Mass spectrometry-based proteomics methods are finding increasing use in structural biology research. Beyond simple interaction networks, information about stable protein-protein complexes or spatially proximal proteins helps to elucidate the biological functions of proteins in a wider cellular context. To shed light on new developments in this field, the Göttingen Proteomics Forum organized a one-day symposium focused on complexome profiling and proximity labeling, two emerging technologies that are gaining significant attention in biomolecular research. The symposium was held in Göttingen, Germany on 23 May, 2019, as part of a series of regular symposia organized by the Göttingen Proteomics Forum. The one-day symposium “Mapping Cellular Microenvironments: Proximity Labeling and Complexome Profiling” organized by the Göttingen Proteomics Forum (GPF) was held at the Max Planck Institute of Experimental Medicine in Göttingen, Germany on 23 May, 2019. Following the example of past GPF symposia to focus on emerging topics in proteomics research, this year’s event was co-organized by the Collaborative Research Centre SFB1190, a local research consortium that focuses on functional protein clusters located at cell compartmental gates and contact sites. Witnessed by more than 100 participants, a range of invited and local speakers presented novel or updated approaches and applications for proximity labeling and complexome profiling, techniques that complement conventional affinity-purification mass spectrometry (AP-MS) for the characterization of biologically relevant protein-protein interactions. Beyond identification of physically interacting proteins, these techniques can provide information about proteins in spatial proximity of a protein of interest (proximity labeling) or the composition, stoichiometry and apparent molecular weight of stable protein complexes (complexome profiling). Both techniques have recently gained significant attention in biomolecular research. Complexome profiling was the topic of the morning session, encompassing a set of methods to characterize the composition of protein complexes on a global scale under near-native conditions. Complex proteome preparations are fractionated for the apparent molecular weight of stable protein complexes by either blue native polyacrylamide gel electrophoresis (BN-PAGE)  or size As first speaker, Uwe Schulte  presented the pioneering work of his group on complexome profiling by BN-PAGE-mass spectrometry . Using pIn direct continuation, Daniel Kownatzki-Danger , as a local speaker, presented an application-oriented approach where BN-PAGE-based complexome profiling was used to compare native cardiac protein complexes in membrane preparations from mouse ventricular cardiomyocytes, as well as in human ventricular tissue biopsies. BN-PAGE was previously proposed as a differential diagnostic tool to study, e.g., OXPHOS defects in cardiac disease . Daniel https://github.com/CCprofiler/. Differential comparison of multiple samples in a clinical context requires faster turnaround, restricting the degree of fractionation and the amount of instrument time available per sample. In addition, differential analysis of multiple samples demands for a very high degree of data completeness, i.e., sample-over-sample consistency in detection and quantitation of peptides. Data-independent acquisition mass spectrometry (DIA-MS) has emerged as a data acquisition strategy that addresses both of these challenges, provided that information about the protein composition of the sample exists to deal with false positive identifications . As the 2O2 activation for generation of reactive biotin-phenoxyl radicals that covalently bind aromatic amino acid residues at proximal proteins within minutes and below [The afternoon session addressed proximity labeling techniques with biotin in living cells. In contrast to complexome analyses, only proteins localizing proximal to a bait protein (tens of nanometers apart) are covalently labeled and subsequently enriched from cell lysates. Captured proteins are then identified with either antibodies (targeted) or mass spectrometry (unbiased). Gene fusions couple bait proteins to enzymes that serve as source of reactive biotin derivatives, either to an engineered soybean ascorbate peroxidase (APEX) or to a modified biotin-ligase (BioID) in order to pursue biotin-based identification of labeled proteins. APEX requires cellular uptake of biotin-phenol and timed Hnd below ,12. Mutand below ,14. MoreSaccharomyces cerevisiae: Ribosomal Gβ-like protein Asc1/RACK1 and membrane-associated PROPPINs. PROPPIN-containing protein complexes are difficult to enrich with classical immunoprecipitation experiments according to their membranous cellular context. Studying membrane-localized bait proteins is feasible using APEX or BioID approaches, as both techniques rely on covalent protein biotin-labeling within living cells, enabling the capture of biotinylated proteins from cell lysates under denaturing conditions afterward, e.g., in the presence of membrane-solving detergents. Both BioID projects made use of Strep-Tactin (IBA GmbH)-based biotinyl enrichment, SILAC-based enrichment-quantification, and unbiased protein identification with mass spectrometry. Strep-Tactin captures enable nondestructive elution of biotinylated proteins through high biotin dosages. Accurate relative enrichment-quantification  against the following two negative controls was emphasized as key requirement for subsequent candidate mining within lists of MS-identified proteins: 1. Biotinyl-enrichment from cells heterologously expressing the foreign biotin ligase birA* without fusion to the bait protein, to account for bait-unspecific biotinylation through birA*; 2. Biotinyl-enrichment from cells without expressing foreign birA*, to account for naturally birA*-independent biotinylated proteins. Biotin site-identification within significantly enriched proteins by mass spectrometry was considered as additional evidence for cellular bait proximity, with potential for an additional gain in structural information concerning the spatial orientation or the accessibility of the bait-ligase fusion protein.Oliver Valerius  and Lena Munzel  presented data from quantitative BioID approaches to characterize the head region of the 40S ribosomal subunit (hr40S) and to identify interaction partners of β-propeller-binding-polyphosphoinositides (PROPPINs) localized to endosomes, vacuoles and autophagic membranes, respectively. Both BioID projects targeted β-propeller proteins of the WD40-repeat domain family in the yeast The RACK1-proxiOME revealed ribosomal proteins known as direct neighbors from ribosome crystal structures, proving the feasibility of the experimental concept . Key feaJulien Béthune  and colleagues have developed a methodology that combines the proximity-labeling technique BioID with the concept of protein fragments complementation assays, referred to as split-BioID . The priAquifex aeolicus biotin ligase [Bacillus subtilis biotin ligase [Escherichia coli birA-R118S [Beyond studying the molecular mechanisms of miRNA-mediated silencing with split-BioID and many other biochemical and genetical approaches, the group of Julien Béthune further developed smaller biotin ligases with improved labeling kinetics that can be used in the split assay format. Already published engineered smaller and/or more efficient biotin ligase variants are BioID2, originated from an n ligase ; BASU, on ligase ; and turrA-R118S . MicroID2O2 results in APEX-mediated biotinylation of the VapB environment that is specific for its localization to the INM. Biotinylated proteins were enriched using neutravidin (deglycosylated avidin to avoid lectin enrichment), identified with mass spectrometry, and relatively quantified against essential controls using stable isotope labeling with amino acids in cell culture (SILAC).In the scientific context of nucleocytoplasmic transport, Ralph Kehlenbach  presented data that originated from a quantitative modified APEX approach utilizing an enhanced ascorbate peroxidase 2 (APEX2)-approach to map compartment-specific protein interactions of the vesicle-associated membrane protein-associated protein B (VapB) . Since VExpression system or strategy for expressing the bait-enzyme fusion protein, including codon-usage. Testing for functionality/localization of the fusion protein. Biotin/biotin-phenol uptake, dosage requirements and subcellular localization, toxicity. 1-based label-free quantitation (LFQ), DIA-MS-based label-free quantitation, in vivo stable isotope labeling with amino acids in cell culture (SILAC), or post-digestion chemical peptide labeling). Enrichment-quantification strategies and negative controls (MSCapture/elution strategies (destructive versus non-destructive) and corresponding protein digestion protocols (in-gel versus in-solution vs. on-bead). Stable versus short-lived protein interactions/proximities. Labeling activity versus background noise (sensitivity versus specificity). Overall proximity capture (unbiased screening) versus context-specific proximity capture (targeted proximity capture).The decision for either APEX, BioID, split-BioID or one of their variants strongly depends on the cellular system or organism used, and on the process/complex/cellular site analyzed. Proximity labeling experiments require significant method establishing and optimization efforts. The following aspects should be considered during that process: Taken together, the symposium provided a balanced overview of both complexome profiling and proximity labeling approaches, two emerging mass spectrometry-based technologies in structural biochemistry that complement traditional affinity purification approaches and thus allow for a much more fine-grained study of cellular organization and cellular function.


