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Genetically engineered human pituitary corticotroph tumor organoids exhibit divergent responses to glucocorticoid receptor modulators

Open AccessPublished:January 11, 2023DOI:https://doi.org/10.1016/j.trsl.2023.01.002

      Abstract

      Cushing's disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that subsequently causes chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist Mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists Pasireotide or Octreotide. In iPSCUSP48 and iPSCUSP8 cultures, Mifepristone induced a predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to Mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to Pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over Mifepristone, supporting its further development for use in the treatment of CD patients.

      Abbreviations:

      ACTH (adrenocorticotropin hormone), BMP4 (bone morphogenetic protein 4), CD (Cushing Disease), CDH23 (cadherin related 23), DMEM/F12 (Dulbecco's modified eagle medium/nutrient mixture F-12), DMSO (dimethylsulfoxide), DPBS (Dulbeccos phosphate buffered saline), ELISA (enzyme-linked immunoassay), FSH (follicle-stimulating hormone), GH (growth hormone), GLI1 (GLI family zinc finger1), GR (glucocorticoid receptor), Hpito (human pituitary organoids), iPSC (induced pluripotent stem cells), MEN1 (multiple endocrine neoplasia link type 1), Mife (mifepristone), NII (nuclear irregularity index), NMA (nuclear morphometric analysis), Oct (octreotide), Pas (pasireotide), PBMCs (peripheral blood mononuclear cells), PBST (PBS with Tween 20), PCR (polymerase chain reaction), PIT1 (pituitary-specific positive transcription factor 1), PitNET (pituitary neuroendocrine tumor), PRL (prolactin), PTCH1 (protein patched homolog 1), Rela (relacorilant), SFDM (serum-free defined media), SHH (sonic hedgehog), SMO (smoothened), SSTR (somatostatin receptor), TPit (t-box transcription factor 19), USP48 (ubiquitin carboxyl-terminal hydrolase 48), USP8 (ubiquitin carboxyl-terminal hydrolase 8)
      BRIEF COMMENTARY
      S. Mallick et al.

      Background

      Cushing's disease (CD), a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) leads to chronic hypercortisolemia. The approved treatment for CD is, Mifepristone (Korlym) is a non-selective glucocorticoid receptor (GR) antagonist with additional competitive binding with progesterone for the progesterone receptor. Relacorilant, an investigational selective GR modulator in development for the treatment of CD, does not bind to the other hormone receptors.

      Translational Significance

      Patient-derived PitNET organoids recapitulate the tumor microenvironment in vitro. PitNET organoids revealed the advantages of relacorilant over Mifepristone, supporting its further development for use in the treatment of CD.

      INTRODUCTION

      Cushing's disease (CD) is a serious endocrine disorder that is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNETs) that leads to excess adrenal cortisol secretion.
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      These disappointing patient outcomes highlight the need for a better understanding of the pathophysiology of CD to enable the development of novel therapeutic approaches for these patients.
      Medical therapy is often considered when initial surgery fails to achieve remission, or when the tumor recurs after apparent surgical remission. Approved treatments include pituitary-targeted drugs, adrenal steroidogenic inhibitors, and a non-selective glucocorticoid receptor (GR) antagonist.
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      There is an inverse relationship between disease duration and reversibility of complications associated with the disease, thus emphasizing the importance of identifying an effective medical strategy to rapidly normalize hypercortisolemia/cortisol activity by directly targeting the pituitary adenoma.
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      Standard of care treatments have low efficacy and/or tolerability, making CD a medical therapeutic challenge. For example, Mifepristone (Korlym), a non-selective GR antagonist approved by the Food and Drug Administration in 2012 for the treatment of hyperglycemia secondary to endogenous Cushing's syndrome, including CD, in adults who have failed surgery or are not candidates for surgery, competes with cortisol binding at the GR and significantly improves hyperglycemia secondary to hypercortisolism in patients with CD.
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      Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome.
      However, while Mifepristone improves the symptoms of CD, it also raises ACTH (and thus cortisol) levels and exhibits significant side effects in some patients. Through its binding to the progesterone receptor, Mifepristone causes side effects including endometrial thickening and vaginal bleeding.
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      Relacorilant is an investigational, selective GR modulator that, unlike Mifepristone, has no detectable binding to the progesterone, estrogen and androgen receptors.
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      did not significantly raise ACTH or cortisol levels, and has been reported to induce tumor regression in some CD patients with pituitary macroadenomas.
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      ,
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      Glucocorticoid Receptor Antagonism Upregulates Somatostatin Receptor Subtype 2 Expression in ACTH-Producing Neuroendocrine Tumors: new insight based on the selective Glucocorticoid receptor modulator Relacorilant.
      Relacorilant is currently under investigation in phase 3 trials for the treatment of patients with endogenous Cushing's syndrome (GRACE (NCT03697109) and GRADIENT (NCT04308590) studies). The mechanisms driving relacorilant-induced tumor regression, and the role of tumor sensitization to endogenous somatostatin, remain unknown due to the lack of advanced multicellular in vitro models that recapitulate the complexity of the PitNET microenvironment. The absence of preclinical models that recapitulate the tumor tissue has prevented us from acquiring the knowledge to develop and implement therapies specifically targeted the tumor with a higher efficacy and tolerability for CD patients.
      Pituitary-directed drugs that target the somatostatin receptor (SSTR), expressed on the surface of corticotroph PitNETs, have been used to treat CD.
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      • et al.
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      Pasireotide is a somatostatin analogue known to bind to SSTR1, 2, 3, and with highest-affinity binding to SSTR5.
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      In 2 large prospective studies, Pasireotide monotherapy normalized urinary free cortisol in up to 42% of patients with CD.
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      Combination therapy may show promise whereby Pasireotide administered together
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      with cabergoline (a dopamine receptor subtype 2 antagonist) and low-dose ketoconazole (a Hedgehog receptor smoothened [SMO] inhibitor that also inhibits cortisol synthesis) induced biochemical remission in 6 of 8 patients at day 80, increasing the number of patients with a complete response to 88%.
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      Pasireotide alone or with cabergoline and ketoconazole in Cushing's disease.
      However, such treatments are designed without clear understanding and consideration for the receptor dynamics that may change during treatment. Cortisol suppresses somatostatin receptor expression, so GR modulation may affect SSTR abundance and/or sensitivity to endogenous somatostatin. Therefore, while somatostatin receptor subtypes (SSTR) 2 and 5 have been targeted to suppress tumor growth and hormone secretion, the therapeutic benefits of somatostatin analogs are limited in patients with CD.
      Given the intriguing preliminary clinical effects of relacorilant on PitNET regression, we sought to identify the mechanism of GR modulation alone or with somatostatin analogs using the PitNET organoids. PitNET organoids were derived from: (1) CRISPR-Cas9 gene editing of patient iPSCs, and (2) CD patient corticotroph PitNETs (hPITOs). These studies demonstrated that SSTR2 and 5 are targets of Hedgehog transcription effector Gli1, and this response is attenuated by activation of the GR pathway. In addition, genetically engineered iPSCs expressing somatic mutations relevant to CD or hPITOs exhibit divergent responses to Mifepristone and relacorilant and may explain the clinical observations made in patients.

      MATERIALS AND METHODS

      Generation, culture and differentiation of Induced Pluripotent Stem Cells (iPSCs) to develop PitNET organoids

      Peripheral Blood Mononuclear Cells (PBMCs) from a healthy individual (JCAZ001) were used to generate iPSCs. Six well culture plates coated with 2 mL/well 0.67% Matrigel (diluted in E8 media, UA iPSC core, 151169-01) were used to initially culture and expand the iPSCs. The iPSC lines were reprogrammed at the University of Arizona iPSC Core using an established protocol.
      • Narsinh K.H.
      • Jia F.
      • Robbins R.C.
      • Kay M.A.
      • Longaker M.T.
      • Wu J.C.
      Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors.
      Once reaching 70% confluency, iPSCs were passaged to Matrigel coated 24 well plates at a ratio of 1:8 and grown to 85%–90% confluency before beginning the directed differentiation schedule (Supplemental Figure 1A) that followed the culture of cells as follows: (1) 0 to 3 with E6 media supplemented with 1% penicillin/streptomycin, 10 μM SB431542 and 5 ng/mL BMP4, (2) at day 3 BMP4 was withdrawn, (3) at day 4 cells were cultured in E6 media, supplemented with 10 μM SB431542, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10ng/mL FGF18 and 50 ng/mL FGF10, and (4) cells were harvested at day 15 using cold E6 media and resuspended in Matrigel (20,000 cells/50 μl Matrigel) . Matrigel domes containing iPSCs were plated in culture dishes and overlaid with differentiation media (E6 media supplemented with 10 μM Y-27632, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10ng/mL FGF18 and 50 ng/mL FGF10, Supplemental Table 1), and cultured for a further 15 days at 37˚C at a relative humidity of 95% and 5% CO2 during which time the 3D organoids developed and used for experiments and analyses. All human iPSC lines were negative for mycoplasma contamination as tested using the Mycoalert Mycoplasma testing kits (LT07-318, Lonza) and no karyotype abnormalities were found (KaryoStat+, Thermo).

