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.

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.

COX-2 can be considered as a clinically relevant molecular target for adjuvant, in particular radiosensitizing treatments. In this regard, using selective COX-2 inhibitors, e.g., in combination with radiotherapy or endoradiotherapy, represents an interesting treatment option. Based on our own findings that nitric oxide (NO)-releasing and celecoxib-derived COX-2 inhibitors (COXIBs) showed promising radiosensitizing effects in vitro, we herein present the development of a series of eight novel NO-COXIBs differing in the peripheral substitution pattern and their chemical and in vitro characterization. COX-1 and COX-2 inhibition potency was found to be comparable to the lead NO-COXIBs, and NO-releasing properties were demonstrated to be mainly influenced by the substituent in 4-position of the pyrazole (Cl vs. H). Introduction of the N-propionamide at the sulfamoyl residue as a potential prodrug strategy lowered lipophilicity markedly and abolished COX inhibition while NO-releasing properties were not markedly influenced. NO-COXIBs were tested in vitro for a combination with single-dose external X-ray irradiation as well as [177Lu]LuCl3 treatment in HIF2α-positive mouse pheochromocytoma (MPC-HIF2a) tumor spheroids. When applied directly before X-ray irradiation or 177Lu treatment, NO-COXIBs showed radioprotective effects, as did celecoxib, which was used as a control. Radiosensitizing effects were observed when applied shortly after X-ray irradiation. Overall, the NO-COXIBs were found to be more radioprotective compared with celecoxib, which does not warrant further preclinical studies with the NO-COXIBs for the treatment of pheochromocytoma. However, evaluation as radioprotective agents for healthy tissues could be considered for the NO-COXIBs developed here, especially when used directly before irradiation.

Pheochromocytomas/paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors.

Hereditary spastic paraplegias (HSPs) cause characteristic gait impairment leading to an increased risk of stumbling or even falling. Biomechanically, gait deficits are characterized by reduced ranges of motion in lower body joints, limiting foot clearance and ankle range of motion. To date, there is no standardized approach to continuously and objectively track the degree of dysfunction in foot elevation since established clinical rating scales require an experienced investigator and are considered to be rather subjective. Therefore, digital disease-specific biomarkers for foot elevation are needed.

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

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.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1's function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor's pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.

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.

Barium-131 is a single photon emission computed tomography (SPECT)-compatible radionuclide for nuclear medicine and a promising diagnostic match for radium-223/-224. Herein, we report on the sufficient production route 133Cs(p,3n)131Ba by using 27.5 MeV proton beams. An average of 190 MBq barium-131 per irradiation was obtained. The SR Resin-based purification process led to barium-131 in high radiochemical purity. An isotopic impurity of 0.01% barium-133 was detectable. For the first time, radiolabeling of the ligand macropa with barium-131 was performed. Radiolabeling methods under mild conditions and reaction controls based on TLC systems were successfully applied. Small animal SPECT/ computed tomography (CT) measurements and biodistribution studies were performed using [131Ba]Ba(NO3)2 as reference and 131Ba-labeled macropa in healthy mice for the first time. Biodistribution studies revealed the expected rapid bone uptake of [131Ba]Ba2+, whereas 131Ba-labeled macropa showed a fast clearance from the blood, thereby showing a significantly (p < 0.001) lower accumulation in the bone. We conclude that barium-131 is a promising SPECT radionuclide and delivers appropriate imaging qualities in small animals. Furthermore, the relative stability of the 131Ba-labeled macropa complex in vivo forms the basis for the development of sufficient new chelators, especially for radium isotopes. Thereby, barium-131 will attain its goal as a diagnostic match to the alpha emitters radium-223 and radium-224.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging.

The widespread use of 68Ga for positron emission tomography (PET) relies on the development of radiopharmaceutical precursors that can be radiolabelled and dispensed in a simple, quick, and convenient manner. The DATA (6-amino-1,4-diazapine-triacetate) scaffold represents a novel hybrid chelator architecture possessing both cyclic and acyclic character that may allow for facile access to 68Ga-labelled tracers in the clinic. We report the first bifunctional DATA chelator conjugated to [Tyr3]octreotide (TOC), a somatostatin subtype 2 receptor (SST2)-targeting vector for imaging and functional characterisation of SSTR2 expressing tumours.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Early detection and treatment of cancers can significantly increase patient prognosis and enhance the quality of life of affected patients. The emerging significance of the tumor microenvironment (TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibroblasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha (FAPα), which are expressed on their surfaces. Targeting FAPα using small-molecule 18F-labeled inhibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using PET. Herein, two potent aryl-fluorosulfate-based FAPIs, 12 and 13, were synthetically prepared, and their inhibition potency was determined using a fluorimetric FAP assay to be IC50 9.63 and 4.17 nM, respectively. Radiofluorination was performed via the sulfur [18F]fluoride exchange ([18F]SuFEx) reaction to furnish [18F]12 and [18F]13 in high activity yields (AY) of 39-56% and molar activities (Am) between 20-55 GBq/µmol. In vitro experiments focused on the stability of the radiolabeled FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET studies of the radioligands were performed in healthy mice to investigate the in vivo biodistribution and 18F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and considerable bone uptake, caused by either 18F defluorination or radioligand accumulation, was observed. In summary, our findings demonstrate the efficiency of [18F]SuFEx as a radiolabeling method as well as its advantages and limitations with respect to PET tracer development.

Although digital mobility outcomes (DMOs) can be readily calculated from real-world data collected with wearable devices and ad-hoc algorithms, technical validation is still required. The aim of this paper is to comparatively assess and validate DMOs estimated using real-world gait data from six different cohorts, focusing on gait sequence detection, foot initial contact detection (ICD), cadence (CAD) and stride length (SL) estimates.