Sc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrPC to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes. Structural diversity of PrPSc strains has been well documented, yet the question of how different PrPSc structures elicit multiple disease phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of PrPSc, are important players in defining strain-specific structures and disease phenotypes. This article introduces a new hypothesis, according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms that form strain-specific patterns of carbohydrate epitopes on PrPSc surface and contribute to defining the disease phenotype and outcomes.Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrP Sc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC. Prions spread between organisms or from cell to cell by recruiting host-encoded PrPC and replicating their disease-specific misfolded structures via a template-assisted mechanism [Sc deposition patterns [C to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes [Sc strains has been well documented [Sc structures elicit multiple disease phenotypes remains poorly understood.Prion diseases or transmissible spongiform encephalopathies represent a class of lethal, transmissible neurodegenerative disorders of humans and animals ,2. Prionechanism . Prion dpatterns . The divsubtypes ,7,8,9,10cumented ,12,13, yC is post-translationally modified with a glycophosphatidylinositol (GPI) anchor and up to two N-linked glycans [C into PrPSc [C expressed in a cell exhibit astonishing heterogeneity, which is attributed to variations in the structure and composition of N-glycans with more than 400 different PrPC glycoforms described [C molecules also display a dramatic range of pIs or net charges at physiological pH [Sc structural and pathological features.PrP glycans ,15,16,17to PrPSc ,19. PrPCescribed ,21. Becaescribed ,21, PrPCgical pH ,23. ConsSc particles proposed in the past assumed that the N-glycans are directed outwards [Sc particles. For testing whether N-glycans are indeed directed outwards of PrPSc particles, brain slices from scrapie-infected animals were stained using Sambucus Nigra lectin (SNA). Animals infected with the S05 strain were selected, as this strain is characterized by deposition of large PrPSc plaques [C and PrPSc, sialic acid residues are attached to galactose via α2-3 or α2-6 linkages with the majority being linked via α2-6 [Sc plaques showed intense staining with SNA . They iiewed in ,32,33,34Sc [C.For addressing this question, a tri-antennary glycan with a size average of those found in PrP sialoglycoforms was used for modeling PrPSc . In in-rSc . ConsideSc . HoweverSc . This mo. identity . TowardC sialoglycoforms selectively, we analyze the composition of sialoglycoforms within PrPSc using 2D gels [Sc was denatured into monomers first, and then monomers were separated according to their sialylation status in horizontal dimension and their glycosylation status in vertical dimension. Then, 2D analysis of multiple strains from two hosts revealed that PrPSc strains exhibit a broad range of strain-specific selectivity with respect to PrPC sialoglycoforms  and achieved in the absence of significant strain-specific variations in folding pattern. Specifically, we propose that for non-selective strains, there is a considerable twist or rotation between neighboring PrP molecules assembled in PrPSc particles  to explore the role of N-linked glycans in prion replication [C molecules were required for propagating the RML strain [C molecules [C molecules were required for replication of the Sc237 strain suggests that N-linked glycans might play a role in stabilizing multimeric structures of non-selective strains via intermolecular interactions.Previous studies employed differentially glycosylated PrPlication . ConsistL strain , which bolecules . The facC, removal of PrPC glycans is expected to impair fidelity of replication. Eliminating the entire glycans from donor PrPSc did not affect strain-specific neurotropism upon transmission of two strains, RML or 301C, to wild type host with unaltered glycosylation status of PrPC [C expressed by a host was altered. Transmission of prions to hosts expressing PrPC deficient in one or both N-glycans was found to change strain-specific characteristics of the 79A strain, yet did not affect considerably strain-specific properties of ME7 or 301C [C was found to have a significant impact on the transmission of prions between different species [C glycans might indeed impair fidelity of prion replication to the extent that, in some cases, result in changes of strain properties and disease phenotype.Prions replicate with high fidelity when transmitted within the same host. Do N-glycans play a role in preserving high fidelity of prion replication? Considering that N-glycans limit diversity of folding patterns accessible to PrP of PrPC . However or 301C . Moreove species . These sSc adapts three- or four-rung solenoid folding patterns, sialylation of N-linked glycans would result in a highly dense negative charge on PrPSc surface . Differresponse ,47. BecaSc surface in triggering CNS response and determining the fate of prions  [Sc is a self-replicating transmissible state [Sc recruits mono- and un-glycosylated PrPC of a host, while strongly excluding di-glycosylated and sialylated PrPC molecules [Sc had the lowest glycosylation and sialylation status [Sc is significantly lower relative to those of conventional PrPSc or brain sialoglycocalyx  ,40,66. Ale state ,67,68, wle state ,40,66,67olecules  (strain n status ,37. Staiycocalyx E,F. Yet,” signal . As a reSc surface and impose considerable structural constraints to PrPSc assembly due to their bulky size and electrostatic repulsion between sialic acid residues. To overcome these restraints, prion strains exhibit a wide range of selectivity in recruiting PrPC sialoglycoforms. While some strains recruit sialoglycoforms proportionally to their representation in PrPC, others avoid diglycosylated and highly sialylated PrPC glycofoms. We propose that strain-specific selectivity in recruiting PrPC sialoglycoforms reports on strain-specific differences in PrPSc quaternary structures and has to be taken into consideration when building realistic PrPSc models. Moreover, we hypothesize that as a result of strain-specific selection, N-linked glycans form individual strain-specific patterns of functional carbohydrate epitopes on PrPSc surfaces and elicit strain-specific responses in CNS. It has been well established that the innate immune system senses terminal carbohydrate groups including galactose and sialic acid residues, which can serve as molecular cues and trigger diverse response programs by glia. Consistent with previous studies on self-associated and pathogen-associated molecular patterns, our work revealed that desialylation of PrPSc undermines survival of prions in vivo. At the same time, recruiting PrPC with low sialylation levels speeds up PrPSc replication, which could be beneficial to prion spread. Therefore, evolution of prion strains might represent a delicate balance between recruiting of highly sialylated glycoforms and generating sufficient density of sialylation for avoiding an “eat-me” response by glia, yet limiting extremely heavily sialylated glycoforms for enabling efficient prion replication. It remains to be tested in future studies whether carbohydrate epitopes on PrPSc surface trigger responses in the CNS in a strain-specific manner.N-glycans are exposed on the PrP


Parkinson’s disease (PD) has a wide spectrum of symptoms including the presence of psychiatric disease. At present, most treatment plans, comprised of dopaminergic drugs, are chronic and complex. Though dopaminergic agents are quite efficient in managing the motor aspects of the disease, chronic pharmacotherapy specifically with dopamine receptor agonists has been highly linked to the occurrence of Impulse Compulsive disorder (ICD), which can be problematic for individual patients.Much of what is known today about PD-related ICD stems from brain imaging studies, however, evidence is not quite conclusive. Research in the field has been focused on identifying the underlying mechanisms of PD-related ICD and understanding the functions of the structures involved in the reward network.This article presents an update of recent findings from key neuroimaging studies in PD-related ICD, discusses results from controversial studies, and identifies areas for future research in the field. Impulse Control Disorder (ICD) is a class of psychiatric disorders that present as alteration of one or more behaviours. In Parkinson’s disease (PD), ICD typically refers to compulsive gambling , hypersexuality (increased pursuit of having sex), binge-eating, excessive shopping, dopamine dysregulation syndrome (compulsivity for PD medication overuse) and punding . Within the spectrum of PD-related ICDs, hobbyism and walkabout are also described . HobbyisThe management of PD-related ICD should be individualized. It is often challenging and most commonly comprises modification of complex dopaminergic regimes . DependiMultiple ICDs are less common in PD (experienced by 8.8% of PD patients versus 1.3% of controls), as compared to single ICD . Though The consensus is that higher ICD prevalence rates are related to dopamine replacement therapy (DRT) and in particular to an increased exposure to DAg , 12. IntObservational epidemiology studies have identified additional risk factors for ICD including younger age at PD onset, male sex, being unmarried, having a history of smoking, depression and a family history of pathological gambling , 18, 19.Most of the recent imaging studies presented here share a rather common study design: PD patients who experience an ICD, abbreviated here as PD(ICD)+, are directly compared to a group of PD patients without a history of ICD, abbreviated as PD(ICD)−. The imaging techniques employed in most published reports include several structural and functional magnetic resonance imaging (MRI) sequences and serial positron emission tomography/single photon emission computed tomography (PET/SPECT) studies. Data from matched healthy control groups are often included for comparison to increase robustness. It should be noted that most studies in the field consider PD-related ICD as one clinical entity and do not differentiate between specific compulsive behaviours. With current knowledge, the study of underlying ICD mechanisms is designed on this basis i.e. patients with one specific ICD are generally not compared against a group of patients with another ICD but to a group of PD(ICD)− patients.Generally, the reward network is composed of dopaminergic, glutamatergic and GABAergic terminals within the cortico-basal ganglia-thalamocortical loop , 26. TheThe ventral striatum is anatomically comprised of the nucleus accumbens, the medio-ventral part of the caudate and putamen nuclei, the olfactory tubercle and the anterior perforated substance , 29. TheIn comparison to PD(ICD)− patients, PD(ICD)+ patients have shown significantly lower blood oxygenation level dependent activity in the ventral striatum during risk taking and significantly reduced resting regional cerebral blood flow in the same region . VentralAt resting state, the anterior putamen appears in PD(ICD)+ patients as functionally disconnected from both anterior cingulate gyrus and inferior temporal gyrus . MoreoveThe amygdala is commonly linked to reward learning and PD(ICD)+ patients with punding have shown lower functional connectivity between the amygdala and the hippocampus . Of inteThe studies above together suggest that in PD-related ICD, connectivity is dysfunctional within and between dopaminergic neuronal circuitries involving important subcortical and limbic-cognitive cortical regions. Among these, the default-mode, the salience and the central-executive networks seem to show consistent involvement.Cortical thickness studies have not been greatly conclusive. PD(ICD)+ patients appear to have higher cortical thickness in the anterior cingulate cortex and rostral pole as well as in orbitofrontal cortex when compared to PD(ICD)− patients , 37. Of PD(ICD)+ patients as well as drug-naïve PD patients have been shown through DTI tractography to have widespread white matter tract damage in particular showing increased radial and axial diffusivity of the genu of corpus callosum and the pedunculopontine tract , 41, 42.18F-Fluoro-2-deoxy-2-d-glucose (18F-FDG) PET during a gambling paradigm (+)-PHNO) PET binding was lower in the ventral striatum of the PD(ICD)+ patients and in negative correlation with clinical measures of ICD severity [11C](+)-PHNO binding was increased in the dorsal striatum across all PD patients in comparison to healthy controls  in the ventral striatum and the putamen, while self-reported severity of ICD positively correlated with 18F-Fallypride binding in the midbrain [18F-fallypride binding was no different between PD(ICD)+ and PD(ICD)− groups [In a more recent study, PD(ICD)+ patients had reduced post-synaptic dopamine receptor densities [as reflected by (S)-N-((1-Allyl-2-pyrrolidinyl)methyl)-5- is considered a relatively robust target to study the integrity of dopaminergic terminals. Nevertheless, given that DAT is highly expressed within the dorsal striatum, DAT-specific imaging in PD has been most commonly used in PD diagnostics and less in studying the non-motor aspects of PD.18F-Fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl)nortropane (18F-FP-CIT) PET, PD(ICD)+ patients have shown significantly higher DAT density in the dorsal posterior cingulate and insular cortices as compared to PD(ICD)− [By using DAT-specific N-(3-PD(ICD)− . HoweverPD(ICD)− , 57. RelPD(ICD)− . In the PD(ICD)− . Taken tPD(ICD)− . Thus, i18F-dopa PET) in nucleus accumbens and impulsivity severity (as reflected by appropriate ICD rating scores), suggesting that nucleus accumbens, which is functionally connected to the rostral anterior cingulate cortex, is significantly affected in PD-related ICD [More recently, an interesting multimodal imaging study combined fMRI with PET data from a large group of PD patients. A negative correlation was found between dopamine synthesis capacity .It appears that ICD develops in some PD individuals from interactions between prescribed dopaminergic drugs and pre-existing structural and functional alterations. Increased dopamine release in the ventral striatum and possibly reduced expression of post-synaptic dopamine receptors form the background mechanisms, on which DAg act to trigger ICD. Constant stimulation by DRT agents may therefore negatively affect the relatively preserved ventral striatum by exacerbating the lack of control within the reward system. In addition, dysfunction in the amygdala and abnormal ventromedial subthalamic nucleus stimulation may contribute to the above dynamics and exacerbate ICD. Future neuroimaging studies should focus on clarifying the complex interactions of all involved neuronal networks and improve the current strategy for managing PD-related ICD.


