Fig 2: Consensus-dominant genes in adenomas causing CDCore and margin sample corticotrophs were separately compared with all other cell types with scran’s scoreMarkers, using patient identity as a blocking factor. Stringent thresholds on effect sizes were used to identify a core set of robustly upregulated markers (STAR Methods).(A) Venn diagram showing the number of core-specific (red), margin-specific (grey), and common (blue) marker genes identified.(B) Dot plot showing the mean expression (marker color) and percentage (marker size) of each cell type expressing core-sample-specific genes (CD signature genes; top group, red in A) and corticotroph marker genes common to core and margin samples, which include canonical marker genes (bottom group, blue in A). Mean expression values are batch corrected across patients using scran correctSummaries. POMC expression was normalized to the second most highly expressed value to better show the levels of other genes (mean POMC: 8.29).(C) UMAP plot of all C cells indicating the CD signature gene score per cell.(D) UMAP plots for all cells from each patient show that cells with high CD signature scores are restricted to cell clusters classified as corticotrophs.(E) UMAP plots for other adenoma subtypes including prolactinoma (PRL) and nonfunctioning pituitary adenoma (NFPA) verify that cells with high CD signature scores are specific to Cushing’s adenomas (C).(F) Independent verification of overexpression of CD signature genes in pairwise bulk RNA-seq analysis of CD adenoma and adjacent normal pituitary gland (P4 and P5). Genes coidentified between bulk RNA-seq and scRNA-seq are color coded as in (B).(G) Bulk RNA-seq comparison of CD and non-CD samples also verified upregulation of several CD signature and corticotroph marker genes.

Fig 3: Non-stem-like proliferating cells localize to the adenoma compartment(A) UMAP plot of corticotroph cells showing cell-cycle gene scores per cell. We defined proliferating cells (Pros) for further study to have a cycle score >0.01.(B) Dot plot showing 12 top expressed genes upregulated in Pros. These cells clearly expressed canonical corticotroph marker genes POMC and TBX19 in addition to cell-cycle genes.(C) UMAP plot showing cell-cycle score across all six scRNA-seq patient samples, showing specificity to the C cluster and some Le cells, as well as localization to the NFPA, and PRL adenoma clusters.(D) SOX2-positive cells are restricted to the cell cluster classified as FS cells and not expressed in the corticotroph compartment.(E) Dot plot of select genes associated with pituitary stem cells (Cox et al., 2017; Fletcher et al., 2019) showing lack of expression in adenoma samples.

Fig 4: Surgical annotation captures canonical pituitary cell classes in post-natal pituitary gland(A) A sublabial transsphenoidal approach was used for resection of microadenomas (white arrowheads) in patients with Cushing’s disease (CD). Coronal, post-gadolinium contrast enhanced magnetic resonance image from patient P1. Adenoma and adjacent normal pituitary gland were separately annotated during surgery and dissociated into a single-cell suspension, followed by GEM embedding, sequencing, and computational analysis. Scale bar: 2 cm.(B) Summary of demographic data and analysis for patients included in the study.(C) UMAP embedding of cells from the 6 patient samples with CD, GH, NFPA, and PRL adenomas used in scRNA-seq analysis. Cells cluster according to dominant secretory phenotype.(D) UMAP embedding showing the cell-type identities cells from the 6 patient samples.(E) UMAP embedding showing CD sample identity for cells from patients P1, P2, and P3.(F) UMAP plot showing CD patient cells clustering by cell-type identity. Cell types: leukocytes (Les), endothelial cells (ECs), pericytes (Pes), folliculostellate cells (FSs), corticotrophs (Cs), gonadotroph (Gs), somatotrophs (Ss), lactotrophs (Ls), ambiguous/somato-lactotrophs (SLs), and POU1F1-positive, hormone-negative (P+H-) cells. GH, growth hormone; NFPA, non-functioning pituitary adenoma; PRL, prolactinoma.

Fig 5: Human adenomas causing wild-type CD have discrepant PMAIP1 transcript and noxa abundance(A) UMAP plot demonstrating PMAIP1 abundance in CD adenomas (C) but not in PRL, G, or NFPA adenomas. PMAIP1 was also detected at lower levels in Les and ECs.(B) UMAP plot showing MYC upregulation in CD corticotrophs but not other hormone-producing cells (left panel). MYC expression was also detected in Pes, ECs, and Les. Middle panel: UMAP plot showing overlap of MYC and PMAIP1 expression is mostly limited to CD corticotrophs (yellow dots). Right panel: UMAP plot showing UCHL1 abundance in most hormone-producing cell types in CD.(C) Bulk RNA-seq of CD and non-CD samples verified overexpression of pro-apoptotic genes including PMAIP1 in CD tissues.(D) DNA methylation levels (beta values) at CpG sites associated with the PMAIP1 promoter methylation demonstrating hypomethylation in CD (n = 3) compared with normal (autopsy-derived, n = 20) pituitary glands. *p < 0.05.(E) Multiplex immunohistochemistry (mIHC) of 5 μm thick sections from a CD adenoma. Insets from the core-margin boundary represented by white dashed lines. White bar: 100 μm. Core-margin boundary identified by overlaying expression of POMC, TBX19, and DAPI. Compared with the margin, core adenoma cells show robust overexpression of POMC, TBX19, and MYC; however, noxa expression is decreased within the adenoma core.(F) Representative image from noxa IHC in independent adenoma/normal pairs (n = 10). Pairwise analysis of noxa deconvoluted IHC images (absorbance = mean pixel intensity count per pixel) showing suppressed noxa signal in CD adenomas compared with normal (margin) tissues (p = 0.0013; 95% confidence interval [CI] −0.027 to −0.007). Scale bar: 100 μM.(G) Expected epithelial growth factor (EGF) signaling upregulation and ERK1/2 phosphorylation were found in human CD adenoma primary cell lines (P6_CD and P26_CD). A, adenoma (core); N, normal (margin) pituitary gland. Noxa was undetectable or decreased in core adenomas.(H) A survey of human primary CD adenoma cell lines revealed variable noxa expression compared with sCD adenoma, GH adenoma, and NFPA. CD, corticotroph adenoma causing Cushing’s disease; sCD, hormonally silent corticotroph adenoma; GH, growth-hormone-secreting adenoma; NFPA, non-functioning pituitary adenoma.