      CRISPR/Cas9 gene editing and validation of iPSCs

      A CRISPR/Cas9 approach was used to genetically engineer iPSCs to express known somatic USP48 and USP8 mutations of CD.
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      • Boikos S.
      • et al.
      The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.
      • Ma Z.Y.
      • Song Z.J.
      • Chen J.H.
      • et al.
      Recurrent gain-of-function USP8 mutations in Cushing's disease.
      • Reincke M.
      • Sbiera S.
      • Hayakawa A.
      • et al.
      Mutations in the deubiquitinase gene USP8 cause Cushing's disease.
      • Reincke M.
      • Theodoropoulou M.
      Genomics in Cushing's Disease: The Dawn of a New Era.
      Mutation sgRNAs for either USP8 (Gene ID: 9101, Double: Y717C (A2150G) & S718P (T2152C)), and USP48 (Gene ID: 84196; M415I (G1245A) and M415V (A1243G), Supplemental Figure 2A) were created using the CRISPR Design Tool (SIGMA VC40007) with the assistance of the SIGMA Bioinformatics Support Team. Single guide RNAs (sgRNAs) targeted specific restriction sites whereby USP8 destroyed a BseRI site and created a BstBI site, and USP48 created a SpeI site. SgRNAs were cloned into a LV01/pLV-U6g-EPCG expression vector (expressing sgRNA and Cas9), and combined with Cas9 protein (SIGMA E120030), warm Opti-MEMTM I Reduced Serum Medium (Thermo Fisher Scientific 31985-070) and TransIT-CRISPR (SIGMA T1706). These TransIT-CRISPR Cas9 Ribonucleotide complexes were then added dropwise to their respective iPSC plates and incubated for 48 hours before starting the initiation of the differentiation schedule.
      Successful gene editing of the iPSCs was validated using restriction fragment length polymorphism (RFLP) analysis. DNA was extracted from iPSCs using the QIAamp DNA Micro Kit (Qiagen 56304) according to manufacturer's protocol, and the concentration was measured using a NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific ND-ONE-W). Amplification of 300 ng of DNA was performed using KOD Hot Start Master Mix (Sigma-Aldrich 71842), forward and reverse primers of the mutations (USP8 Forward: TTG AAG TTT ATC GCC ATT TTA TTC G, Reverse: GCT GGT ATA GCC ATC CAC AGA A; USP48 Forward: TGT TGT TCT AGG TTT GTT CCC CA, Reverse: TCA CCA ACA TTC ACC TTA TGG AA). The samples were amplified in a thermal cycler (Applied Biosystems Veriti 96-Well Thermal Cycler, Thermo Fisher Scientific 4375786) with the following temperature cycles: 95˚ for 2 minutes, 40 cycles of 95˚C for 20 seconds, 48˚C for 10 seconds, 70˚C for 1 minute. 6X Purple Gel Loading Dye (NEB B7025S) was added to the amplicon and the PCR products were loaded onto a 1% agarose gel. After electrophoresis, the bands were visualized using a UV imager, extracted using the GenElute Gel Extraction Kit (SIGMA NA1111) and purified with the GenElute PCR Clean-Up Kit (SIGMA NA1020). 300 ng of the eluted DNA was digested overnight at 37˚C with the restriction enzymes including USP8 – BstBI and USP48 – SpeI. Restriction fragments were visualized on a 2.5% agarose gel alongside undigested DNA (Supplemental Figure 2B, C). PitNET generated organoid lines were analyzed for presence of USP8 and USP48 mutations using the previously mentioned primers.

      Generation and Culture of Human PitNET Tissue Organoids (hPITOs)

      Patients undergoing planned transsphenoidal surgery for PitNETs were identified in the outpatient neuroendocrinology and neurosurgery clinics and collected by the St. Joseph's Hospital and Barrow Neurological Institute Biobank under the collection protocol PHXA-05TS038, and outcomes data protocol PHXA-0004-72-29 with approval of the Institutional Review Board (IRB) and patient consent. De-identified samples were shipped in collection and transport media (Supplemental Table 2) to the Zavros laboratory (University of Arizona) for processing within 24 hours of collection. Organoid development and culture were based on our group's published protocol.
      • Chakrabarti J.
      • Pandey R.
      • Churko J.M.
      • et al.
      Development of human Pituitary Neuroendocrine Tumor Organoids to facilitate effective targeted treatments of Cushing's Disease.
      Resected PitNET tissues were washed with DPBS supplemented with 1X Penicillin/Streptomycin, 1X Kanamycin and 1X Amphotericin/Gentamycin, minced into smaller pieces, and incubated with digestion buffer (DMEM/F12 supplemented with 0.4% Collagenase 2, 0.1% Hyaluronic Acid, 0.03% Trypsin). The tissue was incubated with Accutase (Thermo Fisher Scientific) for a further 5 minutes, and the cells were pelleted and washed with DPBS supplemented with antibiotics. Dissociated cells were then seeded as Matrigel domes in culture dishes and overlaid with growth media (Supplemental Table 3). Media was replaced every 3 days and organoids were harvested for downstream experiments or passaged after 15 days of culture (Supplemental Figure 1B).

      Immunofluorescence

      iPSCs were stained for pituitary specific hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), prolactin (PRL), ACTH, cytokeratin 8/18 (CAM5.2), pituitary transcription factor PIT-1 and neuroendocrine marker Synaptophysin (Supplemental Table 4). Cultures were immunostained for proliferation marker EdU (Click-IT EdU Alexa Fluor 555 Imaging Kit, Thermo Fisher Scientific C10338). Antibody dilutions were determined based on manufacturer guidelines and antibody titration (Supplemental Table 4). Staining was conducted according to previously published protocols.
      • Steele N.G.
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      An Organoid-based preclinical model of human Gastric Cancer.
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      • Feng R.
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      • Kenny S.
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      Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice.
      Briefly, organoids and iPSCs grown in 8-well glass bottom chamber slides were fixed using 3.7% formaldehyde, washed with DPBS, permeabilized with 0.5% Triton X-100 (SIGMA X-100, PBS-T, 20 minute at room temperature), washed with 0.01% PBS-T, and blocked with 2% donkey serum (Jackson Immuno Research 017-000-121) for 1 hour at room temperature. After blocking, primary antibodies were added and incubated overnight at 4˚C. Slides were washed with 0.01% PBS-T and incubated with secondary antibodies (Supplemental Table 4) with Hoechst (Thermo Fisher Scientific H1399) for 1 hour at room temperature. Organoids were washed and stored in PBS at 4˚C until microscopic analysis. Immunofluorescence staining for the same lineage markers was performed using PitNET tissue FFPE sections as a positive control, and normal gastric tissue for a negative control. High content confocal microscopy was performed using the Nikon Ti2-E Inverted Microscope (with a Crest X-Light V2 L-FOV Spinning Disk Confocal) at the University of Arizona Cancer Center, or a ZEISS LSM880 34 channel Laser Scanning Confocal Microscope at University of Arizona Marley Imaging Core. Fluorescence intensity was quantified as the percentage of marker-positive cells vs total cells, using Nikon Elements Software (Version 5.21.05). Proliferation for each iPSC line was quantified as the percentage of EdU-positive cells vs total cells.

      Nuclear Morphometric Analysis (NMA)

      Nuclear Morphometric Analysis (NMA) using treated organoids was performed based on a published protocol that measures cell viability based on the changes in nuclear morphology of the cells using nuclear stain Hoechst or DAPI.
      • Filippi-Chiela E.C.
      • Oliveira M.M.
      • Jurkovski B.
      • Callegari-Jacques S.M.
      • da Silva V.D.
      • Lenz G.
      Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities.
      Images of organoid nuclei were analyzed using the ImageJ Nuclear Irregularity Index (NII) plugin for key parameters that included cell area, radius ratio, area box, aspect, and roundness. Using the published spreadsheet template,
      • Filippi-Chiela E.C.
      • Oliveira M.M.
      • Jurkovski B.
      • Callegari-Jacques S.M.
      • da Silva V.D.
      • Lenz G.
      Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities.
      the NII of each cell was calculated with the following formula: NII = Aspect – Area Box + Radius Ratio + Roundness. The area vs NII of vehicle-treated cells were plotted as a scatter plot using the template and considered as the normal cell nuclei. The same plots were generated for each condition, and the NII and area of treated cells were compared to the normal nuclei, and classified as 1 of the following NMA populations: Normal (N; similar area and NII), Mitotic (S; similar area, slightly higher NII), Irregular (I; similar area, high NII), Small Regular (SR; apoptotic, low area and NII), Senescent (LR; high area, low NII), Small Irregular (SI; low area, high NII), or Large Irregular (LI; high area, high NII). Cells classified as SR exhibited early stages of apoptosis, and cells classified as either I, SI or LI exhibited significant nuclear damage. The percentage of cells in each NII classification category were calculated and plotted as a histogram using GraphPad Prism.

      Immunohistochemistry (IHC)

      PitNET organoids were fixed in 4% paraformaldehyde and embedded in Epredia HistoGel Specimen Processing Gel according to the Manufacturers protocol (Fisher Scientific HG-4000-012). HistoGel embedded organoids were then paraffin embedded and sectioned on slides at a 5-micron thickness. After deparaffinization and antigen retrieval (Antigen Unmasking Solution, Vector Laboratories H-3300-250), endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide/methanol for 20 min. Slides were blocked with 20% normal goat or horse serum for 20 min at room temperature and incubated with primary antibodies overnight at 4°C (Supplemental Table 4). Slides were incubated with biotinylated anti-mouse or anti-rabbit secondary antibodies for 30 minutes. Color was developed with 3,3′-diaminobenzidine (DAB) using the DAB substrate VECTASTAIN Elite Mouse (Vector Laboratories PK-6102) or Rabbit (Vector Laboratories PK-6101). Immunohistochemical slides were dehydrated and mounted using Permount (Fisher Scientific), and images were viewed and captured under light microscopy (Olympus BX60 with Diagnostic Instruments “Spot” Camera; Tokyo, Japan).