Malignant glioblastoma  is the most common and aggressive primary adult brain tumor. The prognosis of GBM patients remains poor, despite surgery, radiation and chemotherapy. The major obstacles for successful remedy are invasiveness and therapy resistance of GBM cells. Invasive glioma cells leave primary tumor core and infiltrate surrounding normal brain leading to inevitable recurrence, even after surgical resection, radiation and chemotherapy. Therapy resistance allowing for selection of more aggressive and resistant sub-populations including GBM stem-like cells (GSCs) upon treatment is another serious impediment to successful treatment. Through their regulation of multiple genes, microRNAs can orchestrate complex programs of gene expression and act as master regulators of cellular processes. MicroRNA-based therapeutics could thus impact broad cellular programs, leading to inhibition of invasion and sensitization to radio/chemotherapy. Our data show that miR-451 attenuates glioma cell migration in vitro and invasion in vivo. In addition, we have found that miR-451 sensitizes glioma cells to conventional chemo- and radio-therapy. Our data also show that miR-451 is regulated in vivo by AMPK pathway and that AMPK/miR-451 loop has the ability to switch between proliferative and migratory pattern of glioma cells behavior. We therefore postulate that AMPK/miR-451 negative reciprocal feedback loop allows GBM cells/GSCs to adapt to tumor “ecosystem” by metabolic and behavioral flexibility, and that disruption of such a loop reduces invasiveness and diminishes therapy resistance. Malignant glioblastoma (GBM), the most common primary brain tumor in adults, is among the most devastating cancers, with a median survival of approximately 15 months  which isGBM presents unique challenges to therapy due to its location, aggressive biological behavior, diffuse infiltrative growth and intratumoral heterogeneity ,6,7. GBMTherapeutic strategies targeting GBM require multi-target molecules to overcome heterogeneous nature of the disease. The deepened understanding of molecular and cellular mechanisms of targeted factors in discovery stage can considerably shorten expensive clinical phase and increase the chance of the development of successful approach. Testing naturally expressed microRNA candidates using heterogeneous, patient-derived cells and clinically relevant model systems to combine their effect with standard therapy is innovative way to turn phenotypic data into robust therapy-relevant discoveries.As we published in Reference , miR-451Control GBM12 (GFP) did not form solid tumors, but rather a diffuse distribution of single cells within the brain. However, GBM12 (GFP-miR-451) cells formed well-circumscribed nodular tumors b. AlthouWe tested an extended panel of GSCs for their invasive potential. From an extensive collection of over 40 GSCs validated for their ability to invade in the brain, we estimate that 20–30% of them are capable of invading in vivo, while others form nodular tumors—both scenarios are shown on Our data on miR-451-mediated suppression of migratory behavior of GBM cells was recently supported by findings pertaining other cancer model as well as GBM. MiR-451 inhibited the migration and invasion in vitro, as well as in vivo metastasis of hepatocellular carcinoma cells through regulating epithelial-mesenchymal transition process . ImportaThe role of miR-451 in drug resistance of cancer cells has been reported in several malignancies. Expression of miR-451 in doxorubicin-resistant breast cancer cells increased their sensitivity to the drug . ImatiniOur data has shown that miR-451 possesses significant anti-migratory effects in GBM cells and that high levels of glucose are required to maintain its expression . AdditioThese findings thus support a unique role of miR-451 the pathophysiology of GBM. Next, we queried both The Cancer Genome Atlas database and our collection of GBM matched samples  for the expression of miR-451. As evident from The adaptation of cancer cells to the continuously changing conditions of their microenvironment as tumor progresses involves dynamic and flexible mechanisms. Tumor cells require an uninterrupted influx of nutrients and oxygen to boost growth and maintain elevated metabolism; yet, supply of blood and energy to nourish these needs is often insufficient. The adaptive mechanisms to insufficient oxygen (hypoxia) have been well described, yet the adaptations to fluctuations in glucose—the major carrier of energy are poorly understood. Elevated glucose metabolism is a hallmark of numerous solid tumors and enhanced glucose uptake was recently demonstrated to be one of the mechanisms of adaptation of glioblastoma cells to dwindled glucose supply . We hypoCells exposed to low glucose experience shortage of ATP, increasing the [AMP]/[ATP] ratio. This event activates the 5′AMP-activated protein kinase (AMPK) complex—a highly conserved energy sensor belonging to a class of serine/threonine kinases. When cellular energy levels are decreased, AMPK is phosphorylated by LKB1 . Our groOct1−/− fibroblasts that showed several-fold diminished levels of miR-451 [Oct1−/− fibroblasts it rescued the expression of miR-451. The reciprocal negative feedback loop between miR-451 and AMPK and the inactivation by phosphorylation of transcription factor promoting miR-451 in conditions of low glucose and AMPK activation, suggested that AMPK may be the kinase directly phosphorylating OCT1. Briefly, we demonstrated that expression of miR-451 inversely correlates with activated AMPK and that AMPK is sufficient for the phosphorylation of OCT1 at S335, thus establishing AMPK as the kinase responsible in shutting-off OCT1-mediated transcription of miR-451 in response to glucose availability [We demonstrated that miR-451 directly targets the 3′-UTR of CAB39 —co-activ miR-451 . Moreove miR-451 . In GBM  miR-451 . Re-intrlability .Recently, it has become increasingly clear that the AMPK complex endows cancer cells with the ability to survive the exposure to stress, (including energy deficiency and genomic damage) , despiteForced expression of miR-451 under stress leads to cytotoxicity , as adapAll standard experimental procedures  were performed as described by us ,62,105. t test from the GraphPad Prism software. Differences were considered statistically significant at p < 0.05. All microscopy-based assays were edited/quantified using ImageJ using the Analyze Particles function of binary images with an automatic threshold. One-way ANOVA followed by Bonferroni’s test was conducted to test for significance among multiple groups. p < 0.05 was considered significant. All significantly deregulated transcripts were visualized via a heatmap and analyzed using dChip software with the Statistical R package. An unpaired, two-tailed t test was used to compare two groups. The list of putative miR-451 target mRNAs was generated based on Target Scan v. 7.1 (http://www.targetscan.org/vert_71/).Data are expressed as mean ± SD. Statistical analyses were performed using the unpaired two-tailed Student’s www.qiagen.com/ingenuity). Experimental and clinical data were analyzed using the GBM-BioDP [https://tcga-data-nci-nih-gov.ezp-prod1.hul.harvard.edu/). Gene expression in the various anatomical regions of glioblastoma was analyzed using the Ivy Glioblastoma Atlas Project . Level 3 microRNA expression data (unc.edu_GBM.H-miRNA_8 × 15 K. Level_3.1.8.0) from 479 glioblastomas were obtained from TCGA [Functional bioinformatics analyses were performed using Qiagen’s Ingenuity Pathway Analysis  expressescribed .


Microfluidic devices have been widely used for cell migration research over the last two decades, owing to their attractive features in cellular microenvironment control and quantitative single-cell migration analysis. However, the majority of the microfluidic cell migration studies have focused on single cell types and have configured microenvironments that are greatly simplified compared with the in-vivo conditions they aspire to model. In addition, although cell migration is considered an important target for disease diagnosis and therapeutics, very few microfluidic cell migration studies involved clinical samples from patients. Therefore, more sophisticated microfluidic systems are required to model the complex in-vivo microenvironment at the tissue or organ level for cell migration studies and to explore cell migration-related clinical applications. Research in this direction that employs organ-on-chip-related approaches for cell migration analysis has been increasingly reported in recent years. In this paper, we briefly introduce the general background of cell migration and organ-on-chip research, followed by a detailed review of specific cell migration studies using organ-on-chip-related approaches, and conclude by discussing our perspectives of the challenges, opportunities and future directions. The physical structure of cells is supported and organized by dynamic functions of the cytoskeleton. Dynamic remodeling of this structure enables cell movements in diverse biological contexts, such as cell–cell migratory interaction, wound healing and transmigration across tissue barriers . The migIn-vitro cell migration assays, such as transwell assays  and otheMicrofluidic devices were rapidly developed over the last two decades and these devices have been increasingly used for cell migration research  22,23].,23.22,23Generally speaking, the construction of organ-on-chip systems is based on microfluidic cell-culture devices, which simulate the key activities and responses of certain tissues or organs. This approach integrates microfluidics technology with tissue engineering, permitting investigation of organ-specific physiological mechanisms and diseases. For example, some studies successfully established “liver-on-chip” and “kidney-on-chip” systems for studying hepatic or renal functions, respectively ,32,33. SIndeed, in this direction, we have observed growing development of cell migration studies involving organ-on-chip-related approaches. In this paper, these studies are broadly defined as meeting one or more of the following criteria: (1) cell migration and interaction studies that involve reconstituting the physiological structures of specific organs using multi-cell co-culture in three-dimensional (3D) microfluidic models; (2) cell migration studies in microfluidic devices that configure complex chemical fields relevant to target organs; and (3) cell migration studies that test clinical cell or/and tissue samples for diagnostic assessment of organ-specific diseases. Under these selection criteria, the rest of this paper is organized to review organ-specific cell migration studies using organ-on-chip-related approaches including “tumor-on-chip”, “lung-on-chip”, “vessel-on-chip”, “lymph node (LN)-on-chip” and “brain-on-chip” . We concCancer cell migration is a critical process during cancer progression and metastasis. During metastasis, tumor cells migrate from their initial locations to distant organs, leading to new tumor formation ,54,55. CAs one example, Kamm and coworkers developed a three-dimensional microfluidic model for investigating endothelial barrier function during tumor cell intravasation A 36]. T. T36]. TAs another example, Wiklund and coworkers established an ultrasonics-based 3D microdevice for studying immune surveillance of natural killer (NK) cells for specific tumors B 37]. B. B37]. BIn another study, Imparato and coworkers developed a breast-cancer-on-chip model to investigate ECM activation during tumor progression C 38]. T. T38]. TFinally, Jiang and coworkers developed a tumor-on-chip model to investigate the interactions between neurons and cancer cells during tumor perineural invasion (PNI) D 39]. T. T39]. TNeutrophils are the largest population among white blood cells, and their migration plays important roles in the human immune system . DisordeE. coli) bacteria were injected into the alveolar epithelial cell layer, which rapidly activated ECs and increased the surface expression of intercellular adhesion molecule 1 (ICAM-1) in the microvascular channel. The results showed that this effect induced neutrophil recruitment to ECs, and stimulated their directional transmigration across the tissue barrier for immune surveillance. This system demonstrated a low-cost on-chip model for cell migration study and provided an alternative option to animal models for drug delivery and toxicity research.Ingber and coworkers engineered a biomimetic lung-on-chip microsystem, which replicated the alveolar–capillary structures of human lung, for investigating bacteria or inflammatory cytokine-induced cell migration. The microfluidic device consisted of two side chambers, and one main channel with two compartments separated by a porous ECM-coated PDMS membrane . The uppInstead of mimicking the lung microenvironment, other studies focused on testing immune cell migration using clinical samples from patients with specific lung diseases for potential diagnostic applications. COPD is a type of lung disease associated with breathing difficulty, which is caused by narrowed airways . A previLeukocyte transendothelial migration (TEM) from blood vessels to inflammatory sites is a critical process for human immune responses. This migration process is mediated by both physical and chemical cues through complex interactions between leukocytes and ECs ,67. SimiN-formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8) were tested for inducing neutrophil chemotaxis. In addition, the number and migration behavior  of neutrophils that transmigrated through both the