Lateralization of dorsal fiber tract targeting Broca's area concurs with language skills during development.

Language is bounded to the left hemisphere in the adult brain and the functional lateralization can already be observed early during development. Here we investigate whether this is paralleled by a lateralization of the white matter structural language network. We analyze the strength and microstructural properties of language-related fiber tracts connecting temporal and frontal cortices with a separation of two dorsal tracts, one targeting the posterior Broca's area (BA44) and one targeting the precentral gyrus (BA6). In a large sample of young children (3-6 years), we demonstrate that, in contrast to the BA6-targeting tract, the microstructural asymmetry of the BA44-targeting fiber tract significantly correlates locally with different aspects of development. While the asymmetry in its anterior segment reflects age, the asymmetry in its posterior segment is associated with the children's language skills. These findings demonstrate a fine-grained structure-to-function mapping in the lateralized network and go beyond our current view of language-related human brain maturation.

Profiling specific cell populations within the inflammatory tumor microenvironment by oscillating-gradient diffusion-weighted MRI.

BACKGROUND: The inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME. METHODS: Sine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining. RESULTS: OGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model. CONCLUSIONS: OGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages.

Signaling Network Response to α-Particle-Targeted Therapy with the 225Ac-Labeled Minigastrin Analog 225Ac-PP-F11N Reveals the Radiosensitizing Potential of Histone Deacetylase Inhibitors.

α-particle emitters have recently been explored as valuable therapeutic radionuclides. Yet, toxicity to healthy organs and cancer radioresistance limit the efficacy of targeted α-particle therapy (TAT). Identification of the radiation-activated mechanisms that drive cancer cell survival provides opportunities to develop new points for therapeutic interference to improve the efficacy and safety of TAT. Methods: Quantitative phosphoproteomics and matching proteomics followed by the bioinformatics analysis were used to identify alterations in the signaling networks in response to TAT with the 225Ac-labeled minigastrin analog 225Ac-PP-F11N (DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) in A431 cells, which overexpress cholecystokinin B receptor (CCKBR). Western blot analysis and microscopy verified the activation of the selected signaling pathways. Small-molecule inhibitors were used to validate the potential of the radiosensitizing combinatory treatments both in vitro and in A431/CCKBR tumor-bearing nude mice. Results: TAT-induced alterations were involved in DNA damage response, cell cycle regulation, and signal transduction, as well as RNA transcription and processing, cell morphology, and transport. Western blot analysis and microscopy confirmed increased phosphorylations of the key proteins involved in DNA damage response and carcinogenesis, including p53, p53 binding protein 1 (p53BP1), histone deacetylases (HDACs), and H2AX. Inhibition of HDAC class II, ataxia-telangiectasia mutated (ATM), and p38 kinases by TMP269, AZD1390, and SB202190, respectively, sensitized A431/CCKBR cells to 225Ac-PP-F11N. As compared with the control and monotherapies, the combination of 225Ac-PP-F11N with the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) significantly reduced the viability and increased the DNA damage of A431/CCKBR cells, led to the most pronounced tumor growth inhibition, and extended the mean survival of A431/CCKBR xenografted nude mice. Conclusion: Our study revealed the cellular responses to TAT and demonstrated the radiosensitizing potential of HDAC inhibitors to 225Ac-PP-F11N in CCKBR-positive tumors. This proof-of-concept study recommends development of novel radiosensitizing strategies by targeting TAT-activated and survival-promoting signaling pathways.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

Cross interactions between Apolipoprotein E and amyloid proteins in neurodegenerative diseases.

Three common Apolipoprotein E isoforms, ApoE2, ApoE3, and ApoE4, are key regulators of lipid homeostasis, among other functions. Apolipoprotein E can interact with amyloid proteins. The isoforms differ by one or two residues at positions 112 and 158, and possess distinct structural conformations and functions, leading to isoform-specific roles in amyloid-based neurodegenerative diseases. Over 30 different amyloid proteins have been found to share similar characteristics of structure and toxicity, suggesting a common interactome. The molecular and genetic interactions of ApoE with amyloid proteins have been extensively studied in neurodegenerative diseases, but have not yet been well connected and clarified. Here we summarize essential features of the interactions between ApoE and different amyloid proteins, identify gaps in the understanding of the interactome and propose the general interaction mechanism between ApoE isoforms and amyloid proteins. Perhaps more importantly, this review outlines what we can learn from the interactome of ApoE and amyloid proteins; that is the need to see both ApoE and amyloid proteins as a basis to understand neurodegenerative diseases.

