Neurodegenerative diseases represent a large group of neurological disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. Although this group of diseases show heterogeneous clinical and pathological phenotypes, they share important pathological features characterized by the age-dependent and progressive degeneration of nerve cells that is caused by the accumulation of misfolded proteins. The association of genetic mutations with neurodegeneration diseases has enabled the establishment of various types of animal models that mimic genetic defects and have provided important insights into the pathogenesis. However, most of genetically modified rodent models lack the overt and selective neurodegeneration seen in the patient brains, making it difficult to use the small animal models to validate the effective treatment on neurodegeneration. Recent studies of pig and monkey models suggest that large animals can more faithfully recapitulate pathological features of neurodegenerative diseases. In this review, we discuss the important differences in animal models for modeling pathological features of neurodegenerative diseases, aiming to assist the use of animal models to better understand the pathogenesis and to develop effective therapeutic strategies. Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington's disease (HD), and Amyotrophic lateral sclerosis (ALS) are incurable and have been one of the most challenging health issues. The common feature of these diseases is a progressive loss of specific populations of neurons in the aging human brain . With inAD, which is the most common form of neurodegenerative diseases, affects about 7–8% people over age 65. The main clinical manifestations of AD include progressive memory loss, cognitive dysfunction, behavioral disorders, and other related impairments. Neuropathologically, AD is characterized by extracellular senile amyloid plaques and intracellular neurofibrillary tangles (NFTs), along with other molecular changes such as neuroinflammation, brain atrophy, synaptic pathologies, and cerebral amyloid angiopathy , 3.More The genetic mutations identified in the neurodegenerative disease genes make it possible to use genetic manipulation for modeling these diseases in animals, which are essential to our understanding of the disease pathogenesis. A variety of genetically modified animal models, mostly in rodents, have been generated to study the pathogenesis and therapeutics for neurodegenerative diseases. The rodent models have provided important insights into the pathogenesis of neurodegenerative diseases. For example, the mouse models can remarkably recapitulate protein misfolding and aggregation seen in the patient brains –14. HoweThe differences in neuropathology between rodent models and patient brains with neurodegenerative diseases could be due to species differences determined by genomic, molecular, and anatomic differences between rodents and humans Table . The braAlthough the modern transgenic methods have been used to establish large animal models, the transgene was randomly introduced into the chromosomes and may not yield the endogenous expression level of mutant genes. Furthermore, the following limitations make it difficult to create large animal models that can mimic germline transmissible mutations: the lack of embryonic stem cells (ESCs) for in vitro genome editing, the low efficiency of homologous recombination, and the long-life cycle. Thus, the recently developed nuclease-mediated genome editing technology (CRISPR/Cas9) that can modify the endogenous genome makes it feasible to expand genetic engineering to many species, especially large animals.CRISPR/Cas9 has now been used for genome editing in non-human primates –24. The For generating monkey models, germline genome editing can be achieved by injection of transgene or CRISPR/Cas9 into the fertilized eggs. However, the long-life cycle and the high costs of the non-human primates prevent the widespread use of this important animal model. Since CRISPR/Cas9 can also target genes in adult neuronal cells –31, it cPig is another commonly used large animal that shares considerable similarities with humans in body size, organ(s) physiology and anatomical dimensions. In comparison to non-human primates, there are several advantages of pigs: generation of multiple piglets (6–12 piglets from a single sow), short gestation time about 120 days), and relatively lower costs . Importa days, anGenetically modified large animal models of HD, PD, ALS, and AD are summarized in Table Transgenic pigs expressing a large fragment of mutant HTT (1–548) containing 124Q were also generated by lentiviral infection of porcine embryos. However, these transgenic HD pigs showed much milder phenotypes and did not die after birth , 40. BecTo overcome the limitation of transgenic approach, a HD knock-in pig model was generated via CRISPR/Cas9 and somatic nuclear transfer, which expresses an expanded polyCAG (150 CAG) in the pig HTT gene and precisely mimics the genetic mutation that occurs in the endogenous HTT gene . ImportaAnimal models developed to investigate the pathogenesis of PD fall into two categories: neurotoxic and genetic models. The neurotoxic models are mainly induced by the neurotoxins 6-hydroxydopamine (6-OHDA)  or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which cause dopamine neuronal loss in the substantia nigra (SN). The neurotoxic PD models are valuable for studying the pathogenesis of PD-associated neurodegeneration , but theAlthough transgenic mouse and monkey models that overexpress mutant alpha-synuclein have been generated for investigating PD pathology and validated the neurotoxicity of mutant proteins –46, the Interestingly, disrupting the PINK1 and Parkin genes in pigs via CRISPR/Cas9 targeting did not produce any neurodegeneration and severe phenotypes either , 53. HowBecause mutations in the nuclear TAR DNA-binding protein 43 (TDP-43) gene cause ALS, animal models carrying TDP-43 mutations were generated to investigate ALS pathogenesis. TDP-43 is a nuclear protein that is involved in a variety of cellular functions including gene transcription, RNA processing, and protein homeostasis –58. In pOur group has previously created the TDP-43 transgenic pig model that displays the cytoplasmic distribution of TDP-43 . We alsoThe human copper/zinc superoxide dismutase 1 (SOD1) mutations also cause familial ALS. However, transgenic mutant SOD1 mouse models do not show the intranuclear inclusions seen in the brains of ALS patient with SOD1 mutations –72. To iAccording to etiology, most AD patients are sporadic, and the familial type of AD only accounts for 5–10% of the total AD cases. The familial type of AD is mostly correlated with the genetic mutations in the genes for amyloid precursor protein (APP), Presenilin-1 (PSEN1), Presenilin-2 (PSEN2) . Until nBecause the non-human primates are much closer to humans, they should be the most biologically relevant model to study Alzheimer’s disease. Interestingly, no case of AD in naturally long-lived non-human primates has been reported . The oldSince ageing is a crucial pathogenic factor in these neurodegeneration diseases, the disease phenotypes might take longer time to appear in large animal models. Species-specific factors are also likely to influence the disease progression. These possibilities should be explored by establishing more large animal models, especially the non-human primate models, that carry the AD genes and are able to show the AD phenotypes.Animal models are an important tool for studying pathogenesis, disease progression, and therapeutic treatments. A successfully established animal model of neurodegenerative disease should adequately recapitulate the clinical characteristics and pathology of patients. However, such an animal model is still lacking for most neurodegenerative disease. Rodents are the most widely used animal model for generating disease models owing to their relatively short life span, easier and rapid breeding, and lower costs. However, rodents have only 48–66% genetic homology with humans and show considerable differences to humans in brain size and development , 91. TheIn addition to gene editing that can induce embryonic mutations in large animals by embryo microinjection, stereotaxic injection of viral expression vectors can be applied to modify genes in specific brain regions in adult animals at different ages to assess brain regional effects or age-dependent effects , 43, 55.The application of gene editing technology, especially the CRISPR/Cas9 system, makes revolutionary changes in modifying genomes in large animals for investigating neurodegenerative diseases. However, there are still many challenges to overcome. One potential challenge of CRISPR/Cas9 system is the mosaic mutations, which may result from various types of mutations created by the Cas9 nuclease. The mosaicism in offspring can be reduced by outcrossing the mosaic founders with wild type animals. However, it will take years to eliminate mosaicism in large animal models, especially for the non-human primates that require 4–5 years of sexual maturity to produce next generation. Thus, the mosaic targeting issue should be considered when evaluating the phenotypes of large animal models, because the funder animals that carry CRISPR/Cas9-mediated mosaic mutations are often used for investigation. The mosaic issue could potentially be improved by shortening the half-life of Cas9 expression after cell division such as tagging Cas9 with ubiquitin proteasomal degradation signals . Other pAlthough the mosaicism often exists in the non-human primate models, it depletes gene expression to various extents, leading to various degrees of cellular phenotypes that are correlated with the extent of genetic elimination. Thus, using imaging and RNAseq analysis at the single cell resolution, one can take the advantage of mosaicism to analyze the correlation of genetic mutation and cellular phenotypes in the large animal models.Possible off-target effects of CRISPR/Cas9 have been considered as another important issue because CRISPR/Cas9 relies on approximately 23 base pair matches . StudiesAn obvious concern for generating large animal models is the high cost and time consumed for such research. For example, pigs have a sexual maturity of 5–8 months and the gestation period of about 114 d. For monkeys, the sexual maturity takes 4–5 years and gestation period is about 165 d with normally delivering only one offspring per year. Alternatively, the direct administration of gRNA/Cas9 into specific brain regions in adult large animals could more rapidly recapitulate brain regional neuropathology. The important information gained from large animal models would be highly valuable for generating more humanized mouse models. For example, once we know the primate-specific factors that critically contribute to neurodegeneration, we can introduce these factors to the rodents to make rodent models to be able to display important pathological features. Such mouse models can be used to rigorously test the effects of therapeutic strategies on neurodegeneration.The newly developed genome editing tool has greatly advanced the progress of generating valuable large animal models for neurodegenerative disease research. These large animal models allowed one to discover important pathological events that otherwise do not occur in small animals. However, the generation and investigation of genetically modified large animal models are still challenging, largely due to the high cost of animals and time consumed experiments. We anticipate that further optimization of the existing genome editing system or the generation of new tools will improve the efficiency and accuracy of genome modification of large animals. Moreover, important insights from large animal models would help establish small animal models that can more faithfully recapitulate important pathological features for investigating pathogenesis and developing effective therapies.