      Organoid drug treatments and dose responses

      Sonic Hedgehog (SHH) was withdrawn from the organoid growth media 16 hours prior to treatment. The experimental groups included treatments of iPSC generated organoids with the following drugs at the specified working concentrations: vehicle (Veh), Mifepristone (Mife, 500nM, Selleckchem S2606), GANT61 (5µM, Stemcell Technologies 73692), ketoconazole (Keto, 10µM, Selleckchem S1353), Pasireotide (Pas, 100nM, Target Mol TP2207-SB001), Octreotide (Oct, 100nM, Selleckchem P1017), Mife plus GANT61, Mife plus Keto, Mife plus Pas, Relacorilant (Rela, 500nM, Corcept Therapeutics CORT125134), Rela plus Pas, Rela plus Oct, or dexamethasone (Dexa, 100nM, SIGMA S4902). Each drug was prepared at a stock concentration of 10mM and diluted with vehicle to the specified working concentration. In the combination treatments, cultures were pretreated with the GANT61, Keto, Pas or Oct for 2 hours prior to treatment with Mife or Rela.
      PitNET tissue derived organoids hPITO36, hPITO37, hPITO38, hPITO39, and hPITO40 were treated with the following experimental conditions: Vehicle (1), Pasireotide (2), relacorilant (3), relacorilant & Pasireotide (4), Mifepristone (5), Mifepristone & Pasireotide (6). The IC50 values used for each individual drug were determined based on dose responses to concentrations of 0, 1, 10, 100, 1000 and 10,000 nM performed on all hPITO lines (Supplemental Figure 3). All organoid cultures were treated for 48 hours prior to analysis. Percent cell viability was measured using an MTS assay (Promega G3580). Absorbance was measured at 490 nm and normalized to the vehicle. Concentrations were plotted in a logarithmic scale, and a nonlinear dose-response curve regression was calculated using GraphPad Prism. An IC50 value for each drug treatment was determined based on the dose response curve using GraphPad Prism analysis software.

      ACTH Enzyme-Linked Immunosorbent Assay (ELISA)

      Organoid conditioned media was collected and ACTH secretion using an ELISA according to the manufacturer's protocol (Novus Biologicals Human ACTH ELISA Kit NBP2-66401). The optical density was measured at a wavelength of 450nm, and ACTH concentration (pg/mL) was interpolated using a standard curve with a 4-parameter logistic regression analysis using GraphPad Prism (v9.3.1).

      Quantitative RT-PCR (qRT-PCR)

      RNA was extracted from PitNET tissue or iPSC generated organoids using TRI Reagent (Molecular Research Center Inc TR118) and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used for cDNA synthesis of RNA following the recommended protocol. For each sample, 60 ng RNA was reverse transcribed to yield approximately 2 μg total cDNA that was then used for the qRT-PCR. Pre-designed real-time polymerase chain reaction assays were purchased for the following genes: TPit (Thermo Fisher Scientific 4331182 Hs00193027), SF1 (Thermo Fisher Scientific 4331182 Hs00610436), SSTR2 (Thermo Fisher Scientific 4331182 Hs00265624_s1), SSTR5 (Thermo Fisher Scientific 4331182 Hs00990407_s1), POMC (Thermo Fisher Scientific 4331182 Hs01596743_m1), hPRT (Thermo Fisher Scientific 4331182 Hs02800695_m1), and FKBP5 (Thermo Fisher Scientific 4331182 Hs01561006_m1). Polymerase chain reaction amplifications were performed in a total volume of 20 μL containing 20X TaqMan Expression Assay primers, 2X TaqMan Universal Master Mix (Applied Biosystems, TaqMan Gene Expression Systems), and cDNA template. Each polymerase chain reaction amplification was performed in triplicate wells in a StepOne Real-Time PCR System (Applied Biosystems) by using the following conditions: 50°C 2 minutes, 95°C 10 minutes, 95°C 15 seconds (denature) and 60°C 1 minute (anneal/extend) for 40 cycles. Fold change was calculated as the following: (Ct–Ct high) = n target, 2ntarget/2nHPRT = fold change where Ct = threshold cycle. The results were expressed as average fold change in or differential gene expression relative to control, with HPRT used as an internal control according to Livak and Schmittgen.
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

      Western blot analysis of iPSCctrl and mutated iPSCs

      Fully differentiated iPSC control and mutated (iPSCUSP48-MI, iPSCUSP48-MV, and iPSCUSP8) organoids were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, 78501) supplemented with protease inhibitors (Roche, 05 892 970 001) according to the manufacturer protocol. The resulting cell lysates were resuspended in 40 μL of Laemmli Loading Buffer containing β-mercaptoethanol (Bio-Rad Laboratories, 1610730). Samples were loaded onto 4%–20% Tris-Glycine Gradient Gels (Invitrogen) and run at 80 volts for 3 hours then transferred to nitrocellulose membranes (Whatman Protran, 0.45 μM) at 105 volts for 1.5 hours at 4°C. After blocking using for 1 hour at room temperature using KPL Detector Block Solution (Kirkegaard & Perry Laboratories, Inc, 718300), membranes were incubated for 16 hours at 4°C with primary antibodies, GAPDH (Thermo Fisher Scientific, 39-8600 1:1000) and GLI1 (Cell Signaling, 2534S 1:500), followed by incubation with secondary antibodies Alexa Fluor 680 donkey anti-rabbit (Thermo Fisher Scientific, A10043) and donkey anti-mouse (Thermo Fisher Scientific, A10038) for 1 hour at room temperature. Blots were imaged using a scanning densitometer and analysis software (Odyssey Infrared Imaging Software System) and the ratio of protein/GAPDH was calculated using ImageJ software.

      Chromatin Immunoprecipitation (ChIP) Assay

      To investigate GLI-bound POMC transcription factor, chromatin immunoprecipitation (ChIP) assay was performed using the Thermo Scientific Pierce Agarose ChIP kit (Thermo Fisher Scientific, 26156) following the manufacturer's protocol.  Briefly, PitNET organoids were treated with vehicle or GLI-inhibitor GANT61 (5µM, Stemcell Technologies 73692) for 48 hours and then fixed with formaldehyde to crosslink and preserve protein-DNA interactions. Protein-DNA complexes were sheared using Micrococcal Nuclease digestion, followed by immunoprecipitation with ChIP-grade antibodies against POMC (R&D AF3324) used for protein-bound DNA sequences. Crosslinking was reversed using NaCl and protein was digested using Proteinase K. GLI-bound POMC expression was then analyzed using qRT-PCR with the POMC primer (Thermo Fisher Scientific 4331182 Hs01596743_m1).

      Spectral Flow Cytometry Using the Cytek Aurora

      The multicolor flow cytometry antibody panel was designed using the Cytek Full Spectrum Viewer online tool to calculate the similarity index. Organoids were harvested in cold Serum-Free Defined Medium media and incubated with Zombie (BioLegend 423107, 1:100) for 15 minutes in the dark at room temperature, followed by incubation with fluorochrome-conjugated/unconjugated primary surface or cytoplasmic antibodies (Supplemental Table 5) at 4˚C for 30 minutes. Cells were then washed with Cell Staining Buffer (BioLegend 420-201) and incubated at 4˚C for 30 minutes with secondary antibodies (Supplemental Table 5). Cells were fixed using Cytofix/CytopermTM Fixation/Permeabilization Solution (BD Biosciences 554714) and washed with Fixation/Permeabilization wash buffer according to the recommended protocol. Intracellular markers were then labeled with fluorochrome-conjugated/unconjugated primary antibodies at 4˚C for 30 minutes, washed and incubated with secondary antibodies at 4˚C for 30 minutes (Supplemental Table 5). UltraComp eBeadsTM, Compensation Beads (Thermo Fisher Scientific 01-2222-42) were stained with the individual antibodies and used as single stain control for compensation and gating. For Zombie dye single cell control, ArC Reactive Beads (Thermo Fisher Scientific A10346A) were stained with Zombie and ArC negative beads (Thermo Fisher Scientific A10346B). Data was acquired using the Cytek Aurora, analyzed using Cytobank Premium software (Beckman Coulter).

      Statistical analysis

      Data collected from each individual study used 4 in vitro experimental or biological replicates unless otherwise stated. Results were analyzed as the mean ± the standard error of the mean (SEM), and data represented as violin or scatter plots, and significance tested using Microsoft Excel and GraphPad Prism (v9.3.1).

      RESULTS

      IPSC-derived pituitary organoids expressing somatic mutations reveal corticotroph PitNET pathology consistent with CD

