8
These authors contributed equally: Barbara Altieri, Ali Kerim Secener.
9
These authors jointly supervised this work: Martin Fassnacht, Cristina L Ronchi, Sascha Sauer.
*Correspondence to:
Prof. Martin Fassnacht

Division of Endocrinology and Diabetes, Department of Internal Medicine I
University Hospital of Würzburg
Oberduerrbacher Str 6
97080 Würzburg (Germany)
Tel: +49-931-201-39700, Email: fassnacht_M@ukw.de
1
bioRxiv preprint doi: https://doi.org/10.1101/2022.08.27.505530; this version posted August 28, 2022. The copyright holder for this preprint
ORCID ID: 0000-0001-6170-6398
Dr. Cristina L. Ronchi

Institute of Metabolism and System Research
University of Birmingham
B15 2TT Edgbaston
Birmingham (United Kingdom)
Phone: +44-121-4146857, Email: C.L.Ronchi@bham.ac.uk
ORCID ID: 0000-0001-5020-2071
Dr. Sascha Sauer

Max Delbrück Center for Molecular Medicine
Robert-Rössle-Straße 10
13092 Berlin
Germany
Phone: +49-30-9406-2995, Email: sascha_sauer_2020@gmx.de
ORCID ID: 0000-0002-4794-3693
2
Abstract
The human adrenal gland is a complex endocrine tissue. Developmental studies on this tissue
have been limited to animal models or human foetus. Here, we present a cell atlas analysis of the
adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial
transcriptome data to reconstruct adrenal gland development and tumourigenesis. We identified
two populations of potential progenitor cells resident within the adrenal cortex: adrenocortical
progenitors NR2F2+-ID1+ cells, located within and underneath the capsule, and medullary
progenitors SYT1+-CHGA- cells, located in islets in the subcapsular region. Using pseudotime
analyses, we provided evidence of the centripetal nature of adrenocortical cell development and
of the essential role played by the Wnt/β-catenin pathway in the adrenocortical self-renewal. By
comparing transcriptional profiles of cells of normal adrenal glands and adrenocortical adenomas
we revealed a high heterogeneity with six adenoma-specific clusters. Overall, our results give
insights into adrenal plasticity and mechanisms underlying adrenocortical tumourigenesis.
Key words: Adrenal cortex, adrenal medulla, stem cells, progenitor cells, adrenocortical
tumours, tumourigenesis, heterogeneity, cortisol secretion, CTNNB1.
3
Introduction
The human adrenal gland is a complex endocrine tissue that maintains homeostasis by responding
to various physiological stimuli by secreting steroid hormones and catecholamines. The adult
adrenal gland consists of two functionally distinct parts, the cortex and the medulla, which
develop from different embryological origins1. According to textbooks, the adrenal cortex is
organized in three zones bearing diverse morphological and functional characteristics. From the
outer to innermost layer, histological analyses identify the zonae glomerulosa (ZG), fasciculata
(ZF) and reticularis (ZR) involved in mineralocorticoid, glucocorticoid, and androgen synthesis
and secretion, respectively. Beyond the cortex, the centre of the adrenal gland is occupied by
catecholamine-secreting medulla, originating from neural crest cells.
Several animal model studies have been performed to reveal the architecture of adrenal glands
and their development. According to these studies, the adrenocortical zonation is driven by
several signalling pathways, including the Wnt/β-catenin, the hedgehog (HH), the cAMP/protein
kinase A (PKA), and the insulin-like growth (IGF) pathways2. Among these, both Wnt/β-catenin
and HH play an important role in the adrenal subcapsular area, where they presumably contribute
to the centripetal renovation of the adrenal cortex3. The dysregulation of these signalling
pathways is likely involved in human adrenal tumourigenesis4. However, having recent studies
mostly been performed in mice, these findings cannot be directly transferred to humans.
Benign tumours deriving from the adrenal cortex (adrenocortical adenomas, ACA) are frequent in
the general population (up to 10% in age > 70 years). They are usually incidentally discovered
and mostly endocrine inactive adenomas (EIA)5,6. However, benign tumours may provoke
autonomous steroid secretion associated with clinically relevant disorders, such as adrenal
Cushing’s syndrome in cortisol-producing adenomas (CPA) or Conn's syndrome in aldosterone-
producing adenomas. On the other side, malignant adrenocortical carcinomas (ACC) are rare
aggressive cancers with overall poor prognosis. In the last years, comprehensive genomics studies
of benign and malignant tumours have identified alterations in signalling pathways involved in
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adrenocortical tumourigenesis (i.e.Wnt/β-catenin, Rb/p53 signalling and/or IGF system) and
autonomous cortisol excess (i.e. cAMP/PKA pathway)5-11. However, the molecular mechanisms
underlying the pathogenesis of a vast percentage of adrenocortical tumours remained largely
unexplained. Therefore, a deeper understanding of the normal adrenal growth, differentiation and
self-maintenance of adrenal cells in the adrenal tumourigenesis is urgently required.
Recently, single-cell/single-nuclei RNA-sequencing (sc/snRNA-seq) and spatial transcriptomics
have provided unprecedented insights into cellular heterogeneity allowing the identification of in
part unexpected cellular identities12-14. Particularly, cell atlases of normal human tissues based on
scRNA-seq data may provide the basis for cellular analysis of pathological human samples - such
as tumours - to gain a deeper understanding of tumourigenesis15-17. Whereas some recent scRNA-
seq based studies focused on the neural compartment of the adrenal gland18, to our knowledge so
far only one mouse study investigated on the single-cell transcriptome level the adrenal cortex by
analysing the effect of stress on the hypothalamic-pituitary-adrenal axis 19. However, adrenal
physiology significantly differs between rodents and humans.
Therefore, we aimed to provide a first comprehensive cell atlas of the cortex of the adult human
normal adrenal gland (NAG) at single nuclei resolution including spatial information. This new
approach allowed us to directly find new cell-specific human biomarkers and gain insights in
molecular processes of adrenal self-renewal and homeostasis in humans. Furthermore, we
integrated our atlas with snRNA-seq datasets from a clinical ACA cohort - comprised of EIA and
CPA patient samples - to reveal human tumour-specific cell subpopulations and potential
treatment targets.
Results
Single-nuclei transcriptome sequencing of NAGs
We sequenced 11,931 nuclei from six NAGs (Table 1 and Supplementary Fig. 1), obtaining an
average depth of 25 million reads per sample. In an unsupervised cluster analysis, we identified
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six main cell clusters with distinct gene expression signatures. The identity of each cluster was
assigned by cross-referencing upregulated transcripts with canonical markers from the literature.
The dominant cluster was populated by classical adrenocortical cell types (Fig. 1A), including
subclusters of ZG, ZF and ZR, as well as a new subcluster named “adreno-medullary progenitors”
(AMP) (Fig. 1B). Highly expressed genes typical for these cortex subclusters are shown in Fig.
1C. The remaining “satellite” clusters were annotated as “Medulla”, “Fibroblasts & Connective
tissue” (FC), “Stem/Progenitor and Vascular Endothelial cells” (Stem/P&VEC), “Myeloid cells”
and “Lymphoid cells” (Fig. 1D). The top 100 differentially expressed genes (DEGs) defining the
individual clusters of the NAG are listed in Supplementary Table 1.
Zonation of the adrenal cortex
Within the adrenal cortex cluster, different cell subpopulations showed high expression of genes
encoding for key steroidogenic enzymes representing the three cortex zones (Fig. 1B,
Supplementary Fig. 2A-C, Supplementary Table 1). Cells from the ZG subcluster showed high
expression of CYP11B2, as well as DACH1 and ANO4, all previously described to be highly
selective for this zone20-23. The ZF was characterized by high expression of CYP17A1, CYP11B1,
and HSD3B2, whereas we observed elevated expression of CYB5A, SULT2A1, and GSTA1 in the
ZR24,25, as expected. Further gene enrichment analyses with DEGs from the three adrenocortical
zones showed a significant overlap with regard to our annotation. In fact, aldosterone synthesis
(fold enrichment (FE) >8), aldosterone-regulated sodium reabsorption (FE >6), endocrine and
other factor-regulated calcium reabsorption (FE >4), and Wnt signalling (FE >2) were
upregulated in the ZG subcluster, while cortisol synthesis and cholesterol metabolism were
clearly overrepresented in the ZF (FE >23 and >13, respectively (Supplementary Fig. 3A-B).
Steroid biosynthesis (FE >27), metabolism of xenobiotics by cytochrome P450 (FE >13), and
estrogen signalling (FE >3) were higly represented in the ZR subcluster (Supplementary Fig. 3C).
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To further analyse the transcriptomic zonation of the adrenal cortex, we performed
immunohistochemistry (IHC) to detect protein expression of selected mRNA-based markers in
the different zones of the adrenal glands (represented via the kernel density26 in the “Uniform
Manifold Approximation and Projection”, UMAP; Fig. 2A). Immuno-staining of DACH1 was
uniform in the subcapsular region while CYP11B2 appeared in specific niches (Fig. 2C: DACH1,
CYP11B2). These findings are consistent with the expression of the corresponding mRNAs in our
single nuclei dataset: CYP11B2 featured a low expression density in ZG but DACH1 covered a
substantial portion of this cluster (Fig. 2A: ZG). Moreover, CYP17A1 protein expression was
detected in the entire cortex region (Fig. 2C: CYP17A1), corresponding to a higher RNA
expression density in ZF with lower expression in the adjacent ZG and ZR clusters (Fig. 2A: ZF).
In contrast, CYB5A protein staining clearly distinguished between ZF and ZR (Fig. 2C: CYB5A,
SULT2A1). Although in the snRNA dataset both CYB5A and SULT2A1 densities highlighted
similar regions of the UMAP (Fig. 2: ZR), SULT2A1 was not selectively expressed in ZR as
CYB5a, but was also expressed (at a lower intensity) in the ZG.
Adrenal progenitor and stem cells
Of note, within the main adrenal cortex cluster we identified a subcluster of sympathoadrenal
lineage cells. These cells were characterised by transcriptional similarities to both neuronal cells
and cells of the “Medulla” cluster (for comprehensive initial snRNA-seq analyses we included
not only the cortex but also the medulla) (Fig. 1B, Supplementary Fig. 2F and Supplementary
Table 1). As in the medulla, these cells specifically expressed genes involved in
neurotransmission, such as SYT1, NRG1, NRXN3, GRIK1, and CADPS. Gene enrichment analysis
further corroborated the chromaffin-like nature of these cells, revealing sets of genes involved in
cholinergic and glutamatergic synapse (FE >7), glutamatergic synapse (FE >5), and axon
guidance (FE >4) (Supplementary Fig. 3F). However, cells within this subcluster did not express
markers of the mature medulla such as CHGA and CHGB, TH, PNMT, and DBH. Due to the
7
expression of neuronal markers and the absence of hints to the mature medulla, we hypothesize
that this subcluster represents early progenitors of the adrenal medulla, henceforth named as the
“Adreno-Medullary Progenitors” (AMP) subcluster. We validated this hypothesis by using IHC
to investigate the protein expression of SYT1 (synaptotagmin 1) and CHGA (chromogranin) (Fig.
3A-B). We observed low SYT1 expression in the entire cortex, whereas the medulla showed
moderate expression. Interestingly, we found groups of cells with stronger SYT1 staining in the