Children's syntax is supported by the maturation of BA44 at 4 years, but of the posterior STS at 3 years of age.

Within the first years of life, children learn major aspects of their native language. However, the ability to process complex sentence structures, a core faculty in human language called syntax, emerges only slowly. A milestone in syntax acquisition is reached around the age of 4 years, when children learn a variety of syntactic concepts. Here, we ask which maturational changes in the child's brain underlie the emergence of syntactically complex sentence processing around this critical age. We relate markers of cortical brain maturation to 3- and 4-year-olds' sentence processing in contrast to other language abilities. Our results show that distinct cortical brain areas support sentence processing in the two age groups. Sentence production abilities at 3 years were associated with increased surface area in the most posterior part of the left superior temporal sulcus, whereas 4-year-olds showed an association with cortical thickness in the left posterior part of Broca's area, i.e. BA44. The present findings suggest that sentence processing abilities rely on the maturation of distinct cortical regions in 3- compared to 4-year-olds. The observed shift to more mature regions involved in processing syntactically complex sentences may underlie behavioral milestones in syntax acquisition at around 4 years.

Timing matters: disentangling the neurocognitive sequence of mentalizing.

A recent electrocorticographic study by Tan et al. makes an important contribution to understanding the processes involved in mentalizing by adding the temporal dimension to the brain network of mentalizing. Combined with multivariate methods, this approach has the potential to unveil the precise representations underlying mentalizing and their functional interplay.

Maturational Indices of the Cognitive Control Network Are Associated with Inhibitory Control in Early Childhood.

Goal-directed behavior crucially relies on our capacity to suppress impulses and predominant behavioral responses. This ability, called inhibitory control, emerges in early childhood with marked improvements between 3 and 4 years. Here, we ask which brain structures are related to the emergence of this critical ability. Using a multimodal approach, we relate the pronounced behavioral improvements in different facets of 3- and 4-year-olds' (N = 37, 20 female) inhibitory control to structural indices of maturation in the developing brain assessed with MRI. Our results show that cortical and subcortical structure of core regions in the adult cognitive control network, including the PFC, thalamus, and the inferior parietal cortices, is associated with early inhibitory functioning in preschool children. Probabilistic tractography revealed an association of frontoparietal (i.e., the superior longitudinal fascicle) and thalamocortical connections with early inhibitory control. Notably, these associations to brain structure were distinct for different facets of early inhibitory control, often referred to as motivational ("hot") and cognitive ("cold") inhibitory control. Our findings thus reveal the structural brain networks and connectivity related to the emergence of this core faculty of human cognition.SIGNIFICANCE STATEMENT The capacity to suppress impulses and behavioral responses is crucial for goal-directed behavior. This ability, called inhibitory control, develops between the ages of 3 and 4 years. The factors behind this developmental milestone have been debated intensely for decades; however, the brain structure that underlies the emergence of inhibitory control in early childhood is largely unknown. Here, we relate the pronounced behavioral improvements in inhibitory control between 3 and 4 years with structural brain markers of gray matter and white matter maturation. Using a multimodal approach that combines analyses of cortical surface structure, subcortical structures, and white matter connectivity, our results reveal the structural brain networks and connectivity related to this core faculty of human cognition.

Parent-Child Play and the Emergence of Externalizing and Internalizing Behavior Problems in Childhood: A Systematic Review.

It has widely been accepted that play has a major role in human development. The play situation is considered a save and controlled space in which children can learn to express their problems and to regulate their emotions, thus promoting emotional and behavioral adjustment. In early childhood, this process is thought to emerge in close interaction with caregivers. Parent-child play is thus viewed as an ideal window for parents to connect with their children and to support them in their social-emotional development. In this preregistered systematic review, we sought to integrate evidence from developmental and clinical psychology to shed more light on the role of parents in the relationship between parent-child play and children's behavioral adjustment as expressed in internalizing or externalizing behavior. Our review revealed that increased harsh control during play interactions as well as a lack of parental responsiveness, warmth and sensitivity were found to be associated with increased behavioral problems. Yet, no protective effect of warmth or responsiveness could be found in the context of risk groups. Moreover, the included studies indicated that positive affect expressed by parents during parent-child play was associated with fewer behavior problems in children, while negative affect was associated with more behavior problems. In general, this review revealed that quality and quantity of playful parent-child interactions were reduced in children with behavioral problems of both domains compared to children without behavioral problems. These findings illustrate the important role of parental characteristics during play interactions and their possible impact on children's behavioral adjustment.

A meta-analytic approach to the association between inhibitory control and parent-reported behavioral adjustment in typically-developing children: Differentiating externalizing and internalizing behavior problems.

