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Somatostatin receptor-targeting endoradiotherapy offers potential for treating metastatic pheochromocytomas and paragangliomas, an approach likely to benefit from combination radiosensitization therapy. To provide reliable preclinical in vivo models of metastatic disease, this study characterized the metastatic spread of luciferase-expressing mouse pheochromocytoma (MPC) cells in mouse strains with different immunologic conditions. Bioluminescence imaging showed that, in contrast to subcutaneous non-metastatic engraftment of luciferase-expressing MPC cells in NMRI-nude mice, intravenous cell injection provided only suboptimal metastatic spread in both NMRI-nude mice and hairless SCID (SHO) mice. Treatment of NMRI-nude mice with anti-Asialo GM1 serum enhanced metastatic spread due to substantial depletion of natural killer (NK) cells. However, reproducible metastatic spread was only observed in NK cell-defective SCID/beige mice and in hairless immunocompetent SKH1 mice bearing disseminated or liver metastases, respectively. Liquid chromatography tandem mass spectrometry of urine samples showed that subcutaneous and metastasized tumor models exhibit comparable renal monoamine excretion profiles characterized by increasing urinary dopamine, 3-methoxytyramine, norepinephrine and normetanephrine. Metastases-related epinephrine and metanephrine were only detectable in SCID/beige mice. Positron emission tomography and immunohistochemistry revealed that all metastases maintained somatostatin receptor-specific radiotracer uptake and immunoreactivity, respectively. In conclusion, we demonstrate that intravenous injection of luciferase-expressing MPC cells into SCID/beige and SKH1 mice provides reproducible and clinically relevant spread of catecholamine-producing and somatostatin receptor-positive metastases. These standardized preclinical models allow for precise monitoring of disease progression and should facilitate further investigations on theranostic approaches against metastatic pheochromocytomas and paragangliomas.

Introduction: Existing mobility endpoints based on functional performance, physical assessments and patient self-reporting are often affected by lack of sensitivity, limiting their utility in clinical practice. Wearable devices including inertial measurement units (IMUs) can overcome these limitations by quantifying digital mobility outcomes (DMOs) both during supervised structured assessments and in real-world conditions. The validity of IMU-based methods in the real-world, however, is still limited in patient populations. Rigorous validation procedures should cover the device metrological verification, the validation of the algorithms for the DMOs computation specifically for the population of interest and in daily life situations, and the users' perspective on the device. Methods and analysis: This protocol was designed to establish the technical validity and patient acceptability of the approach used to quantify digital mobility in the real world by Mobilise-D, a consortium funded by the European Union (EU) as part of the Innovative Medicine Initiative, aiming at fostering regulatory approval and clinical adoption of DMOs.After defining the procedures for the metrological verification of an IMU-based device, the experimental procedures for the validation of algorithms used to calculate the DMOs are presented. These include laboratory and real-world assessment in 120 participants from five groups: healthy older adults; chronic obstructive pulmonary disease, Parkinson's disease, multiple sclerosis, proximal femoral fracture and congestive heart failure. DMOs extracted from the monitoring device will be compared with those from different reference systems, chosen according to the contexts of observation. Questionnaires and interviews will evaluate the users' perspective on the deployed technology and relevance of the mobility assessment. Ethics and dissemination: The study has been granted ethics approval by the centre's committees (London-Bloomsbury Research Ethics committee; Helsinki Committee, Tel Aviv Sourasky Medical Centre; Medical Faculties of The University of Tübingen and of the University of Kiel). Data and algorithms will be made publicly available. Trial registration number: ISRCTN (12246987). We acknowledge support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. SDD, AY and LRo are also supported by the Newcastle Biomedical Research Centre (BRC) based at Newcastle upon Tyne and Newcastle University. CM, BS, LVG and EB are also supported by the Sheffield Biomedical Research Centre (BRC) based at the Sheffield Teaching Hospital and the University of Sheffield. The work was also supported by the NIHR/Wellcome Trust Clinical Research Facility (CRF) infrastructure at Newcastle upon Tyne Hospitals NHS Foundation Trust and the CRF at the Sheffield Teaching Hospital. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care or the funders.This study was co-funded by the European Union’s Horizon 2020 research and innovation programme and EFPIA via the Innovative Medicine Initiative 2 (Mobilise-D project, grant number IMI22017-13-7-820820). The views expressed are those of the authors and not necessarily those of the IMI, the European Union, the EFPIA, or any Associated Partners. We acknowledge the support of Grünenthal GmbH via the funding of a PhD scholarship directly dedicated to the technical validation protocol.

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix–receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.

