Data on GH and prolactin (PRL) co-secreting pituitary neuroendocrine tumors (GH/PRL-PitNETs), a subtype of acromegaly, remain limited. In particular, comparative analyses of clinical outcomes, including remission rates, between GH/PRL-PitNETs and GH-secreting pituitary neuroendocrine tumors (GH-PitNETs) are scarce. This study aimed to characterize and compare the clinical course and remission outcomes of GH/PRL-PitNETs and GH-PitNETs. We retrospectively analyzed 188 patients with acromegaly who underwent surgery between 2018 and 2024. GH/PRL-PitNETs (n = 46) were compared with GH-PitNETs (n = 142) in terms of their clinical, biochemical, and radiological features. Kaplan–Meier analysis was used to assess differences in biochemical remission between the groups, after which Cox proportional hazards modeling—including a time-dependent treatment variable—was used to identify covariates independently associated with remission. GH/PRL-PitNETs were significantly larger and more invasive than GH-PitNETs. The surgical remission rate was lower in the GH/PRL-PitNET group (P = .023). In multivariable Cox analysis, tumor size and cavernous sinus invasion independently predicted inferior outcome in biochemical remission. GH-PitNETs showed a higher incidence of medical comorbidities such as hypertension and thyroid carcinoma. GH/PRL-PitNETs demonstrated greater tumor burden and more pronounced structural invasiveness, resulting in a significantly lower likelihood of endocrinological remission.

Metformin is widely used as a first-line therapy for type 2 diabetes mellitus. However, the molecular mechanisms by which it modulates intestinal glucose metabolism remain incompletely defined. Here metformin was orally administered to male C57BL/6 mice, followed by intraperitoneal glucose tolerance testing and fluorine-18 fluorodeoxyglucose tracing to evaluate glucose homeostasis. To investigate changes in intestinal glucose metabolism, IEC6 and Caco-2 cell lines were used for in vitro analysis, with organoid experiments conducted for further validation. qRT-PCR, western blotting, flow cytometry and immunohistochemistry were performed to elucidate the effects of metformin on glucose metabolism pathways. Metformin enhanced glucose uptake and excretion in the distal intestine, particularly in the ileum and colon. Mechanistically, metformin upregulated the expression and membrane localization of glucose transporter 1 (GLUT1) by downregulating thioredoxin-interacting protein (TXNIP) expression. Consistently, intestinal-specific overexpression of TXNIP abolished metformin-induced improvements in glucose tolerance, while pharmacological inhibition of GLUT1 similarly negated metformin’s glucose-lowering effects. Our findings identified intestinal glucose excretion, mediated through the intestinal TXNIP–GLUT1 regulatory axis, as a previously unrecognized contributor to metformin’s glucoregulatory action. These results highlight a novel intestinal mechanism underlying metformin’s efficacy and provide insights for potential therapeutic strategies beyond traditional glucose regulation. Type 2 diabetes is a common health issue, and metformin is a widely used medication to manage it. Researchers explored how metformin affects glucose handling in the intestines. The researchers gave metformin to mice and observed increased glucose uptake and excretion in the intestines, particularly in the ileum and colon. They found that metformin enhances the activity of a protein called GLUT1, which helps transport glucose across cell membranes. This effect was linked to a decrease in another protein, TXNIP, which usually inhibits GLUT1. The study showed that metformin’s ability to lower blood sugar involves increasing GLUT1 activity by reducing TXNIP levels. This mechanism does not rely on AMPK (a protein involved in energy regulation), suggesting a new pathway for metformin’s action. These findings could lead to better diabetes treatments by targeting the intestinal TXNIP–GLUT1 pathway. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

[This corrects the article DOI: 10.3389/fendo.2025.1527275.].