subcapsular region, which were organized as niches (Fig. 3B.1). In line with snRNA-seq data,
these cells did not show CHGA expression in the double staining SYT1-CHGA (Fig. 3B.2),
confirming that these niches may truly contain progenitor cells of the adrenal medulla.
Interestingly, recent studies in murine models showed that nestin+ medullary progenitor cells
were located not only in the medulla but also in niches of the mouse adrenal capsule, to contribute
to the plasticity of the adrenal cortex. The here found presence of two different progenitor
populations within the adult adrenal cortex might suggest a higher degree of flexibility of tissue
renewal capacity than expected27 to ensure the maintenance of the adult adrenal cortex28-32.
Another cell cluster that includes a significant number of adrenal stem cell markers is the
Stem/P&VEC cluster. Here, classical endothelial genes, such as FLT1 (also known as VEGFR1),
KDR (also known as VEGFR2), and TEK, as well as several genes associated with cell
proliferation and differentiation, including ETS1, ETS2, INSR, and DNASE1L3 were found
(Supplementary Fig. 2E, Supplementary Table 1). Moreover, NOTCH1, a known marker of
adrenal cortical progenitors33 (involved in cell fate specification, differentiation, proliferation, and
survival) and its target gene HES1 (responsible for the regulation of genome-wide glucocorticoid
signalling34) were also both expressed at a high level (Supplementary Fig. 2E). Simultaneously,
low gene expression levels of genes associated to steroidogenesis, i. e. NR5A1 (also known as
SF1) and STAR were found within this cluster. One more interesting finding was the distinct
expression of NR2F2 (also known as COUP-TFII, Fig. 1D), a potential marker of adrenocortical
stem cells in mouse models35,36 and humans37. NR2F2 expression was also observed in the
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"Fibroblasts and connective tissue” cluster (Fig. 3C) that shared a similar transcriptome profile to
the adrenal capsule, expressing COL1A2 and RSPO3 (Supplementary Table 1). Recently, it was
reported that NR2F2 expression is most likely related to RSPO3+ capsular cells and that RSPO3
plays a direct role in the mouse adrenal homeostasis28. In addition to NR2F2, we found ID1, a
known marker of mesenchymal stem cells, exclusively expressed in the same cluster. To gain
more insight in the “stem” identity of this cluster, we performed IHC and microscopy-based
tissue analysis with the undifferentiated protein markers NR2F235,36 and ID1. NR2F2 staining was
found in nuclei of rare, sparsely distributed cells. These cells were mostly localized at the
subcapsular level, whereas few additional NR2F2+ cells were found within the inner cortex,
following the trend of centripetal migration of adrenocortical progenitor cells from ZG to ZF to
ZR (Fig. 3D.1). ID1 nuclei staining showed similar distribution when compared to NR2F2+ cells
(Fig. 3D.1). Via double immunostaining, we found sparse NR2F2+-ID1+ cells mostly localized at
capsular/subcapsular levels that appeared to migrate centripetally within the cortex, suggesting
that ID1 may represent a new marker of stem/progenitor cells (Fig. 3D.2). Interestingly, a
previous study also highlighted the role of ID1 in the tumourigenesis of adrenocortical cancer in
dogs38. Pathway analysis corroborated our hypothesis, revealing significant enrichment of
adherens junction (FE >5) and different signalling pathways, such as mitogen-activated protein
kinase (MAPK, FE >3), Wnt pathway (FE > 2) and signalling regulating the pluripotency of stem
cells (FE >2) (Supplementary Fig. 3E).
Characterisation of additional adrenal cell clusters
As shown in Fig. 1, we detected four other cell types in the adrenal glands. The “Medulla” cluster
showed an expression pattern typical for chromaffin cells with a predominance of CHGA, CHGB,
PNMT, DBH, and TH (Fig. 1D, Supplementary Fig. 2F, Supplementary Table 1). Within the
“Myeloid cells” cluster, we observed a large population of macrophages, mostly polarised into
alternatively activated (M2) macrophages with high expression of MRC1 (also known as CD206)
9
and CD163 together with MSR1 (also known as CD204) (Fig. 1D, Supplementary Fig. 2G,
Supplementary Table 1). Other highly expressed genes were SRGN, a proteoglycan that is
typically expressed in myeloid and lymphoid cells, CSF1R, CD86, and CD14 (Supplementary
Fig. 2G). Furthermore, we observed a second immune cluster of lymphoid cells, which showed
typical T cell markers, such as IL7R and CD96 (Fig. 1D, Supplementary Table 1), as well as
CD247 (part of the T-cell receptor-CD3 complex), RUNX3 and ITK (involved in T cell
differentiation), PRF1 and GZMA (indicators of granule mediated cytotoxic activity of natural
killer and cytotoxic T cells) (Supplementary Table 1, Supplementary Fig. 2H). Of note, only few
markers for B cells were found, e. g. IKZF3 (involved in the regulation of B lymphocyte
proliferation and differentiation) (Supplementary Fig. 2H and Supplementary Table 1).
The “Fibroblasts and connective tissue” cluster was characterised by cells that highly expressed
collagen markers, such as COL1A2 and COL4A1, and markers of the extracellular matrix, such as
MGP, FBLN1, LAMA2 and CCN1 (Fig. 1D, Supplementary Fig. 2I and Supplementary Table 1).
Using IHC, we confirmed protein expression of COL1A2 in the adrenal capsule and MGP in
extracellular matrix and fibroblasts (Supplementary Fig. 4A).
Pathway analyses validated the satellite cluster annotations, revealing enrichment of cluster-
specific gene sets, such as glutamatergic and cholinergic synapse (FE >8 and >5, respectively) for
“Medulla”, phagosome and NF-kappa B signalling (FE >4 for both pathways) for “Myeloid
cells”, Th1 and Th2 cell differentiation (FE >5) and B and T cell receptor signalling pathway (FE
>4) for “Lymphoid cells” and focal adhesion (FE >5) and extracellular matrix-receptor
interaction (FE >4) for “Fibroblasts and connective tissue” cluster (Supplementary Fig. 3F-I).
Adrenal cortex zonation by spatial transcriptomics in NAG
We validated the cellular and tissue assigments of genes based on snRNA-seq data by spatial
transcriptomics using the Visium assay in two consecutive adrenal gland sections deriving from
the exemplary patient sample #NAG-7. As we further focused in this study on the adrenal cortex,
10
the medulla was not part of the analysed tissue sections. To identify each zone, we transferred the
cell type annotation from the microdroplet-based snRNA-seq into the Visium data set using a
cell-to-spot correlation framework (Fig. 4). Mapping of the adrenal cortex zone markers
confirmed the snRNA-seq dataset. ZG appeared as a thin line of cells towards the outer cortex,
while the distribution of spots with high ZF score was rather ubiquitous. Moreover, spots with
higher ZR scores seemed to locate closer towards the center of the tissue section, although a
substantial overlap between ZF and ZR was observed (Fig. 4 and Supplementary Fig. 5).
Regarding the satellite clusters, the label transfer predicted similar mapping for “Fibroblasts and
connective tissue” and “Lymphoid cells” to the region surrounding the cortex, although the latter
displayed a low overall mapping score, probably due to the low abundance of lymphoid cells
(Fig. 4). Of note, “Fibroblasts and connective tissue” markers such as COL1A2 and RSPO3 were
exclusively expressed in this region, while “Lymphoid cells” markers could not be found
(Supplementary Fig. 5). The mapping of the “Myeloid cells” cluster showed a specific expression
of immune markers mostly around the small blood vessels within the adrenal cortex (Fig. 4).
Finally, the Stem/P&VEC mapped with a high prediction score in the outer cortex, specifically
localized in a subcapsular niche, while a medium to low prediction score was observed in the
inner cortex (Fig. 4).
Pseudotime analysis confirms centripetal development of the adrenal cortex
Next, we aimed to understand the developmental relationship between the observed cell clusters
within the adrenal cortex. To predict a differentiation trajectory, we performed pseudotime
analysis39 using the spatial transcriptomics data. The integration of the two NAG replicates
resulted in 5 distinct Louvain clusters (0 =Capsule, 1 =ZG, 2 =ZF-ZR, 3 =blood vessel, 4
=endothelial) that we annotated based on the results of the label transfer (Fig. 5A). As root node
for pseudotime computation we selected cells expressing RSPO3, a well-established marker of
capsule adrenocortical progenitors of mouse adrenal gland23,28 (within the capsule cluster, RSPO3
11
was specifically expressed by a subset of cells (Fig. 5B)). The resulting unsupervised lineage
trajectory of the adrenal cortex started with capsular cells, proceeded through the ZG cluster, and
ended in the ZF-ZR cells, indicating a centripetal development of the adrenal gland.
To further investigate potential changes in global gene expression dynamics along the trajectory,
we applied pseudotime analysis in DEGs of the three main clusters (capsule, ZG, and ZF-ZR)
(Fig. 5C). In total, we found 829 genes that significantly varied across pseudotime. Among them,
RSPO3 was comparatively strongly expressed in capsular, early pseudo-time cells, and weakly
expressed within the inner-zones, late cell types (Fig. 5D). A similar pattern of cellular
development was observed for ID1. INSR gene expression was specifically switched on in ZG
(early time point in development) and its expression diminished throughout the inner cortex (later
time points) (Fig. 5D). The selective activation of these genes at the early stage confirmed the
stem/progenitor nature of the subpopulation of cells within the “Stem/P&VEC cluster” found in
snRNA-seq analysis. Considering the steroidogenic-related genes, CYP11B2 was first activated in
the early ZG cells and significantly decreased throughout the ZG-ZR formation (Fig. 5D). We
further observed an increased gene expression in the late ZG cells - and throughout the
populations of the inner cortex - for CXCR4 and PRKAR1A (cAMP/PKA pathway) and ADGRV1
(cholesterol synthesis pathway) (Supplementary Fig. 6). Of note, the expression of DLK1, which
is a proposed marker of a subcapsular cell population, was observed at two stages of the
trajectory: a first peak appeared around the early development capsular region (Supplementary
Fig 7A-B, indicated by a green box) and a second one at the ZG to ZF-ZR transition zone
(Supplementary Fig. 7A-B, indicated by a blue box). Interestingly, DLK1 expression visualized
on the adrenal sections revealed a similar pattern described by Hadjidemetriou et al.40, i.e as
“clusters of cells” (Supplementary Fig. 7B, regions inside blue boxes).
Moreover, the pseudotime analysis revealed interesting expression of Wnt/β-catenin pathway-
related genes (Supplementary Fig. 6). SFRP1, SFRP2 and SFRP4 were found to be expressed in
early or late capsular cells, but their expression decreased throughout the remaining cells
12
subpopulations. WNT4 was expressed in ZG only. Other genes such as CTNNA1 or CTNNA1L
were specifically switched on in the late ZG subpopulation and continued to be mildly expressed
in the ZF-ZR zones (Supplementary Fig. 6).
Integration of NAG and ACA snRNAseq data reveals adenomas-specific cells
Next, we used our human reference atlas to analyse cell type heterogeneity and identify possible
tumourigenesis processes in our ACA patient samples (Table 2). For this, we sequenced 21,794
single-nuclei transcriptomes from 12 snap-frozen ACA samples (7 CPAs and 5 EIAs) and
integrated this data with 5154 highest scoring NAG nuclei.
The differential expression analyses indicated that several genes involved in cell signalling, tissue
