Exploring stem cell biology in pituitary tumors and derived organoids.

Pituitary tumorigenesis is highly prevalent and causes major endocrine disorders. Hardly anything is known on the behavior of the local stem cells in this pathology. Here, we explored the stem cells' biology in mouse and human pituitary tumors using transcriptomic, immunophenotyping and organoid approaches. In the prolactinoma-growing pituitary of dopamine receptor D2 knock-out mice, the stem cell population displays an activated state in terms of proliferative activity and distinct cytokine/chemokine phenotype. Organoids derived from the tumorous glands' stem cells recapitulated these aspects of the stem cells' activation nature. Upregulated cytokines, in particular interleukin-6, stimulated the stem cell-derived organoid development and growth process. In human pituitary tumors, cells typified by expression of stemness markers, in particular SOX2 and SOX9, were found present in a wide variety of clinical tumor types, also showing a pronounced proliferative status. Organoids efficiently developed from human tumor samples, displaying a stemness phenotype as well as tumor-specific expression fingerprints. Transcriptomic analysis revealed fading of cytokine pathways at organoid development and passaging, but their reactivation did not prove capable of rescuing early organoid expansion and passageability arrest. Taken together, our study revealed and underscored an activated phenotype of the pituitary-resident stem cells in tumorigenic glands and tumors. Our findings pave the way to defining the functional position of the local stem cells in pituitary tumor pathogenesis, at present barely known. Deeper insight can lead to more efficient and targeted clinical management, currently still not satisfactorily.

Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

Organoids from pituitary as a novel research model toward pituitary stem cell exploration.

The pituitary is the master endocrine gland, harboring stem cells of which the phenotype and role remain poorly characterized. Here, we established organoids from mouse pituitary with the aim to generate a novel research model to study pituitary stem cell biology. The organoids originated from the pituitary cells expressing the stem cell marker SOX2 were long-term expandable, displayed a stemness phenotype during expansive culture and showed specific hormonal differentiation ability, although limited, after subrenal transplantation. Application of the protocol to transgenically injured pituitary harboring an activated stem cell population, resulted in more numerous organoids. Intriguingly, these organoids presented with a cystic morphology, whereas the organoids from undamaged gland were predominantly dense and appeared more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes and showed that cystic organoids more resembled the pituitary phenotype, at least to an immature state, and displayed in vitro differentiation, although yet moderate. Organoid characterization further exposed facets of regulatory pathways of the putative stem cells of the pituitary and advanced new injury-activated markers. Taken together, we established a novel organoid research model revealing new insights into the identity and regulation of the putative pituitary stem cells. This organoid model may eventually lead to an interesting tool to decipher pituitary stem cell biology in both healthy and diseased gland.

Major depletion of SOX2+ stem cells in the adult pituitary is not restored which does not affect hormonal cell homeostasis and remodelling.

The pituitary gland contains SOX2-expressing stem cells. However, their functional significance remains largely unmapped. We investigated their importance by depleting SOX2+ cells through diphtheria toxin (DT)-mediated ablation. DT treatment of adult Sox2CreERT2/+;R26iDTR/+ mice (after tamoxifen-induced expression of DT receptor in SOX2+ cells) resulted in 80% obliteration of SOX2+ cells in the endocrine pituitary, coinciding with reduced pituisphere-forming activity. Counterintuitively for a stem cell population, the SOX2+ cell compartment did not repopulate. Considering the more active phenotype of the stem cells during early-postnatal pituitary maturation, SOX2+ cell ablation was also performed in 4- and 1-week-old animals. Ablation grade diminished with decreasing age and was accompanied by a proliferative reaction of the SOX2+ cells, suggesting a rescue attempt. Despite this activation, SOX2+ cells did also not recover. Finally, the major SOX2+ cell depletion in adult mice did not affect the homeostatic maintenance of pituitary hormonal cell populations, nor the corticotrope remodelling response to adrenalectomy challenge. Taken together, our study shows that pituitary SOX2+ fail to regenerate after major depletion which does not affect adult endocrine cell homeostasis and remodelling. Thus, pituitary SOX2+ cells may constitute a copious stem cell reserve or may have other critical role(s) still to be clearly defined.

The Stem Cell Connection of Pituitary Tumors.

Tumors in the pituitary gland are typically benign but cause serious morbidity due to compression of neighboring structures and hormonal disruptions. Overall, therapy efficiency remains suboptimal with negative impact on health and comfort of life, including considerable risk of therapy resistance and tumor recurrence. To date, little is known on the pathogenesis of pituitary tumors. Stem cells may represent important forces in this process. The pituitary tumors may contain a driving tumor stem cell population while the resident tissue stem cells may be directly or indirectly linked to tumor development and growth. Here, we will briefly summarize recent studies that afforded a glance behind the scenes of this stem cell connection. A better knowledge of the mechanisms underlying pituitary tumorigenesis is essential to identify more efficacious treatment modalities and improve clinical management.

An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAFV600E inhibitor-resistant metastatic melanoma cells.

The ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeutic-agent targeting BRAFV600E. Previous studies indicated a tight correlation between heightened melanoma-associated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAFV600E inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosome-mediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAFV600E inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAFV600E inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.

Pituitary stem cell regulation: who is pulling the strings?