      Extensive research has revealed the role of somatic mutations in the development of CD adenomas.
      • Reincke M.
      • Sbiera S.
      • Hayakawa A.
      • et al.
      Mutations in the deubiquitinase gene USP8 cause Cushing's disease.
      ,
      • Chen J.
      • Jian X.
      • Deng S.
      • et al.
      Identification of recurrent USP48 and BRAF mutations in Cushing's disease.
      To study these mechanisms, we generated human pituitary organoids developed from iPSCs. With reference to a published protocol using embryonic stem cells
      • Zimmer B.
      • Piao J.
      • Ramnarine K.
      • Tomishima M.J.
      • Tabar V.
      • Studer L.
      Derivation of diverse hormone-releasing Pituitary cells from Human Pluripotent stem cells.
      together with the knowledge of the growth factor and genetic regulation of pituitary gland development,
      • Zhu X.
      • Gleiberman A.S.
      • Rosenfeld M.G.
      Molecular physiology of pituitary development: signaling and transcriptional networks.
      we optimized our approach for the development of pituitary organoids from human blood-derived iPSCs for gene editing (Supplemental Figure 1A). The iPSC lines were generated to express known somatic mutations in iPSCUSP48 (Gene ID: 84196; M415I [G1245A] and M415V [A1243G]), or iPSCUSP8 (Supplemental Figure 1A, Supplemental Figure 2A).
      • Stratakis C.A.
      • Tichomirowa M.A.
      • Boikos S.
      • et al.
      The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.
      ,
      • Chen J.
      • Jian X.
      • Deng S.
      • et al.
      Identification of recurrent USP48 and BRAF mutations in Cushing's disease.
      ,
      • Ballmann C.
      • Thiel A.
      • Korah H.E.
      • et al.
      USP8 Mutations in Pituitary cushing adenomas-targeted analysis by next-generation sequencing.
      • Sahakitrungruang T.
      • Srichomthong C.
      • Pornkunwilai S.
      • et al.
      Germline and somatic DICER1 mutations in a pituitary blastoma causing infantile-onset Cushing's disease.
      • Cetani F.
      • Pardi E.
      • Cianferotti L.
      • et al.
      A new mutation of the MEN1 gene in an italian kindred with multiple endocrine neoplasia type 1.
      • Marini F.
      • Giusti F.
      • Fossi C.
      • et al.
      Multiple endocrine neoplasia type 1: analysis of germline MEN1 mutations in the Italian multicenter MEN1 patient database.
      • Cazabat L.
      • Bouligand J.
      • Chanson P.
      AIP mutation in pituitary adenomas.
      • Cazabat L.
      • Bouligand J.
      • Salenave S.
      • et al.
      Germline AIP mutations in apparently sporadic pituitary adenomas: prevalence in a prospective single-center cohort of 443 patients.
      USP48 mutations included either M415V or M415I, designating iPSCUSP48MV and iPSCUSP48MI respectively. Successful gene editing was validated using primer specific visualization of a change in the restriction pattern at the site of interest (Supplemental Figures 2B, C). Brightfield images showed morphological variations between the control (iPSCctrl) and mutant iPSCs as early as day 4 and at day 15 (Supplemental Figure 1A). Morphological changes and proliferation were obvious when iPSCs were embedded in Matrigel to generate organoids (Supplemental Figure 1A).
      Expression of PIT1 (pituitary-specific positive transcription factor [1]), ACTH (adrenocorticotropic hormone), GH (growth hormone), FSH (follicle-stimulating hormone), LH (luteinizing hormone), PRL (prolactin) and synaptophysin (synapto) with co-stain Hoechst (nuclei, blue) was measured by immunofluorescence using chamber slides collected at days 4 (D4, Supplemental Figure 4) and 15 (D15, Fig 1) of the differentiation schedules. While pituitary tissue that was differentiated from control iPSCs (iPSCctrl) expressed all major hormone-producing cell lineages (Fig 1A), there was a significant increase in the expression of ACTH and synaptophysin with a concomitant loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated USP48 and USP8 (Fig. 1B–D). Positive immunofluorescence staining of pituitary tumor tissue was used as a positive control and is shown in Fig 1E. Gastric tissue was used as a negative control as is shown in Supplemental Figure 5. Immunofluorescence of iPSCs collected at day 4 of the differentiation schedule revealed no expression of PIT1, ACTH, GH, FSH, LH and PRL in iPSCctrl (Supplemental Figure 4A). Unexpectedly, ACTH expression was increased in iPSCUSP48 at day 4 (Supplemental Figures 4B, C).
      Fig 1
      Fig 1Expression pattern of pituitary hormone-producing cell lineages in iPSCs differentiated to pituitary organoids. Expression of PIT-1 (green), ACTH (green), GH (red), FSH (red), LH (green), PRL (red) and synaptophysin (synapto, green) with co-stain Hoechst (nuclei, blue) was measured by immunofluorescence using chamber slides collected at day 15 (D15) of the differentiation schedule of (A), control iPSCs (iPSCctrl) and iPSCs expressing (B), USP48M415V (iPSCUSP48MV), (C), USP48M415I (iPSCUSP48MI) and (D), USP8 (iPSCUSP8) mutations. Red arrows highlight the increased expression of ACTH and synaptophysin with the concomitant loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing somatic mutations USP48 and USP8. Quantification of the percentage of positive cells is shown in the dot plots for each iPSC line. *P < 0.05 compared to the PIT1, GH, FSH, LH and PRL cell lineages for each line. (E), Expression of PIT-1 (green), ACTH (green), GH (red), FSH (red), LH (green), PRL (red) and synaptophysin (synapto, green) with co-stain Hoechst (nuclei, blue) was measured by immunofluorescence using positive (pituitary tissue) and negative (gastric tissue) controls. Quantification for positive and negative controls is shown to the right. “(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)”
      Immunohistochemistry of formalin fixed and paraffin embedded iPSCctrl, iPSCUSP48, and iPSCUSP8 expressed CAM5.2, T-PIT, synaptophysin and ACTH (Fig 2A, higher magnification of image is shown in the inset). The iPSCctrl line expressed transcription factors TPit (TBX19), PIT1 (POU1F1) and SF1 that corresponded to the differentiation of corticotrophs, somatotrophs and gonadotrophs as documented by positive immunofluorescence staining (Fig 2B). The iPSC lines expressing mutations in USP48MV exhibited significantly elevated transcription factor T-Pit consistent with the skewed differentiation in the corticotroph cell lineage (Fig 2B). Transcription factors PIT1 (somatotrophs) and SF1 (gonadotroph) were significantly decreased in the cultures of iPSC lines expressing USP48MV compared to iPSCctrl (Fig 2B). Culture conditioned media was collected during the differentiation schedule of the iPSCs and analyzed for ACTH secretion by ELISA (Fig 2C). Compared to control lines, iPSC lines expressing mutated USP8 and USP48 secreted significantly greater concentrations of ACTH earlier in the differentiation schedule (Fig 2C).
      Fig 2
      Fig 2Expression of cell linages and transcription factors in iPSC generated pituitary tumor organoids. (A), Immunohistochemistry using FFPE sections prepared from iPSCctrl, iPSCUSP48MV, iPSCUSP48MI, and iPSCUSP8 organoids stained with antibodies specific for CAM5.2, T-Pit, Synaptophysin and ACTH. High magnification images are shown in insets. (B), Differential expression of transcription factors TPit, PIT1 and SF1 and POMC were measured by qRT-PCR. (C), Plot comparing the ACTH secretion (pg/mL), measured by an ELISA using conditioned media of iPSCctrl, iPSCUSP48MV, iPSCUSP48MI, and iPSCUSP8 throughout the directed differentiation schedule. (D), viSNE maps showing concatenated flow cytometry standard files for iPSCctrl, iPSCUSP48MV, iPSCUSP48MI, and iPSCUSP8 organoids 30 days post-directed differentiation. Maps define spatially distinct cell populations using pituitary specific cell lineage, stem cell and transcription factor markers between iPSCctrl organoids and mutant lines. (E), viSNE heatmaps showing the number of cells positive for proliferation marker Ki67. (F), Violin plots comparing the TPit lineage expression, through proliferation marker Ki67 between control and mutated iPSC lines. *P < 0.05 compared to iPSCctrl organoids, n = 3 individual experimental replicates.
      The expression pattern of corticotroph PitNET specific markers was analyzed using Cytek Aurora spectral flow cytometry (Fig 2D). The location of cells that were found in each cluster based on the highly expressed antigens are shown in the representative t-SNE maps (Fig 2D). Compared to iPSCctrl organoids, iPSCUSP48 and iPSCUSP8 contained higher numbers of cells expressing T-Pit cell lineage and ACTH/SSTR2/SSTR5 expressing cells (representative t-SNE map of iPSCUSP48MV shown in Fig 2D). In addition, iPSCctrl organoids expressed a higher percentage of Pit1 cell lineage, GH and PRL positive cells (Fig 2D). Within the iPSCUSP48 and iPSCUSP8 cultures, stem cells expressing SOX2+/nanog+/CD133+ were significantly more abundant compared to the iPSCctrl organoids (Fig 2D). Heatmap dot plots obtained after tSNE analysis showed that both the stem cell and TPit populations within the iPSCUSP48 and iPSCUSP8 organoid cultures expressed higher proliferating cells as measured by Ki67 (Fig 2E–F).
      Differentiated iPSCs were then collected at D15 of the culture schedule and embedded into Matrigel. Proliferation was quantified by EdU uptake of the cells within the pituitary organoids (Supplemental Figure 6A, B). Compared to control pituitary organoids iPSCctrl, organoids expressing mutations in USP8 (iPSCUSP8), and USP48 (iPSCUSP48MV and iPSCUSP48MI) expressed significantly elevated Edu+ proliferating cells (Supplemental Figure 6A, B). Collectively, these data show that iPSCs genetically engineered to express somatic mutations relevant to the development of CD exhibit corticotroph PitNET pathology and function in vitro.