EC monolayer and ECM layer were measured. These results showed that fMLP gradients more strongly attracted neutrophils than IL-8 gradients, and that neutrophil–EC interactions were indispensable to neutrophil TEM during the inflammation process. Despite its utility in the cell migration study, this system can be used as a potential disease model for drug screening as well.Chung and coworkers developed a vessel-on-chip model for investigating neutrophil TEM during inflammatory processes A 35]. T. T35]. TIn another study, Qin and coworkers developed a biomimetic blood-vessel-on-chip model for investigating tumor TEI B 42]. T. T42]. TAs another example, Kamm and coworkers established a microvasculature-on-chip model for investigating tumor cell extravasation C 43]. B. B43]. BBesides these TVM-oriented studies, Neumann and coworkers developed a microvessel-on-chip model for investigating angiogenesis . The devDendritic cells (DCs) are the most-potent antigen-presenting cells (APCs) in the immune system . GeneralFor example, a mimetic LN-on-chip flow device was developed for investigating the interaction between T cells and DCs A 45]. T. T45]. TIn another example, Yarmush and coworkers developed a microfluidic device for evaluating DC chemotaxis and DC–T cell interaction B 46]. B. B46]. BIn another study, Swartz and coworkers established a 3D agarose-based microfluidic device for investigating differential chemotaxis of DCs to CCR7 ligands CCL21 and CCL19 C 47]. T. T47]. TSimilarly, Lin and coworkers used a microfluidic device to configure simple or complex co-existing chemical fields for studying the guidance of CCR7 ligands during T cell migration in LNs D 5]. Th. Th5]. THammer and coworkers also attempted to reconstitute the complex chemical microenvironment in LNs based on a network-shaped microfluidic flow device for investigating differential DC chemotaxis through CCR7 and CXCR4 signaling . Using tNeural cell migration is an essential process during early embryonic brain development and for the central nervous system (CNS). Neural cell migration is regulated by various chemotactic factors such as inflammatory chemokine CXCL12 . FurtherA recent study generated a biomimetic brain-on-chip model for investigating neuronal differentiation and chemotaxis A 49]. T. T49]. TAs another example, Nery and coworkers developed a microfluidic-based method for investigating neuronal migration in embryonic brain explants B 50]. B. B50]. BIn another study, Keenan and coworkers established a microfluidic-based method to investigate human neural stem cell (hNSC) neurogenesis . BrieflyIn contrast to the above studies that focused on neuronal differentiation and migration, Qin and coworkers developed a mimetic organotypic microfluidic system that reproduced the principal structural, functional and mechanical features of the BBB in vivo, in order to investigate brain tumor metastasis C 52]. T. T52]. TUnderstanding the mechanisms of cell migration and developing its applications are of great significance for fundamental research and are important targets for disease diagnosis and therapeutics. Organ-on-chip-related approaches offer a promising new concept, enabling technologies to study cell migration in biomimetic tissue or organ microenvironments and to permit clinical applications. These approaches stem from microfluidics and tissue engineering, but the integration of the two in a highly controlled manner is significantly beyond each individual technology or their simple combination. A considerable number of current cell migration studies using organ-on-chip-related approaches employed similar ideas and techniques to reconstitute various organ structures and environments, suggesting the natural emergence of diverse organ-specific applications into the same conceptual and technological framework. We have observed the active research development of this emerging field and we anticipate its continuing growth in the future.On the other hand, current organ-on-chip technologies for cell migration research are still in their infancy and thus are facing many challenges. Firstly, precise presentation of guiding signals in space and time is an important building block of the organ microenvironment for cell migration. While 3D multi-cell co-culture microfluidic devices more closely mimic physiological tissue or organ microenvironments, the accompanied reduction in their ability to control and manipulate chemical gradients presents a significant drawback compared with 2D systems. In contrast, the 2D microfluidic approach is well established to study cell migration in precisely defined, simple and complex physiologically-relevant chemical fields. While these systems are fundamentally limited to mimic the real in-vivo situations, some cell types evidently show poor or very different migratory behaviors in the 2D systems compared with 3D systems. Therefore, developing organ-on-chip models that effectively integrate the respective advantages of 2D and 3D systems in a balanced manner sets one of the immediate future directions. For example, new techniques must be developed to enable advanced partition of 3D viscous gel zones and soluble flow zones within the microfluidic channels that would allow controlled flow transport over the gel environments. In this direction, 3D bio-printing technology offers a promising approach, owing to its unique ability for controlled and rapid reconstruction of different tissue structures. Recent studies have demonstrated the feasibility of integrating 3D printing with biocompatible micro/nanoparticles for biomimetic bone tissue engineering, which demonstrated the potential of 3D bio-printing for broad organ-on-chip applications ,79,80. SSecondly, while the widely used PDMS microfluidic devices offer advantages in fast prototyping, low cost, biocompatibility and optical transparency, the interactions between various biomolecules and PDMS can be a significant issue for organ-on-chip applications, such as drug testing and ECM configuration. New alternative device fabrication materials are, therefore, being explored. For example, recent studies showed that other elastomers such as polyurethanes offer similar features of PDMS, but higher compatibility with small hydrophobic drugs . Cell miThirdly, increased complexity of organ-on-chip devices limits experimental throughput compared with simple devices. The throughput issue is also reflected by the isolated, single organ-on-chip approach for many current studies, which underscores the difficulty of accurately mimicking the complex properties of the interacting multi-organ environments in the body, wherein tightly regulated and highly dynamic cell migration and inter-organ trafficking occurs. Thus, innovative and optimized device designs to improve experimental throughput and advanced multi-organs-on-chip devices are highly desirable for future cell migration research. The work of Qin’s group that mimicked the structural, functional and mechanical features of the BBB is an excellent example of such integrated multiple organ-mimicking units with microchannel networks that are required for advanced on-chip cell migration monitoring and analysis .Finally, mimicking disease conditions and testing diseased clinical samples will ultimately enable diagnostic and therapeutic applications of the organ-on-chip approaches. On the other hand, disease-oriented studies are facing significant scientific and technological challenges to faithfully recreate the pathological processes on the chip, which presents an important open question for microfluidics and cell migration researchers to address in the future.


Both RGB and RGB-D performed similarly in experiment two (bunch-level), while RGB outperformed RGB-D in experiment three (plant-level). The RGB-D sensor (Kinect) is suited to ideal laboratory conditions, while the robust RGB methodology is suitable for both laboratory and in-situ yield estimation.Vineyard yield estimation provides the winegrower with insightful information regarding the expected yield, facilitating managerial decisions to achieve maximum quantity and quality and assisting the winery with logistics. The use of proximal remote sensing technology and techniques for yield estimation has produced limited success within viticulture. In this study, 2-D RGB and 3-D RGB-D (Kinect sensor) imagery were investigated for yield estimation in a vertical shoot positioned (VSP) vineyard. Three experiments were implemented, including two measurement levels and two canopy treatments. The RGB imagery (bunch- and plant-level) underwent image segmentation before the fruit area was estimated using a calibrated pixel area. RGB-D imagery captured at bunch-level (mesh) and plant-level (point cloud) was reconstructed for fruit volume estimation. The RGB and RGB-D measurements utilised cross-validation to determine fruit mass, which was subsequently used for yield estimation. Experiment one’s (laboratory conditions) bunch-level results achieved a high yield estimation agreement with RGB-D imagery (r Modern-day viticulture has seen an increase in the use of robust scientific methods combined with new technologies to improve overall production . PrecisiYield estimation provides information to the winegrower that can be used to manage the vineyard, optimising quality and yield . AwareneWolpert and Vilas  outlinedA common PRS approach employs 2-D  RGB  imagery, captured with a digital camera for yield estimation; for example ,9,13. Th2 of 0.73. An alternative segmentation approach to image classification uses colour thresholding to differentiate grapes from the background. Dunn and Martin  and LR (approximate r2 = 0.75) treatments in experiment two. Experiment two tested the established methodologies in situ, under both FC and LR canopy treatments. The produced results of experiment two—and experiment three—confirmed the hypothesis of a superior yield estimation agreement under the LR canopy treatment. If good canopy management practices are established early in the season, better yield estimation results than those obtained under the FC treatment could be achieved. Both sensors produced similar results for FC [approximate r2 = 0.877) significantly outclassed the RGB-D results (r2 = 0.487) under the FC treatment, whereas a smaller margin between the RGB (r2 = 0.779) and RGB-D (r2 = 0.594) sensors occurred under the LR treatment. Inference behind the differing results can lead to the following observations. The different lighting conditions influenced the results: the RGB imagery was collected at midday, while the RGB-D imagery was collected immediately before sunset. Additionally, the continuous movement of the Kinect sensor (RGB imagery captured in a stationary position) could have contributed to lowered RGB-D results. Further research is encouraged, using a standardised experimental setup, where feasible, to create more favourable conditions under which both sensors can operate. The success of the two PRS methodologies can be differentiated at plant-level by the results of experiment three. Unlike experiment one, the RGB methodology outperformed the RGB-D methodology for yield estimation. The RGB results . However, this practice can be implemented in specific zones of the vineyards, enabling an approximation of the total yield using a monitoring strategy. The FC results provide a better indication of the commercial readiness of the developed methodologies. Future work should determine an optimal canopy coverage that favours both sensor operationality and commercial farming methods.Overall, the use of the Kinect sensor as a cost-effective RGB-D sensor for vineyard yield estimation, specifically at bunch-level, is supported by the results obtained for experiment one. However, the robustness of the RGB methodology is evident across all three experiments, with substantial plant-level results obtained in situ. A better understanding of the current limitations would allow further improvements to the methodologies. To this end, the results obtained in this study support the potential for operationalisation of both PRS sensors. A refined methodology could see commercially favourable data-acquisition methods implemented on a larger dataset, with fully automated data processing. See for example ,16. The A novel approach to a side-by-side investigation of 2-D and 3-D PRS techniques for successful vineyard yield estimation has been presented. This study assessed RGB imagery captured by a digital camera and RGB-D imagery captured by a Kinect V1 sensor across three experiments, with in-situ measurements obtained under two canopy treatments. Our results show that the Kinect RGB-D sensor produced the highest yield estimation agreement under laboratory conditions (bunch-level). At bunch-level, RGB and RGB-D PRS techniques performed equally under both canopy treatments for in-situ yield estimation. At plant-level, the best in-situ results were obtained using the RGB imagery, which significantly outperformed the RGB-D results. Both sensors support the use of PRS technology and techniques for vineyard yield estimation, with improved accuracies presented. The results of this study confirm the operational potential of 2-D RGB imagery for accurate yield estimation, with the recommendation that future work should investigate a more automated RGB methodology suitable for operational environments. Regarding the presented RGB-D methodology, the Kinect demonstrates the potential for vineyard yield estimation using 3-D RGB-D imagery. Future work should investigate the use of the Kinect V2 sensor coupled with suitable lighting conditions for in-situ yield estimation.