ObjectiveGraves’ orbitopathy (GO) is characterized by increased production of proinflammatory cytokines and hyaluronic acid by fibroblasts and their differentiation into adipocytes in response to immunologic stimuli. The suppressor of cytokine signaling-3 (SOCS3) is an inducible negative regulator of the JAK/STAT pathway, implicated in various inflammatory diseases. In this study, we investigated the role of SOCS3 in the inflammatory and adipogenic pathogenesis of GO.MethodsTranscriptome profiling of orbital tissues obtained from five patients with GO who underwent orbital decompression surgery and four healthy subjects was performed using RNA-sequencing. Among the top-ranked differentially expressed genes, we identified 24 hub genes and found SOCS3 to be the most significantly upregulated gene in GO samples compared with that in healthy tissue based on quantitative real-time polymerase chain reaction. SOCS3 expression was analyzed in IL-1β-, and IGF-1-stimulated orbital fibroblasts using quantitative real-time polymerase chain reaction and western blot analysis. Knockdown of SOCS3 using siRNA transfection was performed to assess the effect of SOCS3 on the production of proinflammatory cytokines and adipogenic phenotype.ResultsWe identified 184 consistently differentially expressed genes—120 upregulated and 64 downregulated— in GO tissues compared to the control. SOCS3 mRNA expression was significantly higher in GO tissues (n = 17) compared with that in control (n = 15). IL-1β and IGF-1 enhanced the expression of SOCS3 at mRNA and protein levels. Silencing of SOCS3 suppressed the levels of IL-1β-induced proinflammatory cytokines, including IL-6, IL-8, and ICAM-1. Phosphorylation of NF-kB and Akt was suppressed and adipogenic differentiation was significantly attenuated by SOCS3 knockdown.ConclusionsSOCS3 was remarkably expressed in the adipose tissues of patients with GO and was induced by IL-1β and IGF-1 in orbital fibroblasts. SOCS3 inhibition attenuated the production of proinflammatory cytokines and adipogenesis, suggesting that SOCS3 may be a therapeutic target for controlling the inflammatory and adipogenic mechanisms in GO.

Background This study aimed to determine the value of 68Ga-DOTATOC positron emission tomography/computed tomography (PET/CT) in localizing adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Methods In this retrospective cohort study, we enrolled 30 patients with Cushing’s disease and positive ACTH immunoreactivity. All patients underwent 68Ga-DOTATOC PET/CT and pituitary magnetic resonance imaging (MRI) before transsphenoidal adenomectomy. Results Twenty-five patients showed 68Ga-DOTATOC uptake in their pituitary glands on PET/CT. Median age, pre-operative ACTH levels, pre-operative cortisol, and tumor size on MRI were comparable irrespective of DOTATOC uptake. 68Ga-DOTATOC PET/CT showed a 77% success rate for localizing adenomas, which was not statistically different from that of MRI. The ACTH level in the successful localization group was significantly higher than that in the failed group (84.41 pg/mL vs. 37.26 pg/mL, P=0.001). The ACTH level was statistically significant predictor of successful localization using 68Ga-DOTATOC PET/CT (P=0.013). The area under the curve was 0.932 with a cutoff of 53.86 pg/mL for ACTH levels to determine successful localization. Pre-operative ACTH levels above 53.86 pg/mL showed the best diagnostic accuracy in predicting the success of localizing adenomas (sensitivity, 91.3%; specificity, 85.7%). Mean and maximum standardized uptake value of adenoma negatively correlated to pre-operative ACTH level. Conclusion Plasma ACTH level is a favorable predictor for the successful localization and negative correlation with 68Ga-DOTATOC uptake of corticotroph adenomas in 68Ga-DOTATOC PET/CT. 68Ga-DOTATOC PET/CT did not improve tumor localization for Cushing’s disease compared with MRI alone.