remodelling, cell replication and RNA transcription were significantly upregulated in ACAs
compared with NAGs (Fig. 6A.1-2). Particularly genes like IGF2R (average fold-change for gene
expression, avg-log2FC >4.2, p<1e-6 in both EIA and CPA), SLBP (avg-log2FC >3.4, p<1e-6 in
both EIA and CPA), GAS2 (avg-log2FC >3.4, p=1.04e-272 in EIA and avg-log2FC >2.0,
p=8.85e-153 in CPA), and SP100 (avg-log2FC >1.8, p=2.45e-208 in EIA and avg-log2FC >1.6,
p=9.33e-184 in CPA) were strongly expressed in both EIA and CPA (Fig. 6A.3). Genes like
MMP26 (avg-log2FC >3.6, p<1e-6) and ADGRG2 (avg-log2FC > 1.5, p=2.27e-138) were
strongly expressed in EIAs, whereas RBFOX1 (avg-log2FC > 3.1, p<1e-6) and HMGCS1 (avg-
log2FC >1.6, p=4.51e-134) were predominantly expressed in CPAs (Fig. 6A.4).
In line with the UMAP results of NAGs, in ACAs we found a central main cluster and 5 satellite
clusters comprised of myeloid, lymphoid, endothelial, stromal (mainly composed of fibroblasts
and connective tissue) and medullary cells (Fig. 6B). The central major cluster showed a complex
organization including the three cortex-specific ZG, ZF and ZR and seven other subclusters (Fig.
6B-C). The “Cholesterol-and Steroid-Enriched Metabolism” (CSEM) subcluster was also found
in a very small percentage in NAGs (~1%). The six remaining subclusters were specifically
expressed in ACAs and were named as “Adenoma MicroEnvironment” (AME), “ZG-like”, and
13
“adenoma-specific clusters” (AC1-4). These subclusters were mostly distributed in the periphery
of the central major cluster, except for AME that occupied a large area in the central region (Fig.
6B). Top 100 DEGs defining the individual clusters of the integration of NAG and ACA are
listed in Supplementary Table 2. In short, RNA markers specific for the ZF and ZR clusters of the
NAG were largely expressed within the central cluster, and RNA markers such as CYP17A1 and
CYP11B1 were rather ubiquitously expressed at low to medium levels across the tumour clusters
(Fig. 6C), confirming the adrenocortical nature of these cells.
ACA-specific clusters distribution according to subtype and mutational status
We further explored the cluster/cell type composition across the 18 samples by grouping our
samples according to tissue entity (CPA, EIA or NAG) and mutational status – available from
previous next-generation DNA or Sanger sequencing of bulk tumour tissues (no driver mutation,
CTNNB1 mutations, and PRKACA/GNAS mutations) (Fig. 6D).
The ZG-like and the AME clusters showed a homogeneous distribution, without significant
differences among the samples (Fig. 6D). Within the “ZG-like” cluster, common ZG markers,
such as DACH1, ANO4, and NR4A2 (also known as NURR1) were dominantly expressed (Fig.
7A), indicating the presence of normal adrenocortical cells in ACAs. However, unlike ZG, the
ZG-like cluster was characterised by lower to null expression of CYP11B2 (avg-log2FC >-2,
p=8.69e-36), CALN1 (avg-log2FC >-2.3, p=9.60e-82) and MC2R (avg-log2FC >-1.9, p=1.17e-
31). Moreover, ZG-like cluster showed also significant expression of non-ZG specific genes, such
as CYP11B1 (avg-log2FC >1.2, p=2.05e-16), IGF2R (avg-log2FC >4.4, p=3.79e-78) and GAS2
(avg-log2FC >2.4, p=.82e-12). Pathway analyses revealed enrichment of several pathways
involved in cell signalling, such as protein phosphorylation (FE >6), as well as pathways involved
in calcium signalling (FE >3) and aldosterone secretion (FE >2) (Fig. 7B and Supplementary Fig.
8).
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Cells of the AME overexpressed genes like APOE, ADIPOR2 and RARRES2 involved in the
lipoprotein’s metabolism, as well as IGF1R and NDRG2 (Fig. 6C and 7A). IGF1R is a
transmembrane tyrosine kinase receptor of the IGF family that activates a mitogenic signalling
pathway, stimulating cell proliferation, division, and translation, and inhibiting apoptosis41.
NDRG2 regulates the Wnt signalling pathway by modulating CTNNB1-target genes42. Pathway
analyses revealed enrichment of several pathways involved in cell signalling, such as AMPK (FE
>4) and insulin signalling pathways (FE >3), cholesterol trafficking (FE >4), and pathways
associated with different tight, focal and adherens junctions (FE >2) (Fig. 7B and Supplementary
Fig. 8).
The CSEM cluster was significantly over-represented in the CPA cohort compared to EIAs
(p=0.03; Fig.8A), with high expression of genes related to cholesterol metabolism, including
HMGCS1, SQLE, and INSIG1, but also KCND2and POLE2 (Fig. 7A), which are involved in
DNA repair and replication. The enrichment pathway analyses confirmed this finding, showing
several pathways involved in cholesterol metabolism and steroidogenesis, among which a very
high enrichment of cholesterol and steroid biosynthesis (FE >9), pathways involved in
ubiquitination processes, including protein ubiquitination (FE >5) and cellular response to
oxidative stress (FE >2.5) (Fig. 7B and Supplementary Fig. 8).
The remaining clusters presented a slightly different distribution within the tumour subtypes and
mutational status. Specifically, AC1 was mostly found in two CTNNB1-mutated samples, one
EIA (EIA-1, ~48%) and one CPA (CPA-4, ~46%) (Fig 6D and Fig. 8B), with a high expression
of the β-catenin target genes LEF1, AFF3, and ISM1 (Fig 7A, Supplementary Table 2). Both
AFF3 and ISM1 are known to be overexpressed in benign and malignant adrenocortical tumours
with CTNNB1 mutation43-45. Also, we found a high expression of FTO, a N6-methyladenosine
that promotes cells proliferation, migration and chemo-radiotherapy resistance by targeting
CTNNB1 in different cancer types46,47 (Fig. 6C and Fig. 7A). In addition, other genes involved in
cell proliferation, including GAS2 and TERB1, and long non-coding RNAs, such as CASC16 and
15
C9orf84, were found to be highly expressed within this cluster (Fig. 7A). The enrichment analysis
revealed a very high expression of different pathways involved in spliceosome (FE >10), cell
proliferation and tissue development (FE >3), and extracellular cell-matrix adhesion (FE >5) (Fig.
7B and Supplementary Fig.8).
AC2 cluster was more abundant in two EIA samples (EIA-2 and EIA-5) (Fig. 6D) without
correlation with the mutational status (Fig. 8B). The AC2 cluster was characterised by an
overexpression of genes like MMP26, ZNF98 and SP100 (Fig. 6C and 7A) associated with
tumour promotion. Enrichment analysis showed also for AC2 a high expression of spliceosome
pathway (FE >6) and ubiquitin-related pathways, including SUMOylation (FE >5) and cell cycle
(FE >2) (Fig 7B and Supplementary Fig. 8).
In contrast, AC3 was significantly over-represented in CPAs with PRKACA/GNAS mutation
compared to those with CTNNB1-mutated (p=0.02) (Fig. 6D and 8B). Particularly, it was very
abundant in the single CPA with GNAS mutation (CPA-7, ~23%). Of note, we observed a high
expression of PDE8A within this cluster (Fig. 7A). Also, a high expression of SAE1, associated to
SUMOylation, GRB14 and FBLN1, associated with cell growth and extracellular matrix,
respectively, was found (Fig. 6C and 7A). Enrichment analyses confirmed a high enrichment in
genes of the SUMOylation pathway (FE >4), cell adhesion molecular binding (FE >3), but also
spliceosome (FE >2) (Fig 7B and Supplementary Fig. 8). Interestingly, AC1-3 showed a high
enrichment of elements associated to spliceosome, sometimes differently expressed among the
three. As such, splicing pathways were observed at very high levels in AC1 and decreased
through AC2 and AC3, where they were found at lower levels.
Looking into the AC4 cluster, its distribution was similar among all tumour samples except for
one CPA with CTNNB1 mutation (CPA-3), where it represented ~36% of the cells (Fig. 8B).
Within this cluster we found a high expression of the proto-oncogene MYBL2, as well as of
CGGBP1, which regulates cell cycle in cancer cells (Fig. 6C and 7A). Pathway analyses showed
significant enrichment of several pathways involved in extracellular matrix, cell-cell
16
communication (FE >2) or different types of cells junctions (FE >2) and oxidative stress induced
senescence (FE >3) (Fig. 7B and Supplementary Fig. 8).
Immune clusters
We observed a decrease in quantity of both lymphoid and myeloid cells from NAGs to ACAs.
Particularly, the lymphoid cluster was predominantly enriched in NAGs compared to EIAs
(p=0.02) and by trend compared to CPAs (p=0.09) (Fig 8C). No difference was observed among
the ACAs according to the mutational status. The expression of UTY significantly decreased from
NAGs to both EIAs (avg-log2FC >-2, p=1.92e-08) and CPAs (avg-log2FC > 1.5, p=5.32e-09)
(Supplementary Fig. 9A). Other genes like HLA-A, HLA-B, HLA-C, and CCL5 followed a trend
to decreased expression in CPA compared to NAG (avg-log2FC >-1.3, p=2.84e-08 for HLA-A,
avg-log2FC >-1.2, p=6.55e-07 for HLA-B, avg-log2FC >-1.7, p=5.58e-07 for HLA-C, avg-
log2FC >-1.5, p=1.05e-06 for CCL5) (Supplementary Fig. 9A).
The myeloid cluster was similarly represented in NAGs when compared to EIAs, but less
represented in CPA samples (p=0.07) (Fig. 8D). Interestingly, the distribution of the myeloid
cluster among the ACA samples changed according to the mutational status. Particularly, ACA
samples with CTNNB1 mutation revealed a higher abundance of myeloid cells compared to
samples with no driver (p=0.03) and PRKACA/GNAS mutations (p=0.08) (Fig. 8D). Among
differentially expressed genes (Supplementary Fig. 9B), MSR1 was significantly downregulated
in EIAs (avg-log2FC >-1.2, p=2.5e-19) and CPAs (avg-log2FC =-0.9, p=4.49e-12) compared to
NAGs. Similarly, SRGN decreased in EIA (avg-log2FC >-1.2, p=5.24e-27) and CPA (avg-
log2FC >-0.6, p=3.57e-11) compared to NAGs. Genes like CSF1R and CD74 were less expressed
in CPAs compared to both NAGs (avg-log2FC >-0.9, p=2.07e-07 for CSF1R and avg-log2FC -
0.9, p=1.87e-10 for CD74) and EIA (avg-log2FC >-1.0, p=3.04e-10 for CSF1R and avg-log2FC
>-1.3, p=4.38e-24 for CD74) (Supplementary Fig. 9B).
17
Discussion
In this study, we provide a high-resolution, comprehensive transcriptional cell atlas of the normal
adult human adrenal gland including spatial RNA and protein expression information. We could
distinguish characteristic zones of the adrenal cortex (ZG, ZF, ZR), as well as the adrenal medulla
and capsule. In addition, we found different satellite clusters, including lymphoid, myeloid and
vascular cells. Moreover, by comparing our single-cell transcriptome atlas of the adult human
normal adrenal glands to a cohort of adrenocortical adenomas, we revealed seven adenoma-
specific cell types that were involved in adrenocortical tumourigenesis.
One of the key findings of our study was the identification of two different populations of
potential progenitor cells within the adult human NAG: the adrenocortical progenitor-like
NR2F2+-ID1+ cells, located within and underneath the capsule, and the medullary progenitor-like
SYT1+-CHGA- cells, gathering in niches in the subcapsular region of the adrenal cortex. To our
knowledge, we here show for the first time the presence of NR2F2+ progenitor cells in adult
human adrenal glands. Moreover, we also found the co-expression of NR2F2 with ID1 (a
mesenchymal stem cell marker) in few cells that were predominantly located under the capsule.
As observed for progenitor cells in animal models, also in human adrenal glands these
subcapsular cells showed a centripetal migratory pattern. The high differentiation potential of
ID1+ cells is supported by high expression within the same “Stem/P&VEC cluster” of genes
associated with cell proliferation and differentiation (ETS1, INSR, HES1, DNASE1L3,
NOTCH1)33. Moreover, the trajectory analysis of spatial transcriptomic data enabled us to
