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.

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.

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.

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.

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.

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.

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.

Glioma are clinically challenging tumors due to their location and invasiveness nature, which often hinder complete surgical resection. The evaluation of the isocitrate dehydrogenase mutation status has become crucial for effective patient stratification. Through a transdisciplinary approach, we have developed an 18F-labeled ligand for non-invasive assessment of the IDH1R132H variant by using positron emission tomography (PET) imaging. In this study, we have successfully prepared diastereomerically pure [18F]AG-120 by copper-mediated radiofluorination of the stannyl precursor 6 on a TRACERlab FX2 N radiosynthesis module. In vitro internalization studies demonstrated significantly higher uptake of [18F]AG-120 in U251 human high-grade glioma cells with stable overexpression of mutant IDH1 (IDH1R132H) compared to their wild-type IDH1 counterpart (0.4 vs. 0.013% applied dose/µg protein at 120 min). In vivo studies conducted in mice, exhibited the excellent metabolic stability of [18F]AG-120, with parent fractions of 85% and 91% in plasma and brain at 30 min p.i., respectively. Dynamic PET studies with [18F]AG-120 in naïve mice and orthotopic glioma rat model reveal limited blood-brain barrier permeation along with a low uptake in the brain tumor. Interestingly, there was no significant difference in uptake between mutant IDH1R132H and wild-type IDH1 tumors (tumor-to-blood ratio[40-60 min]: ~1.7 vs. ~1.3). In conclusion, our preclinical evaluation demonstrated a target-specific internalization of [18F]AG-120 in vitro, a high metabolic stability in vivo in mice, and a slightly higher accumulation of activity in IDH1R132H-glioma compared to IDH1-glioma. Overall, our findings contribute to advancing the field of molecular imaging and encourage the evaluation of [18F]AG-120 to improve diagnosis and management of glioma and other IDH1R132H-related tumors.

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.

S100A4, a member of the S100 protein family of EF-hand calcium-binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild-type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375-hS100A4) or human receptor for advanced glycation endproducts (A375-hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum-Golgi-dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4-RAGE interaction and its blockade on A375, A375-hS100A4, A375-hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.

Ketoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) that also exhibits cytotoxic activity against various cancers. This makes ketoprofen an attractive structural lead for the development of new NSAIDs and cytotoxic agents. Recently, the incorporation of carboranes as phenyl mimetics in structures of established drugs has emerged as an attractive strategy in drug design. Herein, we report the synthesis and evaluation of four novel carborane-containing derivatives of ketoprofen, two of which are prodrug esters with an nitric oxide-releasing moiety. One of these prodrug esters exhibited high cytostatic activity against melanoma and colon cancer cell lines. The most pronounced activity was found in cell lines that are sensitive to oxidative stress, which was apparently induced by the ketoprofen analogue.

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).

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell- (PSCA) or/and prostate specific membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

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.

In a previous study, EphB4 was demonstrated to be a positive regulator of A375-melanoma growth but a negative regulator of tumor vascularization and perfusion. To distinguish between EphB4 forward and ephrinB2 reverse signaling, we used the commercially available EphB4 kinase inhibitor NVP-BHG712 (NVP), which was later identified as its regioisomer NVPiso. Since there have been reported significant differences between the inhibition profiles of NVP and NVPiso, we compared the influence of NVP and NVPiso on tumor characteristics under the same experimental conditions. Despite the different inhibitory profiles of NVP and NVPiso, the comparative study conducted here showed the same EphB4-induced effects in vivo as in the previous investigation. This confirmed the conclusion that EphB4-ephrinB2 reverse signaling is responsible for increased tumor growth as well as decreased tumor vascularization and perfusion. These results are further substantiated by microarrays showing differences between mock-transfected and EphB4-transfected (A375-EphB4) cells with respect to at least 9 angiogenesis-related proteins. Decreased expression of vascular endothelial growth factor (VEGF), angiotensin 1 (Ang-1), and protein kinase B (Akt/PKB), together with the increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta-2 (TGF-β2), is consistent with the impaired vascularization of A375-EphB4 xenografts. Functional overexpression of EphB4 in A375-EphB4 cells was confirmed by activation of a variety of signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT), rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen activated protein kinase kinase (Ras/Raf/MEK), and nuclear factor kappa-B (NFkB) pathways.

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.

Transglutaminase 2 (TG2) is a protein expressed in many tissues that exerts numerous, sometimes contradictory, intra- and extracellular functions, under both physiological and pathophysiological conditions. In the context of tumor progression, it has been found to be involved in cell adhesion, DNA repair mechanisms, induction of apoptosis, and mesenchymal transdifferentiation, among others. Here, we hypothesized that TG2 also contributes to the radioresistance of two human melanoma cell lines, A375 and MeWo, which can be seen to differ in their basal TG2 biosynthesis by examining their proliferation and clonal expansion after irradiation. For this purpose, cellular TG2 biosynthesis and TG2 activity were modulated by transfection-induced overexpression or TG2 knock-out and application of TG2-selective inhibitors. Proliferation and clonal expansion of TG2-overexpressing cells was not enhanced over wildtype cells, suggesting that increased TG2 biosynthesis does not further enhance the radioresistance of melanoma cells. Conversely, TG2 knock-out in A375 cells reduced their proliferation, as well as clonal and spheroidal expansion after irradiation, which indicates a contribution of TG2 to the radioresistance of melanoma cells. Since TG1, TG3, and partly also, TG6 biosynthesis was detectable in A375 and MeWo cells, it can be assumed that these other members of the TG family may exert a partially compensatory effect.