Thyrogen, a recombinant human thyroid-stimulating hormone (rhTSH), has a short half-life in the bloodstream, which necessitates multiple doses during treatment. Therefore, we developed a new long-acting rhTSH using anti-serum albumin Fab-associated (SAFA) technology to validate its biological activity through in vitro assays, pharmacokinetic studies in healthy mice and pharmacodynamics studies in a thyroid-stimulating hormone (TSH)-suppressed mouse model. SAFA-TSH was produced using a Chinese hamster ovary expression system. To verify its biological activity, we generated Nthy-ori 3-1 cells stably overexpressing TSHR and measured the production of cyclic adenosine monophosphate (cAMP). In a rat study, slow-release triiodothyronine (T3) pellets were implanted 3 days before administering Thyrogen or SAFA-TSH to measure the amount of thyroxine (T4) release alone resulting from exogenous administration. SAFA-TSH increased cAMP production dose-dependently, but less effectively than Thyrogen at similar concentrations. SAFA-TSH required six times the dose of Thyrogen to achieve similar cAMP levels, likely due to differences in molecular weight and relative bioactivity. In a rat study, SAFA-TSH produced elevated thyroid hormone levels well after the decline in the response to Thyrogen. SAFA-TSH had significantly higher cumulative effects on T4 and free T4 levels compared with Thyrogen, as observed by a more than two-fold higher average area under the effect curve of 262.56 vs 118.89 μg × h/dL and 127.47 vs 60.75 μg × h/dL, respectively. SAFA technology created successful long-acting TSH that demonstrated bioactivity. These findings endorse the continued development of SAFA-TSH for clinical use, highlighting its potential as a significant advancement treating thyroid cancer patients.

OBJECTIVE:Dopamine agonist (DA) treatment is effective for hyperprolactinemia and reduces tumor size in patients with prolactinoma; however, prolonged DA administration without prolactinoma causes fibrosis around tumor tissues. Therefore, we aimed to identify circulating microRNAs (miRNAs) as potential biomarkers to predict prolactinoma in patients with hyperprolactinemia and pituitary tumors. DESIGN:Plasma samples were collected from 3 comparison groups: (1) patients clinically diagnosed with prolactinoma vs nonfunctioning pituitary adenoma (NFPA) based on response to cabergoline treatment, (2) patients with surgically confirmed prolactinoma vs NFPA, and (3) patients before and after cabergoline treatment. Candidate miRNAs from the initial nCounter assay were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in a larger cohort of 247 patients with hyperprolactinemia and 37 controls. METHODS:The nCounter assay was used for miRNA expression profiling, and the qRT-PCR validated the candidate miRNAs in the plasma and tumor tissue samples. Total RNA sequencing was conducted on pituitary tumor tissues to identify transcriptomic alterations. Furthermore, candidate miRNA target genes and their biological roles were analyzed using prolactinoma cell lines. RESULTS:Three miRNA candidates (miR-20a-5p, miR-424-5p, and miR-514a-5p) were selected by analyzing 3 sets of expression comparisons between the 2 groups. Furthermore, the relative miR-20a-5p expression significantly increased in prolactinoma compared with that in normal pituitary glands, NFPA, growth hormone-secreting pituitary adenoma, and adrenocorticotropic hormone-secreting pituitary adenoma. In MMQ and GH4 cells, miR-20a-5p inhibition decreased prolactinoma cell proliferation and prolactin secretion. CONCLUSIONS:Circulating miR-20a-5p is a potential biomarker for prolactinoma, which could be associated with responsiveness to DAs.

Abstract Disclosure: Y. Kim: None. Introduction: Age-related decline in endocrine function has been associated with multiple physiological changes. However, aging-associated molecular shifts in the pituitary gland have not yet been systematically investigated. This study aimed to investigate the mechanism underlying these age-related molecular changes in young and old mice model using single cell transcriptomics. Method: Single-cell RNA sequencing (scRNA-seq) were conducted in young (2-month) and old (20-month) mouse. We used systems biology techniques to build a specific protein interaction network for somatotrophs, integrating validated interactions and gene expression data. By simulating drug effects on this network, we predicted changes in transcription factor activities due to aging. We identified potential therapeutic targets for preventing aging in somatotrophs by assessing the anti-aging effects of chemical perturbations, similar to methods used in the Connectivity Map project. Results: We identified 14 distinct cell types including follicular stellate cells (FSCs), somatotrophs, corticotrophs, gonadotrophs, lactotrophs, and melanotrophs. We found that the effect of aging on transcription was cell type dependent. FSC subtypes are heterogeneously regulated by aging at the transcriptomic level. However, somatotroph subtypes shared functional alterations with aging in terms of mRNA surveillance and spliceosome pathways. Our systems biology approach identified SENP1(Sentrin specific peptidase gene), as a critical node in the regulatory network affecting somatotroph aging. The senescence-associated beta-galactosidase staining (SAbgal) showed that Momordin (Mc), a SENP1 inhibitor, had anti-aging effects on somatotrophs, regardless of age-promoting doxorubicin. Western blot analysis indicated a two-fold increase in growth hormone levels in aged GH3 cells treated with Mc compared to vehicle. Conclusion: Our data and analysis provide promising resources to better understand the molecular shifts of pituitary aging at the transcriptomic level and help the community generate interesting hypotheses to develop anti-aging strategies for the pituitary gland. We suggest that SENP1 inhibitors may represent potential anti-aging targets in somatotroph. Presentation: 6/3/2024