      Genetically engineered iPSCs expressing somatic mutations relevant to CD identify differential effects of relacorilant and Mifepristone on SSTR expression, and tumor cell proliferation and apoptosis

      The treatment of iPSC organoids with Mifepristone or relacorilant resulted in a significant induction in the expression of SSTR2 and 5 (Fig. 3A–B). Mifepristone led to a significantly greater induction in SSTR2 expression when compared to relacorilant (Fig 3A). In contrast, relacorilant induced greater SSTR5 expression (Fig 3B).
      Fig 3
      Fig 3Differential expression of SSTR2 and SSTR5 in iPSC generated pituitary tumor organoids in response to Mifepristone and Relacorilant. Differential expression of (A), SSTR2 and (B), SSTR5 in iPSCctrl, iPSCUSP48MI, iPSCUSP48MV and iPSCUSP8 organoids in response to vehicle (Veh), Mifepristone (Mife, 500nM) or relacorilant (Rela, 500nM). *P < 0.05 compared to vehicle-treated organoids, #P < 0.05 compared to Mifepristone treated organoids, n = 3 experimental replicates/organoid line. (C), Differential expression of SSTR2 and SSTR5 in iPSCctrl organoids treated with vehicle (Veh), Mifepristone (Mife), GANT61 (GANT, 5μM), Mife+GANT, ketoconazole (Keto, 10μM), Mife+Keto, or dexamethasone (Dexa, 100nM). *P < 0.05 compared to iPSCctrl organoids treated with Veh, n = 3 experimental replicates/organoid line. (D), Differential expression of SSTR2 and SSTR5 in iPSCctrl organoids treated with vehicle (Veh), relacorilant (Rela), GANT61 (GANT), Rela+GANT, ketoconazole (Keto), Rela+Keto, or dexamethasone (Dexa). *P < 0.05 compared to iPSCctrl organoids treated with Veh, n = 3 experimental replicates/organoid line. (E), Mutations in USP48 and USP8 in PitNETs are believed to enhance corticotropin releasing hormone (CRH)-induced production coherent with the Hh signaling pathway. Hh ligand, Sonic Hedgehog (Shh), binds to Patched (Ptch1) that relieves suppression of Smoothened (SMO) and subsequently Gli1 activation. Crosstalk between Shh and CRH at the Gli1 level stimulates POMC transcription and ACTH secretion. (F), Differential expression of POMC in iPSCctrl, iPSCUSP48MI, iPSCUSP48MV and iPSCUSP8 organoids. *P < 0.05 compared to iPSCctrl organoids, n = 3 experimental replicates/organoid line. (G), Representative western blots of the expression of Gli1 relative to GAPDH in iPSCctrl, iPSCUSP48MI, iPSCUSP48MV and iPSCUSP8 organoids. (H), Quantification of western blots shown in G. *P < 0.05 compared to iPSCctrl organoids, n = 3 experimental replicates/organoid line. (I), Mutations in USP8 and USP48 detected in hPITO cultures (hPITO1 and 7) were also expressed in the patient's matched PitNET tissue. (J), Human PITO cultures expressing USP8 and USP48 mutations were used for gene ChIP analysis after treatment with vehicle or GANT61. *P < 0.05 compared to iPSCctrl organoids, n = 4 experimental replicates/organoid line.
      To identify the role Hedgehog signaling as a mediator of Mifepristone or relacorilant regulated SSTR expression, iPSCctrl organoids were used in combination with inhibitors GANT61 (GLI inhibitor) and ketoconazole (a SMO inhibitor that also inhibits cortisol synthesis) (Fig. 3C–D). Mifepristone significantly induced the differential expression of both SSTRs 2 and 5, and this increase was reduced with GANT61 pretreatment of the organoid cultures (Fig 3C). In contrast to the inhibitory effect of GANT61 on Mifepristone-induced SSTR expression, ketoconazole pretreatment had no effect on this induction (Fig 3C). GANT61 and ketoconazole alone had no effect on the differential expression of SSTRs 2 and 5, while dexamethasone, a known GR agonist, significantly decreased expression of these receptors (Fig 3C). Similar responses were observed with relacorilant with or without GANT61 and ketoconazole (Fig 3D). These results suggest that Mifepristone and relacorilant induce SSTR2 and SSTR5 via a SMO-independent non-canonical Hh signaling pathway.
      Organoids expressing both the USP8 and USP48 mutations exhibited characteristics consistent of corticotroph subtype PitNETs (Fig 1, Fig 3). Based on initial evidence in the literature, USP48 and USP8 mutations in PitNETs are believed to enhance corticotropin releasing hormone (CRH)-induced production coherent with the Hh signaling pathway (Fig 3E).
      • Reincke M.
      • Sbiera S.
      • Hayakawa A.
      • et al.
      Mutations in the deubiquitinase gene USP8 cause Cushing's disease.
      ,
      • Sbiera S.
      • Perez-Rivas L.G.
      • Taranets L.
      • et al.
      Driver mutations in USP8 wild-type Cushing's disease.
      • Zhou A.
      • Lin K.
      • Zhang S.
      • et al.
      Gli1-induced deubiquitinase USP48 aids glioblastoma tumorigenesis by stabilizing Gli1.
      • Vila G.
      • Papazoglou M.
      • Stalla J.
      • et al.
      Sonic hedgehog regulates CRH signal transduction in the adult pituitary.
      • Vila G.
      • Theodoropoulou M.
      • Stalla J.
      • et al.
      Expression and function of sonic hedgehog pathway components in pituitary adenomas: evidence for a direct role in hormone secretion and cell proliferation.
      • Xia R.
      • Jia H.
      • Fan J.
      • Liu Y.
      • Jia J.
      USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization.
      • Pyczek J.
      • Buslei R.
      • Schult D.
      • et al.
      Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland.
      Hh ligand, Sonic Hedgehog (Shh), binds to Patched (Ptch1) that relieves suppression of Smoothened (Smo) and subsequently Gli1 activation (Fig 3E). Crosstalk between Shh and CRH at the Gli1 level stimulates POMC transcription and ACTH secretion. Our data is consistent with the hypothesis that mutations in USP48 lead to increased levels of Gli1 and enhancing POMC transcription via an unknown a mechanism yet to be revealed (Fig 3E).
      • Vila G.
      • Papazoglou M.
      • Stalla J.
      • et al.
      Sonic hedgehog regulates CRH signal transduction in the adult pituitary.
      USP8 was shown to promote SMO activity that may subsequently also lead to POMC transcription and ACTH secretion (Fig 3E).
      • Xia R.
      • Jia H.
      • Fan J.
      • Liu Y.
      • Jia J.
      USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization.
      Our proposed mechanism is supported by significantly greater differential expression of POMC (Fig 3F) in the iPSCUSP48 and iPSCUSP8 organoids when compared to the control cultures. In addition, GLI1 protein expression was significantly increased in the iPSCUSP48 and iPSCUSP8 organoids compared to the iPSCctrl cultures (Fig. 3G and H). Mutations in USP8 and USP48 detected in hPITO cultures (hPITO1 and 7) were also expressed in the patient's matched PitNET tissue (Fig 3I). Gene ChIP assay revealed that the transcriptional regulation of POMC by Gli1 was blocked by GANT61 treatment of cultures (Fig 3J).
      Analysis of proliferation using EdU uptake in iPSCctrl and iPSCUSP48MV organoid cultures showed that Mife induced a significant increase in cell proliferation (Fig. 4A and B and Fig 5A and B). While the proliferative response to Mife was inhibited by pretreatment with Oct, Pas had no effect (Fig 4A and B and Fig 5A and B). While a similar response was observed in Rela treated cultures with pretreatment with Pas or Oct, Rela alone did not induce organoid proliferation (Fig 4A and B and Fig 5A and B). The nuclear irregularity index (NII) was measured based on the quantification of the morphometric changes in the nuclei in response to the treatment groups in the iPSCctrl (Fig 4C–F, Supplemental Figure 7) and iPSCUSP48MV organoids (Fig 5C–F, Supplemental Figure 8). While Rela induced a significant expression pattern of nuclear morphology consistent with increased apoptotic cells in the iPSCUSP48MV cultures (Fig 5C–F, Supplemental Figure 8), this was not observed in the iPSCctrl organoids (Fig 4C–F, Supplemental Figure 7). The magnitude of the apoptotic response to Rela was not observed with Mife in either the iPSCctrl (Fig 4C–F, Supplemental Figure 7) or iPSCUSP48MV (Figure 5C–F, Supplemental Figure 8) organoids.
      Fig 4
      Fig 4Changes in pituitary tumor cell proliferation, viability and ACTH secretion in iPSCctrl organoids in response to Mifepristone and relacorilant. (A), Immunofluorescence images of EdU expression in iPSCctrl organoids in response to vehicle (Veh), Mifepristone (Mife, 500nM), Pasireotide (Pas, 100nM), Octreotide (Oct, 100nM), relacorilant (Rela, 500nM), Mife+Pas, Mife+Oct, Rela+Pas, Rela+Oct, dexamethasone (Dexa, 100nM). (B), Quantification of EdU positive cells of iPSCctrl and mutant organoids. *P < 0.05 compared to iPSCctrl organoids, n = 4 individual organoids quantified per culture. (C), Representative Hoechst staining of iPSCctrl organoids in response to experimental treatments for the calculation of nuclear irregularity index (NII). Nuclear morphometric analysis of iPSCctrl organoids in response to experimental treatments with quantification shown for (D), Veh, (E), Mife, or (F), Rela treatments. Morphometric classification of NII was based on the normal (N), small (S), small regular (SR), short irregular (SI), large regular (LR), large irregular (LI) and irregular (I) nuclear morphology. (G), An ELISA was performed using conditioned media collected from iPSCctrl cultures in response to treatments for the measurement of ACTH secretion (pg/mL). *P < 0.05 compared to Veh treatment, #P < 0.05 compared to Mife or Rela alone, n = 4 individual organoids quantified per culture.
      Fig 5
      Fig 5Changes in pituitary tumor cell proliferation, viability and ACTH secretion in iPSCUSP48MV organoids in response to Mifepristone and relacorilant. (A), Immunofluorescence images of EdU expression in iPSCUSP48MV organoids in response to vehicle (Veh), Mifepristone (Mife, 500nM), Pasireotide (Pas, 100nM), Octreotide (Oct, 100nM), relacorilant (Rela, 500nM), Mife+Pas, Mife+Oct, Rela+Pas, Rela+Oct, dexamethasone (Dexa, 100nM). (B), Quantification of EdU positive cells of iPSCUSP48MV and mutant organoids. *P < 0.05 compared to iPSCUSP48MV organoids, n = 4 individual organoids quantified per culture. (C), Representative Hoechst staining of iPSCctrl organoids in response to experimental treatments for the calculation of nuclear irregularity index (NII). Nuclear morphometric analysis of iPSCUSP48MV organoids in response to experimental treatments with quantification shown for (D), Veh, (E), Mife, or (F), Rela treatments. Morphometric classification of NII was based on the normal (N), small (S), small regular (SR), small irregular (SI), large regular (LR), large irregular (LI) and irregular (I) nuclear morphology. (G), An ELISA was performed using conditioned media collected from iPSCUSP48MV cultures in response to treatments for the measurement of ACTH secretion (pg/mL). *P < 0.05 compared to Veh treatment, #P < 0.05 compared to Mife or Rela alone, n = 4 individual organoids quantified per culture.
      Within the iPSCctrl and iPSCUSP48MVl organoid cultures, the magnitude of ACTH secretion induced by Mifepristone was significantly greater than the effect of relacorilant (Fig 4G and 5G). Pas and Oct significantly reduced ACTH secretion in response to Mifepristone, although the inhibition by Octreotide was greater that Pasireotide (Fig 4G and Fig 5G). Pasireotide reduced hormone secretion in combination with relacorilant at a greater magnitude compared to that of Octreotide plus relacorilant in iPSCctrl and iPSCUSP48MV organoid cultures (Fig 4G and Fig 5G).
      Heatmap dot plots obtained after tSNE analysis of iPSCUSP48MV organoids showed the relative expression of the SSTR2, SSTR5, ACTH and zombie positive cells in the different phenotypic clusters in response to Rela and Mife (Fig 6). Quantification based on the gated identified cell clusters revealed that both Rela and Mife induced a significant increase in percentage of SSTR2 (Fig 6A and E) and SSTR5 (Fig 6B–F) positive cells. However, the magnitude of Mife induction of SSTR2 was significantly greater than that of Rela (Fig 6A and E). Similarly, the magnitude of Rela induction of SSTR5 was significantly greater than that of Mife (Fig 6B and F). In contrast to Rela, Mife led to a significant induction in the number of ACTH positive cells (Fig 6C and G). Rela did not significantly increase ACTH, and Pas + Rela significantly reduced ACTH expression in cultures (Fig 6C and G). Rela, or Rela plus Pas, clearly induced iPSCUSP48MV cell death as measured by the significant increase in Zombie positive cells that co-expressed ACTH and stem cell markers, a response not observed with Mife (Fig 6D and H).
      Fig 6
      Fig 6Changes in SSTR2, SSTR5 and ACTH expression, and pituitary tumor cell death in iPSCUSP48MV organoids in response to Mifepristone and relacorilant. Fluorescent intensity of (A), SSTR2, (B), SSTR5, (C), ACTH, and (D), zombie of viSNE heatmaps for iPSCUSP48MV organoids in response to vehicle (Veh), Pasireotide (Pas, 100nM), relacorilant (Rela, 500nM), Rela+Pas, Mifepristone (Mife, 500nM), and Mife+Pas. iPSCUSP48 = 15000 events. Quantification of percentage of SSTR2 (E), SSTR5 (F), ACTH (G), or Zombie (H), positive cells within iPSCUSP48MV organoid cultures in response to experimental treatments. *P < 0.05 compared to Veh.
      To assess GR modulation, the GR target gene FKBP5 was measured in the PitNET organoids. While Mifepristone and relacorilant significantly reduced the differential expression of FKBP5, dexamethasone caused a significant induction in gene expression (Supplemental Figure 9A-C). Compared to Mifepristone, the magnitude of FKBP5 inhibition was significantly greater in response to relacorilant (Supplemental Figure 9A–C). This confirmed GR modulation in the organoid cultures in response to Mifepristone and relacorilant.
      Collectively, these data demonstrate that, compared to relacorilant, Mifepristone preferentially induced the expression of SSTR2, ACTH secretion and PitNET organoid proliferation. In contrast, relacorilant predominantly induced SSTR5 expression and PitNET organoid apoptosis.