Impairments in inhibitory control (IC) are traditionally seen as a vital aspect in the emergence and course of maladaptive behavior across early childhood. However, it is currently unclear whether this view applies to both the externalizing and internalizing domain of parent-reported behavioral adjustment. Furthermore, past (meta-analytic) developmental research and theory characterizing this association have largely neglected the vast heterogeneity of IC measures and conceptualizations. The present meta-analyses examined the association of IC with parent-reported externalizing (N = 3160, 21 studies) and internalizing (N = 1758, 12 studies) behavior problems, assessed with the Child Behavior Checklist (CBCL), in non-clinical populations of children aged 2-8 years. They further investigated the moderating effects of a priori IC categorization, according to a recently proposed two-factor model of IC ("Strength/Endurance" account, Simpson & Carroll, 2019). In line with previous research in the clinical domain, the current results corroborate the notion of a robust, but small association between IC and externalizing behavior problems (r = -0.11) in early childhood. However, although frequently proposed in the literature, no significant linear association could be identified with internalizing behavior problems. Furthermore, in both meta-analyses, no significant moderating effects of IC categorization could be revealed. These findings enhance our knowledge about the cognitive underpinnings of early-emerging maladaptive behavior, indicating that different subtypes of IC are statistically related with externalizing, but not internalizing behavior problems. Overall, the small association of IC ability with behavior problems in non-clinical populations provokes broader questions about the role of IC in behavioral adjustment.

Positive emotion enhances conflict processing in preschoolers.

The rapid detection and resolution of conflict between opposing action tendencies is crucial for our ability to engage in goal-directed behavior. Research in adults suggests that emotions can serve as a "relevance detector" that alarms attentional and sensory systems, thereby leading to more efficient conflict processing. In contrast, previous research in children has almost exclusively stressed the impeding influence of emotion on the attentional system, as suggested by the protracted development of performance in "hot" executive function tasks. Do preschool children show a facilitative effect of emotion on conflict processing? We addressed this question applying a modified version of a color flanker task that either involved or did not involve positive emotional stimuli in preschool children (N = 43, with preregistered Bayesian sequential design, aged 2.8-7.0 years). Our results show a robust conflict effect with higher error rates in incongruent compared to congruent trials. Crucially, conflict resolution was faster in emotional compared to neutral conditions. Furthermore, while efficient conflict processing increases with age, we find evidence against an age-related change in the influence of positive emotion on conflict processing. Taken together, these findings provide indication that positive emotion can trigger efficient control processes already from early on in life. In contrast to the predominant view in developmental psychology, this indicates that, depending on the role that emotion has in conflict processing, emotion may show a facilitative or impeding effect already in the preschool period.

Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone.

G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward ß-arrestin-1 and ß-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting ß-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.

Humor in Psychiatry: Lessons From Neuroscience, Psychopathology, and Treatment Research.

Humor is a ubiquitous human characteristic that is socially motivated at its core and has a broad range of significant positive effects on emotional well-being and interpersonal relationships. Simultaneously, however, impairments in humor abilities have often been described in close association with the occurrence and course of neuropsychiatric disorders, such as schizophrenia, social anxiety, or depression. In the past decade, research in the neuroimaging and psychiatric domain has substantially progressed to (i) characterize impaired humor as an element of psychopathology, and (ii) shed light on the neurobiological mechanisms underlying the role of humor in neuropsychiatric diseases. However, (iii) targeted interventions using concepts of positive psychology have revealed first evidence that a systematic training and/or a potential reactivation of humor-related skills can improve rehabilitative outcome in neuropsychiatric patient groups. Here, we sought to integrate evidence from neuroscience, as well as from psychopathology and treatment research to shed more light on the role of humor in psychiatry. Based on these considerations, we provide directions for future research and application in mental health services, focusing on the question of how our scientific understanding of humor can provide the basis for psychological interventions that foster positive attitudes and well-being.

Antagonism between brain regions relevant for cognitive control and emotional memory facilitates the generation of humorous ideas.