The pituitary gland plays a pivotal role in the endocrine system, steering fundamental processes of growth, metabolism, reproduction and coping with stress. The adult pituitary contains resident stem cells, which are highly quiescent in homeostatic conditions. However, the cells show marked signs of activation during processes of increased cell remodeling in the gland, including maturation at neonatal age, adaptation to physiological demands, regeneration upon injury and growth of local tumors. Although functions of pituitary stem cells are slowly but gradually uncovered, their regulation largely remains virgin territory. Since postnatal stem cells in general reiterate embryonic developmental pathways, attention is first being given to regulatory networks involved in pituitary embryogenesis. Here, we give an overview of the current knowledge on the NOTCH, WNT, epithelial-mesenchymal transition, SHH and Hippo pathways in the pituitary stem/progenitor cell compartment during various (activation) conditions from embryonic over neonatal to adult age. Most information comes from expression analyses of molecular components belonging to these networks, whereas functional extrapolation is still very limited. From this overview, it emerges that the 'big five' embryonic pathways are indeed reiterated in the stem cells of the 'lazy' homeostatic postnatal pituitary, further magnified en route to activation in more energetic, physiological and pathological remodeling conditions. Increasing the knowledge on the molecular players that pull the regulatory strings of the pituitary stem cells will not only provide further fundamental insight in postnatal pituitary homeostasis and activation, but also clues toward the development of regenerative ideas for improving treatment of pituitary deficiency and tumors.

Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.

The endometrium, which is of crucial importance for reproduction, undergoes dynamic cyclic tissue remodeling. Knowledge of its molecular and cellular regulation is poor, primarily owing to a lack of study models. Here, we have established a novel and promising organoid model from both mouse and human endometrium. Dissociated endometrial tissue, embedded in Matrigel under WNT-activating conditions, swiftly formed organoid structures that showed long-term expansion capacity, and reproduced the molecular and histological phenotype of the tissue's epithelium. The supplemented WNT level determined the type of mouse endometrial organoids obtained: high WNT yielded cystic organoids displaying a more differentiated phenotype than the dense organoids obtained in low WNT. The organoids phenocopied physiological responses of endometrial epithelium to hormones, including increased cell proliferation under estrogen and maturation upon progesterone. Moreover, the human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the morpho-histological and molecular levels. Together, we established an organoid culture system for endometrium, reproducing tissue epithelium physiology and allowing long-term expansion. This novel model provides a powerful tool for studying mechanisms underlying the biology as well as the pathology of this key reproductive organ.

Regeneration in the Pituitary After Cell-Ablation Injury: Time-Related Aspects and Molecular Analysis.

We recently showed that the mouse pituitary holds regenerative competence. Young-adult GHCre/iDTR mice, expressing diphtheria toxin (DT) receptor in GH-producing cells, regenerate the GH(+) cells, as ablated by 3-day DT treatment (3DT), up to 60% after 5 months. The pituitary's stem cells participate in this restoration process. Here, we characterized this regenerative capacity in relation to age and recovery period and started to search for underlying molecular mechanisms. Extending the recovery period (up to 19 mo) does not result in higher regeneration levels. In addition, the regenerative competence disappears at older age, coinciding with a reduction in pituitary stem cell number and fitness. Surprisingly, prolonging DT treatment of young-adult mice to 10 days (10DT) completely blocks the regeneration, although the stem cell compartment still reacts by promptly expanding, and retains in vitro stem cell functionality. To obtain a first broad view on molecular grounds underlying reparative capacity and/or failure, the stem cell-clustering side population was analyzed by whole-genome expression analysis. A number of stemness factors and components of embryonic, epithelial-mesenchymal transition, growth factor and Hippo pathways are higher expressed in the stem cell-clustering side population of the regenerating pituitary (after 3DT) when compared with the basal gland and to the nonregenerating pituitary (after 10DT). Together, the regenerative capacity of the pituitary is limited both in age-related terms and final efficacy, and appears to rely on stem cell-associated pathway activation. Dissection of the molecular profiles may eventually identify targets to induce or boost regeneration in situations of (injury-related) pituitary deficiency.

Pituitary tumors contain a side population with tumor stem cell-associated characteristics.

Pituitary adenomas cause significant endocrine and mass-related morbidity. Little is known about the mechanisms that underlie pituitary tumor pathogenesis. In the present study, we searched for a side population (SP) in pituitary tumors representing cells with high efflux capacity and potentially enriched for tumor stem cells (TSCs). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the purified SP (pSP) that is depleted from endothelial and immune cells, is enriched for cells that express 'tumor stemness' markers and signaling pathways, including epithelial-mesenchymal transition (EMT)-linked factors. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered to be a property of TSCs. These sphere-initiating cells were recovered in the pSP. Because benign pituitary adenomas do not grow in vitro and have failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it to be more tumorigenic than the non-SP 'main population'. Of the two EMT regulatory pathways tested, the inhibition of chemokine (C-X-C motif) receptor 4 (CXCR4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth, whereas the activation of TGFß had no effect. The human adenoma pSP also showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2(-/-)) mice that bear prolactinomas contain more pSP, Sox2(+), and colony-forming cells than WT glands. In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. The present study adds new elements to the unraveling of pituitary tumor pathogenesis and may lead to the identification of new therapeutic targets.