      Relacorilant induces tumor cell death and reduces ACTH in combination with Pasireotide in organoids generated from CD patient PitNETs

      As part of a previous study, we generated an initial biobank of organoids generated from CD patient PitNETs. Human PitNET tissue was harvested during endoscopic transsphenoidal pituitary surgery from 40 patients to generate organoids (full list published in.
      • Chakrabarti J.
      • Pandey R.
      • Churko J.M.
      • et al.
      Development of human Pituitary Neuroendocrine Tumor Organoids to facilitate effective targeted treatments of Cushing's Disease.
      These cultures are referred to as human PitNET derived organoids (hPITOs). Table 1 summarizes the neuropathology reports and clinical diagnosis from cases used to generate hPITOs 37, 38, 39, 40 reported in the current study. Bright-field microscopy images of hPITOs that were generated from corticotroph PitNETs from patients diagnosed with CD revealed morphological diversity, including size, density, and regularity, among the organoid lines between individual patients and between Crooke's cell adenoma and sparsely granulated subtypes (Fig 7A). Immunofluorescence confocal microscopy showed the expression of ACTH and CAM5.2 (cytokeratin 8/18) pituitary corticotroph markers (representative image of Crooke's cell hPITO37 shown in Fig 7B, Supplemental video 1). Consistent with the immunofluorescence, immunohistochemistry of FFPE sections of hPTO37 organoid line and the patient's matched PitNET showed positive staining for both ACTH and CAM5.2 (Fig 7B). These data were consistent with the neuropathology report (Table 1).
      Table 1Clinical characteristics of pituitary adenoma samples used for the generation of organoids
      Organoid lineGenderAgeNeuropathology reportClinical diagnosisPrior treatment
      hPITO37F53Crooke cell adenoma. T-PIT+, ACTH+, CAM5.2+, CK20+, synaptophysin+, MIB (Ki67) LI: <1%Cushing's diseaseNone
      hPITO38F43Corticotroph subtype, sparsely. T-PIT+, ACTH+, CAM5.2+, CK20+, synaptophysin+, MIB (Ki67) LI: <1%Cushing's diseaseNone
      hPITO39M62Corticotroph subtype, sparsely granulated, T-PIT+, ACTH+, CAM5.2+, CK20+, synaptophysin+, MIB (Ki67) LI: <1%Cushing's diseaseNone
      hPITO40F28Corticotroph subtype, Crooke cell adenoma, T-PIT+, ACTH+, CAM5.2+, CK20+, synaptophysin+, alpha subunit+, MIB (Ki67) LI: 2.5%Cushing's diseaseNone
      Fig 7
      Fig 7Changes in SSTR2, SSTR5 and ACTH expression, and pituitary tumor cell death in hPITOs in response to Mifepristone and relacorilant. (A), Brightfield images of organoid cultures generated from patients with CD (hPITOs 37, 38, 39, 40). (B), Immunofluorescence staining using antibodies specific for CAM5.2 (red) and ACTH (green) of hPITO37. Immunohistochemical staining using antibodies specific for ACTH and CAM5.2 of FFPE sections prepared from embedded organoid line (left panels, hPITO37), and patient's matched PitNET tissue (Pt37, right panels). (C), viSNE maps showing concatenated flow cytometry standard files for hPITO37 line defining the spatially distinct cell populations using pituitary specific cell lineages, stem cell and transcription factor markers. (D) Differential expression of SSTR2 and SSTR5 measured in hPITO36, 37, 38, 39 and 40 lines. Fluorescent intensity of (E, F), SSTR2, (G, H), SSTR5, (I, J), ACTH, and (K, L), zombie of viSNE heatmaps for hPITO37 organoids in response to vehicle (Veh), Pasireotide (Pas, 837.5nM), relacorilant (Rela, 382nM), Rela+Pas, Mifepristone (Mife, 2.85nM), and Mife+Pas. hPITO = 5000 events. Quantification of percentage of SSTR2, SSTR5, ACTH, or Zombie positive cells within hPITO cultures in response to experimental treatments is shown in F, H, J and L. *P < 0.05 compared to Veh.
      To further test the similarity in cell lineages identified between the organoid line and the patient's tumor, we compared the immunohistochemistry from the neuropathology reports of the patients detailed in Table 1, to the expression pattern of pituitary tumor specific markers that were measured using Cytek Aurora spectral flow cytometry (Fig 7C). Flow cytometric analysis using Cytobank revealed that hPITOs derived from patients with CD expressed increased stem and progenitor cell markers including CXCR4, SOX2, and CD133 (Fig 7C). Using a gating strategy for the TPit positive cells, we identified ACTH positive cell populations co-expressing SSTR2 and SSTR5 (Fig 7C). Diversity in the differential expression of SSTR2 and SSTR5 among the different hPITO lines was observed when SSTR2 and SSTR5 gene expression was measured (Fig 7D).
      The calculation of the total area under the curve (Fig 8) based on the dose response curve (Supplemental Figure 3) for each organoid line showed divergent responses to Mifepristone, Pasireotide and relacorilant in individual cultures (Fig 8A–I). While differential IC50 values were observed among the individual PitNET organoid lines (Fig. 8A, D, G), this divergence in sensitivity was not reported in cultures prepared from the matched adjacent normal pituitary tissues (Fig. 8B, E, H). Organoid line hPITO37 (Crooke's cell adenoma subtype) expressed significantly lower SSTR2 and SSTR5 expression levels compared to the other cultures (Fig 7D) and was insensitive to Pas (Fig 8, Supplemental Figure 3). Therefore, we used this hPITO line to identify the differential effects of Mife and Rela on receptor expression and cell viability. Heatmap dot plots obtained after tSNE analysis of hPITO cultures showed the relative expression of the SSTR2, SSTR5, ACTH and zombie positive cells in the different phenotypic clusters in response to Rela and Mife (Fig 7E–L). Quantification based on the gated identified cell clusters revealed that both Rela and Mife induced a significant increase in percentage of SSTR2 and SSTR5 positive cells (Fig 7E–H). However, the magnitude of Mife induction of SSTR2 was significantly greater than that of Rela (Fig 7E and F). While both Rela and Mife induced a significant increase in percentage SSTR5 positive cells, the magnitude of Rela induction of SSTR5 was significantly greater than that of Mife (Fig 7G and H). In contrast, Mife significantly increased the number of ACTH expressing cells in culture, a response that was not inhibited by Pas pretreatment (Fig 7I and J). Rela clearly induced hPITO cell death as measured by the significant increase in Zombie positive cells in response to Rela or Rela plus Pas, a response not observed with Mife (Fig 7K and L). In contrast to Mife, Rela also induced significant cell death in cell populations expressing stem cell markers SOX2, CXCR4 and nestin, and an epithelial/mesenchymal hybrid cell population that co-expressed CK20, vimentin and CXCR4 (Fig 7K and L). Therefore, Rela sensitizes CD PitNET tissue derived organoids to Pasireotide and induces cell death in several different morphologically diverse cell populations.
      Fig 8
      Fig 8Analysis of Area-Under-Curve (AUC) for PitNET organoids treated with Pasireotide, Mifepristone or relacorilant. Dose response curves using PitNET organoid lines (hPITO36, 37, 38, 39 and 40) treated with (A), Pasireotide, (D), Mifepristone or (G), relacorilant. Dose response curves using PitNET organoid lines generated from adjacent normal tissue (hPITO37N, 38N) treated with (B), Pasireotide, (E), Mifepristone or (H), relacorilant. (C, F, I), Calculated AUC and IC50 values. n = 3 experimental replicates performed per organoid line.