Neutrophils have been extensively described in the pathophysiology of autoimmune and infectious diseases. Accumulating evidence also suggests the important role of neutrophils in cancer progression through their interaction with cancer and immune cells in blood and in the tumor microenvironment (TME). Most studies have described neutrophils as key drivers of cancer progression, due to their involvement in various tumor promoting functions including proliferation, aggressiveness, and dissemination, as well as in immune suppression. However, such studies were focusing on late-stages of tumorigenesis, in which chronic inflammation had already developed. The role of tumor-associated neutrophils (TANs) at early stages of tumor development remains poorly described, though recent findings indicate that early-stage TANs may display anti-tumor properties. Beyond their role at tumor site, evidence supported by NLR retrospective studies and functional analyses suggest that blood neutrophils could also actively contribute to tumorigenesis. Hence, it appears that the phenotype and functions of neutrophils vary greatly during tumor progression, highlighting their heterogeneity. The origin of pro- or anti-tumor neutrophils is generally believed to arise following a change in cell state, from resting to activated. Moreover, the fate of neutrophils may also involve distinct differentiation programs yielding various subsets of pro or anti-tumor neutrophils. In this review, we will discuss the current knowledge on neutrophils heterogeneity across different tissues and their impact on tumorigenesis, as well as neutrophil-based therapeutic strategies that have shown promising results in pre-clinical studies, paving the way for the design of neutrophil-based next generation immunotherapy. Neutrophils represent 50–70% of all circulating leucocytes in humans, while they account for 10–25% in mice 1 immunotherapies provides new hope in cancer management. However, due to an overall response rate below 40% , 30 and Cancer-associated systemic inflammation often characterized by a high NLR, associated with a poor prognosis, was thought to occur only at late stages of tumorigenesis , 39. EviDiscrepancies across studies may be due to the level of heterogeneity of the populations studied , limitations of retrospective reports, wide variation in NLR cut-off values, as well as in the dynamic assessment of NLR during treatment, and/or a lack of specificity of NLR (neutrophil count gathering immature neutrophils that might be released in the context of inflammation and expected circulating mature neutrophils). Interestingly, studies assessing the prognostic and/or predictive values of NLR in a wide variety of conditions  argue in favor of its utility. This correlation of NLR with clinical outcome suggests that changes in NLR may be linked with broader modifications beyond the tumor microenvironment. Hence, beyond being a relevant clinical biomarker, increase in NLR with disease progression highlights the importance of considering the systemic environment and not simply the tumor for a deeper understanding of biological mechanisms underlying cancer progression. It is possible that the tumor itself secretes factors into the bloodstream, which thereby act on bone marrow to skew hematopoiesis toward granulocytic lineages. Tumor may also release danger/damaged-associated molecules (DAMP) that might be a target for infiltrating neutrophils. Hence, the means by which an increase in NLR promotes cancer progression remains to be elucidated.in vitro. These T-cell suppressive neutrophils are classically termed granulocytic myeloid-derived suppressor cells (G-MDSCs) for both humans and mice. G-MDSCs were documented to expand in tumor-bearing hosts compared to healthy subjects (+ CD14− CD66b+ CD15hi expressing-cells that are enriched in the low-density neutrophils (LDNs) fraction present within the peripheral blood mononuclear cell (PBMCs) ring, unlike normal density neutrophils (NDNs), which are found in the granulocyte pellet of ficolled blood  in immune deficient NSG-M3 mice accelerated osteosarcoma tumor growth compared with the transfer of committed monocyte progenitors  and Arginase 1 are the two most extensively described neutrophil-derived T-cell suppressive factors , 52, 55.An emerging tumor-promoting function of circulating neutrophils has recently been unveiled. Neutrophils were shown to entrap circulating tumor cells (CTCs) at metastatic sites to facilitate their extravasation thus contributing to metastasis –60. ReceTaken together, in addition to the NLR, there is a strong rationale for routinely monitoring CTC-neutrophil clusters with the aim of evaluating their prognostic impact and predictive value in cancer patients.+ Ly6Cint Ly6Ghi, whereas in humans, they are identified as CD11b+ CD14− CD66b+ CD15hi cells (Neutrophils are able to infiltrate tumor tissue and are termed tumor-associated neutrophils (TANs). In mice, TANs express CD11bhi cells .The clinical relevance of evaluating pro- and anti-tumor functions of TANs is highly pertinent in cancer patients, since TAN infiltration was reported to predict either poor –66 or goTAN infiltration can be also quantified through immunostaining, using antibodies against markers of neutrophils, such as CD66b and CD15. Neutrophils and eosinophils share numerous markers as they are closely related ontologically speaking, and very few studies took this cofounding effect into account. Evidence showed that CD66b is expressed at the same level between eosinophils and neutrophils in peripheral blood of patients with rheumatoid arthritis . For CD1Bioinformatics approaches inferring the fractions of tumor-infiltrating immune cells from bulk tumor RNAseq data have recently emerged . The mos+ myeloid cells secrete interleukin-1 receptor antagonist (IL-1RA), which were shown to antagonize the anti-tumor effects of cellular senescence in a murine model of PTEN−/− prostate cancer  by sustaining the expression of the EMT transcription factor Snail in cancer cells  of neutrophils but also by differences in staining methods such as haematoxylin/eosin (HE), neutrophil elastase (NE), Arginase, MPO, CD66b, or CD15 surface markers. New evidence suggest that the tumor stage may also explain such differences  vs. early-stage tumors (stage I–II), suggesting that pro-tumor PD-L1+ neutrophils only emerge at a later-stage of gastric tumorigenesis. Further investigations will be needed to confirm the predominance of pro-tumor TANs in late-stage gastric tumors compared to early-stage tumors. In sharp contrast, in lung cancer patients, TANs, not peripheral blood neutrophils, from early-stage tumors (stages I–II), were reported to enhance the proliferation of autologous T-cells stimulated with anti-CD3/CD28 antibodies , Phorbol 12-myristate 13-acetate (PMA), N-Formylmethionyl-leucyl-phenylalanine (fMLF formerly termed fMLP)  , 122 in ed fMLP) . The meced fMLP) . The enged fMLP)  or Fc-gaed fMLP)  was alsoc sepsis  and in mc sepsis . Neutropc sepsis , 126. Thc sepsis . Interesc sepsis . Activatc sepsis .in vitro  into pro-tumor SiglecF+ TANs  requiredive TANs . Splenecive TANs . Collect+ neutrophils from G-CSF-treated healthy donors display T-cell suppressive properties ex vivo, whereas those from untreated healthy donors did not. Interestingly, CD10+ neutrophils from untreated healthy donors treated with G-CSF in vitro did not display any suppressive activities  . Accumul studies . The bioin vivo. Pre-clinical studies in the mouse have already reported therapeutic effects of neutrophil depletion using the specific neutrophil-depleting antibody, anti-Ly6G , was first developed to treat cancer patients with alterations affecting the c-MET pathway in cancer cells due to activating mutations, overexpression, gene amplification, and translocations . Interesmor TANs  in diffein vitro and to potentiate the effect of CTLA-4 immune checkpoint blockade in vivo. A recent first-in human clinical trial evaluated the clinical impact of targeting TRAIL-R2. In cancer patients with elevated levels of LDNs before treatment, the TRAIL-R2 agonist antibody selectively depleted LDNs without impacting the number of other peripheral blood myeloid and lymphoid cells, nor showing dose-limiting toxicities  (−/− or Casp1−/− mice or wild-type mice treated with the IL-1 receptor antagonist (IL-1Ra)  was reported to have anti-tumor effects M-MDSCs) . In this(IL-1Ra) . TherefoTaken together, studies aiming at evaluating the neutrophil-depleting effects of already-approved therapies remain contrasted. These discrepancies are likely due to differences in doses, timing of administration, location of neutrophil sampling, time of neutrophils detection, as well as mouse and tumor models , 173. Fuin vitro that neutrophil-derived arginase 1 suppresses T-cell proliferation  patients, the elevated percentage of the T-cell suppressive CD15t manner . Studiest manner , 188. Indication . HoweverThe C5a receptor  becomes fairly well known for its role in immunosuppressive activity of myeloid cells. Lung cancer cells were found to produce C5a which could bind to C5aR expressed by myeloid cells to increase their immunosuppressive functions . ConcentSTAT3 transcription factor is well known to be important for the tumor-promoting activities of myeloid cells such as immunosuppression –197 or tin vitro  encoding the very long-chain acyl-CoA synthetase, specifically in neutrophils using the S100a8-cre mice, abrogated tumor growth in different tumor models  and IgG-Fc-Receptors , evidence showed that neutrophils are more effective at mediating ADCC through IgA antibodies than IgG antibodies in vitro , 110. Nuin vitro . Due to Although immunotherapies held great promise for oncology, only a small percentage of cancer patients showed benefits from these treatments. The search for new therapeutic targets is thus critically needed. Current immunotherapies mostly rely on the adaptive immune system, involving adaptive immune checkpoints such as CTLA-4, PD-1, and PD-L1. Recent pre-clinical studies suggest that innate immunity could offer new therapeutic opportunities. Inhibition of the innate immune checkpoints Tyro3, Axl, and Mertk tyrosine kinase receptors, mostly expressed by TAMs, displayed additional therapeutic effects when combined to the anti-PD-1 monoclonal antibody in a murine model of triple-negative breast cancer . Accumulex vivo functional analyses also support the concept of an anti-tumor role for neutrophils in early-stage human tumors. Although challenging, access to early-stage tumors will be original and fundamental to gain new insights into potentially novel yet undiscovered phenotypes and functions of TANs. With such diverse roles, the ideal targeting of neutrophils in oncology would be to promote the enrichment of anti-tumor neutrophils while depleting pro-tumor ones without altering antibacterial neutrophils.While the elimination of neutrophils seems to provide therapeutic effects in established