Background: Recombinant human follicle-stimulating hormone (rhFSH) is commonly used to treat female infertility, but its short half-life necessitates multiple doses. Even corifollitropin alfa, with an extended half-life, requires supplementary injections of rhFSH after 7 days. This study aimed to develop and evaluate a long-acting follicle-stimulating hormone (FSH) formulation using anti-serum albumin Fab-associated (SAFA) technology to avoid additional injections and enhance ovarian function.Methods: SAFA-FSH was synthesized using a Chinese hamster ovary expression system. Its biological efficacy was confirmed through assays measuring its ability to stimulate cyclic adenosine monophosphate (cAMP) production, estradiol synthesis, and the expression of human cytochrome P450 family 19 subfamily A member 1 (hCYP19α1) and human steroidogenic acute regulatory protein (hSTAR) in human ovarian granulosa (KGN) cells. To evaluate the effects of SAFA-FSH, we compared its impact on serum estradiol levels and ovarian weight increase with that of rhFSH in Sprague-Dawley (SD) rats using the modified Steelman-Pohley test.Results: The results indicated that SAFA-FSH induces cAMP synthesis in KGN cells and upregulates the expression of hCYP19α1 and hSTAR in a dose-dependent manner. Female SD rats, aged 21 days, receiving daily subcutaneous human chorionic gonadotropin injections for 5 days exhibited a significant increase in serum estradiol levels and ovarian weight when administered SAFA-FSH on the first day or when given nine injections of rhFSH over 5 days. Notably, the group receiving SAFA-FSH on the first and third days demonstrated an even greater rise in serum estradiol levels and ovarian weight.Conclusion: These findings suggest that SAFA-FSH presents a promising alternative to current rhFSH treatments for female infertility. However, further research is essential to thoroughly assess its safety and efficacy in clinical contexts.

Abstract Disclosure: K. Kim: None. The role of the spliceosome in the development of pituitary tumors is not well understood, despite preliminary studies indicating abnormal expression of oncogenic splicing variants in these neuroendocrine tumors. We aimed to identify differentially expressed spliceosome components in growth hormone (GH)-secreting pituitary tumors and investigate their roles in pathogenesis.We performed transcriptome analysis of 20 somatotroph adenomas and 6 normal pituitary tissues to select dysregulated spliceosome components. Clinical characteristics were analyzed based on gene expression in 64 patients with acromegaly. GH secreting pituitary adenomas were confirmed by 75 g oral glucose tolerance and immunohistochemical staining. Proliferation, invasion, and hormonal activity of GH secreting pituitary adenoma cells were investigated after modulating gene expression.TCERG1 expression was significantly higher in somatotroph adenomas than in normal cells (log2 fold change 0.59, adjusted P=0.0002*). Genotype-phenotype analysis revealed that patients with higher TCERG1 expression had lower surgical remission rates than those with lower expression (63.64% vs. 95.45%, P=0.009*). TCERG1 expression was significantly higher in groups with cavernous sinus (CS) invasion or Ki67 index over 1.5 (2.06 vs. 1.65, P=0.011*; 2.02 vs. 1.66, P=0.043*). TCERG1 overexpression led to a 16.91% increase in proliferation after 72 h (P<0.001*) and a 249.47% increase in invasion after 48 h in GH3 cells (P=0.026*). Conversely, TCERG1 silencing significantly decreased cell proliferation (10.15% at 72 h, P<0.001*) and invasion (96.87% at 48 h, P=0.029*). Vimentin was increased, but E-cadherin was decreased in both TCERG1 overexpressed GH3 cells and somatotroph adenomas. And TCERG1 silence reversed the expression of the genes (CDH2, SNAI1, ZEB2, and VIM) in GH3 cells. Additionally, GH secretion increased when TCERG1 was overexpressed in GH3 cells. Spliceosome machinery provide novel insights into the pathogenesis of GH-secreting pituitary tumor and highlight the potential role of TCERG1 as a therapeutic target. Presentation: 6/2/2024