      DISCUSSION

      Lack of effective medical therapies targeted directly to the corticotroph PitNETs is potentially attributed to the lack of human patient-relevant model systems that recapitulate the cellular complexity of the tumor. Our studies contribute to overcoming this challenge by generating a PitNET organoid model system that is applied to identifying the mechanisms of action of Mifepristone and relacorilant (alone and in combination with other medical therapies for CD). As part of our studies, PitNET organoids were generated from: (1) CRISPR-Cas9 gene editing of patient iPSCs, and (2) CD patient corticotroph PitNETs (hPITOs).
      • Chakrabarti J.
      • Pandey R.
      • Churko J.M.
      • et al.
      Development of human Pituitary Neuroendocrine Tumor Organoids to facilitate effective targeted treatments of Cushing's Disease.
      Using these advanced in vitro culture systems, we demonstrated that SSTR2 and SSTR5 are targets of Hedgehog transcription effector Gli1 and this response is attenuated by activation of the GR pathway. In these experiments, we have shown that Mifepristone and relacorilant exerted differential effects on the induction of SSTR2 and SSTR5 expression, ACTH secretion and PitNET organoid proliferation and apoptosis.
      While both Mifepristone and relacorilant significantly induced the expression of SSTR2 and SSTR5, the magnitude of induction of these receptors was different. While Mifepristone predominantly induced SSTR2 expression, relacorilant predominantly induced SSTR5. The SSTRs expressed on the surface of corticotroph PitNETs have often been targeted for the treatment of CD.
      • Pivonello R.
      • Ferrigno R.
      • De Martino M.C.
      • et al.
      Medical treatment of Cushing's disease: an overview of the current and recent clinical trials.
      Multiple studies suggest that SSTR5 is consistently overexpressed in corticotroph PitNETs,
      • Pivonello R.
      • Ferrigno R.
      • De Martino M.C.
      • et al.
      Medical treatment of Cushing's disease: an overview of the current and recent clinical trials.
      however, treatments with somatostatin analogues (Octreotide and Pasireotide) are designed without considering variations in receptor subtype expression among individual patients with CD. We clearly show the presence of variation in the SSTR2 and SSTR5 expression amongst individual patients with CD, and, importantly, SSTR5 was significantly upregulated by relacorilant and subsequently sensitized organoid tumor cells to suppression of ACTH by Pasireotide. Our findings are consistent with the knowledge that Pasireotide has high-affinity binding to SSTR5.
      • Gatto F.
      • Barbieri F.
      • Arvigo M.
      • et al.
      Biological and biochemical basis of the differential efficacy of first and second generation somatostatin receptor ligands in Neuroendocrine Neoplasms.
      In fact, Pasireotide monotherapy normalizes cortisol in up to 42% of patients with CD but assumes that all patients express this receptor subtype.
      • Feelders R.A.
      • de Bruin C.
      • Pereira A.M.
      • et al.
      Pasireotide alone or with cabergoline and ketoconazole in Cushing's disease.
      Combination treatment using Pasireotide administered together with cabergoline and ketoconazole may increase the efficacy of the somatostatin analogue, but there is no clear consideration for changes in SSTRs during treatment.
      • Feelders R.A.
      • de Bruin C.
      • Pereira A.M.
      • et al.
      Pasireotide alone or with cabergoline and ketoconazole in Cushing's disease.
      Therefore, our findings suggest that treating patients with CD with relacorilant may increase the efficacy of Pasireotide (or, potentially, endogenous somatostatin) suppression of ACTH secretion from corticotroph PitNETs.
      In our studies we showed that Hedgehog (Hh) signaling mediates SSTR2 and 5 expression that is induced by Mifepristone and relacorilant through a non-canonical Hh signaling pathway. In the iPSC organoids, GLI inhibitor GANT61 inhibited both Mifepristone and relacorilant stimulated SSTR2 and SSTR5. The SMO inhibitor ketoconazole (which also inhibits cortisol synthesis) failed to block the induction of both SSTR2 and SSTR5 by both compounds. Upon ligand binding to the transmembrane receptor Patched-1 (PTCH1), the repression of PTCH1 on transmembrane transducer SMO is released. De-repressed SMO triggers the activation of GLI zinc finger transcription factors GLI1, GLI2 and GLI3. Activated GLI1 protein translocates into the nucleus, where several target genes are regulated.
      • Brennan D.
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      Noncanonical Hedgehog signaling.
      Our studies suggest that within the corticotroph PitNETs, SSTR2 and SSTR5 are transcriptional targets of GLI. The failure of the SMO inhibitor ketoconazole to inhibit Mifepristone and relacorilant induction of SSTR expression suggests that the signaling pathway is non-canonical (Supplemental Figure 10). Non-canonical Hh signaling pathways have been classified as (1) Type I pathways involving signaling through PTCH1 independent of SMO, (2) Type II pathways involving signaling through SMO independent of GLI, and (3) SMO-independent activation of GLI such as that observed in the current study.
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      Noncanonical Hedgehog signaling.
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      Canonical and non-canonical Hedgehog signalling and the control of metabolism.
      In support of our findings, crosstalk between GLI and GR (NR3C1) signaling has been reported in T cell acute lymphoblastic leukemia.
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      Crosstalk between Hedgehog pathway and the glucocorticoid receptor pathway as a basis for combination therapy in T-cell acute lymphoblastic leukemia.
      In addition, studies in the adult stomach demonstrated that the Hh pathway is critical for the regulation of somatostatin and SSTR signaling.
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      Sonic Hedgehog contributes to gastric mucosal restitution after injury.
      ,
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      Loss of Parietal Cell Expression of Sonic Hedgehog Induces Hypergastrinemia and Hyperproliferation of surface Mucous Cells.
      While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland,
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      Hedgehog signaling is required for pituitary gland development.
      this is the first report demonstrating that Hh signaling via Gli1 regulates the expression of SSTR2 and SSTR5 in PitNET organoids.
      The expression of USP8 and USP48 mutations increased the differentiation of the iPSC generated organoids consistent with a corticotroph subtype PitNET phenotype. Our data and evidence in the literature shows that corticotroph subtype PitNETs expressing USP8 and USP48 mutations target members of the Hh signaling pathway including SMO and Gli1 respectively. Published studies show that Hh ligand Shh and the activation of the signaling pathway is a key regulator of the stem/progenitor cell proliferation in both normal pituitary and PitNETs that may promote the development of corticotroph subtype PitNETs.
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      Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland.
      ,
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      SOX2 mediates crosstalk between Sonic Hedgehog and the Wnt/beta-catenin signaling pathway to promote proliferation of pituitary adenoma cells.
      ,
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      Hedgehog signaling in endocrine and folliculo-stellate cells of the adult pituitary.
      Thus, a plausible explanation for the tumorigenesis of corticotroph subtype PitNETs is that Hh signaling drives the increased proliferation and POMC expression of stem/progenitor cells.
      In contrast to Mifepristone, relacorilant induced PitNET cell death in treated organoid cultures without significant stimulation of ACTH secretion. In concurrence with the organoid in vitro studies, tumor regression in 2 patients with macroadenomas treated with relacorilant for 3 months have been reported in an initial study based on standard of care imaging of the pituitary gland.
      • Massimo Terzolo M.
      • Iacuaniello Davide
      • Pia Anna
      • Adriano Priola
      • Moraitis Andreas
      • Pivonello Rosario
      SUN-463 Tumor shrinkage with preoperative relacorilant therapy in two patients with Cushing Disease due to Pituitary Macroadenomas.
      This unexpected finding is further being investigated in an ongoing Phase 3 study of relacorilant in patients with Cushing's syndrome (GRACE Study, NCT03697109). Such macroadenoma regressions have not been observed with Mifepristone. Based on the negative feedback mechanism regulating the hypothalamic-pituitary-adrenal (HPA) axis, we propose the following mechanisms to explain the differential effects between Mifepristone and relacorilant. First, Mifepristone predominantly induced SSTR2 expression. However, unlike relacorilant, Mifepristone treatment significantly increased ACTH secretion from PitNET both in vivo and in vitro (Supplemental Figure 10). Our organoid experiments are supported by patient data showing that Mifepristone increases ACTH,
      • Fleseriu M.
      • Biller B.M.
      • Findling J.W.
      • et al.
      Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome.
      ,
      • Feiteiro J.
      • Mariana M.
      • Verde I.
      • Cairrao E.
      Genomic and Nongenomic Effects of Mifepristone at the Cardiovascular Level: a review.
      with a concomitant increase in cortisol secretion without tumor growth.
      • Pivonello R.
      • Ferrigno R.
      • De Martino M.C.
      • et al.
      Medical treatment of Cushing's disease: an overview of the current and recent clinical trials.
      ,
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      Changes in plasma ACTH levels and corticotroph tumor size in patients with Cushing's disease during long-term treatment with the glucocorticoid receptor antagonist mifepristone.
      In contrast, relacorilant predominantly induced SSTR5 expression without an increase in ACTH secretion and tumor cell death (Supplemental Figure 10). A plausible explanation for PitNET regression in response to relacorilant treatment may be the known heterodimerization between SSTR2 and SSTR5 that occurs following the selective activation of SSTR2 but not human SSTR5 or concurrent stimulation.
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      Cell growth inhibition and functioning of human somatostatin receptor type 2 are modulated by receptor heterodimerization.
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      Somatostatin receptor biology in neuroendocrine and pituitary tumours: part 1–molecular pathways.
      Heterodimerization between SSTR2 and SSTR5 leads to an increased recycling rate and a greater propensity of SSTR2 to induce tumor growth inhibition.
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      Somatostatin receptor biology in neuroendocrine and pituitary tumours: part 1–molecular pathways.
      ,
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      • et al.
      Coexpression of somatostatin receptor subtype 5 affects internalization and trafficking of somatostatin receptor subtype 2.
      Thus, in vivo, the significant effect of Mifepristone on SSTR2 and increased receptor signaling offsets the proliferative effects of this antagonist and may explain the lack of tumor growth in treated patients. Relacorilant may also induce PitNET apoptosis independently of SSTR activation (Supplemental Figure 10). In support of this notion, studies in several cancers that are driven by the dysregulation of these receptors, including leukemia, breast, prostate, lung and ovarian cancers, show promise of targeting the GR as a strategy for combination treatment.
      • Ahmad N.
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      Steroid hormone receptors in cancer development: a target for cancer therapeutics.
      ,
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      • Fleming G.F.
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      Overcoming Taxane resistance: preclinical and Phase 1 studies of Relacorilant, a selective Glucocorticoid Receptor Antagonist, with Nab-Paclitaxel in Solid Tumors.
      In addition, relacorilant induces apoptosis in vitro using pancreatic and ovarian cancer cell lines.
      • Greenstein A.E.
      • Hunt H.J.
      Glucocorticoid receptor antagonism promotes apoptosis in solid tumor cells.
      The pro-apoptotic effects of GR modulators are tissue specific and may be a result of targeting the apoptotic/cycle signaling pathways and genes such as Bcl2.
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      Tissue-specific actions of glucocorticoids on apoptosis: a double-edged sword.
      The mechanism of action of relacorilant on PitNET cell apoptosis requires further investigation. Collectively, these studies support broader investigation of relacorilant alone or in combination with somatostatin analogs as pre- or post-operative medical treatment in patients with CD.
      While many investigators have proposed using organoids in personalized medicine for the targeted treatment of cancers,
      • Steele N.G.
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      An Organoid-based preclinical model of human Gastric Cancer.
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      Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as pre-clinical models of Pancreatic Ductal Adenocarcinoma.
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      Organoid models of human and mouse ductal pancreatic cancer.
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      Organoid profiling identifies common responders to Chemotherapy in Pancreatic Cancer.
      this study is the first to execute this approach for the potential treatment of corticotroph type PitNETs associated with the development of CD. Existing medical therapy for CD remains suboptimal with negative impact on health and quality of life, including the considerable risk of therapy resistance and tumor recurrence. Our data demonstrate that organoids derived from corticotroph type PitNETs consist of differentiated cell lineages, stem/progenitor cells, and stromal cells that replicate much of the patient's own tumor pathology and function clearly documented by extensive high-throughput flow cytometry. Previously published in vitro experiments in the field of CD research were performed using pituitary cell lines or spheroids, aggregates and tumoroids that do not replicate the primary pituitary tumor microenvironment due to cell transformation and/or unphysiological 2D culture conditions.
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      Bombesin stimulates prolactin secretion from cultured rat pituitary tumour cells (GH4C1) via activation of phospholipase C.
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      • et al.
      The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells.
      Pituitary research has largely been conducted using cell culture techniques using rat (GH3) or mouse (AtT20) pituitary-like cell lines lacking a multicellular identify reflective of the PitNET.
      • Ikeda H.
      • Mitsuhashi T.
      • Kubota K.
      • Kuzuya N.
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      Epidermal growth factor stimulates growth hormone secretion from superfused rat adenohypophyseal fragments.
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      Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time.
      Recent advances have led to the development of pituitary tissue generated organoids, but these are limited to the use of transgenic mouse models as the source.
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      Organoids from pituitary as a novel research model toward pituitary stem cell exploration.
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      Nys et al.
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      reported the generation of human pituitary tumor organoids from a single stem cell as claim of true organoids due to the clonality. Unlike our studies, the multicellular complexity was not validated by the protein expression or hormone secretion from pituitary cell lineages in these cultures.
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      • Roose H.
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      It is fundamental to note that according to the National Cancer Institute (NCI, NIH), an ‘organoid’ is defined as “A tiny, 3-dimensional mass of tissue that is made by growing stem cells (cells from which other types of cells develop) in the laboratory.” Our cultures begin with both single and 3–4 cell clusters isolated from the native CD patient PitNET tissue that harbor the stem cells and begin a process of ‘budding’, growth and differentiation as documented by supplemental videos 2 and 3 and the comprehensive spectral flow cytometry analysis documenting functional cell lineages. Our PitNET organoids are consistent with gastrointestinal tissue derived organoids including that begin from cell clusters, crypts or glands.
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      ,
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      Establishment of Gastrointestinal Epithelial Organoids.
      ,
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      The use of murine-derived fundic organoids in studies of gastric physiology.
      Importantly, the videos show a different process than what would be an expected observation for the formation of a tumoroid which is the migration and adhesion of initially separate cells to form an aggregate. There are the reports of mouse nonadherent spheres with stem/progenitor characteristics,
      • Shintani A.
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      Isolation of PRRX1-positive adult pituitary stem/progenitor cells from the marginal cell layer of the mouse anterior lobe.
      and human embryonic stem cell generated spheroids or patient derived tumoroids that also lack a multicellular identify and consist of poorly differentiated cells.
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      Hypothalamic contribution to pituitary functions is recapitulated in vitro using 3d-cultured human iPS cells.
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      • Zhou Y.
      • Wilson R.R.A.
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      • McLemore J.
      • Sadri-Ardekani H.
      • Criswell T.
      Pituitary lineage differentiation from human-induced pluripotent stem cells in 2D and 3D cultures.
      • Zhang D.
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      A human ACTH-secreting corticotroph tumoroid model: novel human ACTH-Secreting Tumor Cell in vitro Model.
      Therefore, the findings reported here are significant because we offer an advanced in vitro approach that reveals cell populations expressing stem cell markers that potentially contribute to the support of tumor growth and targeted to prevent tumor recurrence. The ability of relacorilant to induce PitNET cell death in a multicellular culture system is the first step in the development of effective targeted therapies for patients with CD and potentially, other PitNET subtypes.