The retention of the heavy metal, gadolinium, after a Gadolinium-Based Contrast Agent-assisted MRI may lead to a symptom cluster termed Gadolinium Deposition Disease. Little is known of the disorder’s underlying pathophysiology, but a recent study reported abnormally elevated serum levels of pro-inflammatory cytokines compared to normal controls. As a calcium channel blocker in cellular plasma and mitochondrial membranes, gadolinium also interferes with mitochondrial function. We applied to sera from nine Gadolinium Deposition Disease and two Gadolinium Storage Condition patients newly developed methods allowing isolation of plasma neuron-derived extracellular vesicles that contain reproducibly quantifiable levels of mitochondrial proteins of all major classes. Patients’ levels of five mitochondrial functional proteins were statistically significantly lower and of two significantly higher than the levels in normal controls. The patterns of differences between study patients and controls for mitochondrial dynamics and mitochondrial proteins encompassing neuronal energy generation, metabolic regulation, ion fluxes, and survival differed from those seen for patients with first episode psychosis and those with Major Depressive Disorder compared to their controls. These findings suggest that mitochondrial dysfunction due to retained gadolinium may play a role in causing Gadolinium Deposition Disease. Larger samples of both GDD and GSC patients are needed to allow not only testing the repeatability of our findings, but also investigation of relationships of specific mitochondrial protein deficiencies or excesses and concurrent cytokine, genetic, or other factors to GDD’s neurological and cognitive symptoms. Studies of neuronal mitochondrial proteins as diagnostic markers or indicators of treatment effectiveness are also warranted. The retention of the heavy metal gadolinium (Gd) after an MRI with a Gadolinium-Based Contrast Agent may lead to a symptom cluster termed Gadolinium Deposition Disease (GDD) that includes neuropathic pain, new onset frequent headaches, muscle fatigue, and cognitive complaints . Little 3+ increased mitochondrial membrane fluidity, decreased membrane potential, and increased cytochrome c release in rodent liver cells and isolated mitochondria, suggesting ionic Gd can enter cells and bind to mitochondria. ; and muscle twitching are very common GDD symptoms . Althougin vitro studies cited above, we undertook the current study of exosomes reflecting brain mitochondrial function to investigate the possibility that impaired mitochondrial function could be related these symptoms.Newly developed methods allow isolation of human plasma neuron-derived extracellular vesicles (NDEVs) containing reproducibly quantifiable levels of mitochondrial proteins of all major classes. Specific profiles of abnormal levels have been observed in first episode psychosis (FEP)  and majoEleven study participants were enrolled from March 2019 to January 2020 at a Chapel Hill, NC medical clinic where they had come for Ca-DTPA chelation to remove Gd. All signed a Stanford University Medical Center IRB-approved Informed Consent for use of their blood samples and data. Since the benefit or lack of benefit of off-label Ca-DTPA chelation was not under study, consent for this treatment was given to the treating physician as part of ordinary medical practice and was not part of the Stanford IRB form. All diagnoses were made by the treating physician, who is an author of the first peer-reviewed articles describing GDD  and GadoTwo patients with GSC, in which gadolinium has been retrained without accompanying symptoms , were alhttps://doctorsdata.com/licensing). The DDI upper limit of normal (95th percentile) is ≤0.6 μg/24 h for women, and ≤1.0 μg/24 h for men, based on unprovoked urine samples from 336 women and 204 men asked to refrain from a GBCA-enhanced MRI for ≥48 h before starting urine collection.The 24-h urine specimens for Gd analysis were collected the 24 h before and during approximately 24 h after Ca-DTPA chelation. Doctor’s Data, Inc. (DDI) determined the Gd amounts via inductively coupled plasma mass spectrometry .The reasons for patients’ MRIs were back pain (3), abdominal pain (2), throat pain (1), post-hysterectomy scan (1), breast CA , prostate CA , enlarged liver (1), and headache (1).No clear relationship was seen between the pre- or post-chelation 24-h urine amounts and the time since the most recent GBCA-assisted MRI.Of the twelve proteins within the four mitochondrial functional classes, plasma levels of five proteins were statistically significantly lower and of two higher in patients than in controls. Plasma levels of the remaining five proteins showed no statistically significant difference between GDD/GSC patients and controls .For five proteins, the direction and approximate magnitude of the differences in GDD/GSC patients’ plasma level versus the levels in controls were the same as for comparisons in earlier studies between FEP patients  and MDD Our findings of abnormal function in brain-derived mitochondria of GDD patients are the first reports utilizing plasma exosome analysis. They suggest that mitochondrial dysfunction may be involved in the pathophysiology of the neuropathic pain, “brain fog,” and muscle fatigue reported by GDD patients .The fact that similar mitochondrial changes were found in the two GSC patients, who lacked these symptoms, suggests that mitochondrial dysfunction is not a sufficient cause. It may be unrelated, but more probably, is a necessary or contributing cause. Mitochondrial dysfunction may join with Gd’s generation of abnormally elevated levels of specific cytokines as compared to levels in both normal controls  and a smResults for individual mitochondrial proteins suggest a variety of possible pathophysiological pathways to GDD symptom formation. NDEV levels of MY06 were higher in GDD/GSC patients than in controls. MY06 is an unconventional myosin responsible for ruffled neuronal membrane structure and the tethering of mitochondria and some membrane vesicles to neuronal presynaptic microfilaments. The increased MY06 levels in plasma NDEVs of GDD/GSC subjects suggests that adherence of their mitochondria to neuronal microfilaments is altered, with consequently increased turnover of neuronal presynaptic but not of axonal mitochondria since NDEV levels of SNPH are normal . By ATP ++ fluxes directly or indirectly by influencing LETM-1 actions in maintaining mitochondrial membrane structure and respiratory functions , were significantly lower in the patients. NMNAT2  is vital for mitochondrial energy generation via establishing mitochondrial concentrations of NADH and NAD+, which are as important in energy generation as the NADH/NAD+ ratio . SARM1 iLevels of the metabolic regulatory peptide Humanin were also significantly lower. Humanin helps protect neurons from stressors, restrains apoptosis, reduces neuroinflammation, preserves synaptic proteins, and assists in glucose metabolism. Reduced levels suggest that retained Gd lowers mitochondrial resistance to adverse stressors, including possibly the increased levels of pro-inflammatory cytokines reported in GDD . Again, PGC1alpha and NRF2 both promote mitochondrial DNA replication, termed biogenesis, and several functional activities . Their lThe lower levels of NMNAT2, Humanin, PGC1alpha and NRF2, and the resultant impairment in mitochondrial energy production, and the decreased restraint of neuronal apoptosis, protection from neuroinflammation, and regulation of ROS have plausible physiological pathway connections to the GDD complaints of neuropathic pain  and cognThe GDD patients’ post-chelation 24-h urine Gd amounts were 6–59 times the DDI laboratory norm for individuals not exposed to GBCAs. This suggests that the amount of retained Gd needed to contribute to evoking GDD symptoms in susceptible individuals may be lower than these multiples. The absence of a relationship between pre- and post-chelation 24-h urine Gd amounts may reflect differences in the number of MRIs received and in the stability constants and excretion patterns of the different GBCAs utilized . The timThe pattern of differences between GDD/GSC patients and normal controls for twelve mitochondrial proteins encompassing neuronal energy generation, metabolic regulation, and ion fluxes were distinctive, i.e., these patterns differed from the patterns of differences seen for FEP  and MDD This study is limited by small sample size and by the absence of independent rater quantitative ratings of the severity of patients’ symptoms. Future studies should utilize validated symptom rating scales to obtain these measures. Larger samples of both GDD and GSC patients are needed to allow not only testing the repeatability of our findings, but also to allow identification of additional factors involved in the development of GDD symptoms. Larger studies may elucidate relationships of specific mitochondrial protein deficiencies or excesses and concurrent cytokine, genetic, or other factors to the neurological, cognitive and skeletal muscle symptoms of GDD.The pathophysiology of GDD symptoms remains to be definitively established. Still, the mitochondrial functional abnormalities reported here, as well as the reported cytokine abnormalities, deserve further exploration and elaboration. Mitochondrial function markers might prove useful for diagnosis or as markers of treatment effectiveness. If mitochondrial dysfunction is proven to be involved in causing GDD symptoms, studies in GDD of treatments that lessen this dysfunction in other disorders will deserve pursuit.