observe the selective activation of some genes at the early stages of pseudotime, confirming the
progenitor nature of this subpopulation. In addition, the microenviroment of this cluster is rich in
adherens junctions and vascular cells, which are essential in cell-cell communication, cell renewal
and differentiation48,49. All these findings suggest that these ID1+ cells might represent a new
progenitor population in the adult adrenal cortex.
18
In addition, we observed a small subcluster of cells, which expressed neuronal-like gene markers,
including SYT1, NRG1, NRXN3, GRIK1 and CADPS as found in chromaffin cells of the adrenal
medulla. The subcluster was void of markers of mature medullary cells such as CHGA. Using
IHC we validated the presence of SYT1+- CHGA- cells that form clusters in the subcapsular
region of the cortex, as had been observed for the nestin+ medullary progenitor cells in mouse.
However, we could not identify nestin itself, potentially due to the differences in gene expression
between mice and humans. Nevertheless, we cannot exclude that the rarity of these progenitors in
adult adrenal glands or their presence only in early-stage adrenal development is responsible for
this fact. The neuronal-like nature of the expressed markers within this subcluster raised the
question if SYT1+- CHGA- cells could represent a progenitor population of the adrenal medulla
that is located in the subcapsular region of the adult human adrenal cortex, as had been observed
in animal models50. Moreover, in line with the undifferentiated nature of NR2F2+-ID1+ and
SYT1+-CHGA- cells, both potential progenitor clusters showed little to no expression (< 5% in
both clusters) of mature cells markers at transcriptional levels, including NR5A1 and STAR in the
Stem/P & VEC cluster, and CHGA, CHGB, TH, PNMT, and DBH in the AMP cluster.
Interestingly, by combining pseudotime and spatial transcriptomic analysis, we corroborated the
centripetal nature of adrenocortical cell development. However, it still remains to be
demonstrated ex vivo whether NR2F2+-ID1+ and SYT1+-CHGA- cells represent populations
capable to undergo steroidogenic or chromaffin differentiation. Future lineage tracing
experiments will enable us to functionally characterise these progenitor subpopulations, this being
beyond the scope of this pilot study.
The trajectory and pseudotime analyses allowed us to look more deeply into the transition zone
between the capsular and sub-capsular region, where the progenitor cells are mostly located. Here
we showed, for the first time, a selective activation of members of the Wnt/β-catenin pathway
(i.e. SFRP2 and SFRP4), which play an important role in adrenocortical renewal and
differentiation51, as well as in adrenocortical tumourigenesis11,52,53. Particularly, both SFRP2 and
19
SFRP4 were expressed in human mesenchymal stem cells isolated from bone marrow54, and a
decreased expression of SFRP2 was associated with aldosterone-producing adenoma
development55. These findings corroborated an essential role of the Wnt/β-catenin pathway for
progenitor cell formation and function throughout the adrenal development. Intriguingly, DLK1
expression was observed in cells organised as clusters in the subcapsular region, as previously
described40. Pseudotime analyses revealed that DLK1 expression exhibits two peaks (first during
the capsule-ZG transition and second during ZG to ZF-ZR transition), strengthening its potential
role in adrenocortical remodelling.
Recently, it was proposed that adrenal renewal is sexually dimorphic36,56. In our study, we were
not able to find any significant transcriptomic differences between the two sexes, potentially due
to the small sample size (tissue from two females versus four males) and the limited number of
(few hundreds of) detected expressed genes per nucleus. Similarly, age-specific differences were
not observed, as all samples of this cohort belonged to adult subjects (ranging from 49 to 81 years
of age).
Our transcriptome atlases of healthy adult human adrenal glands will be an important tool as
reference to investigate adrenal-related disease. In a first step, we interrogated benign
adrenocortical tumours (adenomas), which were further classified according to glucocorticoid
secretion and mutational status. Using this approach, a high intratumoural heterogeneity was
observed, confirming for the first time at single-nuclei level previous morphological and genetic
findings in bulk tumours57-59.
The comparative analysis of the transcriptional profiles of NAGs and ACAs revealed the
presence of seven adenoma-specific clusters. First, our results revealed an adenoma
microenvironment (AME) cluster, which was quite homogeneously distributed among the
samples. This represents a lipid-enriched milieu, similar to the tumour microenvironment
described in other solid tumours60. It has been demonstrated that such an enrichment in adipocyte
and lipid metabolism related genes plays a key role in aberrant cell growth and cancer
20
progression, as well as in immune escape and treatment-resistance60. Furthermore, as reported for
the tumour microenvironment in colon cancer61, a high expression of RARRES2, a key modulator
of tumour growth and inflammation, was detected within this cluster. This finding is in line with a
previous study reporting a higher RARRES2 expression in benign compared to malignant
adrenocortical tumours62. Within the AME, also a high expression of IGF1R, which plays an
important role in the adrenocortical tumourigenesis, was observed41. Interestingly, an
overepression of IGF1R within the tumour microenvironment was reported to promote tumour
initiation and progression in lung cancer and medulloblastoma63,64.
The “cholesterol- and steroid-enriched metabolism” cluster, which showed a high expression of
cholesterol pathway genes (i. e. HMGCR, MSMO1, INSIG1, HMGCS1), was, as expected, largely
represented in CPAs65. Furthermore, in the single CPA with GNAS mutation (CPA-7), we
observed a high prevalence of AC3, characterised by an overexpression of PDE8A. These results
confirm previous reports that highlighted the potential role of the phosphodiesterase 8 family
members in the pathogenesis of Cushing’s syndrome11,66. On the contrary, the AC2 cluster was
significantly abundant in two out five EIA samples (EIA-2 and EIA-5), where a high enrichment
of post transcriptional modifications (SUMOylation, deubiquitination) was detected.
A high morphological heterogeneity was particularly observed in CTNNB1-mutated adenomas, as
recently reported in hepatocellular carcinomas harbouring mutations in the β-catenin gene67,68.
Based on the transcriptome profile, we observed three types of CTNNB1-mutated ACA: one with
considerable abundance of AC1 (more than 45% of cells, including one EIA and one CPA); one
CPA with abundance of AC4 (more than 35% of cells); and the remaining three EIAs without
preference for a specific adenoma cluster. Of note, in the CPA with abundance of AC1 we have
observed a high expression of AFF3, ISM1 and LEF1, all known CTNNB1 target genes in benign
and malignant adrenocortical tumours43-45. Particularly, it has been demonstrated that AFF3
mediates the oncogenic effects of β-catenin in ACC cell line NCI-H295R by acting on
transcription and RNA splicing44. Similarly, in the AC4 abundant CPA, a high expression of
21
MYBL2 was found. It has been shown that MYBL2 is a Wnt/β‐catenin target gene in different
types of cancer-xenograft models69. Furthermore, MYBL2 regulates cell proliferation, survival and
differentiation and its overexpression is associated with poor outcome in different cancers types70,
including ACC71. These findings might support the previously reported hypothesis of a potential
adenoma-carcinoma sequence at least in a subgroup of CTNNB1-mutated adrenocortical
tumours11.
Another notable finding emerging from the transcriptional profiles of adenomas was the
enrichment of elements associated with spliceosome observed within three ACs (AC1-3),
suggesting a potential role of spliceosome pathway in adrenocortical tumourigenesis, as
demonstrated in other benign and malignant tumours72-74.
Interestingly, a different distribution of immune clusters emerged among tissue entities. Both
lymphoid and myeloid clusters were better represented in normal adrenals than in adenomas.
Although this trend was more evident for lymphoid cells, the median number of myeloid cells
showed a tendency to decrease in CPA compared to EIA and NAG. This finding is in line with
the known interplay between glucocorticoids and tumour-immune infiltration observed in
malignant adrenocortical tumours75, suggesting the inhibition of tumour-infiltrating immune cells
also in CPAs. In addition, we demonstrated for the first time a high prevalence of M2-like
macrophages within the myeloid cluster in normal adrenal. M2-like macrophages have anti-
inflammatory properties and promote cell homeostasis, confirming the potential trophic function
of adrenal macrophages76. This shifted macrophage polarisation towards M2-like phenotype and,
in particular, towards M2c subgroup, characterised by a high expression of the scavenger
receptors MRC1 and CD16377, could be related to the autocrine action of glucocorticoids secreted
by the adrenal gland. To note, in adenomas, the mutational status significantly affected the
myeloid cluster, which was highly enriched in CTNNB1-mutated adenomas. As observed in
NAG, adenoma-associated macrophages presented a prominent M2-like polarisation,
corroborating previous studies reporting that Wnt/β-catenin signalling promotes M2-like
22
macrophage in tumours78 and that the M2 signature is highly prevalent in CTNNB1-mutated
samples79.
Short summary and outlook
In conclusion, using snRNA-seq and spatial RNA and protein expression analyses of adult human
adrenal glands, we provide a first comprehensive cell atlas to identify complex mechanisms of
homeostasis and self-renewal of the normal adrenal gland. Moreover, the present reference atlas
allowed us already to analyse molecular processes of adrenocortical tumourigenesis and
autonomous cortisol secretion in adenomas. In the future, further improved snRNA-seq
technologies that will generate significantly more sequences per nucleus and provide data from
more nuclei per analysis will help to ameliorate transcriptional data analysis, for example to
deeper characterise developmental processes of the two newly identified progenitor cell types
(SYT1+-CHGA-- and NR2F2+-ID1+-cells) in NAGs. Furthermore, ex vivo experiments will also
be needed to analyse in more detail the influence of markers such as DLK1 and ID1 on cellular
differentiation and specification.
Materials and Methods
Tissue sample selection
For single-nuclei RNA-seq (snRNA-seq), nuclei were isolated from six snap-frozen samples
collected from NAGs deriving from the tissue surrounding EIA or from adrenalectomies
performed during surgery for renal cell carcinoma (Supplementary Fig. 1). Demographic data and
details of detected cellular transcriptomes are summarized in Table 1. Additionally, single nuclei
were isolated from 12 fresh-frozen ACA samples, including 7 CPAs and 5 EIAs. Among these
samples, 4 tissues were included in two previous studies on whole-exome sequencing10 or bulk
RNA-sequencing11. Clinical characteristics, hormonal secretion, and detected cellular
transcriptomes are summarized in Table 2 and Supplementary Table 3. For validation by
23
immunohistochemistry (see below), 16 formalin-fixed paraffin-embedded (FFPE) tissue sections
of NAGs were analysed (Supplementary Table 4). FFPE tissues included 3 out of 6 NAGs
evaluated by snRNA-seq and one sample used for the Visium analysis.