tumor models, emerging evidence favors an anti-tumor role for neutrophils in early-stage tumors in mice , 115. EvThe current dogma in cancer immunotherapy mainly relies on restoring anti-tumor T-cell response. However, a high tumor infiltration by anti-tumor lymphocytes is not sufficient to eradicate all tumor clones during the course of metastasis as for example in colorectal cancer patients, shedding light on tumor-intrinsic and tumor-extrinsic mechanisms of escape that remain to be discovered . Resistain vitro studies. It remains to be known if such neutrophil-mediated T-cell suppression occurs in blood in vivo. Growing evidences also highlight the importance of distant microenvironment away from tumor bed such as the bone marrow and the spleen to educate pro-tumor TANs (Beyond their role at tumor site, neutrophils seem to promote tumor progression by acting systemically as exemplified by the ability of blood neutrophils to escort circulating tumor cells within the bloodstream and facilitate their extravasation to metastatic site –61. Moremor TANs , 131. FuPL, MS, MP, and JM wrote the original draft of manuscript. PL performed original figures and overall the supervision of writing. LK, CC, NB-V, and M-CM revised and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In clomiphene-citrate-resistant anovulatory women with polycystic ovary syndrome (PCOS) and no other infertility factors, either metformin combined with clomiphene citrate or gonadotrophins could be used as a second-line pharmacological therapy, although gonadotrophins are more effective. Gonadotrophins could also be used as a second-line pharmacological therapy in anovulatory women with PCOS and clomiphene-citrate-failure. Laparoscopic ovarian surgery can also be used as a second-line therapy for ovulation induction in anovulatory women with clomiphene-citrate-resistant PCOS and no other infertility factors. The usefulness of letrozole as a second-line pharmacological treatment for ovulation induction in clomiphene-citrate-resistant women with PCOS requires further research. In terms of improving fertility, both pharmacological anti-obesity agents and bariatric surgery should be considered an experimental therapy in anovulatory women with PCOS and no other infertility factors. Where first- or second-line ovulation induction therapies have failed, in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) could be offered as a third-line therapy in women with PCOS in the absence of an absolute indication for IVF/ICSI. For women with PCOS undergoing IVF/ICSI treatment, the gonadotropin-releasing hormone (GnRH) antagonist protocol is preferred and an elective frozen embryo transfer strategy could be considered. In assisted conception units with sufficient expertise, in-vitro maturation (IVM) of oocytes could be offered to women with PCOS.  Polycystic ovary syndrome (PCOS), with a prevalence of between 8% and 13%, depending on the population studied and definitions used, is the most common endocrinopathy affecting reproductive-aged women. PCOS is a complex condition with reproductive, metabolic and psychological features . Infertility is a prevalent presenting feature of PCOS with 75% of these women suffering infertility due to anovulation, making PCOS the most common cause of anovulatory infertility . The treThe aim of this narrative review is to provide a summary and brief update of the best available and most current research evidence that evaluates the second- and third-line treatment of PCOS-related infertility and which has informed the recommendations in the assessment and treatment of infertility section of the International Evidence-Based Guideline on PCOS ,7. The mLetrozole, an aromatase inhibitor, was first proposed as an ovulation-inducing drug in anovulatory PCOS women in 2001 . Letrozop = 0.324) and clinical pregnancy  rates but a higher ovulation rate  per woman with letrozole in clomiphene-citrate-resistant PCOS women. There were no cases of miscarriage or multiple pregnancies in the RCT.The recently published international evidence-based guideline on PCOS identified a single published randomised controlled trial (RCT) comparing letrozole versus placebo or no treatment with timed intercourse in PCOS . This RCThe use of letrozole as a second-line pharmacological treatment for ovulation induction in anovulatory women with clomiphene-citrate-resistant PCOS and no other infertility factors has been shown to improve ovulation rates. Further large, adequately powered, well conducted and reported RCTs are required comparing letrozole versus placebo or no treatment in anovulatory women with PCOS with clomiphene citrate resistance or failure to properly evaluate letrozole as a second-line treatment agent.Metformin, an insulin-sensitizing drug, was first reported as a treatment for PCOS in 1994 where it was found to facilitate normal menses and pregnancy at doses of 1500 mg daily . ClomiphThe most recent systematic reviews and pairwise meta-analyses of RCTs that have compared the combination of metformin plus clomiphene citrate versus clomiphene citrate alone in women with PCOS who were clomiphene-citrate-resistant showed that metformin combined with clomiphene citrate had a higher live birth, pregnancy and ovulation rate compared with clomiphene citrate alone ,18. TherThis Cochrane systematic review and pairwise meta-analysis of RCTs also compared metformin combined with clomiphene citrate versus letrozole  and locaMetformin combined with clomiphene citrate could be used as second-line pharmacological therapy in anovulatory women with PCOS and no other infertility factors, as it is superior to clomiphene citrate alone in clomiphene-citrate-resistant women. Ovulation induction with gonadotrophins began in the 1960s and there is a large body of observational evidence supporting the use of gonadotrophin ovulation induction in clomiphene citrate resistant or clomiphene-citrate-failure PCOS women, with the use of the low dose step-up protocol with typical starting doses of follicle-stimulating hormone (FSH) 50–75 IU being recommended in order to reduce the risk of multiple pregnancy and ovarian hyperstimulation syndrome . The evidence is applicable to the both urinary and recombinant gonadotrophins including human menopausal (urinary) gonadotrophins (HMG), highly-purified urinary FSH (HP-uFSH) and recombinant FSH (rFSH). The recently published international evidence-based guideline on PCOS  and the p = 0.0124) and reduced time to conception leading to live-birth  favouring gonadotrophins and the addition of IUI did not increase live-births compared with timed intercourse . There was no evidence of a difference in multiple pregnancy rates  [A recently published multi-centre Dutch RCT compared gonadotrophins with timed intercourse or intra-uterine insemination (IUI) versus clomiphene citrate with timed intercourse or IUI over six cycles in 666 infertile WHO Group 2 anovulatory women with clomiphene-citrate-failure (ovulatory for six clomiphene citrate 150 mg maximum dose ovulation induction cycles but not conceived) and found a higher live-birth rate  1.24; 95% CI 1.05–1.46; = 0.826) .I2 85%), as evidenced by a systematic review and pairwise meta-analysis of RCTs [However, gonadotrophins are superior to metformin combined with clomiphene citrate for ovulation induction in women with PCOS who are clomiphene-citrate-resistant in terms of live-birth/ongoing pregnancy , clinical pregnancy  and ovulation rates , ongoing pregnancy  and clinical pregnancy  rates with no evidence of a difference in miscarriage , multiple pregnancy , or ovarian hyperstimulation syndrome (OHSS)  rates in clomiphene-citrate-resistant PCOS women with anovulatory infertility in a Cochrane systematic review and pair-wise meta-analysis of five RCTs [Gonadotrophins could be used as a second-line pharmacological therapy in cases where first-line pharmacological therapy has failed in anovulatory women with PCOS and no other infertility factors, as they are more effective than placebo/no treatment , clomiphene citrate alone (clomiphene-citrate-failure women), and metformin combined with clomiphene citrate (clomiphene-citrate-resistant women). The addition of metformin to gonadotrophins could be considered in clomiphene-citrate-resistant PCOS women as it is superior to gonadotrophins alone. Laparoscopic ovarian surgery (LOS) was first described in women with PCOS by Gjonnaess in 1984 . Both laLaparoscopic ovarian surgery is a second-line therapy in clomiphene-citrate-resistant women with PCOS with no evidence of a difference compared to 3–6 treatment cycles of gonadotrophin ovulation induction in terms of the live birth rate per woman , pregnancy rate per woman , ovulation rate per woman , miscarriage rate per pregnancy  and OHSS rate per woman  but with a lower risk of multiple pregnancy per pregnancy  and lower cost .p < 0.03) and a higher ovulation rate per cycle (50.8% (131/258) vs. 33.5% (94/281); p < 0.001) with LOS with no evidence of a difference in THE miscarriage rate per pregnancy whilst the RCT by Palomba and colleagues reported a lower live-birth rate per pregnancy (64.5% (20/31) vs. 82.1% (32/39); p < 0.05), lower pregnancy rate per cycle (13.4% (31/231) vs. 18.6% (39/210); p < 0.05) and higher miscarriage rate per pregnancy (29% (9/31) vs. 15.4% (6/39); p < 0.05) with LOS. There are two RCTs that compare LOS versus metformin in clomiphene-citrate-resistant women with PCOS which show conflicting findings ,31. The Laparoscopic ovary surgery versus clomiphene citrate in clomiphene-citrate-failure women with PCOS has been compared in a single RCT which demonstrated no evidence of a difference in live-birth or pregnancy rates per patient between the two interventions and increased miscarriage rates per pregnancy with LOS .I2 78%). There was insufficient evidence to determine if there was a difference in pregnancy, ovulation, miscarriage and multiple pregnancy rates [A Cochrane systematic review and meta-analysis has compared LOS versus metformin combined with clomiphene citrate in clomiphene-citrate-resistant women with PCOS and showed a decrease in the live-birth rate with LOS  in 1999 and is typically taken at doses of 120 mg with each main meal up to three times daily . No published RCTs on pharmacological anti-obesity agents  in PCOS were identified by the recently published international evidence-based guideline on PCOS . TherefoPharmacological anti-obesity agents, for the purpose of improving fertility, should be considered an experimental therapy in anovulatory women with PCOS and no other infertility factors. 