We have recently reported a correlation between the accumulation of specific T > C and T > G mutations and the chromosomal instability in cells of Barrett's esophagus (BE), which represents a premalignant condition of esophageal adenocarcinoma. Additionally, we identified seven marker genes that facilitate the distinction of individual BE stages by histopathological examination. Patients with IM are believed to have a higher risk of EAC development. The different BE stages are columnar epithelium without IM, non-dysplastic BE (NDBE), low-grade dysplasia (LGD) and high-grade dysplasia (HGD). No curative treatment for BE exists, and the only option is to endoscopically resect more advanced BE stages such as LGD and HGD. The distinction of the individual stages relies on histopathological assessment of morphological anomalies and remains challenging.The incidence of Barrett's esophagus (BE) increased in the last decades and affects up to 11% of all adults. Chronic gastroesophageal reflux is considered a major risk factor, which leads to the replacement of the healthy multi-layered esophageal epithelium with a single-layered columnar epithelium in the distal esophagus. BE is detected during routine endoscopy, and its diagnosis is verified by histology. BE is a premalignant condition of esophageal adenocarcinoma (EAC) and is classified into different stages.2 or did not specify the respective BE stages,3 we performed scRNAseq experiments with biopsies corresponding to different BE stages.4 Our analysis identified two sets of marker genes that distinguish between BE with and without IM, and CLDN2 as a marker gene for dysplastic BE stages. Next to scRNAseq, we used, to our knowledge for the first time, single-cell DNA-sequencing (scDNAseq) to detect chromosomal instabilities (CINs) and DNA mutations at single-cell resolution in BE. Interestingly, only cells with CINs accumulated specific T > C and T > G mutations known as the COSMIC mutational signature SBS17. This finding adds to our current knowledge of BE pathology by identifying an early mutational event, which likely contributes to later tumor development.Different approaches were applied to identify stage-specific marker genes for histological assessment, although with little success. Single-cell RNA-sequencing (scRNAseq) is an attractive approach to identify such marker genes. While others focused their analyses on NDBE biopsies5 We wondered if all cells in the BE tissue are equally affected by the underlying mutagenic mechanism. We therefore performed scDNAseq experiments with fourteen biopsies from eight patients.4 Our high-quality scDNAseq data facilitated the determination of megabase-wide chromosomal rearrangements and the identification of genome-wide mutational patterns, while the detection of gene-specific mutations was impossible. All NDBE biopsies were chromosomally stable (CS) and were void of SBS17 signatures. One of the two LGD biopsies and all analyzed HGD samples revealed multiple cell clusters, which carried different degrees of CINs, while one cluster was always CS. The observed clonal heterogeneity is consistent with previously published WGS experiments.6 Interestingly, DNA mutations corresponding to SBS17 were only observed in the CIN clusters in five of six biopsies but never in CS cell clusters. This difference was most obvious, when analyzing NDBE and HGD biopsies from the same patients, as SBS17-specific mutations were detected only in CIN cells of the HGD biopsy but not in NDBE cells. The identification of this relationship was possible due to the single-cell resolution of the scDNAseq method4 and could not be observed by WGS analyses.6,7 As the mutagenic process causing the SBS17-specific mutations is still unknown, it is tempting to speculate that chronic gastroesophageal reflux may be involved, although it cannot explain the selective mutagenesis observed in only a subset of cells. Either the accumulation of SBS17-specific mutations is uncoupled from the gastroesophageal reflux or it acts together in concert with a second, still unknown mechanism.Initially, we established organoid cultures from different anatomic locations of the same patient, including the non-diseased esophageal epithelium, gastric cardia, corpus, pylorus and LGD BE. These cultures were clonally expanded for whole-genome sequencing (WGS), which revealed SBS17-associated mutations only in BE organoids but not in organoids from the non-diseased control tissues. The mechanism causing the SBS17-specific mutations is still poorly understood. Notably, these alterations are primarily detected in gastric and esophageal cancers.8 BE cells revealed the best transcriptional overlap with gastric epithelium as also shown by others.3 Unexpectedly, BE cells did not form clusters according to their BE stages.4 Some clusters were even biopsy-specific, which may reflect differences in the underlying CIN patterns. Although we attempted to correct for these CIN biases, we failed in identifying novel BE stage-specific marker genes. A plausible explanation is that the expression of genes in chromosomal stable regions is indirectly influenced by the deregulation of genes located in CIN regions. Since our dataset was composed of all the different BE stages, we selected seventeen genes from different clusters and analyzed their expression by RNA in situ hybridization experiments on histological sections from endoscopic resections containing distinct BE stages within the same slide. Seven genes proved suitable to distinguish between BE stages. Selective expression was observed for PSCA, SLC5A5 and LIPF in columnar epithelium without IM, for ANPEP, CEACAM6 and REG4 in BE with IM and for CLDN2 in dysplastic BE stages. Notably, our RNA in situ hybridization method is more sensitive and specific than previous antibody staining, which yielded controversial results.9,10 Our gene expression data will need to be confirmed in a larger patient cohort to corroborate the utility of these new marker genes for histopathological assessment.To identify stage-specific marker genes, we performed single-cell RNA sequencing (scRNAseq) experiments with replica plates corresponding to the scDNAseq experiments and additionally with new biopsies. We analyzed eighteen biopsies from fourteen patients and compared them to our previously published scRNAseq data of healthy control epithelium.