      Author contributions are as follow

      Saptarshi Mallick: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation, Software, Formal Analysis, Reviewing and Editing; Jayati Chakrabarti: Methodology, Software, Data curation, Writing – Original draft preparation, Validation, Formal Analysis, Reviewing and Editing; Jennifer Eschbacher: Resources, Validation, Reviewing and Editing; Andreas G. Moraitis: Conceptualization, Resources, Reviewing and Editing; Andrew Greenstein: Conceptualization, Resources, Reviewing and Editing; Jared Churko: Methodology, Validation, Formal Analysis, Reviewing and Editing; Kelvin W. Pond: Methodology, Validation, Formal Analysis, Reviewing and Editing; Antonia Livolsi: Methodology; Curtis Thorne: Resources; Andrew S. Little: Conceptualization, Writing – Original draft preparation, Validation, Reviewing and Editing, Resources, Visualization; Kevin Yuen: Conceptualization, Writing, Original draft preparation, Validation, Reviewing and Editing, Resources, Visualization; Yana Zavros: Conceptualization, Methodology, Software, Data curation, Writing – Original draft preparation, Software, Validation, Formal Analysis, Reviewing and Editing, Resources, Visualization, Investigation, Supervision.

      Conflicts of Interest

      All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare.

      Acknowledgments

      This research was supported by Department of Cellular and Molecular Medicine (University of Arizona College of Medicine) startup funds (Zavros). We acknowledge Maga Sanchez in the Tissue Acquisition and Cellular/Molecular Analysis Shared Resource (TACMASR University of Arizona Cancer Center) for assistance with embedding and sectioning of organoids. We would also like to acknowledge Patty Jansma (Marley Imaging Core, University Arizona) and, Douglas W Cromey (TACMASR imaging, University of Arizona Cancer Center) for assistance in microscopy. Research reported was partly supported by the National Cancer Institute of the National Institutes of Health under award number P30 CA023074 (Sweasy). We would like to acknowledge the assistance of Tina Schlafly (Corcept) for help with manuscript editing. Finally, the authors thank the patients who consented to donate tissues and blood for the development of the organoids. Without their willingness to participate in the study, this work would not be possible.

      Appendix. Supplementary materials

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