2 or for patients with a BMI of ≥35 kg/m2 with at least one obesity-related comorbidity who have failed conservative treatment. Bariatric surgery contributes to weight loss by either restriction  or malabsorption . Common bariatric surgeries include vertical sleeve gastrectomy (VSG), laparoscopic adjustable gastric banding, Roux-en-Y gastric bypass, duodenal switch with biliopancreatic diversion and several minimally invasive devices including intra-gastric balloons [Bariatric surgery is usually indicated for patients with a body mass index (BMI) of ≥40 kg/mballoons .No published RCTs on bariatric surgery in PCOS were identified by the recently published international evidence-based guideline on PCOS . TherefoBariatric surgery, for the purpose of improving fertility, should be considered an experimental therapy in anovulatory women with PCOS and no other infertility factors. The world’s first IVF child, Louise Brown, was born in the United Kingdom on 25 July 1978 . The recently published international evidence-based guideline on PCOS did not identify any published RCTs comparing IVF treatment with ovulation induction treatments or placebo/no treatment in PCOS . In the The gonadotropin-releasing hormone (GnRH) antagonist protocol has been compared with the GnRH agonist long protocol in women with PCOS undergoing IVF or ICSI in a recently published systematic review and meta-analysis . There wp = 0.27) or clinical pregnancy  rates per patient, total dose of gonadotrophins  and ovarian hyperstimulation syndrome . However, Human Menopausal Gonadotrophin had a longer duration of ovarian stimulation , lower maximum serum E2 level  and lower number of eggs collected  compared to recombinant FSH [There has been a single RCT comparing Human Menopausal Gonadotrophin versus recombinant FSH in women with PCOS undergoing IVF using a GnRH agonist long protocol, demonstrating no evidence of a difference in live-birth  Chinese RCT has compared fresh versus elective frozen embryo transfer (FET) in 1508 infertile women with PCOS undergoing their first IVF cycle using the GnRH antagonist protocol with urinary hCG trigger [p = 0.004), singleton birth weight  and risk of pre-eclampsia  with a lower clinical pregnancy loss rate  and risk of moderate or severe ovarian hyperstimulation syndrome . There was no evidence of a difference between FET and fresh ET groups in terms of biochemical , clinical  and ongoing pregnancy  rates.A multi-centre ( trigger . The intIn vitro fertilization/ICSI could be offered as third-line therapy in women with PCOS, where first- or second-line ovulation induction therapies have failed, in the absence of an absolute indication for IVF/ICSI. In women with PCOS undergoing IVF/ICSI treatment, the GnRH antagonist protocol is preferred over the GnRH agonist long protocol, either Human Menopausal Gonadotrophin or recombinant FSH can be used and elective frozen embryo transfer strategy could be considered. If using the GnRH agonist long protocol, adjunct metformin therapy could be used to improve clinical outcome.Where IVF is indicated in PCOS, OHSS risks are increased with gonadotrophin stimulation. IVM of oocytes limits or omits ovarian stimulation prior to oocyte retrieval, with maturation of oocytes post retrieval, avoiding OHSS risk .No published RCTs comparing IVM treatment with IVF/ICSI or ovulation induction treatments or placebo/no treatment in women with PCOS were identified by the recently published international evidence-based guideline . TherefoIn vitro maturation, where an embryo is generated, then vitrified and thawed and transferred in a subsequent cycle, could be offered to women with PCOS in assisted reproduction units with sufficient expertise. The evidence and summaries for second- and third-line ovulation induction in this review are applicable to women with PCOS who have anovulatory infertility and no other infertility factors. The usefulness of letrozole as a second-line pharmacological treatment for ovulation induction in clomiphene-citrate-resistant PCOS requires further study. Metformin combined with clomiphene citrate could be used as second-line pharmacological therapy in clomiphene-citrate-resistant women, although gonadotrophins are more effective. Gonadotrophins could also be used as second-line pharmacological therapy in clomiphene-citrate-failure women. Laparoscopic ovarian surgery can also be used as second-line ovulation induction therapy in women with clomiphene citrate resistance. Both pharmacological anti-obesity agents and bariatric surgery, with the aim of improving fertility, should be considered as an experimental therapy. In the absence of an absolute indication for IVF/ICSI, IVF/ICSI could be offered as third-line therapy in women with PCOS, where first- or second-line ovulation induction therapies have failed. In women with PCOS undergoing IVF/ICSI treatment, the GnRH antagonist protocol is preferred and an elective frozen embryo transfer strategy could be considered. IVM could be offered to women with PCOS in assisted reproduction units with sufficient expertise.


Background: Human bone marrow mesenchymal stem cell-derived hepatocyte-like cells (hBMSC-HLCs) are a promising alternative for primary human hepatocytes (HHs) for treating liver disease. However, the molecular characteristics of HLCs remain unclear. Here, we aimed to clarify the transcriptome characteristics of hBMSC-HLCs for future clinical application.Materials and Methods: hBMSCs were isolated from the bone marrow of healthy volunteers and differentiated into hepatocytes. mRNA sequencing was used in the transcriptome profiling of hBMSC-HLCs, with hBMSCs and HHs as controls.Results: hBMSC-HLCs exhibited a polygonal morphology, glycogen accumulation and albumin expression. A total of 630 upregulated and 1082 downregulated genes were observed in hBMSC-HLCs and HHs compared with undifferentiated hBMSCs. The upregulated genes were mainly involved in hepatic metabolism and inflammatory and immune responses. The downregulated genes were mainly associated with stem cell characteristics . Confirmatory qRT-PCR of 9 upregulated and 9 downregulated genes with log2 fold changes > 5 showed similar results. In vivo transdifferentiation of hBMSCs in pigs with fulminant hepatic failure confirmed the similarly upregulated expression of 5 hepatogenic genes , showing a 150-fold change in liver tissues at 7 days after hBMSC transplantation. These 5 genes mainly contributed to liver metabolism and inflammation.Conclusion: hBMSC-HLCs possess a hepatic transcriptome profile and express hepatic-specific genes in vitro and in vivo, which might be useful for future clinical applications. The five upregulated genes identified herein could be potential biomarkers for the characterization of hBMSC-HLCs. Bone marrow mesenchymal stem cell (BMSC)-dervied hepatocyte-like cells (BMSC-HLCs) have the potential to overcome the limitations of primary human hepatocytes (HHs) for clinical application in treating liver disease and for drug development in vitro differentiation of hESCs to a hepatic lineage involves a sequential epithelial-mesenchymal-epithelial transition (EMT-MET) and the participation of differentially expressed genes involved in proliferation, extracellular matrix-related functions, hepatic metabolism and normal liver functions Although several strategies are available for generating BMSC-HLCs, these cells are still less effective than HHs in repopulating the liver, and the transcriptome mechanism is still unclear TDO2, HP, SERPINA3, LBP and SAA1) validated in hBMSC transplantation with a fulminant hepatic failure (FHF) model in pigs could be potential biomarkers for the characterization of hBMSC-HLCs.Herein, we report a gradual loss of pluripotency and gain of hepatic characteristics during human BMSC (hBMSC) hepatic differentiation using a whole-genome mRNA sequencing (mRNA-seq) analysis and compared the results with the mRNA-seq data for HHs. Finally, the five genes  density-gradient centrifugation and cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum  at 37°C in 5% COThe phenotypic identification of cultured hBMSCs was performed by standard flow cytometry methods with antibodies against human CD45 , CD34, CD29, and CD90 , as described in our previous work To detect the multilineage differentiation potential of hBMSCs, the cells were cultured separately in a commercially available osteogenic and adipogenic differentiation medium . On day 21, the cells that underwent adipogenic induction were stained with Oil red O for lipid droplets. On day 28, the cells that underwent osteogenic induction were stained with alizarin red for calcium compounds.Hepatogenic differentiation was performed by using a two-step protocol ALB, α-fetoprotein (AFP), tryptophan 2,3-dioxygenase (TDO2) and tyrosine aminotransferase (TAT)] on days 0 (D0), 10 (D10) and 20 (D20) after hepatogenic differentiation. The primer sequences for the above genes are provided in Table GAPDH) was used as an internal control. HHs, used as a positive control, were isolated from human liver tissue specimens that were harvested from therapeutic partial hepatectomies performed on three male patients with hepatic haemangioma  who exhibited no sign of hepatic dysfunction, hepatitis virus infection or hepatic cancer. Signed informed consent was obtained and approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. The isolation of HHs was performed as previously described After 20 days of induction, the cells were characterized for hepatic function with immunohistochemistry (IHC) staining for albumin (ALB) and periodic acid-Schiff (PAS) staining for glycogen accumulation. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the hepatic-specific genes , with one DEG showing the opposite trend [CACNB2 (decreased 2.06-fold)] and the other 3 DEGs  were not significantly changed  and HHs, indicating that these cells are hardly fit for clinical applications, which requires further improvement. Therefore, we focused on the transcription changes from D0 to D20 and found that the direction of hepatic transformation during induction is clearly precise, as shown by the clustering analysis of mRNA-seq data, especially by the downregulated DEGs  exhibited the same expression trend in our in-house hBMSC-transplanted FHF pigs, in which hBMSCs had differentiated into hepatocytes within 7 days (Fig.  in vitro had gene expression patterns similar to those of hBMSC-HLCs developed in vivo.Based on the rationale that the identification of suboptimal gene networks represents a tractable target for improving cell phenotype, the major hurdle in the path to stem cell-derived hepatocyte-like cells is the control mechanisms for the above biological processes; thus, adjusting the involved gene expression levels to those of HHs should be helpful for generating functional hBMSC-HLCs. Interestingly, 5/9 upregulated DEGs (ays Fig.  and havein vitro, the above 5 DEGs may not be hepatocyte-specific, but they could be potential biomarkers for the characterization of HLCs, which should be validated during hepatic differentiation from other stem cell sources in the future.In conclusion, mRNA transcriptome profiling indicated the mechanical direction of hBMSC differentiation into hepatocyte-like cells. Considering the immature HLCs differentiated Supplementary figure.Click here for additional data file.