Purpose We aimed to identify differentially expressed spliceosome components in growth hormone (GH)-secreting pituitary tumors and investigate their roles in pathogenesis. Methods We performed transcriptome analysis of 20 somatotroph adenomas and 6 normal pituitary tissues to select dysregulated spliceosome components. Clinical characteristics were analyzed based on gene expression in 64 patients with acromegaly. Proliferation, invasion, and hormonal activity of GH secreting pituitary adenoma cells were investigated. Results TCERG1 expression was significantly higher in somatotroph adenomas than in normal pituitaries (log2 fold change 0.59, adjusted P = 0.0002*). Genotype-phenotype analysis revealed that patients with higher TCERG1 expression had lower surgical remission rates than those with lower expression (63.64% vs. 95.45%, P = 0.009*). TCERG1 expression was significantly higher in groups with cavernous sinus (CS) invasion or Ki67 index over 3 (all P>0.05*). TCERG1 overexpression led to a 29.60% increase in proliferation (P<0.001*) and a 249.47% increase in invasion after 48 h in GH3 cells (P = 0.026*). Conversely, TCERG1 silencing significantly decreased cell proliferation (25.76% at 72 h, P<0.001*) and invasion (96.87% at 48 h, P = 0.029*). E-cadherin was decreased, but vimentin was increased in both TCERG1 overexpressed GH3 cells and somatotroph adenomas. And TCERG1 silence reversed the expression of the genes (CDH2, SNAI1, ZEB2, and VIM) in GH3 cells. Conclusions Spliceosome machinery provide novel insights into the pathogenesis of GH-secreting pituitary tumor and highlight the potential role of TCERG1 as a biomarker for tumor aggressiveness.

This study investigated differential gene expression between granulation patterns in growth hormone (GH)-secreting pituitary tumors, aiming to elucidate novel transcriptomes that explain clinical variances in patients with acromegaly. Transcriptome analysis was conducted on 6 normal pituitary tissues and 15 GH-secreting pituitary tumors, including 9 densely granulated somatotroph tumors (DGSTs) and 6 sparsely granulated somatotroph tumors (SGSTs). We identified 3111 differentially expressed genes (DEGs) in tumors compared to normal pituitaries, with 1117 DEGs unique to a specific granulation within tumors. SGST showed enrichment of neuronal development and acute inflammatory response pathways, along with a significant enhancement of JAK-STAT, phosphatidylinositol 3-kinase, and MAPK signaling. The results suggest that granulation-specific gene expression may underpin diverse clinical presentations in acromegaly, highlighting the potential for further investigation into these transcriptomic variations and their roles in disease pathology, particularly the involvement of genes linked to neuronal development, inflammatory response, and JAK-STAT signaling in SGST.