Pheochromocytomas and paragangliomas (PCC/PGLs) are rare, mostly catecholamine-producing neuroendocrine tumors of the adrenal gland (PCCs) or the extra-adrenal paraganglia (PGL). They can be separated into three different molecular clusters depending on their underlying gene mutations in any of the at least 20 known susceptibility genes: The pseudohypoxia-associated cluster 1, the kinase signaling-associated cluster 2, and the Wnt signaling-associated cluster 3. In addition to tumor size, location (adrenal vs. extra-adrenal), multiplicity, age of first diagnosis, and presence of metastatic disease (including tumor burden), other decisive factors for best clinical management of PCC/PGL include the underlying germline mutation. The above factors can impact the choice of different biomarkers and imaging modalities for PCC/PGL diagnosis, as well as screening for other neoplasms, staging, follow-up, and therapy options. This review provides a guide for practicing clinicians summarizing current management of PCC/PGL according to tumor size, location, age of first diagnosis, presence of metastases, and especially underlying mutations in the era of precision medicine.

Sporadic mononeuropathies without trauma or compression are challenging to diagnose. Nerve ultrasound has recently proven its usefulness in the diagnosis of traumatic neuropathies, tumors and polyneuropathies. However, its role in mononeuropathies currently remains unclear. We describe ultrasonography follow-up results in 12 patients with suggested spontaneous, monophasic mononeuritis without signs of generalization. Nerve conduction studies (NCS), ultrasonography of the affected nerves and the contralateral side, laboratory analysis, and if possible magnetic resonance imaging (MRI) of the affected nerves were established in all patients at onset. In one patient, additive nerve biopsy was performed. In all patients, ultrasonography was repeated after immunotherapy. An infectious pathogen of neuritis was not found in any patient. All but one patient showed predominant axonal nerve damage in NCS, whereas ultrasonography and MRI revealed fascicular and/or overall cross-sectional area (CSA) enlargement or T2 hyperintensity of the affected nerve segments, suggesting an inflammatory background of the neuropathy. Most patients showed significant clinical amelioration of symptoms under treatment (75.0 %) and consequently a decrease in CSA/fascicle enlargement over time (77.8 %). Ultrasonography and MRI of the nerves revealed enlargement in patients with mononeuropathy of axonal NCS pattern of unknown origin. Ultrasonography can facilitate the therapeutic decision for immunotherapy. Next to nerve trauma, nerve tumors and nerve entrapments, ultrasonography reliably shows nerve enlargement in the case of inflammation and therefore could further enrich neurophysiology. Nerve imaging might serve as a follow-up tool by observing a decrease in nerve enlargement and improved function. Die Diagnosefindung bei sporadischen Mononeuropathien ohne Trauma oder Kompression in der Vorgeschichte ist eine Herausforderung. Der Nervenultraschall hat sich zwar in der Vergangenheit bei der Diagnostik von Nervenverletzungen, -tumoren und Polyneuropathien bewährt, seine Rolle bei sporadischen Mononeuropathien ist jedoch völlig unklar. Im Folgenden beschreiben wir 12 Fälle spontaner, vermutlich immunvermittelter sensibler, schmerzhafter und/oder motorischer Mononeuropathien, die keine Hinweise auf eine Generalisierung aufwiesen. Alle Patienten erhielten Elektrophysiologie, Nervenultraschall und wenn möglich ein Nerven-MRT der betroffenen und der Gegenseite sowie ausgedehnte Laboruntersuchungen. Ein Patient erhielt eine Nervenbiopsie. Die Untersuchungen wurden bei allen Patienten nach Immuntherapie wiederholt. Infektiöse Erreger einer Neuropathie fanden sich bei keinem Patienten; alle Patienten zeigten mit einer Ausnahme ein elektrophysiologisch axonales Schädigungsmuster. Alle Patienten zeigten fokale, faszikulär begrenzte oder ausgedehnte Nervenschwellungen, korrelierend mit T2-Hyperintensitäten im Magnetresonanztomogramm (MRT), hinweisend auf einen entzündlichen Prozess. Unter Immunbehandlung zeigten die meisten Patienten (75,0 %) eine klinische Besserung und einen Rückgang der Nervenschwellungen (77,8 %). Die periphere Bildgebung konnte Nervenschwellungen bei Patienten mit axonaler Neuropathie unklaren Ursprungs zeigen und somit die Entscheidung für eine immunmodulatorische Therapie erleichtern. Neben seiner Wertigkeit bei Nerventraumata, Nerventumoren und Engpass-Syndromen kann der Nervenultraschall in der Diagnostik entzündlicher Mononeuropathien die Elektrophysiologie somit sehr bereichern. Ferner kann er als Verlaufsbiomarker für ein Therapieansprechen genutzt werden.