The review also outlines the techniques used to identify epithelial stem/progenitor cells, specifically in vitro functional assays and in vivo lineage tracing. We will also discuss their known interactions and hierarchy and known roles in endometrial dynamics across the menstrual or estrous cycle including re-epithelialization at menses and regeneration of the tissue during the proliferative phase. We also detail their potential role in endometrial proliferative disorders such as endometriosis.The human endometrium undergoes approximately 450 cycles of proliferation, differentiation, shedding and regeneration over a woman’s reproductive lifetime. The regenerative capacity of the endometrium is attributed to stem/progenitor cells residing in the basalis layer of the tissue. Mesenchymal stem cells have been extensively studied in the endometrium, whereas endometrial epithelial stem/progenitor cells have remained more elusive. This review details the discovery of human and mouse endometrial epithelial stem/progenitor cells. It highlights recent significant developments identifying putative markers of these epithelial stem/progenitor cells that reveal their  The endometrium is a unique tissue that undergoes monthly cycles of proliferation, differentiation, breakdown, shedding and repair under the control of fluctuations in circulating ovarian hormones, 17 β-estradiol and progesterone . The endWhilst the outward manifestation of menstruation, vaginal bleeding, may be experienced for 5 days or longer in some women, repair processes are initiated at the beginning of this process. Immediate post-menstrual repair involving re-epithelization of the luminal epithelium commences within 48 h of onset  in a stePost menstruation, the functionalis endometrium grows from the remaining basalis which has a thickness of 0.5 mm, reaching a maximum thickness of 7–8 mm by the mid-proliferative phase . This reSimilar to human, the endometrial epithelium of the adult mouse uterus consists of luminal (LE) and glandular (GE) epithelia, two histologically and functionally distinct cell types . The simDuring proestrus, under the influence of increasing concentrations of circulating estrogens, uterine water content, height of LE cells and GE proliferation increases . During Menstruation can be mimicked in a mouse using exogenous steroids and inducing artificial decidualization via delivery of sesame oil into the lumen of the uterine cavity . Post-paWhilst the cyclical changes in the mouse endometrium during the estrous cycle are not as dynamic as in human endometrium, putative stem/progenitor populations have been identified in the mouse endometrium, which likely support cell turnover and endometrial repair and regeneration post-partum . While dStem/progenitor cells are rare cells present in most postnatal tissues and organs, where they function in maintaining cellular homeostasis of the tissue or organ . Stem/prin vitro  and used predominantly in Drosophila. This review will focus on the Cre-loxP recombination system used in mice. In this site-specific recombination system, one mouse line contains a tissue or cell-specific promotor expressing Cre recombinase, with the enzyme’s activity temporally controlled through its fusion with an estrogen receptor , and the permanent genetic labeling of the tissue or cell population and their progeny. The use of low ligand doses allows for the labeling of individual cells and their clones, with subsequent lineage tracing potentially leading to the identification of a stem/progenitor cell population.Today, the predominant method of lineage tracing is cell marking by genetic recombination. Here, the expression of a cell or tissue specific recombinase enzyme leads to the subsequent expression of a conditional reporter gene. This allows for the permanent genetic labeling of a cell and its future progeny. There are two widely used recombination systems: one adapted from bacteriophage P1 (Cre-loxP) and used predominantly in mice, and the other adapted from receptor  or progereceptor . Activatreceptor . This Crin vitro clonogenicity assays or transplantation. Another advantage of single-cell lineage tracing is that it can be performed in any cell type without knowing the specific gene markers of this cell type (Lgr5)  . Initialntestine . Similarse liver . Using apulation .+ epithelial cells obtained from hysterectomy tissue which includes the basalis layer   , 35–45 pal cells . SP cellal cells . While tAXIN2 was discovered using a gene microarray approach, comparing highly purified EpCAM+ epithelial cells isolated from pre- and post-menopausal hysterectomy endometrium  endometrial epithelial cells were more clonogenic than N-cadherin– epithelial cells, showed greater self-renewal and more population doublings by serial cloning. They also differentiated into cytokeratin-expressing organoids in 3D culture. N-cadherin-immunostained epithelial cells were located in the bases of the glands adjacent to the myometrium in pre- and post-menopausal endometrium , in the deep basalis, with 78% of N-cadherin+ cells showing colocalization -fucosyltransferase 4] catalyzing protein glycosylation associated with the expression of SSEA-1 were identified in the gene profiles, suggesting the basalis epithelium was not sampled. It is also not known whether ciliated cells are part of the putative endometrial epithelial hierarchy. Nor is it clear how this epithelial hierarchy are located in 3D endometrium, given the degree of gland branching and rhizome formation in the basalis glands  have been used to visualize cell lineages in the intestine . In the salis GE . Multiplsalis GE . Individsalis GE . Reassesal cells . This hoal cells .in vivo lineage tracing of passenger mutations (as above), new tissue clearing methods, slice cultures (see below), organoid models and molecular (sequencing) will enable investigation of the epithelial hierarchy in 3D and at the single cell level. These approaches will inform and may build on the current models.The current model of the putative stem/progenitor cell populations in human endometrium are based on 2D imaging with its inherent limitations. Future research using +SOX9+ cells described above, as the functionalis layer regenerates from the re-epithelialized basalis, and the luminal epithelium retains the SSEA-1+SOX9+ phenotype as it is pushed ever upwards during endometrial growth. It is also possible that residual SSEA-1+ luminal epithelial cells may be activated at menstruation to support rapid re-epithelialization during piecemeal shedding of the functionalis, given the adhesive and migratory properties of SSEA-1+ cells  are implicated in tumor initiation, progression, metastasis and recurrence. Endometrial CSCs are thought to originate through several mechanisms including; genetic mutation or epigenetic alteration of epithelial stem/progenitor cells residing in the tissue, de-differentiation of endometrial epithelial cells which form a CSC progenitor, or via EMT of endometrial Side Population cells . EndometAt birth, the murine uterus lacks endometrial glands and consists of a tube lined with a simple luminal epithelium supported by undifferentiated mesenchyme. The LE forms buds which invade the mesenchyme to initiate the development of GE around post-natal day 5 (P5). Around P7, histologically distinct uterine glands appear in the endometrium , which c+ non-LRC. This molecular difference may be used to characterize their identity and function  are absent or very rare after a 3- to 4-week chase in postnatal and prepubertal mouse models and predominantly found in the LE along with rare LRC in the GE . These Lfunction . LRCs infunction . By applfunction . Howeverfunction . Thus, tfunction .mTert-GFP or Chβ-actin-GFP reporter mice demonstrated no contribution of bone marrow-derived cells to endometrial epithelial lineages, but contributed to immune cells which were likely misidentified in previous studies (Mouse telomerase reverse transcriptase (mTert) marks stem cells in the intestine  and was  studies .CreERT2-LoxP–based single-cell lineage tracing system in the adult mouse uterus to functionally identify epithelial stem cells resulted from characterizing stem cell clones in vivo  promotor positioned next to an inducible Cre recombinase was crossed with the Rosa26-YFP reporter strain to lineage label epithelial cells. By quantifying distinct cellular