Endocrine Abstracts

JOINT822

Background: Primary aldosteronism is frequently caused by aldosterone-producing adenomas (APAs). Aldosterone-producing micronodules (APMs) are distinct histopathological lesions consisting of subcapsular clusters of zona glomerulosa (zG) cells. APMs have been proposed as potential APA precursors, but molecular evidence supporting this hypothesis remains limited.

Objective: This study aimed to create a comprehensive cellular atlas of APAs and APMs and elucidate their developmental mechanisms through integrated single-cell, single-nucleus and spatial transcriptomics analyses.

Methods: We performed single nucleus (sn) RNA-seq on two APA specimens and integrated publicly available single cell (sc) RNA-seq data from two APMs. To map spatial gene expression, we conducted spatial transcriptomics analyses on 12 captured areas from formalin-fixed paraffin-embedded adrenal glands from five patients with primary aldosteronism. Functional in vitro studies were performed in human adrenocortical cells (HAC15).

Results: Integration of sn and scRNA-seq transcriptome data revealed distinct phenotypic profiles and variable frequencies of major cell types between APM and APA. APAs exhibited an immunosuppressive tumour microenvironment, potentially driven by enhanced VISFATIN pathway activity promoting M2 macrophage polarisation. Gene regulatory network analysis identified APA-specific transcription factors LEF1 and FOXO1, while JUN and FOS expression in APMs suggested their role in early tumour responses. Trajectory analyses revealed two main developmental pathways: direct progression from zG to APA, and stepwise progression from zG through APM to APA. Within APAs, we identified two distinct cellular states with different differentiation potentials: a progenitor-like state (low differentiation potential) and a mature state (high differentiation potential). Compared to the progenitor state, the mature state was characterised by increased PCP4 and CYP11B2 expression and high copy number variation. Functional enrichment analysis revealed a dynamic regulatory environment during APA development, involving pathways of oxidative stress (including mechanisms leading to cell death by ferroptosis) and focal adhesion (influenced by cell density). We demonstrate that RSL3-induced ferroptosis sensitivity is modulated by cell density. TAZ knockdown in low-density HAC15 cells conferred resistance to ferroptosis, mimicking the effect observed in high cell densities.

Conclusion: Our findings reveal an immunosuppressed tumour microenvironment in APAs, characterised by increased M2 macrophages. We demonstrate dynamic changes in molecular profiles and cell cluster composition during APA initiation and progression. These insights enhance our understanding of APA pathobiology and may inform the development of novel diagnostic and therapeutic strategies for primary aldosteronism.