Abstract Disclosure: C. Ku: None. C. Kang: None. J. Oh: None. E. Wang: None. H. Song: None. K. Park: None. B. Kim: None. K. Kim: None. S. You: None. S. Park: None. E. Lee: None. Background: Forty to sixty percent of acromegalic patients undergo medical treatment, utilizing somatostatin analogues, dopamine agonists, and GH receptor (GHR) antagonists. The GHR antagonist, pegvisomant exhibits the most robust efficacy in reducing IGF-1 levels in acromegalic patients. However, pegvisomant faces several clinical limitations, such as poor compliance due to the necessity of daily injections. This study aimed to develop a novel, long-acting fusion GHR antagonist with optimized therapeutic efficacy and duration. Method: Twelve mutant proteins with alterations in the C-terminal and N-terminal regions of GHR were generated to confirm the GHR binding affinity and inhibitory potency compared with pegvisomant, which was evaluated with STAT5b luciferase reporter assay. Several carriers were engineered into selected mutant GHRs to increase the therapeutic duration. For in vivo experiments, single or repetitive injection of GHR antagonists were applied to acromegalic mouse model (somatotroph specific Aip knock out (sAIPKO) mice). Pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated in New Zealand White (NZW) rabbits. Result: Four out of the 12 GHR mutants exhibited higher affinity and stronger inhibitory potency toward human GHR when compared to pegvisomant. Specifically, three GHR mutants significantly reduced serum IGF-1 levels in sAIPKO mice following daily injections of 20 mg/kg for 7 days (52.3±6.0 to 5.9±1.3, 50.3±3.5 to 32.3±2.3, and 49.8±3.7 to 13.9±4.3 ng/mL; all p < 0.05). Among them, S4020N exhibited the lowest EC50 value for GHR binding affinity in in vitro analysis containing human GHR (0.594 nM) and mouse GHR (0.891 nM). Following fusion with each long-acting carrier, S4020N with the Fc carrier protein (ALT-B5) demonstrated the highest binding affinity and the strongest inhibitory potency toward human GHR, surpassing pegvisomant by 14 and 25 times, respectively. Administration of ALT-B5 (80 mg/kg) and pegvisomant (24 mg/kg) at a 3-day interval revealed the superior efficacy of ALT-B5 over pegvisomant (1879±219.4 vs. 3800±355.9 ng*hr/mL IGF-1 AUC for 5 days in ALT-B5 and pegvisomant, respectively; p < 0.01). In PK/PD studies, ALT-B5 was administered at doses of 3 mg/kg and 10 mg/kg in NZW rabbits, and PK/PD parameters were compared with the same molar concentration of pegvisomant. ALT-B5 exhibited the longest therapeutic duration and the highest efficacy compared to pegvisomant. Conclusion: ALT-B5 exhibited the longest duration and highest effectiveness in lowering IGF-1 levels. ALT-B5 emerges as a potential novel therapeutic option for acromegaly. Presentation: 6/1/2024

Prolactinomas are the most prevalent type of pituitary neuroendocrine adenomas, primarily affecting women of reproductive age. Unlike other pituitary tumors, the first-line management has traditionally been pharmacological rather than surgical. This preference is due to the effectiveness of dopamine agonists (DAs), which typically reduce tumor size and normalize prolactin levels in most patients. However, this does not imply that there is no room for improvement; the duration of treatment and medication side effects often lead to compliance issues among patients. Recent advances in surgical techniques and molecular biology have paved the way for the development of precision medicine, allowing for more flexible and personalized treatment strategies for prolactinomas. This review aims to enhance clinical decision-making and patient care for endocrinologists by focusing on several key factors: predictive markers of DA sensitivity, clinical characteristics and suitability for transsphenoidal adenomectomy as a potential first-line treatment, factors determining the successful withdrawal of DAs after prolonged use, safety concerns during pre/post-pregnancy and breastfeeding, and determinants of tumor aggressiveness. Through tailored therapy—a patient-focused, multidisciplinary approach— we aim to improve the management of prolactinoma patients.

Cushing's disease is associated with increased morbidity and mortality. Osilodrostat, a potent oral 11β-hydroxylase inhibitor, provided rapid, sustained mean urinary free cortisol (mUFC) normalization in Cushing's disease patients in two Phase III studies (LINC 3, NCT02180217; LINC 4, NCT02697734). Here, we evaluate the efficacy and safety of osilodrostat in Cushing's disease in patients of Asian origin compared with patients of non-Asian origin. Pooled data from LINC 3 and LINC 4 were analyzed. Outcomes were evaluated separately for Asian and non-Asian patients. For the analysis, 210 patients were included; 56 (27%) were of Asian origin. Median (minimum-maximum) osilodrostat dose was 3.8 (1-25) and 7.3 (1-47) mg/day in Asian and non-Asian patients, respectively. mUFC control was achieved at weeks 48 and 72 in 64.3% and 68.1% of Asian and 68.2% and 75.8% of non-Asian patients. Improvements in cardiovascular and metabolic-related parameters, physical manifestations of hypercortisolism, and quality of life were similar in both groups. Most common adverse events (AEs) were adrenal insufficiency (44.6%) in Asian and nausea (45.5%) in non-Asian patients. AEs related to hypocortisolism and pituitary tumor enlargement occurred in more Asian (58.9% and 21.4%) than non-Asian patients (40.3% and 9.1%). Of Asian and non-Asian patients, 23.2% and 13.6%, respectively, discontinued because of AEs. Asian patients with Cushing's disease generally required numerically lower osilodrostat doses than non-Asian patients to achieve beneficial effects. Hypocortisolism-related AEs were reported in more Asian than non-Asian patients. Together, these findings suggest that Asian patients are more sensitive to osilodrostat than non-Asian patients.