phenotypes  and the proliferative ability (KI67+) of the endometrial epithelium, different clonal populations were identified in the mouse endometrial epithelium. The founder cells of mixed clones, originating from the intersection zone of the LE and GE, were identified as endometrial epithelial stem cells  following 1 week of maximally labeled Axin2+ GE cells, the contribution of Axin2+ GE to the LE is minimal. Thus, Axin2+ GE has a very limited contribution to LE after multiple cycling or post-partum injury, but a high contribution to GE, suggesting Axin2+ GE supply to LE is insufficient to maintain homeostasis or renewal of LE  for these cell populations in human and they are still to be identified in the mouse. This limits the identification of their microenvironment on a cellular and molecular level. There is currently insufficient published knowledge to provide a detailed understanding of the endometrial epithelial stem cell niche in this review. However, it is anticipated that new studies in the next few years will generate discoveries on the endometrial epithelial stem cell niche.Wnt7a mRNA coinciding with decreasing concentrations of stromal Wnt4 mRNA suggesting stem/progenitor activity and MET. In a postpartum repair model, a group of stromal cells expressing Anti Mullerian Hormone Receptor type II contributed to epithelial repair and regeneration via MET (Stem cell contributions to re-epithelization can be studied using mouse models of menstruation  or pseud via MET . A compr via MET .+ LE cells were not observed prior to endometrial breakdown, most likely due to the high exogenous progesterone support. Following progesterone withdrawal and induction of menses, LE mTert+ clusters were identified in the repairing epithelium (+ cells are identified, suggesting that GE expression of mTert may be estrogen dependent and may have a role in endometrial regeneration. LE mTert+ cells were located adjacent to clusters of KI67+ cells suggesting that mTert+ cells may undergo asymmetrical division to form TACs, which proliferate to form new LE cells (SP cells have been found in the stroma in postpartum mice but not in the normal cycling endometrium . The ideithelium , suggestLE cells . SimilarLE cells . These cWhilst the bone marrow does not appear to contribute to the endometrial epithelium during the estrous cycle, bone marrow transplantation studies under pathological conditions have revealed a limited contribution of bone marrow-derived cells to endometrial regeneration .Axin2+ (Lgr5+ (Axin2+ and Lgr5+ GE progenitors in future studies. It is possible that the epithelial stem cells in the intersection area differentiate into either Axin2+ or Lgr5+ GE progenitors with significant overlap between these 2 progenitor cells given the role of both markers in the Wnt signaling pathway.Lineage tracing has provided direct evidence that the epithelial stem cells in the intersection zone between LE and GE are capable of life-long maintenance of the self-renewing endometrial epithelium and post-partum regeneration of epithelial lineages . Axin2+ Axin2+  or Lgr5++ (Lgr5+  progenitAccumulating reports of endometrial epithelial stem/progenitor cells have revealed their important roles in homeostasis and regeneration of the endometrial lining of the uterus in both humans and mice. Increasing knowledge of endometrial stem/progenitor cell biology and their niches provides new understanding of the remarkable regenerative capacity of mouse and human endometrium. It also contributes new insight into endometrial proliferative disorders, offering potential for new therapies targeting the epithelial stem/progenitor cells. In mice, lineage tracing single cells in the whole uterus reliably tracks the behavior and fate of the endometrial epithelial stem/progenitor cells, by which, potency, location and markers of endometrial epithelial stem/progenitor cells have been advanced. In humans, previous and recent studies applying functional stem cell assays including organoid formation and specific surface marker identification have enabled characterization of the location of endometrial epithelial stem/progenitor cells. The surface markers identified for human endometrial epithelial stem/progenitor cells allow their isolation and future manipulation for treatment of infertility or miscarriage caused by an inadequate endometrial proliferation. Single cell sequencing comparing basalis, functionalis and luminal epithelium will likely increase our understanding of the epithelial hierarchy in human endometrium in relation to the newly discovered unique structural differences between these endometrial zones. The regulatory mechanisms of self-renewal and differentiation of endometrial epithelial stem/progenitor cells are the scientific premise needed to decode aberrations in these cells and their role in the development of endometrial diseases such as endometriosis and endometrial cancer. Only then can effective treatments be developed that target abnormal endometrial epithelial stem/progenitor cells. The promise of endometrial epithelial stem/progenitor cells for regenerative medicine, their markers and their regulatory mechanisms of self-renewal and differentiation should ensure further research in these areas are pursued.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Bifidobacterium bifidum TMC3115 (TMC3115) supplementation using a sub-chronic and mild social defeat stress murine model in this study. TM3115 supplementation maintained body weight gain and alleviated a polydipsia-like symptom induced by the stress. In the analyses of fecal and cecal bacterial profiles, expansions of Proteobacteria in stressed mice and increases in Actinobacteria and Bifidobacterium in mice supplemented with TMC3115 were observed. There was no marked difference in the diversity of cecal bacteria between the tested mice. Elevated serum levels of inflammatory markers such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were observed in the stressed mice, while TMC3115 only reduced the IL-6 level. These findings suggest that TMC3115 supplementation confers tolerance to psychosocial stress in the host through modulation of the gut microbiota and alleviation of stress-induced inflammatory responses. Furthermore, it may be expected to exert prevention and treatment of disorders related to peripheral IL-6, including depression.With the accumulation of knowledge on the relation between psychological stress and gut microbiota, there is growing interest in controlling stress and/or mood disorders via probiotic supplementation. We aimed to examine the effect of probiotic  Stressful life events often lead to a variety of disorders, such as depression, post-traumatic stress disorder, and cardiovascular disease . Stress As one of the definitions of probiotics is “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” , the curThe chronic social defeat stress (CSDS) model is often used for studying depression because mice subjected to CSDS display various depressive-like features . SeveralBifidobacterium bifidum TMC3115 (TMC3115) is a probiotic bacterium originally isolated from healthy infant stools _Desulfovibrionaceae and Desulfovibrio. In contrast, there was no correlation between the serum IL-6 level and these genera, as estimated from the fact that TMC3115 supplementation did not affect the abundance of Proteobacteria. Additionally, no correlation between the serum IL-6 level and even the genus Bifidobacterium was observed. These findings indicate that TMC3115 modulates IL-6 production without involving gut microbiota. Suzuki et al., reported that TMC3115 might suppress IL-6 production from preadipocytes via stimulating mononuclear cells from Peyer’s patch using a clonal porcine intramuscular preadipocyte line [Stress may be associated not only with inflammation, as mentioned above, but also with the abundance of l stress . Proteobhenotype . In factto sCSDS . Thus, wto sCSDS . As a reyte line . TherefoIn conclusion, oral supplementation of probiotic TMC3115 was associated with stress tolerance in the host. The possible mechanisms were the increase in bifidobacteria and the anti-inflammation associated with a low peripheral IL-6 level. In addition, the effect of reducing the peripheral IL-6 level shows potential for the prevention and treatment of depression in the future. Further studies are required to confirm these findings.


