Integrated Transcriptomics Reveals Evolutionary Trajectories and Cell Density‐Dependent Mechanisms in Aldosterone‐Producing Adenomas

Figures

• 

  Figure 1. Global cellular landscape in human APA and APM tissues. A) Schematic overview of the experimental design and analytical workflow. B) Integrative analysis of 13 APA tissues and 2 APM samples, visualized using a common UMAP embedding for individual samples (left) and cell clusters (right). C) Bubble plot of representative cell‐type specific markers across all clusters. D) UMAP visualization of major cell types. E) Cell‐type distribution across APA (left) and APM (right) samples, visualized separately using UMAP. F) Changes in composition across all compartments and sample fractions are visualized as cell density on a unified embedding plot. G) Statistical assessment of cell density differences between APA and APM. A two‐sided Wilcoxon test was performed, with results visualized as Z scores. Red indicates increased cell abundance in APA, while blue indicates decreased cell abundance in APM. H) Changes in cell composition were assessed using Compositional Data Analysis. The x‐axis represents the separation coefficient for each cell type, where positive values indicate increased abundance in the APA and negative values signify decreased abundance. Boxplots and individual data points…

• 

  Figure 2. Characterization of the immune microenvironment in APAs. A) UMAP plot demonstrating myeloid subpopulations. B) Violin plots showing the average expression levels of M1 and M2 macrophage signature gene panels across different macrophage populations. Statistical significance was determined using a two‐sided Wilcoxon rank‐sum test. C) Changes in the composition of the myeloid compartment between APA and APM visualized as cell density on the joint embedding. D) CYP11B2 immunohistochemistry staining (left) and H&E staining (right) of one spatial transcriptomics section. E) Spatial cluster distribution in one section. F) UMAP plot depicting the 7 spatial cell types (left) and co‐expression of CYP11B2 with CD163 in the same section. G) UMAP visualization of 10 major spatial cell types integrated across 12 tissue sections using Spatial RNA‐seq Technology. H) Bubble plot of representative cell‐type specific markers across all cell types. I) Violin plots illustrating the expression of CD163 and CD206 in APA and APM cell types. Statistical significance was determined using a two‐sided Wilcoxon rank‐sum test. J) Violin plot displaying TREM2 expression across all cell types. K) Violin plot…

• 

  Figure 3. Complex cell–cell communication networks in APA. A) Weighted total interaction strength. Line thickness denotes interaction strength, while color corresponds to different cell types. B) Heatmap showing the strength of outgoing and incoming interaction events between cell types. Bars at the top of the heatmaps summarize the total intensity of outgoing and incoming events per cell type, which are indicated at the bottom of the heatmaps. Rows are ordered by contributing signaling pathways. Color intensity represents interaction strength. C) Bubble plots depicting intercellular communication patterns among different cell types. Left: outgoing contributions; Right: incoming contributions. Bubble color represents distinct cell types; bubble size indicates the strength of communication. D) Bubble plots showing gene expression levels of receptor‐ligand pairs involved in interactions between cell types. Bubble color indicates average relative expression; bubble size indicates percentage of relative expression. E) Communication probabilities of important ligand‐receptor pairs from myeloid cells to individual APA and APM cells, with bubble color indicating communication probability and size…

• 

  Figure 4. Cell‐type‐specific regulon activity analysis and identification of combinatorial regulon modules. A) Rank for regulons in each cell type based on regulon specificity score (RSS). The “regulon” refers to the regulatory network of transcription factors (TFs) and their target genes. B) Combinatorial regulation modules are identified based on regulatory connection specificity index (CSI) matrices, along with representative transcription factors, corresponding binding motifs, and associated cell types. C) Interaction network for the combinatorial regulation modules based on similarity of regulon activities. D) Heatmap of regulon activity analysed by single‐cell regulatory network inference and clustering with default thresholds for binarization. “On” indicates active regulons; “Off” indicates inactive regulons. E) Regulatory networks consisting of shared transcription factors and their target genes. APA, aldosterone‐producing adenoma; APM, aldosterone‐producing micronodule; zF, zona fasciculata; zG, zona glomerulosa; zR, zona reticularis.

• 

  Figure 5. Pseudotime analysis of zG, APM, and APA cells in three spatial transcriptome sections. A) UMAP plot illustrating the distribution of zG, APM, and APA cells at the integrated statical transcriptome (ST) level. B) UMAP plot showing cell differentiation status using CytoTRACE. Color scale from dark green (low stemness) to dark red (high stemness) indicates differentiation levels. C) Bar plot showing genes correlated with the highest and lowest differentiation levels based on their correlation with CytoTRACE scores. D,E) Functional annotation of regulatory genes driving heightened cell stemness (D) and reduced cell stemness (E). F) Reconstructed pseudotime evolution trajectory of zG, APM, and APA cells inferred by Monocle2. The trajectory plot showing the three cell types assigned by different colors (top) and the direction of developmental time points arranged by pseudotime (bottom). The arrows indicate the likely course of evolution within the trajectory. G) Heatmap of gene dynamics along the reconstructed cell trajectories, with columns representing cells and rows denoting genes. Genes are ranked and divided into four clusters based on expression trends on the left, accompanied by…

• 

  Figure 6. Trajectory analysis of biological process during APA progression. A) UMAP plot illustrating two distinct APA cell differentiation states based on the CytoTRACE score: progenitor‐like (high differentiation potential, CytoTRACE >0.5) and mature (low differentiation potential, CytoTRACE ≤0.5) states. B) UMAP plot showing the distribution of cell stemness scored each cell by AUCell. C) Violin plot comparing stemness scores between progenitor‐like and mature APA cell states. Statistical significance was determined using a two‐sided Wilcoxon rank‐sum test. D) Heatmap showing large‐scale copy number variation (CNV) profile of each APA cell. Red and blue colors represent high and low CNV levels, respectively. zG cells are defined as reference cells. E) Violin plot showing the CNV scores between progenitor‐like and mature APA cell states. Statistical significance was determined using a two‐sided Wilcoxon rank‐sum test. F) Volcano plot showing differentially expressed genes between progenitor‐like and mature APA cell states, with gene colors according to cell subtypes. G) Functional annotation analysis depicting the Gene Ontology (GO) biological process enriched in progenitor‐like and…

• 

  Figure 7. Hippo pathway effectors and ferroptosis in human adrenal cells. A) Bright‐field images of HAC15 cells cultured at low or high densities treated with DMSO or 4 µM RSL3. Representative data of at least three independent experiments. Scale bar, 200 µm. B,C) Cell death analysis of HAC15 cells cultured at low or high densities treated with DMSO or 4 µM RSL3 by immunofluorescence (B) and flow cytometry (C). Representative data of at least three independent experiments. Scale bar, 100 µm. D) TfR1 immunofluorescence in HAC15 cells cultured at low or high densities treated with DMSO or 4 µM RSL3. Representative data of three independent experiments. Scale bar, 100 µm. E) Cell viability of HAC15 determined by WST‐1 assay following treatment with 4 µM RSL3 or Lip‐1 + 4 µM RLS3 for 4 h (n = 5 independent experiments). Statistical analysis was performed using two‐way ANOVA. F) Volcano plot showing differentially expressed genes of HAC15 cells treated with 4 µM RSL3 cultured for 3 h at low versus high cell densities. G) Gene Ontology (GO) biological process of differential gene expression between HAC15 cells treated with 4 µM RSL3 at low and high cell densities. H) Western blot analysis of YAP…