B lymphocytes play a pivotal role in the adaptive immune system by facilitating antibody production. Young B cell progenitors originate in the bone marrow and migrate to the spleen for antigen-dependent maturation, leading to the development of diverse B cell subtypes. Thus, tracking B cell trajectories through cell type distinction is essential for an appropriate checkpoint assessment. Despite its significance, monitoring specific B cell subclasses in live states has been hindered by a lack of suitable molecular tools. In this study, we introduce CDoB as the first mature B cell-selective probe, enabling real-time discrimination of three classified stages in B-cell development: progenitor, transitional, and mature B cells, through a single analysis using CyTOF. The selective mechanism of CDoB, elucidated as gating-oriented live-cell distinction (GOLD), targets SLC25A16, identified through systematic screening of SLC-CRISPRa and CRISPRi libraries. CDoB selectively brightens mature B cells in the mitochondrial area using SLC25A16 as the main gate, and the staining intensity correlates positively with the expression level of SLC25A16 along the B cell maturation continuum. In spleen tissues, CDoB demonstrates selective marking in mature B cell areas in live tissue status, representing the first performance achieved by a small-molecule fluorescent probe.

Pituitary adenoma-induced excess endocrine growth hormone (GH) secretion can lead to breast cancer development and metastasis. Herein, we used an acromegaly mouse model to investigate the role of excess endocrine GH on triple-negative breast cancer (TNBC) growth and metastasis. Additionally, we aimed to elucidate the molecular mechanism of transcription factor 20 (TCF20)/nuclear factor erythroid 2-related factor 2 (NRF2) signaling-mediated aggressiveness and metastasis of TNBC. Excess endocrine GH induced TCF20 activates the transcription of NRF2 and NRF2-target genes to facilitate TNBC metastasis. Inhibition of GH receptor (GHR) and TCF20 activity using the GHR antagonist or small-interfering RNA-induced gene knockdown resulted in reduced tumor volume and metastasis, suggesting that excess endocrine GH stimulates TCF20/NRF2 pathways in TNBC and promotes metastasis to the lung. GHR inhibitors present an effective therapeutic strategy to prevent TNBC cell growth and metastasis. Our findings revealed functional and mechanistic roles of the GH-TCF20-NRF2 signaling axis in TBNC progression.