TrkA splice variant TrkAIII, which exhibits exon 6, 7 and 9 skipping and oncogenic-activity that depends upon deletion of the extracellular D4 Ig-like domain. In contrast to fully spliced TrkA, which exhibits tumour suppressor activity in NB and associates with good prognosis, TrkAIII associates with advanced stage metastatic disease, post therapeutic relapse and worse prognosis, induces malignant transformation of NIH-3T3 cells and exhibits oncogenic activity in NB models. TrkAIII induction in NB cells is stress-regulated by conditions that mimic hypoxia or perturbate the ER with potential to change TrkA tumour-suppressing signals into oncogenic TrkAIII signals within the stressful tumour microenvironment. In contrast to cell surface TrkA, TrkAIII re-localises to intracellular pre-Golgi membranes, centrosomes and mitochondria, within which it exhibits spontaneous ligand-independent activation, triggering a variety of mechanisms that promote tumorigenicity and malignant behaviour, which impact the majority of cancer hallmarks. In this review, we present updates on TrkAIII detection and association with human malignancies, the multiple ways TrkAIII exerts oncogenic activity and potential therapeutic approaches for TrkAIII expressing cancers, with particular reference to NB.Oncogenes derived from the neurotrophin receptor tropomyosin-related kinase TrkA act as drivers in sub-populations of a wide-range of human cancers. This, combined with a recent report that both adult and childhood cancers driven by novel oncogenic TrkA chimeric-fusions exhibit profound, long-lived therapeutic responses to the Trk inhibitor Larotrectinib, highlights the need to improve clinical detection of TrkA oncogene-driven cancers in order to maximise this novel therapeutic potential. Cancers potentially driven by TrkA oncogenes include a proportion of paediatric neuroblastomas (NBs) that express the alternative  The neurotrophin receptor tropomyosin-related kinase A (TrkA) regulates responses to the neurotrophins NGF and NT3 in a wide variety of normal tissues and is critical for normal development and function of both nervous and immunological systems , 2.The first human TrkA-derived oncogene, Trk-oncogene, was identified in colon cancer as a novel chimeric fusion between truncated tropomyosin and protein tyrosine kinase sequence . This prTrkA splice variant, TrkAIII. TrkAIII exhibits exon 6–7 skipping and is subject to spontaneous ligand-independent intracellular activation, dependent upon deletion of the extracellular receptor D4 Ig-like domain, encoded within exons 6 and 7, which acts as a spontaneous-activation prevention domain [Cancers potentially driven by TrkA oncogenes include paediatric neuroblastomas (NBs), a significant proportion of which express the oncogenic alternative n domain . In contn domain , 19–22, TrkA splice variant that exhibits skipping of exons 6, 7 and 9, resulting in deletion of the receptor extracellular D4 Ig-like domain and several N-glycosylation sites , may prsing NBs . Alternasing NBs , suggestsing NBs –42, may , comparable to TrkT3 oncogene, and also augments osteolytic bone metastasis and non-bone metastasis formation in a nude mouse xenograft metastasis model, confirming that TrkAIII promotes both primary tumorigenicity and metastatic capacity. TrkAIII promotion of osteolytic bone metastasis associates with osteoclast differentiation and expression of the osteoclast-activating cytokine RANKL [TrkAIII induces malignant transformation of NIH 3 T3 cells  in a sinne RANKL , 23, 29.In NB transfectants, NGF activation of fully spliced TrkA receptors results in receptor tyrosine phosphorylation and signaling through both PI3K/Akt/NF-κB and RAS/MAPK pathways, resulting in neuronal differentiation, characterised by inhibition of proliferation and neuritogenesis , 19. In In contrast to fully spliced TrkA expression, which reduces NB xenograft tumorigenicity, tumour-associated angiogenesis and angiogenesis factor expression by NB cells , 45, xenPI3K/Akt/NF-кB signaling is also important for cell survival, and TrkAIII transfectants exhibit enhanced resistance to genotoxic- (cisplatin), oxidative-  and ER-stress  , 32, 49.Increased mitochondrial SOD2 expression and activity in TrkAIII transfectants attenuates constitutive and induced  mitochondrial free-radical ROS production and increases resistance to agents that induce mitochondrial free radical-mediated death, identifying a novel TrkAIII/SOD2 mitochondrial protection axis .TrkAIII intracellular re-localisation and oncogenic behaviour supports the growing concept that mislocalization of TrkA-derived oncogenes underpins oncogenic signaling , 40, 50.Spontaneous TrkAIII activation within ERGIC/COPI membranes, confirmed by co-localisation with ERGIC and COPI markers in purified membrane fractions and also by confocal microscopy, is prevented by the COPI vesicle ARF-inhibitor Brefeldin A and indicates that the ERGIC/COPI membrane compartment is more permissive for TrkAIII activation . This adIn addition to pro-survival and pro-angiogenic PI3K/Akt/NF-кB signaling, TrkAIII expression also results in partial activation of a survival-adapted UPR, characterised by ATF6 but not Ire1a/Xbp-1 activation, and increased expression of the UPR chaperone Grp78/Bip and the apoptosis inhibitor Mcl-1 , 50, 58.Retrograde MT minus-end transport not only results in TrkAIII transport back to the ER but also translocation of activated TrkAIII to the centrosome, where it localises in complexes with γ- and α-tubulin , 51. TrkTherefore, retrograde TrkAIII transport and recycling between the ER and ERGIC/COPI compartments in addition to reinforcing ERGIC/COPI-associated TrkAIII activation, pro-survival and pro-angiogenic PI3K/Akt/NF-кB signaling and a UPR survival-adaption also results in accumulation at the centrosome, promoting centrosome amplification, chromosome instability, micronuclei formation and MTOC activity, increasing metastatic potential and hindering differentiation.In addition to increasing chromosomal instability, TrkAIII transfectants also exhibit increased levels of sister chromatid exchanges, suggesting that TrkAIII may also promote replication-stress either by elevating levels of homologous recombination and/or the burden of single strand DNA breaks . As for TrkA expression is a prerequisite for NB cell differentiation and NB regression, via RAS/MAPK-dependent inhibition of proliferation and induction of neuritogenesis , 19, 36.2+ interplay, providing conditions necessary for activation of mitochondrial Omi/HtrA2, which is responsible for eliminating the remaining N-terminal spontaneous activation-preventing sequences from TrkAIII, which together with ROS inactivation of mitochondrial PTPases facilitates the spontaneous activation of IMM-associated cleaved TrkAIII. The activation of IMM-associated TrkAIII results in the tyrosine phosphorylation of mitochondrial proteins including pyruvate dehydrogenase kinase-1, an inhibitor of the pyruvate dehydrogenase complex, resulting in a metabolic switch to aerobic glycolysis [Continuous TrkAIII recycling between ER and ERGIC membranes  also leaycolysis . This idycolysis –67 er drugs , 50. As Considering the NB tumour suppressing activity of fully spliced TrkA, reversal of alternative TrkAIII splicing back to fully spliced TrkA expression may represent an important therapeutic goal. This could be achieved using new generation lentiviral vectors engineered to express TrkAIII inhibitory siRNAs and the fully spliced TrkA coding sequence, mutated in wobble sites to prevent off-target inhibition by TrkAIII-specific siRNA, without altering the TrkA amino acid sequence. This approach would block TrkAIII oncogenic activity and reinstate TrkA tumour-suppressing activity, with potential to slow advanced disease progression, enhance chemotherapeutic-sensitivity and increase the potential for spontaneous regression and post-therapeutic event-free survival, all of which associate with fully spliced TrkA expression , 20, 36.TrkAIII promotion of pro-survival and pro-angiogenic PI3K/Akt/NF-кB signaling exhibited by TrkAIII transfectants suggests that inhibitors of PI3K/Akt/NF-кB signaling could significantly slow tumour progressions, reduce tumour-associated angiogenesis and enhance chemotherapeutic-sensitivity. This could be achieved either by plant-derived PI3K inhibitors (reviewed in ), syntheTrkAIII induces partial activation of a survival-adapted UPR, which preconditions cells to better survive and resolve acute episodes of ER stress, in an important “non-oncogene addiction” mechanism. Agents  that reduce expression of the major UPR regulator Grp78/Bip, overexpressed by TrkAIII transfectants may target this mechanism to render TrkAIII-expressing tumour cells more sensitive to acute ER-stress-induced death. Down-regulation of Grp78/Bip expression could be achieved using the cancer drug OSU-03012, developed from Celecoxib, that supresses Grp78/Bip expression by > 90% in many tumour cell lines and significantly increases tumor cell killing . CombiniTrkAIII enhances NB cell resistance to genotoxic-, oxidative- and ER-stress by increasing the expression and mitochondrial-localisation of anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 proteins , 58. TheTrkAIII augments the expression of mitochondrial SOD-2, enhancing resistance to mitochondrial ROS-mediated death . Down reThe original pre-clinical promise of pro-apoptotic TRAIL/TRAIL-R signaling as a tumour-specific immunotherapy has been disappointing. Recombinant soluble TRAIL exhibits limit efficacy, short half-life and rapid clearance in vivo and first generation TRAIL-receptor agonists, designed to reduce toxicity, exhibit limited efficacy. Furthermore, many cancers including NB exhibit initial or acquired resistance to TRAIL-induced apoptosis and exogenous TRAIL may enhance the proliferation, invasive and metastatic behaviour of TRAIL-resistant tumour cells. These setbacks, however, have resulted in the development of innovative reagents and novel ways to increase half-life and overcome resistance, with major improvements in TRAIL and TRAIL receptor-agonists, delivery and ways to overcome TRAIL-resistance (reviewed in –80).TrkAIII exhibits a potential therapeutic “Achilles heel” by sensitizing transfectants to one-way TRAIL-induced pro-apoptotic crosstalk between the TRAIL receptor signaling pathway and TrkAIII, resulting in delayed apoptosis and abrogation of tumorigenic activity in vitro . In cellHsp90 stabilises and promotes the activity of receptor tyrosine kinase (RTK) oncogenes, many of which are inhibited and/or induced to degrade by the Hsp90 inhibitor GA, prompting therapeutic evaluation and development of GA-analogues for use in adult and paediatric cancers, including NB . TrkAIIITrkA splicing, such as TrkAIII. The capacity of alternative splicing to unlock TrkA oncogenic potential, characterises sequence encoded within exons 6–7 in preventing oncogenic activation, the deletion of which causes receptor re-localization and spontaneous activation within altered intracellular substrate contexts, resulting in a plethora of “addiction” and “non-addiction” oncogenic mechanisms that impact upon a majority of the Hallmarks of Cancer (Schematized in Figs. TrkA splicing may not be limited to TrkAIII [TrkA splice variants may be required to fully exploit the therapeutic potential of Trk tyrosine kinase inhibitors and/or inhibitors of post receptor signaling.The profound, long-term therapeutic responses reported for TrkA fusion oncogene-driven cancers to Trk inhibitory therapy, highlights the need to better identify cancers driven by TrkA oncogenes, including TrkA oncogenes activated by aberrant alternative  TrkAIII , 22, sug