Pheochromocytomas can usually be confirmed or excluded using currently available biochemical tests of catecholamine excess. Follow-up tests are, nevertheless, often required to distinguish false-positive from true-positive results. The glucagon stimulation test represents one such test; its diagnostic utility is, however, unclear.

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (Mɸ) and granulocytes (Gɸ) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only Mɸ altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.

Hydrogels based on gelatin have evolved as promising multifunctional biomaterials. Gelatin is crosslinked with lysine diisocyanate ethyl ester (LDI) and the molar ratio of gelatin and LDI in the starting material mixture determines elastic properties of the resulting hydrogel. In order to investigate the clinical potential of these biopolymers, hydrogels with different ratios of gelatin and diisocyanate (3-fold (G10_LNCO3) and 8-fold (G10_LNCO8) molar excess of isocyanate groups) were subcutaneously implanted in mice (uni- or bilateral implantation). Degradation and biomaterial-tissue-interaction were investigated in vivo (MRI, optical imaging, PET) and ex vivo (autoradiography, histology, serum analysis). Multimodal imaging revealed that the number of covalent net points correlates well with degradation time, which allows for targeted modification of hydrogels based on properties of the tissue to be replaced. Importantly, the degradation time was also dependent on the number of implants per animal. Despite local mechanisms of tissue remodeling no adverse tissue responses could be observed neither locally nor systemically. Finally, this preclinical investigation in immunocompetent mice clearly demonstrated a complete restoration of the original healthy tissue.

Von Hippel-Lindau (VHL)-associated hemangioblastomas (VHL-HB) arise in the central nervous system (CNS), and are a leading cause of morbidity and mortality in VHL disease. Currently, surgical resection is the most effective way to manage symptomatic VHL-HBs. Surgically unresectable VHL-HBs or those in frail patients are challenging problems. Therapies targeting oncologic and vascular endothelial growth factor (VEGF) pathways have failed to demonstrate tumor control. Our experience and previous reports on VHL-HB avidity to somatostatin analogues suggested somatostatin receptor (SSTR) expression in VHL-HBs, offering an alternative therapeutic strategy. We explored this possibility by demonstrating consistent histologic expression of SSTR1, 2a, 4, and 5 in VHL-HBs. We found that somatostatin analogue octreotide induces apoptosis in VHL-HB stromal cells in a dose-dependent fashion by BAX - caspase-3 pathway unrelated to canonical VHL pathway. When administered to a patient with unresectable symptomatic suprasellar hemangioblastoma, octreotide resulted in tumor volume reduction, symptom stabilization, and tumor cytopenia on repeat 68Ga-DOTA-TATE positron emission tomography (PET) within 6 months, suggesting tumor infarction. We conclude that VHL-HBs harbor multiple SSTR subtypes that offer actionable chemo-therapeutic strategy for management of symptomatic, unresectable tumors by somatostatin analogue therapy.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells, adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging, anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

Combining stem cells with biomaterial scaffolds provides a promising strategy for the development of drug delivery systems. Here we propose an innovative immunotherapeutic organoid by housing human mesenchymal stromal cells (MSCs), gene-modified for the secretion of an anti-CD33-anti-CD3 bispecific antibody (bsAb), in a small biocompatible star-shaped poly(ethylene glycol)-heparin cryogel scaffold as a transplantable and low invasive therapeutic machinery for the treatment of acute myeloid leukemia (AML). The macroporous biohybrid cryogel platform displays effectiveness in supporting proliferation and survival of bsAb-releasing-MSCs overtime in vitro and in vivo, avoiding cell loss and ensuring a constant release of sustained and detectable levels of bsAb capable of triggering T-cell-mediated anti-tumor responses and a rapid regression of CD33+ AML blasts. This therapeutic device results as a promising and safe alternative to the continuous administration of short-lived immunoagents and paves the way for effective bsAb-based therapeutic strategies for future tumor treatments.

