Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

Internal tandem duplications within the juxtamembrane region of the FMS-like tyrosine kinase receptor FLT3 (FLT3-ITD) represents one of the most common mutations in patients with acute myeloid leukemia (AML) which results in constitutive aberrant activation, increased proliferation of leukemic progenitors and is associated with an aggressive clinical phenotype. The expression of CD97, an EGF-TM7 receptor, has been linked to invasive behavior in thyroid and colorectal cancer. Here, we have investigated the association of CD97 with FLT3-ITD and its functional consequences in AML.Higher CD97 expression levels have been detected in 208 out of 385 primary AML samples. This was accompanied by a significantly increased bone marrow blast count as well as by mutations in the FLT3 gene. FLT3-ITD expressing cell lines as MV4-11 and MOLM-13 revealed significantly higher CD97 levels than FLT3 wildtype EOL-1, OCI-AML3 and HL-60 cells which were clearly decreased by the tyrosine kinase inhibitors PKC412 and SU5614. CD97 knock down by short hairpin RNA in MV4-11 cells resulted in inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock down of CD97 led to an altered mechanical phenotype, reduced adhesion to a stromal layer and lower wildtype FLT3 expression.Our results, thus, constitute the first evidence for the functional relevance of CD97 expression in FLT3-ITD AML cells rendering it a potential new theragnostic target.

Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4'-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.

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.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

Adoptive transfer of chimeric antigen receptor (CAR)-equipped T cells have demonstrated astonishing clinical efficacy in hematological malignancies recently culminating in the approval of two CAR T cell products. Despite this tremendous success, CAR T cell approaches have still achieved only moderate efficacy against solid tumors. As a major obstacle, engineered conventional T cells (Tconvs) face an anti-inflammatory, hostile tumor microenvironment often infiltrated by highly suppressive regulatory T cells (Tregs). Thus, potent CAR T cell treatment of solid tumors requires efficient activation of Tconvs via their engrafted CAR to overcome Treg-mediated immunosuppression. In that regard, selecting an optimal intracellular signaling domain might represent a crucial step to achieve best clinical efficiency. To shed light on this issue and to investigate responsiveness to Treg inhibition, we engrafted Tconvs with switchable universal CARs (UniCARs) harboring intracellularly the CD3ζ domain alone or in combination with costimulatory CD28 or 4-1BB. Our studies reveal that UniCAR ζ-, and UniCAR BB/ζ-engineered Tconvs are strongly impaired by activated Tregs, whereas UniCARs providing CD28 costimulation overcome Treg-mediated suppression both in vitro and in vivo. Compared to UniCAR ζ- and UniCAR BB/ζ-modified cells, UniCAR 28/ζ-armed Tconvs secrete significantly higher amounts of Th1-related cytokines and, furthermore, levels of these cytokines are elevated even upon exposure to Tregs. Thus, in contrast to 4-1BB costimulation, CD28 signaling in UniCAR-transduced Tconvs seems to foster a pro-inflammatory milieu, which contributes to enhanced resistance to Treg suppression. Overall, our results may have significant implications for CAR T cell-based immunotherapies of solid tumors strongly invaded by Tregs.

Tyrosine is an attractive target for chemo- and site-selective protein modification. The particular chemical nature of tyrosine residues allows bioconjugation chemistry with reactive aryl diazonium salts via electrophilic aromatic substitution to produce diazo compounds. In this work, we describe the preparation of 64Cu- and 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-diazonium salts as building blocks for azo coupling chemistry with tyrosine and tyrosine-containing peptides and proteins under mild conditions. 2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA) was used to form the corresponding 64Cu- and 68Ga-labeled complexes, followed by diazotization with NaNO2 in the presence of HCl. 64Cu- and 68Ga-labeled NOTA complexes were prepared in high radiochemical yields >80% starting from 20 μg of p-NH2-Bn-NOTA. Conversion of p-NH2-Bn-NOTA complexes into diazonium salts followed by azo coupling with l-tyrosine afforded 64Cu- and 68Ga-labeled tyrosine in radiochemical yields of 80 and 56%, respectively. Azo coupling with tyrosine-containing hexapeptide neurotensin NT(8-13) afforded 64Cu- and 68Ga-labeled NT(8-13) in radiochemical yields of 45 and 11%, respectively. Azo coupling of 64Cu-labeled NOTA-diazonium salt with human serum albumin (HSA) gave 64Cu-labeled HSA in radiochemical yields of 20%. The described azo coupling chemistry represents an innovative and versatile bioconjugation strategy for selective targeting of tyrosine residues in peptides and proteins.

Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain.

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.

Chronic pelvic pain (CPP), despite its high prevalence, is still relatively poorly understood mechanistically. This study, as part of the Translational Research in Pelvic Pain (TRiPP) project, has used a full quantitative sensory testing (QST) paradigm to profile n = 85 women with and without CPP (endometriosis or bladder pain specifically). We used the foot as a control site and abdomen as the test site. Across 5 diagnostically determined subgroups, we found features which are common across different aetiologies, eg, gain of function in pressure pain threshold (PPT) when assessing responses from the lower abdomen or pelvis (referred pain site). However, disease-specific phenotypes were also identified, eg, greater mechanical allodynia in endometriosis, despite there being large heterogeneities within diagnostic groups. The most common QST sensory phenotype was mechanical hyperalgesia (>50% across all the groups). A "healthy' sensory phenotype was seen in <7% of CPP participants. Specific QST measures correlated with sensory symptoms assessed by the painDETECT questionnaire (pressure-evoked pain [painDETECT] and PPT [QST] [ r = 0.47, P < 0.001]; mechanical hyperalgesia (painDETECT) and mechanical pain sensitivity [MPS from QST] [ r = 0.38, P = 0.009]). The data suggest that participants with CPP are sensitive to both deep tissue and cutaneous inputs, suggesting that central mechanisms may be important in this cohort. We also see phenotypes such as thermal hyperalgesia, which may be the result of peripheral mechanisms, such as irritable nociceptors. This highlights the importance of stratifying patients into clinically meaningful phenotypes, which may have implications for the development of better therapeutic strategies for CPP.

Genetic lesions of IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution.

Simultaneous positron emission tomography (PET) and magnetic resonance imaging (MRI) is a new whole-body hybrid PET/MR imaging technique that combines metabolic and cross-sectional diagnostic imaging. Since the use of MRI in imaging of soft-tissue sarcoma is extremely beneficial, investigation of the combined PET/MRI is of great interest. In this paper, we present three cases and first data. Combined PET/MRI technique can support the process of clinical decision-making and give answers to some meaningful questions when treating patients with STS. Therefore, the combined modality of simultaneous PET/MRI offers new pieces to the puzzle of sarcoma treatment.

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.

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.

Radiotherapy is one of the most frequently applied treatments in oncology. Tissue-absorbed ionizing radiation damages not only targeted cells but the surrounding cells too. The consequent long-term induced oxidative stress, irreversible tissue damage, or second malignancies draw attention to the urgent need of a follow-up medical method by which personalized treatment could be attained and the actually dose-limiting organ could be monitored in the clinical practice. We worked out a special hemisphere irradiation technique for mice which mimics the radiation exposure during radiotherapy. We followed up the changes of possible brain imaging biomarkers of side effects, such as cerebral blood flow, vascular endothelial function, and cellular metabolic processes for 60 days. BALB/c mice were divided into two groups (n=6 per group) based on the irradiation doses (5 and 20 Gy). After the irradiation procedure arterial spin labeling (ASL), diffusion-weighted imaging (DWI) in magnetic resonance modality and [18F]fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) scans of the brain were obtained at several time points (3, 7, 30, and 60 days after the irradiation). Significant physiological changes were registered in the brain of animals following the irradiation by both applied doses. Elevated standard uptake values were detected all over the brain by FDG-PET studies 2 months after the irradiation. The apparent diffusion coefficients from DWI scans significantly decreased one month after the irradiation procedure, while ASL studies did not show any significant perfusion changes in the brain. Altogether, our sensitive multimodal imaging protocol seems to be an appropriate method for follow-up of the health status after radiation therapy. The presented approach makes possible parallel screening of healthy tissues and the effectiveness of tumor therapy without any additional radiation exposure.

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.