Tissue samples were selected from the Würzburg Adrenal Biomaterial Archive, part of the
BMBF-funded Interdisciplinary Bank of Biomaterials and Data Würzburg (IBDW) applying the
highest standards for biobanking80 (details in Supplementary Material).
The study was approved by the ethics committee of the University of Würzburg (No. 93/02 and
88/11) and written informed consent was obtained from all subjects.
Sequencing of driver mutations in adenoma tissues
Known driver hot-spot mutations in CTNNB1 (exon 3), PRKACA (p.Leu206) and GNAS
(p.Arg201 and p.Gln227)10,11 were evaluated by Sanger sequencing in bulk tumour tissues (n=8).
Four samples were analysed by Whole Exome Sequencing or RNA-seq as part of previous studies
from our group10,11. DNA was isolated from snap-frozen ACA tissues with the Maxwell® 16
Tissue DNA Purification Kit (#AS1030, Promega, Madison, WI, USA) according to the
manufacturer’s instructions. The mutations were genotyped by Polymerase Chain Reaction
(PCR), as previously reported81 (details in Supplementary Material).
Clinical data collection
For ACAs, the endocrine workup was conducted according to the adrenal incidentalomas
guidelines issued by the European Society of Endocrinology/European Network for the Study of
Adrenal Tumors5 and the Endocrine Society guidelines for the diagnosis of Cushing’s
syndrome82. Hormone levels were measured using commercially available analytical procedures
(details in Supplementary Material), as previously reported83. CPA derived from patients with
overt Cushing's syndrome and unequivocal laboratory results (for details see Supplementary
Material).
24
Single-nuclei RNA sequencing (snRNA-Seq)
Single-nuclei isolation: We applied a previously published protocol for nuclei isolation from
snap-frozen tissue84 (Supplementary Fig. 1 and Supplementary Material). The nuclei integrity and
purity were checked under a microscope and the yield was quantified using a Neubauer chamber.
Single-nuclei RNA sequencing: We used the inDropTM method consisting of three major steps:
Silanization, Reverse Transcription and Library preparation (1CellBioTM, USA) following
manufacturer’s protocols. First, chips were silanized, primed with the inDrop hydrogel beads,
droplet making oil, RT/lysis mix and the nuclei. After completing the run protocol, cell
encapsulations were collected and exposed to UV light to release photocleavable primers from
hydrogel beads. Subsequently, reverse transcription was performed, and the beads filtered out.
From there on, the library preparation step followed the CEL-Seq 2 protocol85: the RT product
was digested by ExoI and HinFI and purified using AMpure XP beads (Beckman CoulterTM,
USA). Then, the second strand cDNA synthesis and in vitro transcription (IVT) steps were
carried out. The resulting RNA was subjected to fragmentation and then reverse-transcribed using
random hexamers. The cDNA was ultimately purified using AMpure XP beads and quantified by
qPCR assay86 (Roche Light Cycler 480 Instrument II, Switzerland).
After quantification, the 6 normal adrenal gland (NAG), 2 endocrine inactive adenoma (EIA-3,
EIA-4) and 2 cortisol producing adenoma (CPA-1, CPA-2) libraries were pooled together and
sequenced using an Illumina HiSeq 2000 instrument with 60 bases for read (R1), 6 for the
Illumina index and 50 for the read 2 (R2). The 6 samples were integrated using the standard
Seurat (version 3.0) integration pipeline87 (freely available software): The resulting data is shown
in Supplementary Fig. 1C. Cells from the two subtypes showed a similar clustering pattern,
suggesting that the data were well integrated. Of note, data derived from either NAG-EIA or
NAG-RCC patients showed correlation with a high Pearson correlation score of 0.99
(Supplementary Fig. 1D), indicating that NAGs could be merged in one single group.
25
Moreover, the remaining 8 adrenocortical adenoma (ACA) libraries were pooled together and
sequenced on two NovaSeq S1 flow cell at 2 x 100 configuration.
Generation of count matrices and core processing: The resulting base calls (BCL) files were
converted to FASTQ files using bcl2fastq tool (Illumina). These raw sequencing reads were
processed with the zUMI88 (version 2.5 – default parameters) pipeline. The count matrices from
reads spanning both introns and exons (inex) were used for subsequent analysis. Prior to pre-
processing, the genes/features were converted from Ensembl (ENSG) IDs to gene symbols using
a custom script. The same cut-off was used to filter nuclei and genes likewise from individual
samples (min.features= 200 and min.cells =3, CreateSeuratObject). The fraction of reads
mapping to the mitochondrial chromosome (MT-) was calculated (PercentageFeatureSet).
Data normalization and integration using Seurat: The filtered data was log-normalized
(NormalizeData) and 2000 highly variable genes (HVG) were selected. The six samples were
then integrated (1. FindIntegrationAnchors, 2. IntegratedData)89. The integrated data was then
scaled (ScaleData) and the effect of mitochondrial genes was regressed out.
Dimensionality Reduction and Clustering: Thirty principal components (PCs) were computed
(RunPCA) and first 15 were used to generate the UMAP reduction (RunUMAP). Subsequently,
the k-nearest neighbours (knn) were determined (FindNeighbors) and clustering was performed
(resolution=0.01, FindClusters). The resulting clusters were annotated using known marker
genes and differential gene expression (DGE – FindAllMarkers) analysis.
Module Scoring: A list of top DEGs and prior knowledge was curated for each cluster, and
combined gene expression scores were calculated (AddModuleScore). The Seurat object was
further filtered using these scores, with the following thresholds: ZG >0.3, ZF >0.8, ZR >0.5, MC
>0, LC >0, FC >0, Stem/P&VEC >0, AM >0, AMP >0. This resulted in a count matrix of 5154
nuclei that was then integrated with the ACA dataset. A Shiny implementation (R) was set up to
easily navigate within this single-nuclei atlas.
26
Gene enrichment analysis: We used pathfindR90 to perform the gene enrichment analysis using
the FindAllMarkers output table from Seurat. The analysis was performed using the function
run_pathfindR for both NAG and ACA samples. For the NAGs, KEGG pathways was used as
reference. To better cover tumour heterogeneity in ACAs, three different databases (KEGG,
Reactome and GO-All) were used as reference. For each ACA cluster, similar terms across the
three databases were grouped together under broader categories (Splicing, Extracellular matrix,
Cell proliferation and tissue development, Ubiquitin related, Senescence, Cell Cycle, Receptor
tyrosine kinases, Cell signalling, Cholesterol trafficking, ABC transporters, Calcium signalling,
Aldosterone synthesis, Steroidogenesis, Cholesterol metabolism, and Other) based on shared
genes.
Visium (10x Genomics) spatial gene expression assay
Frozen adrenal gland samples (NAGs) were embedded in OCT (TissueTek) and cryo-sectioned
(Thermo Scientific, CryoStar NX50) with a 10 µm thickness. The sections were then placed on
tissue optimization and gene expression slides. 20 minutes was determined as the optimal
permeabilisation temperature. Bright field H&E images were taken with a 10X objective (Leica
DMi8 – A). After recovery of cDNA from the slide, the libraries were prepared following the
gene expression user guide (10x Genomics – CG000239 Rev A). Subsequent to pooling, libraries
were loaded at 200 pM and sequenced on a HiSeq-4000 (Illumina).
Generation of Visium count matrices. The base call (BCL) files generated from the raw
sequencing data were converted to FASTQ reads using bcl2fastq tool (Illumina). The FASTQ
reads were mapped to human reference dataset GRCh38 (build 2020-A; refdata-gex-GRCh38-
2020-A) using Space Ranger v1.1.0 count pipeline. On average the two slides captured each 1269
spots under the tissue, and both yielded 1973 median genes and 225,346 mean reads per spot.
Seurat Processing of Visium count data. The two Visium Seurat objects were created using
default parameters (Load10X_Spatial), and were subsequently normalised (SCTransform). Thirty
27
PCs were computed and first 20 were used to generate the UMAP reduction. Knn of each spot
was then determined (FindNeighbors) and clustering was performed (resolution =0.2,
FindClusters), yielding 6 spatial clusters.
Label transfer from snRNA-seq data onto Visium. Label transferring was performed according
to the label transfer workflow in Seurat. Anchors, or cell-spot pairwise comparisons were
computed (FindTransferAnchors), and subsequently a prediction assay was generated
(TransferData), containing the prediction scores for each cell type within each spot. Spatially
variable features list were generated and included in both objects (assay=“SCT”,
selection.method=“markvariogram”,FindSpatiallyVariableFeatures).
Trajectory analysis
Both Visium objects were integrated with the SCTransform integration framework using default
parameters. The Louvain clustering was performed (resolution=0.2) and those were annotated
with prior knowledge from the label transfer analysis. The SCT adjusted count matrix (“counts”
slot in SCT assay) was extracted from the integrated object, and a “cell data set” object was
generated (new_cell_data_set) within Monocle 339. Subsequently, the cds object was normalized
(preprocess_cds) and seurat computed UMAP coordinates were added into reducedDims slot.
The trajectory was then fit (close_loop =F, use_partition =F, learn_graph) and pseudotime was
computed (order_cells) by setting the root to RSPO3+ cells. After getting the genes that vary as a
function of pseudotime (neighbor_graph="principal_graph", graph_test), they were grouped into
modules (resolution = 0.02, find_gene_modules). Genes from top scoring modules (0 for
Capsule, 1 for ZG and 2 for ZF_ZR) were combined to construct the top gene list that
differentially change across pseudotime.
28
ACA-NAG data integration
Count matrix merging and normalization. Count matrices from 12 ACA (37,037 nuclei: 7 CPA
and 5 EIA) and highest scoring 5154 NAG nuclei (See Module Score section above) were
merged. The Seurat object was then generated (min.features=200, min.cells=3,
CreateSeuratObject), cell cycle (S.Score, G2M.Score) and mitochondrial genes were scored
(PercentageFeatureSet) and regressed out during the normalisation step with SCTransform91.
Integration using Harmony: Post normalisation, sample integration was performed within the
Harmony framework (1. RunPCA, 2. RunHarmony with 40 PCs)92. Using the Harmony
dimensions, knns were computed (FindNeighbors) and clustering was done (resolution=0.2,
FindClusters). Finally, a UMAP reduction was performed on the Harmony dimensions
(RunUMAP). Clusters with less than 5 differentially expressed features were removed.
Analysis of DEG among the ACA clusters: Logistic regression test was used within the
FindMarkers framework implemented in Seurat for computing DEGs across subtype (NAG, EIA,
CPA) and clusters. The results were visualised as volcano plots (EnhancedVolcano v1.6.0) and
heatmaps (ComplexHeatmap v2.4.3)
Cluster distribution among tissue samples: The distribution of the clusters among the samples
were evaluated considering the type of the tissue (NAG vs EIA vs CPA) and the mutational status
(no driver vs CTNNB1-mutation vs PRKACA/GNAS-mutation) for clusters selectively expressed
only in ACA. To this aim, the percentage of the number of cells within the cluster per sample was
considered. Data were analysed using the non-parametric Student´s T-tests and Mann-Whitney U
test, as appropriate. A p-value <0.05 was considered statistically significant. The statistical
analysis was performed with GraphPad Prism version 9 (GraphPad Software, San Diego, CA,
USA).
29