Background: Kidney perfusion and oxygenation are two important determinants of kidney graft function. In kidney transplantation, repeated graft hypoperfusion may occur during hip flexion, for example in the sitting position, due to the progressive development of fibrotic tissue around iliac arteries. The aim of this study was to assess the changes in oxygenation and perfusion of kidney grafts during hip flexion and extension using a new functional magnetic resonance imaging (fMRI) protocol.Methods: Nineteen kidney graft recipients prospectively underwent MRI on a 3T scanner including diffusion-weighted, blood oxygenation level dependent (BOLD), and arterial spin labeling sequences in hip positions 0° and >90° before and after intravenous administration of 20 mg furosemide.Results: Unexpectedly, graft perfusion values were significantly higher in flexed compared to neutral hip position. Main diffusion-derived parameters were not affected by hip position. BOLD-derived cortico-medullary R2* ratio was significantly modified during hip flexion suggesting an intrarenal redistribution of the oxygenation in favor of the medulla and to the detriment of the cortex. Furthermore, the increase in medullary oxygenation induced by furosemide was significantly blunted during hip flexion (p < 0.001).Conclusion: Hip flexion has an acute impact on perfusion and tissue oxygenation in kidney grafts. Whether these position-dependent changes affect the long-term function and outcome of kidney transplants needs further investigation. Kidney transplantation is the therapy of choice for patients with end-stage kidney disease conferring survival benefit regardless of graft source . HoweverIn spite of advanced operative techniques, vascular complications after kidney transplantation remain of concern , 6. SurgIntriguingly, lower extremity claudication has been reported in professional cyclists due to kinking of iliac arteries during hip flexion visualized by magnetic resonance (MR) angiography , 9. KinkLittle is known about dynamic perfusion changes in kidney grafts depending on body posture. Kidney grafts typically placed into iliac fossa might be exposed to similar position-dependent perfusion problems. Specifically, kinking or narrowing of the transplant renal or iliac artery may occur during hip flexion due to tethering by adjacent fibrotic tissue leading to iterative hypoperfusion episodes as well as chronic ischemic graft damage and IFTA in the long-term. Considering common sedentary lifestyle majorly in the sitting position, this influence could be significant. Furthermore, pre-existing endovascular lesions in this high-cardiovascular-risk population or subclinical vascular anastomotic problems may have an additional effect. To the best of our knowledge, this question has not been addressed so far.Today, novel functional MR imaging (fMRI) techniques allow for the non-invasive investigation of renal tissue oxygenation, perfusion, and diffusion with the use of blood oxygenation level dependent (BOLD)-MRI, arterial spin labeling (ASL)-MRI, and diffusion-weighted imaging (DWI) .The aim of this prospective interventional study was therefore to assess the influence of hip flexion on kidney graft oxygenation and perfusion using fMRI techniques in kidney transplant recipients. Our hypothesis was that hip flexion >90°  would lead to an instant and temporary reduction of renal tissue oxygenation and perfusion as measured by BOLD-MRI, ASL-MRI, and DWI compared to neutral hip position. To correlate results with vascular anatomy , non-contrast-enhanced time-of-flight (TOF) angiography during hip flexion and duplex-ultrasound scans (DUS) were performed. Additionally, we analyzed the correlation of oxygenation and perfusion changes with clinical parameters.The protocol of this prospective single-center interventional study was approved by the local ethics committee  and conducted in accordance with the Declarations of Helsinki and Istanbul , 15.2 according to Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)-equation and preserved faculties of judgment were eligible.Patients ≥18 years having received a kidney graft ≥6 months ago into the iliac fossa with a stable graft function  and an estimated glomerular filtration rate (eGFR) ≥30 ml/min/1.73 mExclusion criteria were pregnancy; New York Heart Association stage IV dyspnea or orthopnea; acute infection; active neoplasia; surgical intervention, severe trauma, or acute ischemic or thromboembolic event within the preceding 2 months; inability of ipsilateral hip flexion; classical contraindications to MRI; body weight >200 kg; as well as any implanted metallic material without prior 3T-MRI after implantation.From June 2015 through January 2016, 97 consecutive patients were screened at the outpatient University Clinic for Nephrology and Hypertension in Bern, 19 of whom fulfilled eligibility criteria and accepted to participate. Written informed consent was obtained from each participant prior to inclusion. A standardized hydration protocol was followed  and diuretics were held on the study day and the preceding day if considered safe by the treating nephrologist . A lightThere were four study phases during one patient's session: anatomical MRI, DWI, BOLD-MRI, and ASL-MRI were first performed during neutral hip position and then repeated during maximally achievable hip flexion (≥90° from bed level); to account for position-induced confounding effects on the BOLD signal as well as to include a functional test, the same measurements were repeated 10 min after the intravenous administration of 20 mg furosemide  –21. At tMRI data were acquired on a 3.0 T whole body MR Scanner . To facilitate hip flexion >90°, measurements were performed in the lateral decubitus position requiring subject positioning far off-center. Maintenance of hip flexion was assured by an MR-safe belt.*; outcome measure is the transverse relaxation rate R2*  which correlates to tissue oxygen content when confounding factors such as blood volume or hydration state are excluded. Rather than providing absolute values of oxygenation, this technique is especially useful to demonstrate relative changes in response to various interventions .ASL-MRI allows quantitative perfusion measurements using magnetically labeled water as an endogenous diffusible tracer , 23, 24.D and the perfusion fraction FP (2), two repetitions, TR of 3300 ms, TE of 56 ms, slice thickness of 5 mm, matrix of 128 × 128 and FOV of 300 × 300 mm2.DWI allows the quantification of diffusion parameters and microperfusion yielding ADCction FP , 22. DWITOF angiography is an MR technique permitting the visualization of vascular flow without the need for contrast agents. Based on the phenomenon of flow-related enhancement of spins entering into an imaging slice, vascular anatomy can be reconstructed in a three-dimensional view.A maximum of six regions of interest (ROIs) were analyzed in every slice  for each of the four measurements . ROIs were manually defined by the same blinded investigator on images handed out in a random fashion (mixed between patients and study phases). ROIs containing approximately 10 voxels were traced in the medulla and cortex. All images were co-registered facilitating comparable ROI position for all methods. Data were analyzed using in-house custom-scripts written in IDL® and MATLAB®.Serum creatinine was measured by an enzymatic creatinine assay .Doppler studies were performed according to clinical routine in the outpatient clinic of the University clinic for Nephrology and Hypertension in Bern on an Acuson® 2000S device  including graft morphology, urinary outflow, and perfusion .* values, in medullary to cortical R2* ratio (MCR R2*) and in R2* ratio without/with (wo/w) furosemide during hip flexion compared to neutral hip position. Secondary outcomes were the changes in mean ASL perfusion values and mean FP by DWI during hip flexion compared to neutral hip position, the presence of renal transplant/iliac artery kinking visualized by TOF angiography during hip flexion, the correlation of oxygenation  and perfusion changes with the presence of functional kinking, endovascular lesions of transplant renal/iliac artery (evidenced by DUS) and with clinical parameters , as well as correlation of these clinical parameters with functional kinking.The primary outcome was the change in mean medullary and cortical R2*; the diffusion coefficient ADCD; and perfusion value of <7, 4, and 15%, respectively, could be detected in 19 patients on a significance level of 0.05 and 80% statistical power  received an incomplete dose of furosemide due to venous access problems. A mean scanning time per study phase of 15–20 min was met, resulting in an overall measurement time for all four scans <90 min including a break leaving the magnet and the time for furosemide injection. Overall visual image quality was judged good despite unusual lateral decubitus in both positions and intermediate for TOF angiography, however, without performing a formal image quality analysis . BOLD, A* values as a marker of tissue oxygenation level  are shown in * values were significantly higher in the medulla. Medullary R2* decreased significantly after furosemide administration, as reported for native kidneys .To investigate determinants of position-induced oxygenation and perfusion changes, correlation testing was performed between fMRI and selected clinical parameters . To reduce the number of comparisons, only significant changes between positions were tested. Two significant correlations were found:* from neutral to flexed position without furosemide correlated positively with eGFR . This might indicate a stronger cortical R2* increase (oxygenation decrease) during flexion in patients with reduced graft function.First, in BOLD, the change in MCR R2p = 0.041) and transplant vintage  as well as with CNI use after furosemide .Second, in ASL, the increase in cortical perfusion from neutral to flexed position without furosemide correlated negatively and significantly with age  Hip flexion diminishes the increase in oxygenation after furosemide. At last, our study demonstrates for the first time the feasibility and reliability of a multiparametric functional MRI protocol with examination of kidney graft oxygenation