Single-nucleus RNA sequencing reveals the cellular composition and the mechanism underlying adrenal myelolipoma

Purpose: Adrenal myelolipoma (AML) is a benign tumor composed of intermixed adipose and hematopoietic tissues, but its detailed cellular composition remains unclear. CXCL12-abundant reticular (CAR) cells have been found to promote hematopoiesis during AML development; however, the mechanism of adipogenesis in AML remains unclear. This study aimed to characterize the cellular composition of AML and elucidate the potential mechanisms underlying its development, with a particular focus on adipocyte origin. Methods: Four AML specimens and matched adjacent adrenal tissues were subjected to single-nucleus RNA sequencing, and an additional ten paired samples were analyzed using immunostaining. Complementary in vitro experiments were performed to validate the proposed mechanisms of AML development. Results: Unsupervised clustering revealed that AML is composed predominantly of T cells, B cells, neural-like cells, CAR cells, adipocytes, and nearly all types of myeloid cells. RNA velocity analysis suggested that CAR cells might be the lineage source of adipocytes. Consistently, CAR cells isolated from AML tissues demonstrated adipogenic capacity in vitro. Expression analysis showed high levels of adrenocorticotropic hormone receptor and androgen receptor in adrenal cortical cells and endothelial cells respectively. CellChat analysis further revealed extensive paracrine signals from adrenal cortical cells and endothelial cells to CAR cells. Overexpression of androgen receptor in endothelial cells transcriptionally upregulated key regulators of adipogenesis including COL4A1 and PDGFD. Conclusions: AML comprises a heterogeneous population of immune and stromal cells, with CAR cells likely serving as the primary source of adipocytes. Moreover, androgen may regulate CAR cells to induce adipocytes via endothelial cells.