Immunohistochemistry
IHC was used to validate the expression and spatial context of the proteins of selected genes from
the transcriptome analysis. This assay was performed with FFPE material from 16 NAGs, as
previously described93,94. Primary antibodies are summarised in Supplementary Table 5.
Moreover, double immunostaining was applied to validate new markers of adrenocortical
stem/progenitors and adrenomedullary progenitors. The combination of NR2F2/ID1 and
CHGA/SYT1 was used to test FFPE consecutive section from 9 NAGs. Details of the IHC assay
including image acquisition is reported in the Supplementary material.
Data availability
Raw gene expression counts can be found under “Supplementary Data” a the website
accompanying this paper (https://.....). Sequencing data was submitted to the ArrayExpress
database of the European Bioinformatics Institute (EBI); accession number: E-MTAB-12129.
Visium data can be retrieved from here: https://doi.org/10.5281/zenodo.5289292.
Acknowledgements
The authors are grateful to Sonja Steinhauer, Antonia Lorey, and Martina Zink for excellent
technical support. We are also thankful to Celso E. Gomez-Sanchez who provided selective
antibodies detecting human CYP11B2. We thank Thomas Conrad for administrative and
technical support.
This work has been supported by the Deutsche Forschungsgemeinschaft (DFG) (project FA-
466/8-1, RO-5435/3-1 and 405560224 (M.F., C.L.R., and S.S.)) and within the CRC/Transregio
(project number: 314061271 – TRR 205), and the Deutsche Krebshilfe (70113526 M.F. and
C.L.R.). This work has been carried out with the help of the Interdisciplinary Bank of
Biomaterials and Data of the University Hospital of Würzburg and the Julius Maximilian
30
University of Würzburg (IBDW), supported by the Federal Ministry for Education and Research
(Grant number FKZ: 01EY1102).
Author contributions
C.L.R., M.F. and S.S. designed and coordinated the study and provided funding. B.A., L.S.L.,
and C.L.R. collected the tissues samples. A.K.S., P.A., S.N.V. and C.B. prepared the samples for
the sequencing and generated sequencing data. A.K.S., So.Sa. and C.F. performed bioinformatics
analysis. A.K.S. performed Visium experiments. B.A. and Si.Sb. performed
immunohistochemistry experiments. S.H. evaluated the mutational status of the included
adenomas. B.A. and A.K.S. performed statistical analysis and generated figures. Data
interrogation and interpretation were carried out by B.A., A.K.S., So.Sa., C.F., Si.Sb., M.F.,
C.L.R., and S.S. B.A., A.K.S., C.L.R. and S.S. wrote the manuscript and Si.Sb., L.S.L. and M.F.
provided critical feedback. All other authors read and approved the manuscript.
31
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Figure legends
Fig 1. A comprehensive adult human normal adrenal gland cell atlas at single-nuclei
resolution.
A. Left: Transverse section depicting the three major adrenal zones (capsule, adrenal cortex, and
medulla). Right: UMAP (Uniform Manifold Approximation and Projection) representation of the
six integrated single-nuclei transcriptomic datasets (clusters were annotated by using known
marker genes and DEG analysis). B. Adrenal cortex clusters: left: UMAP representation of the
adrenal cortex clusters with complete zonation inferred by module scoring (see Fig. 2); right:
feature expression plots representing genes specific for zonae reticularis, fasciculata, glomerulosa
and adreno-medullary progenitors (scale represents log normalized expression values). C.
Anatomical sketch of the adult human normal adrenal gland: the displayed markers for each
identified cell type are based on DGE analysis in single-cell transcriptome data, available
literature (see text for references) and in-situ validations by immunohistochemistry. D. Satellite
clusters. Feature expression plots representing genes used in annotation of following clusters:
“Medulla”, “Myeloid cells”, “Lymphoid cells”, “Stem/P&VEC“ (Stem/Progenitor & Vascular
Endothelial cells) and “Fibroblasts & Connective tissue” (scale represents log normalized
expression values).
Fig 2. Correlation of single-nuclei transcriptome clusters with the zonation of the adrenal
cortex
A. Kernel density estimation of selected features (in frames) for each of the three adrenocortical
zones: ZG (CYP11B2, DACH1), ZF (CYP17A1) and ZR (CYB5A, SULT2A1). B. Example of
hematoxylin and eosin (H&E) staining that served to distinguish the different zones of the normal
adrenal gland (at 10x enlargement all the three zones of the cortex (glomerulosa, ZG; fasciculata,
ZF; reticularis, ZR) together with the capsule (C) and the adrenal medulla (M) were observed). C.
39
Consecutive adrenal sections were stained by immunohistochemistry (IHC) for the markers that
are indicated in the boxes in A. CYP11B2 staining was confirmed to be expressed in the ZG as
sporadic and scattered cells underneath the adrenal capsule or as clusters of cells (previously
termed as aldosterone-producing cell clusters). DACH1 was expressed in the nuclei of the ZG
cells. CYP17A1 was strongly expressed in the cytoplasm of adrenocortical cells of ZF and ZR,
but not in the ZG. CYB5A1 was selectively stained in the cytoplasm of cells of the ZR, whereas
SULT2A1 was stained in both ZF and ZR, although in the ZR a stronger staining was observed.
All images were acquired by Leica Aperio Versa brightfield scanning microscope (Leica,
Germany). 1x pictures with an enlargement of 10x of the same area of consecutive slides were
used to obtain the IHC pictures.
Fig. 3. Adreno-medullary and adrenocortical progenitor cells in adult human normal
adrenal glands.
A. RNA expression of SYT1 and CHGA in the AMP cluster. B.1. SYT1 and CHGA
immunostaining. A low SYT1 protein expression was observed in cells of the entire cortex,
whereas cells of the medulla showed moderate expression. Interestingly, groups of cells localized
in the subcapsular region (organized as niches) showed a strong SYT1 staining. CHGA staining
was found to be pronounced in medullary cells but was very low in cells of the inner part of the
adrenal cortex and no staining was observed in the outer part of the adrenal cortex and in the
adrenal capsule. B.2. CHGA/SYT1 double staining. At 2x enlargement, cells of the medulla
showed a strong CHGA staining (fuchsin-red color). At 10x enlargement, a SYT1 strong positive
niche (starker green-blue color) was marked with a yellow line. All images were acquired by
Leica Aperio Versa brightfield scanning microscope (Leica, Germany). A 1x picture with an
enlargement of 10x and 20x of the same area of consecutive slides were used to obtain the IHC
pictures. C. Expression of NR2F2 and ID1 genes in the Stem/P&VEC cluster. D.1. NR2F2 and
ID1 immunostaining. IHC analysis revealed sparse, rare cells mostly localized at the subcapsular
40
levels that showed a high nucleus staining of NR2F2 or ID1 (marked with yellow circles). Those
cells located under the capsule seemed to centripetally migrate from ZG to ZF and ZR (orange
arrows represented the centripetal migration in adrenal glands). D.2. NR2F2/ID1 double staining.
Nuclei of cells positive for both NR2F2 and ID1 were stained in dark blue (because of the sum of
fuchsin-red + blue-green colours) and were mostly located under the capsule. Double positive
NR2F2/ID1 nuclei were indicated with red circles at 10x enlargement and red arrows at 20x
enlargement. Orange arrows at 10x enlargement represent the centripetal migration in adrenal
glands.
Fig. 4. Validation of adrenal zonation by spatial transcriptomics in adult human normal
adrenal glands.
A pairwise (cell-to-spot) score is calculated within the label transfer framework in Seurat (V3)
and projected onto sections. Two sections are represented for each mapped cluster. Medulla and
AMP clusters were rendered transparent as the label transfer did not detect the presence of these
cell types in the Visium assay.
Fig. 5: Trajectory analysis of spatial transcriptomics data in adult human normal adrenal
glands.
A. Top: Integrated UMAP (Uniform Manifold Approximation and Projection) from both tissue
sections analysed using the Visium assay (10x Genomics). Trajectory was visualised as a black
line. Bottom: clusters identified in the integrated object transferred to the tissue (Visium)
sections. B. Pseudotime estimation both for the UMAP and the tissue sections: white circles
represent the root node (chosen based on RSPO3 expression, indicated with a black arrow (right
panel)). C. Heatmap of 829 differentially expressed genes across pseudotime: cells were ordered
by pseudotime (columns), genes were ordered by expression and pseudotime (rows). Top genes
are highlighted. D. Expression variation of RSPO3, ID1, INSR and CYP11B2 over pseudotime:
41
smoothed lines were generated based on the scatter profile of each gene (95% confidence interval
displayed in grey around the line). Vertical lines represent the transition zones from 0-Capsule
(red) to 1-ZG (green) and 1-ZG to 2-ZF-ZR (ochre).
Figure 6. Identification of adenoma-specific clusters in adrenocortical adenoma (ACA)
A. 1. Volcano plot depicting differentially expressed genes between NAG and EIA. Genes
characteristic of EIA subtype are written in bold. The x axis represents fold change (Log2FC) and
y axis represents P values (-Log10P). 2. Volcano plot depicting differentially expressed genes
between NAG and CPA. Genes characteristic of CPA subtype are written in bold. 3. Violin plots
showing log normalized expression values of common signatures across the three subtypes
(NAG, EIA, CPA). 4. Left: Violin plots showing log normalized EIA specific gene expression
values. Right: Violin plots showing log normalized CPA specific gene expression values.
B. UMAP (Uniform Manifold Approximation and Projection) representation of the 18 integrated
samples (normal adrenal glands (NAG, n=6); cortisol-producing adenomas (CPA, n=7);
endocrine inactive adenomas (EIA, n=5) coloured by cluster identity. The UMAP could be
divided into two groups: the “central cluster” containing previously identified cortex cell types
(ZG, ZF and ZR) as well as ZG-like, Adenoma specific (AC1, AC2, AC3, and AC4),
Cholesterol-and steroid-enriched metabolism (CSEM) and adenoma microenvironment (AME)
clusters, and the “satellite clusters”, which are Medulla, Myeloid cells, Lymphoid cells ,
Endothelial and Stromal. NAG cell type distribution in the UMAP is represented within the top
right box. C. Scaled average expression of selected markers for each cluster (dot size represents
the percentage of cells in each cluster expressing the marker). D. Subtype and sample specific
composition coloured by cluster identity: samples (x axis) are ordered by mutational signatures.
Figure 7. Transcriptomic profile of the newly identified ACA clusters
A. Heatmap representing top identified features for the 7 newly identified adenoma clusters.
42
Colour scale represents Z-score computed from log normalized expression matrix.
B. Top 5 significant pathways for each cluster combining results from 3 separate databases
(KEGG, Reactome and GO-All (GO-MF, GO-BP, GO-CC))
Figure 8. Cluster distribution in ACA samples across subtype and mutational status
Distribution of A. Cholesterol-and steroid-enriched metabolism (CSEM) cluster in cortisol
producing adenomas (CPA) versus endocrine inactive adenomas (EIA; upper panel) and across
mutational status (lower panel); B. Adenoma clusters (AC1-4) across mutational status; C.
Lymphoid cluster across subtypes (upper panel) and mutational status (lower panel); D. Myeloid
cluster across subtypes (upper panel) and mutational status (lower panel). Significant cluster
enrichments are encircled in red.
43
Fig. 1
Fig. 2
A Markers UMAP C IHC staining
CYP11B2 CYP11B2 CYP11B2 DACH1