and perfusion during two different body positions in a single session.The main findings of this study are the following: (1) hip flexion induces a consistent increase in renal perfusion as measured by ASL; (2) hip flexion causes a redistribution of renal tissue oxygenation with a significant decrease of the medulla/cortex ratio R2Our initial hypothesis was that hip flexion would lead to an instant and temporary reduction of graft perfusion associated with a decrease in graft oxygenation. To our surprise, perfusion values measured by ASL-MRI consistently increased during hip flexion. ASL technique involves the magnetic labeling of inflowing blood with the creation of a subtraction image of the kidney with and without labeling . In modeThe first is an increase in arterial inflow due to the partial obstruction distally to the graft leading to increased flow through the external and internal iliac artery. In line with this hypothesis, maximal hip flexion has been shown to induce a shortening, bending, and twisting of iliac arteries in healthy subjects , 34. In A second hypothesis is that hip flexion causes an increase in arterial blood pressure secondary to a global increase in systemic vascular resistance. Analogously, the squatting position classically adopted by patients with cyanotic heart disease leads to blood pressure elevations by increasing venous return and systemic arterial resistance. Yet, this effect might be restricted to the standing position , 36. HowThe increase in renal perfusion could also represent a counter-regulatory mechanism secondary to the changes in tissue oxygenation. Indeed, the scan order putting BOLD before ASL sequences might explain inverse trends in BOLD and ASL values. Thus, in a recent study, sympathetic stimulation by handgrip exercise has been reported to decrease renal artery flow and increase medullary oxygenation measured by BOLD-MRI. A decreased reabsorptive workload due to reduced distal sodium delivery was hypothesized by the authors .Venous outflow obstruction with backflow to the graft during hip flexion is still another mechanism whereby hip flexion could increase graft perfusion. Venous obstruction might actually explain both the increased renal perfusion and the reduced oxygenation response to furosemide. In this situation, the marked prolonged position change might have overcome the hurdle of venous valves. The hydration protocol inducing a larger plasma volume may have increased this effect. Similarly, the two subjects with ipsilateral graft implantation (with a risk of arterio-venous crossing) showed a particularly reduced perfusion ratio neutral/flexed hip position possibly pointing to outflow obstruction. Importantly, potential changes in arterial inflow may have been masked in this case. Thus, in this view, the positive correlation between age, transplant vintage and CNI use on one hand and the cortical perfusion ratio neutral/flexed hip position on the other may be explained by a reduced arterial component of overall flow. However, this hypothesis remains speculative.The perfusion increase in the cortex and medulla during hip flexion after furosemide is augmented by the observed  lower baseline perfusion values during neutral hip position after furosemide compared to the values before the administration of furosemide. This is in line with previous microelectrode measurements in rats and ASL-MRI measurements in humans , 39.* ratio, suggesting a redistribution of intrarenal tissue oxygenation toward the medulla. The decrease of MCR R2* during hip flexion correlated with lower eGFR values. MCR R2* has previously been defined as the normalized intrarenal oxygen bioavailability . The patients/participants provided their written informed consent to participate in this study.LYM participated in research design, performance of the research, data analysis, and writing of the manuscript. MS participated in research design, performance of the research, data collection, and revision of the manuscript. FN participated in data analysis and the revision of the manuscript. DT participated in data analysis and the revision of the manuscript. GD participated in data analysis. PM contributed the arterial spin labeling–magnetic resonance imaging sequence and participated in the revision of the manuscript. MB participated in the writing and revision of the manuscript. BV participated in the research design, performance of the research, and revision of the manuscript. PV participated in the research design, performance of the research, data collection, data analysis, and revision of the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The metastatic cancer disease represents the real and urgent clinical need in oncology. Therefore, an understanding of the complex molecular mechanisms sustaining the metastatic cascade is critical to advance cancer therapies. Recent studies highlight how redox signaling influences the behavior of metastatic cancer cells, contributes to their travel in bloodstream from the primary tumor to the distant organs and conditions the progression of the micrometastases or their dormant state. Radical oxygen species not only regulate intracellular processes but participate to paracrine circuits by diffusion to nearby cells, thus assuming unpredicted roles in the communication between metastatic cancer cells, blood circulating cells, and stroma cells at site of colonization. Here, we review recent insights in the role of radical oxygen species in the metastasis formation with a special focus on extravasation at metastatic sites.The online version contains supplementary material available at 10.1007/s10456-021-09779-5. Metastasis spreading from the primary lesion to secondary sites causes over 90% of human cancer deaths . FortunaMetastasis diffusion is a multiple step process with different bottlenecks, which renders the journey of cancer cells in bloodstream highly demanding with few chances to get to the final destination. Although, primary tumors can discard millions of cells into the capillaries every day, a metastatic event occurs rarely. The cancer population accountable for the metastatic process is defined metastatic cancer cell (MCC), the protagonist of cancer progression. The first step is the intravasation of MCCs into the tumor vascular bed. Then, MCCs move into the blood as a single cell or clusters, possibly surrounded by platelets or polymorphonuclear cells (PMNs). Patterns of MCCs in the bloodstream and features of the vasculature in each organ influence the efficacy of the metastatic process. Furthermore, there are increasing evidences suggesting a role of primary tumor secretome in dictating the selection of metastatic sites. For example, primary tumors release angiocrine molecules or chemokines able to directly or indirectly address bone marrow-derived hematopoietic progenitors at site of metastatic spreading to induce an immune-suppressive environment . PrimaryWhen MCCs stop at the capillary surface of targeted tissues, they extravasate into the parenchyma and settle within an area near the capillaries termed “metastatic niche ”, where This scenario is well supported by a huge amounts of in vitro and in vivo data , 7. NeveThis review will focus on the role of redox state and radical oxygen species (ROS) in influencing the success rate of metastatic process analyzing the extravasation and the settlement of MCCs in distant organs , 8. Low Furthermore, recent observations extended their regulatory roles from intracellular mechanisms to signal circuits occurring between single cells. ROS might be generated within cells before being released in a paracrine manner diffusing to nearby target cells, or ROS-generating systems might be delivered through exosomes to affect the behavior of neighboring cells .Endogenous free radicals, both ROS and reactive nitrogen species (RNS), are produced in several sub-cellular organelles  and according to their concentrations and half-time exert both signaling and oxidative damage on lipids, nucleic acids and proteins.2 to superoxide ion (O2•−). Almost 95% of O2•− is unspecifically produced along the electron transfer through the complexes of the mitochondrial electron transport chain. The residual production is catalyzed by NADPH oxidase (NOX) family, which transfers electrons from NADPH to O2. Superoxide is then detoxified to generate other ROS molecules, which include hydroxyl radical, aloxyl, peroxyl, singlet oxygen and non-radical H2O2, which is the primary mediator of ROS-driven cellular signaling , catalase, thioredoxin (Trx) reductase and glutathione (GSH) peroxidase-GSH reductase system , which converts superoxide into H−, which mediates the nitrosylation or the oxidation of amino acid residues such as tyrosine, cysteine, methionine, and tryptophan.ROS metabolism is strictly connected with RNS and in particular with nitric oxide (NO), which is synthesized by various isoforms of nitric oxide synthase (NOS). NO can further react with superoxide to form peroxynitrite ONOO++, Cu+) undergoing oxidation and the enzymatic reductive systems [Evolution has developed highly organized systems to quench the dangerous effect of the excessive ROS production or fine-tune their physiologic role . These a systems . Further systems .There is growing evidence that tumorigenesis is fueled by a minute population of cells endowed with unique self-renewal potential and arising when transit-amplifying cells with mutant genomes differentiate and enter the stem cell state. These cells are operationally classified as initiating cancer cells . By theiA typical feature of MCCs is the ability to show a metastable phenotype encompassing epithelial and mesenchymal traits . The celOxidative stress plays a contributive role in this context, being a driver of malignant transformation and fostering a highly metastatic phenotype . NeverthThe higher content of ROS is ensured by increased basal metabolic activity, mitochondrial dysfunction paralleled to hypoxia or mitophagy, uncontrolled cytokines signaling, and oncogene activity. Several known ROS sources, including NOXs, cyclooxygenases, or lipoxygenases, show an intense enzymatic activity contributing to keep ROS levels higher than in normal cells. Different signaling pathways associated with oxidative stress