Adrenocortical cell and macrophage heterogeneity at single-cell spatial resolution in KCNJ5-mutant aldosterone-producing adenomas

KCNJ5-mutant aldosterone-producing adenomas (APAs) represent a primary cause of primary aldosteronism, leading to severe secondary hypertension. However, the adrenal cellular heterogeneity and microenvironmental landscape of KCNJ5-mutant APAs remain to be characterized. Using single-cell RNA sequencing and spatial transcriptomics, we analyzed three paired KCNJ5-mutant APAs and distal adrenal tissues (DATs), with experimental validation by immunohistochemistry and immunofluorescence. In DATs, we identified a previously unrecognized TSPAN8-positive zona glomerulosa (ZG) cell population. Pseudotime and functional enrichment analyses indicate that these cells represent an intermediate state between ZG and zona fasciculata (ZF). Within APAs, adrenocortical cells exhibited remarkable heterogeneity. The key enzyme mediating aldosterone synthesis, CYP11B2, was predominantly expressed in ZF-like cells, suggesting that targeting ZF-like cells may be critical for controlling aldosterone overproduction in APAs. Furthermore, CYP11B2-positive cells displayed two distinct functional states: Fate 1 (aldosterone-producing cells) specialized in mitochondrial metabolism and steroid hormone synthesis, while Fate 2 (tumor growth-promoting cells) participated in anti-apoptotic pathways that may drive APA cell accumulation. In contrast, CYP11B2-negative tumor cells demonstrated enhanced proliferative and differentiation potential, potentially playing a more active role in APA tumorigenesis. Notably, we discovered a unique subset of APA-associated macrophages (AAMs) within the tumor microenvironment. These AAMs were immunosuppressive and communicated with APA cells via the SPP1-(ITGAV/ITGB1) axis, likely promoting tumor proliferation. These findings provide novel insights into the cellular complexity of KCNJ5-mutant APAs, highlighting adrenal cortical cell plasticity and tumor-associated macrophages as critical determinants of APA pathogenesis.