LGR5
DACH1
ZG CACNB2
DACH1
CALN1
MCOLN3
B
H&E staining
MC2R
ANO4
CYP17A1 CYP17A1
HSD3B2 CYP17A1
FDX1
ZF CYP11B1
POR
NOV M
ZR
LEF1
ZF
CYB5A C
CYB5A SULT2A1
ZG
SULT2A1
CYB5A
TSPAN12
ZR SULT2A1
PAPSS2
CYP17A1
HSD3B2
Low High
Density
C
Fig. 3
ID1
SYT1
NR2F2
CHGA
ZG ZG ZG ZG
ZF ZF ZF ZF
ZR ZR ZR ZR
AM AM AM AM
AM AM AM AM
P P P P
M M M M
St C St C St C St C
em em em em
/P L /P L /P L /P LC
& C & C & C &
VE VE VE VE
C C C C
FC FC FC FC
B
D
1
ID1
SYT1
NR2F2
CHGA
20x
20x
2
2
NR2F2 + ID1
CHGA + SYT1
20x
10x
2x
10x
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Supplementary figures
Supplementary Fig. 1
A Fibroblast & connective tissue
H&E
COL1A2 MGP
M Stem/P&VEC Medulla
ZR
ZF
C
ZG
ENTPD1 CHGA
B C
Miscellaneous
Wnt/β-cat
NAG EIA CPA
A
Lymphoid cells
Expression
1
0.5
-0.5
B -1
Myeloid cells