Abstract Disclosure: J. Oh: None. C. Kang: None. E. Wang: None. S. Lee: None. J. Nam: None. C. Ku: None. E. Lee: None. Introduction Acromegaly is a rare disease characterized by chronic growth hormone (GH) and insulin-like growth factor-1 hypersecretion. In previous studies, it is known that the incidence of breast cancer increases in patients with acromegaly. For breast cancers, autocrine GH and GHR are expressed in primary lesion, which is significantly increased in metastatic one. To date, however, the precise mechanism of endocrine GH in breast cancer metastasis has not been defined. This study aimed to investigate the role of endocrine excess GH facilitating breast cancer metastasis in acromegaly mice model. Method We used Aiplox/lox (Con) and rGHp-Cretg/+; Aiplox/lox mice (Acro), which was previously established to model the phenotype of human acromegaly. To generate orthotopic xenograft model, we used C57BL/6 female mouse derived breast cancer cell line Py230 cells. Py230 cells were injected into mammary gland and tumor volumes were measured every 2 days. Tumor growth and metastasis were confirmed by bioluminescent in vivo imaging system (IVIS). Also, we investigated the effects of GH on triple negative breast cancer cell lines, which have the higher expression of GHR. To define GH induced pathway, we performed RT2 array. Cell proliferation was investigated with MTT assay and cell migration was investigated with wound healing assay and transwell invasion assay. Results In mouse orthotopic xenograft studies, tumor volumes of Acro mice were higher than that of Con mice (increase of 310%, 846.04±290.7 vs. 205.97±33.7 mm3, p=0.001). The tumor volume and serum GH level showed positive correlation (R2= 0.4739, p=0.027). After 14 weeks, the Acro mice showed significantly increased bioluminescence activity in the lung (increase of 1023%, 494666.7±104668.2 vs. 44037.7±6641.9 AU, p=0.0127). When GHR antagonist was treated, tumor growth and lung metastasis were decreased. We excised primary tumor and lung metastatic foci and performed RT2 array. We found Tcf-20 gene, which is upregulated in both primary tumor and lung metastatic burden (increase of 216%, p=0.0153). We confirmed GH induced Tcf-20 gene induction (increase of 57% (p=0.043), 41% (p=0.05), 67% (p=0.0021) in Hs578T, Py230 (p=0.05), and Hcc1806 cells each). We knocked down Ghr or Tcf-20 genes and observed decrease of invasion and migration compared to GH treated siCON HCC1806 (decrease of 64.5% in siGHR, decrease of 83.3% in siTCF-20, compared to siCON cells, p=0.000819977) and Py230 cells (decrease of 47.3% in siGHR transfected, decrese of 49.3% in siTCF-20 transfected, compared to siCON cells, p=0.0012). Conclusion Excess GH in acromegaly possess a strong potential to promotes tumor development and metastasis. We identified a molecular mechanism by which GH-induced TCF-20 promote tumor aggression and metastasis. These findings can aid in the development of improved strategies for managing cancer in acromegalic patients. Presentation: Thursday, June 15, 2023

Abstract Disclosure: D. Kim: None. Y. Bao: None. M. Kang: None. Y. Cho: None. Y. Kim: None. Y. Hwang: None. C. Ku: None. E. Lee: None. Objectives: Over years, various approaches have been used to model human Graves’ disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunizations with the extracellular A subunit of the human thyrotropin receptor (hTSHRa). However, these models require a lot of effort for multiple dosing over several months and the results are very heterogeneous, limiting their use as a platform for the development of new therapeutics. Thus, we developed a novel Cre-loxP based mouse model for Graves' disease. Methods: To overcome the limitation of existing animal models, we made ESC-derived KI mouse expressing hTSHRa that will act as an antigen in a conditional manner by inserting the CAG-loxP-STOP-loxP-hTSHRa cassette into the mouse Rosa26 locus. TAT-Cre, a recombinant cell-permeant fusion cre-recombinase protein, was used for site-specific recombination to avoid leaky expression which may induce immune tolerance. Various concentrations of TAT-Cre were tested to avoid neutralization and produce adequate hyperthyroidism. Serum TSHR Ab and T4 levels were measured. Immunohistochemical examinations of the thyroid gland and orbits were performed. Results: Treatment with TAT-Cre within the range of 250-750U produced the most efficient TSHR Ab and significantly increased T4. Four weeks after TAT-Cre 500U administration, TSHR Ab levels were already significantly produced in all mice and T4 levels started to increase. At 8 and 12 weeks, TSHR Ab levels were produced at a very high levels in all mice and T4 levels were increased to a very high levels in over 80% of mice. After 3 months, MRI images did not show any significant protrusion. However, there was an enlargement in the volume of the orbital muscles. We also found thyroid enlargement and hypertrophy, orbital macrophage infiltration and fibrosis in Immunohistochemical examinations. Conclusions: A new mouse model for Graves' disease was successfully established by using the Cre-loxP system. After being optimized for orbitopathy induction, this model can be used as a platform for the development of novel therapeutics. Presentation: Friday, June 16, 2023