The ability to generate humor gives rise to positive emotions and thus facilitate the successful resolution of adversity. Although there is consensus that inhibitory processes might be related to broaden the way of thinking, the neural underpinnings of these mechanisms are largely unknown. Here, we use functional Magnetic Resonance Imaging, a humorous alternative uses task and a stroop task, to investigate the brain mechanisms underlying the emergence of humorous ideas in 24 subjects. Neuroimaging results indicate that greater cognitive control abilities are associated with increased activation in the amygdala, the hippocampus and the superior and medial frontal gyrus during the generation of humorous ideas. Examining the neural mechanisms more closely shows that the hypoactivation of frontal brain regions is associated with an hyperactivation in the amygdala and vice versa. This antagonistic connectivity is concurrently linked with an increased number of humorous ideas and enhanced amygdala responses during the task. Our data therefore suggests that a neural antagonism previously related to the emergence and regulation of negative affective responses, is linked with the generation of emotionally positive ideas and may represent an important neural pathway supporting mental health.

WIPI1 promotes fission of endosomal transport carriers and formation of autophagosomes through distinct mechanisms.

Autophagosome formation requires PROPPIN/WIPI proteins and monophosphorylated phosphoinositides, such as phosphatidylinositol-3-phosphate (PtdIns3P) or PtdIns5P. This process occurs in association with mammalian endosomes, where the PROPPIN WIPI1 has additional, undefined roles in vesicular traffic. To explore whether these functions are interconnected, we dissected routes and subreactions of endosomal trafficking requiring WIPI1. WIPI1 specifically acts in the formation and fission of tubulo-vesicular endosomal transport carriers. This activity supports the PtdIns(3,5)P2-dependent transport of endosomal cargo toward the plasma membrane, Golgi, and lysosomes, suggesting a general role of WIPI1 in endosomal protein exit. Three features differentiate the endosomal and macroautophagic/autophagic activities of WIPI1: phosphoinositide binding site II, the requirement for PtdIns(3,5)P2, and bilayer deformation through a conserved amphipathic α-helix. Their inactivation preserves autophagy but leads to a strong enlargement of endosomes, which accumulate micrometer-long endosomal membrane tubules carrying cargo proteins. WIPI1 thus supports autophagy and protein exit from endosomes by different modes of action. We propose that the type of phosphoinositides occupying its two lipid binding sites, the most unusual feature of PROPPIN/WIPI family proteins, switches between these effector functions.Abbreviations: EGF: epidermal growth factorEGFR: epidermal growth factor receptorKD: knockdownKO: knockoutPtdIns3P: phosphatidylinositol-3-phosphatePtdIns5P: phosphatidylinositol-5-phosphatePtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphateTF: transferrinTFRC: transferrin receptorWT: wildtype.

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis.

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P2 , and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.

Characterizing the theory of mind network in schizophrenia reveals a sparser network structure.

Impaired social functioning is a hallmark of schizophrenia and altered functional integration between distant brain regions are expected to account for signs and symptoms of the disorder. The functional neuroarchitecture of a network relevant for social functioning, the mentalizing network, is however poorly understood. In this study we examined dysfunctions of the mentalizing network in patients with schizophrenia compared to healthy controls via dynamic causal modelling and an interactive social decision-making game. Network characteristics were analyzed on a single subject basis whereas graph theoretic metrics such as in-degree, out-degree and edge-connectivity per network node were compared between the groups. The results point to a sparser network structure in patients with schizophrenia and highlight the dorsomedial prefrontal cortex as a disconnected network hub receiving significantly less input from other brain regions in the network. Further analyses suggest that integrating pathways from the right and the left temporo-parietal junction into the dorsomedial prefrontal cortex were less frequently found in patients with schizophrenia. Brain and behavior analyses further suggest that the connectivity-intactness within the entire network is associated with functional interpersonal behavior during the task. Thus, the neurobiological alterations within the mentalizing network in patients with schizophrenia point to a specific integration deficit between core brain regions underlying the generation of higher-order representations and thereby provide a potential treatment target.

Distance-Dependent Cellular Uptake of Oligoproline-Based Homobivalent Ligands Targeting GPCRs-An Experimental and Computational Analysis.

Tumor targeting with bivalent radiolabeled ligands for GPCRs is an attractive means for cancer imaging and therapy. Here, we studied and compared the distance dependence of homobivalent ligands for the human gastrin-releasing peptide receptor (hGRP-R) and the somatostatin receptor subtype II (hSstR2a). Oligoprolines were utilized as molecular scaffolds to enable distances of 10, 20, or 30 Å between two identical, agonistic recognition motifs. In vitro internalization assays revealed that ligands with a distance of 20 Å between the recognition motifs exhibit the highest cellular uptake in both ligand series. Structural modeling and molecular dynamics simulations support an optimal distance of 20 Å for accommodating ligand binding to both binding sites of a GPCR dimer. Translation of these findings to the significantly higher complexity in vivo proved difficult and showed only for the hGRP-R increased tumor uptake of the bivalent ligand.

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of ß-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.