Purpose To wavelet transform the electromyograms of the vastii muscles and generate wavelet intensity patterns (WIP) of runners. Test the hypotheses: 1) The WIP of the vastus medialis (VM) and vastus lateralis (VL) of one step are more similar than the WIPs of these two muscles, offset by one step. 2) The WIPs within one muscle differ by having maximal intensities in specific frequency bands and these intensities are not always occurring at the same time after heel strike. 3) The WIPs that were recorded form one muscle for all steps while running can be grouped into clusters with similar WIPs. It is expected that clusters might have distinctly different, cluster specific mean WIPs. Methods The EMG of the vastii muscles from at least 1000 steps from twelve runners were recorded using a bipolar current amplifier and yielded WIPs. Based on the weights obtained after a principal component analysis the dissimilarities (1-correlation) between the WIPs were computed. The dissimilarities were submitted to a hierarchical cluster analysis to search for groups of steps with similar WIPs. The clusters formed by random surrogate WIPs were used to determine whether the groups were likely to be created in a non-random manner. Results The steps were grouped in clusters showing similar WIPs. The grouping was based on the frequency bands and their timing showing that they represented defining parts of the WIPs. The correlations between the WIPs of the vastii muscles that were recorded during the same step were higher than the correlations of WPIs that were recorded during consecutive steps, indicating the non-randomness of the WIPs. Conclusions The spectral power of EMGs while running varies during the stance phase in time and frequency, therefore a time averaged power spectrum cannot reflect the timing of events that occur while running. It seems likely that there might be a set of predefined patterns that are used upon demand to stabilize the movement.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [(177)Lu]Lu-DOTA-(Tyr(3))octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [(64)Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [(177)Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [(177)Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated small-animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth–dependent increases (P < .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P < .001) and strongly with tumor fluorescence intensity (rs = 0.946), and urinary outputs of dopamine (rs = 0.952), methoxytyramine (rs = 0.947), norepinephrine (rs = 0.756), and normetanephrine (rs = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors.

Pheochromocytoma (PHEO) are potentially lethal tumors of adrenal and extra-adrenal chromaffin cells. Available treatment strategies for malignant PHEO are limited. Interestingly, peptide hormone receptors are frequently overexpressed on PHEO and can be used as specific targets for highly effective cytotoxic and radiolabeled anti-tumor peptide analogs. Here, we focused on the establishment and characterization of a novel preclinical fluorescence-based mouse model of PHEO in order to evaluate combined therapies of malignant pheochromocytoma. In previous in vitro studies we demonstrated strong anti-tumor effects of various peptide analogs specifically targeting SSTR2 and LHRH-R in mouse pheochromocytoma cell lines. These neuropeptide receptors were found to be abundantly expressed also on human PHEO samples studied. Furthermore, we examined the expression of these receptors as potential anti-tumor targets in vivo and ex vivo. To this end we established a subcutaneous mouse model of PHEO based on the injection of mCherry-expressing MPCs into the shoulders of athymic nude mice. Tumor development was followed by multimodal in vivo imaging, including fluorescence, PET, CT and MRI. Additionally, we analyzed urinary catecholamines by recently established LC-MS/MS. Both the SSTR2 and LHRH-R are still abundantly expressed in the tumors and could thus be used as therapeutic targets in ongoing pre-clinical studies. In summery, our current work resulted in the establishment of both an appropriate PHEO model system and methodology for our planned combined peptide receptor cytotoxic/radionuclide therapy studies.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor. The prognosis for malignant pheochromocytoma is particularly poor and there are currently no effective treatments. Interestingly, peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by highly effective anti-tumor peptide analogs. Our actual study focuses on two endocrinologically relevant mouse pheochromocytoma cell lines, one more malignant and one more benign one as well as on mouse models of pheochromocytoma, which our group currently establishes. First, we could demonstrate significant anti-tumor effects mediated by cytotoxic somatostatin analogs AN-162 and AN-238, by LHRH antagonist Cetrorelix and cytotoxic LHRH analog AN-152 as well as by GHRH antagonist MIA-602 targeting their respective receptors on mouse pheochromocytoma cells (MPC). Furthermore, similar anti-tumor effects were evidenced also for AN-152 and MIA-602 on more aggressive tumor tissue-derived (MTT) cells. Second, we established a subcutaneous mouse model of pheochromocytoma for optimization of parametric tumor imaging in vivo. Tumor volume, morphology, metabolic activity and peptide receptors are evaluated using PET/MR and PET/CT imaging as well as fluorescence and bioluminescence imaging after injection of mCherry or luciferase transduced MPC cell lines. Furthermore, we are establishing an intravenous mouse model of pheochromocytoma to test the most effective peptide analogs in vivo. Ex vivo characterization of mouse pheochromocytomas demonstrated that peptide hormone receptors are still expressed during tumor growth in mice. Altogether, our current investigation provides further evidence for the usefulness of targeted peptide hormone receptor therapy as a potential new option for future treatment of malignant pheochromocytoma