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bioRxiv preprint doi: https://doi.org/10.1101/2022.08.27.505530; this version posted August 28, 2022.

The copyright holder for this preprint

Panagiota Arampatzi5, Sabine Herterich6, Laura-Sophie Landwehr1, Sarah N. Vitcetz2,3, Caroline

Braeuning2, Martin Fassnacht1,6,9*, Cristina L. Ronchi1,7,9*, Sascha Sauer2,3,5,9*

Hospital, University of Würzburg, Würzburg (Germany).

Prof. Martin Fassnacht

ORCID ID: 0000-0001-6170-6398

Dr. Cristina L. Ronchi

Dr. Sascha Sauer

transcriptome data to reconstruct adrenal gland development and tumourigenesis. We identified

insights into adrenal plasticity and mechanisms underlying adrenocortical tumourigenesis.

tumours, tumourigenesis, heterogeneity, cortisol secretion, CTNNB1.

catecholamine-secreting medulla, originating from neural crest cells.

Cushing’s syndrome in cortisol-producing adenomas (CPA) or Conn's syndrome in aldosterone-

adrenocortical tumourigenesis (i.e.Wnt/β-catenin, Rb/p53 signalling and/or IGF system) and

underlying the pathogenesis of a vast percentage of adrenocortical tumours remained largely

self-maintenance of adrenal cells in the adrenal tumourigenesis is urgently required.

Recently, single-cell/single-nuclei RNA-sequencing (sc/snRNA-seq) and spatial transcriptomics

as tumours - to gain a deeper understanding of tumourigenesis15-17. Whereas some recent scRNA-

physiology significantly differs between rodents and humans.

molecular processes of adrenal self-renewal and homeostasis in humans. Furthermore, we

Single-nuclei transcriptome sequencing of NAGs

individual clusters of the NAG are listed in Supplementary Table 1.

Zonation of the adrenal cortex

To further analyse the transcriptomic zonation of the adrenal cortex, we performed

immunohistochemistry (IHC) to detect protein expression of selected mRNA-based markers in

Adrenal progenitor and stem cells

“Adreno-Medullary Progenitors” (AMP) subcluster. We validated this hypothesis by using IHC

following the trend of centripetal migration of adrenocortical progenitor cells from ZG to ZF to

dogs38. Pathway analysis corroborated our hypothesis, revealing significant enrichment of

cells (FE >2) (Supplementary Fig. 3E).

Characterisation of additional adrenal cell clusters

proliferation and differentiation) (Supplementary Fig. 2H and Supplementary Table 1).

extracellular matrix and fibroblasts (Supplementary Fig. 4A).

Adrenal cortex zonation by spatial transcriptomics in NAG

inner cortex (Fig. 4).

Pseudotime analysis confirms centripetal development of the adrenal cortex

within the adrenal cortex. To predict a differentiation trajectory, we performed pseudotime

resulted in 5 distinct Louvain clusters (0 =Capsule, 1 =ZG, 2 =ZF-ZR, 3 =blood vessel, 4

for pseudotime computation we selected cells expressing RSPO3, a well-established marker of

“clusters of cells” (Supplementary Fig. 7B, regions inside blue boxes).

Moreover, the pseudotime analysis revealed interesting expression of Wnt/β-catenin pathway-

in the ZF-ZR zones (Supplementary Fig. 6).

Integration of NAG and ACA snRNAseq data reveals adenomas-specific cells

integrated this data with 5154 highest scoring NAG nuclei.

log2FC >1.6, p=4.51e-134) were predominantly expressed in CPAs (Fig. 6A.4).

clusters comprised of myeloid, lymphoid, endothelial, stromal (mainly composed of fibroblasts

(Fig. 6C), confirming the adrenocortical nature of these cells.

ACA-specific clusters distribution according to subtype and mutational status

CTNNB1 mutations, and PRKACA/GNAS mutations) (Fig. 6D).

(avg-log2FC >2.4, p=.82e-12). Pathway analyses revealed enrichment of several pathways

oxidative stress (FE >2.5) (Fig. 7B and Supplementary Fig. 8).

with CTNNB1 mutation43-45. Also, we found a high expression of FTO, a N6-methyladenosine

that promotes cells proliferation, migration and chemo-radiotherapy resistance by targeting

7B and Supplementary Fig.8).

(FE >2) (Fig 7B and Supplementary Fig. 8).

In contrast, AC3 was significantly over-represented in CPAs with PRKACA/GNAS mutation

enrichment of elements associated to spliceosome, sometimes differently expressed among the

significant enrichment of several pathways involved in extracellular matrix, cell-cell

senescence (FE >3) (Fig. 7B and Supplementary Fig. 8).

log2FC >-1.5, p=1.05e-06 for CCL5) (Supplementary Fig. 9A).

>-1.3, p=4.38e-24 for CD74) (Supplementary Fig. 9B).

normal adrenal glands to a cohort of adrenocortical adenomas, we revealed seven adenoma-

Braeuning2, Martin Fassnacht1,6,9, Cristina L. Ronchi1,7,9, Sascha Sauer2,3,5,9*