Gsα couples multiple receptors, including the melanocortin 4 receptor (MC4R), to intracellular cAMP generation. Germline inactivating Gsα mutations lead to obesity in humans and mice. Mice with brain-specific Gsα deficiency also develop obesity with reduced energy expenditure and locomotor activity, and impaired adaptive thermogenesis, but the underlying mechanisms remain unclear.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel-Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare, neuroendocrine tumors derived from adrenal or extra-adrenal chromaffin cells, respectively. Metastases are discovered in 3-36% of patients at the time of diagnosis. Currently, only suboptimal treatment options exist. Therefore, new therapeutic compounds targeting metastatic PHEOs/PGLs are urgently needed. Here, we investigated if anthracyclines were able to suppress the progression of metastatic PHEO. We explored their effects on experimental mouse PHEO tumor cells using in vitro and in vivo models, and demonstrated that anthracyclines, particularly idarubicin (IDA), suppressed hypoxia signaling by preventing the binding of hypoxia-inducible factor 1 and 2 (HIF-1 and HIF-2) to the hypoxia response element (HRE) sites on DNA. This resulted in reduced transcriptional activation of HIF target genes, including erythropoietin (EPO), phosphoglycerate kinase 1 (PGK1), endothelin 1 (EDN1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and vascular endothelial growth factor (VEGFA), which consequently inhibited the growth of metastatic PHEO. Additionally, IDA downregulated hypoxia signaling by interfering with the transcriptional activation of HIF1A and HIF2A. Furthermore, our animal model demonstrated the dose-dependent suppressive effect of IDA on metastatic PHEO growth in vivo. Our results indicate that anthracyclines are prospective candidates for inclusion in metastatic PHEO/PGL therapy, especially in patients with gene mutations involved in the hypoxia signaling pathway.

The purpose of this study was to compare components of cardiometabolic risk and health behaviors of 20 family caregivers of allogeneic hematopoietic stem cell transplant patients to those of age, gender, and race/ethnicity-matched controls. A prospective, repeated measures design was used to compare cardiometabolic risk and health behaviors in caregivers and controls at three time-points: pre-transplantation, discharge, and six weeks post-discharge. Measures included components of metabolic syndrome, Reynolds Risk Score, NMR serum lipoprotein particle analyses, and the Health-Promoting Lifestyle Profile II (HPLP-II). Mixed-model repeated measure analyses were used. There were no between or within group differences in LDL cholesterol, HDL cholesterol, and triglycerides. There was a significant interaction effect between time and role in large VLDL concentration (VLDL-P) (F (2, 76) = 4.36, p = .016), with the trajectory of large VLDL-P increasing over time in caregivers while remaining stable in controls. Within caregivers, VLDL particle size (VLDL-Z) was significantly larger at time-point three compared to time-points one (p = .015) and two (p = .048), and VLDL-Z was significantly larger in caregivers than in controls at time point three (p = .012). HPLP-II scores were lower in caregivers than controls at all time-points (p < .01). These findings suggest that caregiving may have a bigger impact on triglycerides than on other lipids, and it is through this pathway that caregivers may be at increased cardiometabolic risk. More sensitive measurement methods, such as NMR lipoprotein particle analyses, may be able to detect early changes in cardiometabolic risk.

Proteasome inhibitors have been frequently used in treating hematologic and solid tumors. They are administered individually or in combination with other regimens, to prevent severe side effects and resistance development. Because they have been shown to be efficient and are pharmaceutically available, we tested the first Food and Drug Administration-approved proteasome inhibitor bortezomib alone and in combination with another proteasome inhibitor, salinosporamid A, in pheochromocytoma cells. Pheochromocytomas/Paragangliomas (PHEOs/PGLs) are neuroendocrine tumors for which no definite cure is yet available. Therefore, drugs with a wide spectrum of mechanisms of action are being tested to identify suitable candidates for PHEO/PGL treatment. In the current study, we show that bortezomib induces PHEO cell death via the apoptotic pathway in vitro and in vivo. The combination of bortezomib with salinosporamid A exhibits additive effect on these cells and inhibits proliferation, cell migration and invasion, and angiogenesis more potently than bortezomib alone. Altogether, we suggest these proteasome inhibitors, especially bortezomib, could be potentially tested in PHEO/PGL patients who might benefit from treatment with either the inhibitors alone or in combination with other treatment options.

Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.

The risk factors for severe COVID-19 are diverse, yet closely resemble the clinical manifestations of catecholamine excess states (eg, hypertension, cardiovascular disease, immune dysregulation, and hyperglycaemia), suggesting a potentially common basis for disease. Unfortunately, severe illness (eg, respiratory failure, compromised cardiac function, and shock) incurred by COVID-19 hinders the direct study of catecholamines in these patients, especially among those on multiple medications or those on adrenaline or noradrenaline infusions, or both. Phaeochromocytoma and paraganglioma (PPGL) are tumours that secrete catecholamines, namely adrenaline and noradrenaline, often in excess. PPGL are well studied disease processes in which the effects of catecholamines are easily discernible and therefore their potential biochemical and physiological influences in patients with COVID-19 can be explored. Because catecholamines are expected to have a role in patients with critical illness, patients on vasopressor infusions, and patients who sustain some acute and chronic physical stresses, the challenges involved in the management of catecholamine excess states are directly relevant to the treatment of patients with COVID-19. In this Personal View, we discuss the complex interplay between catecholamines and COVID-19, and the management of catecholamine excess states, while referencing relevant insights derived from the study of PPGL.

Succinate dehydrogenase subunit B (SDHB) deficiency frequently occurs in cluster I pheochromocytomas and paragangliomas (PCPGs). SDHB-mutated PCPGs are characterized by alterations in the electron transport chain, metabolic reprogramming of the tricarboxylic cycle, and elevated levels of reactive oxygen species (ROS). We discovered that SDHB-deficient PCPG cells exhibit increased oxidative stress burden, which leads to elevated demands for glutathione metabolism. Mechanistically, nuclear factor erythroid 2-related factor 2 (NRF2)-guided glutathione de novo synthesis plays a key role in supporting cellular survival and the proliferation of SDHB-knockdown (SDHBKD) cells. NRF2 blockade not only disrupted ROS homeostasis in SDHB-deficient cells but also caused severe cytotoxicity by the accumulation of DNA oxidative damage. Brusatol, a potent NRF2 inhibitor, showed a promising effect in suppressing SDHBKD metastatic lesions in vivo, with prolonged overall survival in mice bearing PCPG allografts. Our findings highlight a novel therapeutic strategy of targeting the NRF2-driven glutathione metabolic pathway against SDHB-mutated PCPG.

We previously identified a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia. In these patients, polycythemia occurs long before any tumor develops, and tumor removal only partially corrects polycythemia, with recurrence occurring shortly after surgery. Genetic mosaicism of gain-of-function mutations of the EPAS1 gene (encoding HIF2α) located in the oxygen degradation domain (ODD), typically p.530-532, was shown as the etiology of this syndrome. The aim of the present investigation was to demonstrate that these mutations are necessary and sufficient for the development of the symptoms. We developed transgenic mice with a gain-of-function Epas1A529V mutation (corresponding to human EPAS1A530V), which demonstrated elevated levels of erythropoietin and polycythemia, a decreased urinary metanephrine-to-normetanephrine ratio, and increased expression of somatostatin in the ampullary region of duodenum. Further, inhibition of HIF2α with its specific inhibitor PT2385 significantly reduced erythropoietin levels in the mutant mice. However, polycythemia persisted after PT2385 treatment, suggesting an alternative erythropoietin-independent mechanism of polycythemia. These findings demonstrate the vital roles of EPAS1 mutations in the syndrome development and the great potential of the Epas1A529V animal model for further pathogenesis and therapeutics studies.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.