influence metastatic phenotype.TGF-β increases ROS production by activating the transcription of NOX-2 and -4 and suppresses antioxidant enzymes, leading to a redox imbalance . ROS, in2O2 [Wnt is a complex signaling pathway that controls cell differentiation and proliferation in embryo and adults by a canonical or non-canonical pathway. Canonical pathway is characterized by the nuclear translocation of β-catenin, while the non-canonical pathway signals through Frizzled receptors and triggers calcium-mediated biological functions including cell polarity and stemness . ROS hav2O2 . Further2O2 , and sta2O2 .Accumulating evidences have suggested that ROS also function as second messengers modulating stem cell self-renewal and differentiation by regulation of intricate signaling networks.Differently from cancer cells, cancer stem cells produce low amount of ROS .To maintain low level of ROS, the metabolic set point of cancer stem cells is set-up to favor the reductive metabolism and the production of antioxidants. This peculiarity is mainly governed by hypoxia inducible factor (HIF)  and NRF-HIF induces glycolytic genes and pyruvate dehydrogenase kinase 1, which phosphorylates and inhibits pyruvate dehydrogenase to switch cells from oxidative to glycolytic metabolism. Furthermore it triggers mitophagy  and reprInterestingly, NRF-2, the master gene of redox system, is often mutated in cancers  and is hA holistic and fruitful interpretation of the experimental data available can be obtained by considering the involvement of redox metabolism in metastasis diffusion as a dynamic process finalized to maintain a balanced redox state a little bit shifted towards reducing equivalents finalized to maintain ROS levels consistent with the cellular fitness. The analysis of melanoma patient-derived xenografts elegantly demonstrates that metastatic activity is related to the concomitant increase of ROS production and antioxidant molecules, such as NADPH and GSH . The invTo successfully and safely navigate into the blood circulation, extravasate and colonize a distant site from the primary tumors MCCs need the collaboration of other cell types, including endothelial cells (ECs), PMNs and platelets. These cells have specific metabolic peculiarities, which impact on oxidoreductive system and indeed on MCC circulation.2 levels into the blood.Redox balance is crucial for endothelial fitness and its homeostasis gets worse during life. Endothelial oxidative stress is involved in many pathological events, including vasoconstriction, vascular remodeling, inflammation, angiogenesis and fibrosis . In this2O2 instead of NO when the availability of arginine or the cofactor tetrahydrobipterin is low. Superoxide reacts with NO forming peroxynitrite that further oxidizes tetrahydrobipterin creating a vicious circle and more NOS3 uncoupling [2O2 and O2, together with the mitochondrial electron transport chain are involved in ROS production during pathological conditions [2S, a gas involved in endothelial functions, including angiogenesis, and primarily produced by the trans-sulfuration enzyme cystathionine-γ-lyase. H2S exists as hydrosulphide anions (HS–) and protons (H+) with the remaining third being in the form of H2S undissociated gas. The mechanism supporting H2S functions requires ROS-dependent activation of polysulphides , which 2 and the requirement of a judicious ROS level, ECs have developed different strategies to avoid oxidative stress and in particular they utilize diversified reducing systems. The use of glycolysis to produce ATP instead of mitochondrial respiration is the first method exploited by quiescent and angiogenic ECs to keep ROS production in a narrow and low range [Considering the almost unlimited availability of Oow range . In nascow range . This prow range . Of noteow range .To reduce the risk of oxidative stress, quiescent ECs show a high rate of fatty acid oxidation to fuel tricarboxylic acid cycle (TCA). The activation of this pathway does not support ATP production or biomass synthesis but assures redox homeostasis. The TCA metabolite malate can be exported to the cytosol via the dicarboxylate carrier and oxidized to pyruvate by the NADP-dependent malate dehydrogenase. NADPH in turn supports GSH synthesis resulting in ROS quenching . Of noteFurthermore, other metabolic pathways contribute to generate an efficient and antioxidant system in ECs. Supplemental Table 1 summarizes the most important effects induced by the alteration of the endothelial reductive pathways to frame the role of vasculature in the metastatic process Fig. .Fig. 4EnIn the metastatic process, ECs represent both a physical barrier to be crossed by MCC and a support to the formation of the metastatic niche . LeukocyLeukocyte diapedesis occurs at site of tissue injury, where inflammatory cytokines induce the expression of endothelial selectins, which orchestrate the ‘first contact’. Locally accumulated chemokines instruct leukocytes to polarize and acquire a motile phenotype. This step needs the interaction between leukocyte and their endothelial ligands ICAM-1 and VCAM-1. Subsequently, the cells form protrusions to cross endothelial barrier, through EC intercellular junctions or by penetrating EC body .The arrest of MCC on vascular surface depends on the physical restriction of capillaries , or on aAs demonstrated for leukocytes, MCC starts to crawl on endothelial surface by the formation of weak adhesions by the engagement of endothelial E-selectin  or CD44 ROS are produced during leukocyte diapedesis by ECs  and reguPMNs, cells of the innate immune system, are recruited at site of infections to phagocytize and kill pathogens. They are professional ROS producers to exert their antimicrobial defense function. PMN phagocytosis or chemo-attraction depends on ROS release, mostly produced by NOX-2 function and by myeloperoxidases, specific enzymes of PMNs. NOX-2 activation and its cooperation with myeloperoxidase occur both outside PMN plasma membrane and in phagosome. The former characterizes PMN activation by soluble inflammatory stimuli  with the concomitant release of azurophil granules, containing myeloperoxidase. The latter is typical of phagocytosis, in which PMN membrane embeds the pathogen and fuses with azurophil granules enabling ROS synthesis inside the phagosome . To remop47phox subunit of NOX-1 and-2 leading to ROS generation [Platelets show a basal production of ROS, which rapidly increases when platelets start to adhere to injured vessel walls, to one another or to other circulating cells –48. Thisneration .Besides influencing integrin-mediated adhesive process , ROS migThe MCC journey in bloodstream is unsafe. They circulate as single cell or clusters . Indeed,in trans cell–cell stimulation results in the release of inflammatory cytokines  by MCCs that stimulate PMNs [Xenopus embryos [Increasing evidences indicate that MCCs might circulate in bloodstream surrounded by PMNs . Interesate PMNs . It is pate PMNs . Of noteate PMNs . These o embryos . In this embryos  Fig. 6)in trans Very recently, clusters of MCCs, PMNs and myeloid-derived suppressor cells (MDSC) have been described in melanoma and breast cancer patients . This trPlatelets interact with MCCs activating themselves and releasing their granules  in bloodTGFβ accumulated in platelet α granules and the direct platelet-tumor cell contacts synergistically activate Smad and NF-kB pathways in MCCs, resulting in their transition to an invasive mesenchymal-like phenotype and enhanced metastasis in vivo . A similadherens junctions and a retraction of ECs [p47phox subunit of NOX [MCC extravasation mirrors leukocyte diapedesis and requires strict and private interactions with ECs. They might extravasate as single cell or as clusters formed at the primary site, during the intravasation step, the travel in the bloodstream or at the secondary site arrest (reviewed in ). The MCn of ECs . It is pn of ECs . All then of ECs . Invadopt of NOX . Importat of NOX , indicatAlternatively, extravasation occurs after MCC arresting on EC surface and their intravascular proliferation without penetrating the capillary wall. The tumor mass then mechanically disrupts capillaries leading to MCC invasion . FinallyPMNs and platelets surrounding MCCs are not only limited to serve as bodyguard during their travel in the circulation, but they are instrumental to cross endothelial wall. PMNs adherent on endothelial layer stop MCCs on the site of possible colonization by favoring their seeding . PMN ROSIn vivo experimental models have demonstrated that MCC adhesion to ECs is increased by platelets . FurtherAfter crossing the vascular wall, MCCs organize themselves to colonize the new organ starting from the “metastatic niches”, which are reminiscent of the stem cell niche firstly described in hematopoiesis. Features of the metastatic niche show a dynamic behavior alongside disease progression as well as the mechanism sustaining its formation, that precede MCC seeding under the influence of molecules released by the primary tumor (see above) or molecules becoming active on cancer cell arrival . Metastahi) with cancer promoting functions mediated by macrophage M2 polarization and ROS mediated immune suppression [+ γδ T cells [PMNs represent a relevant source of ROS. In a preclinical model of lung carcinoma, cancer cells signal to bone-resident osteoblasts through the systemic release of the soluble receptor for advanced glycation end. Stimulated osteoblasts in turn promote the generation of a specific subset of tumorigenic PMNs . ROS inreg), and M2-polarized macrophages [reg. Actually, it has been reported that macrophages lacking NOX displayed hampered Treg induction and T-cell suppression [phox- deficient patients, which have an impairment in ROS production, showed a reduced number of circulating Treg [reg exert their suppressive activity by releasing exosomes containing NOX-2. These microvesicles are up-taken by T-cells where they produce ROS, which inhibit T cell receptor signal transduction by reducing phosphorylation of the T cell receptor-associated tyrosine kinase ZAP70 [Metastatic niche is characterized by immune suppressive conditions that induce MCCs to escape from immune surveillance. This immunotolerance state is promoted by MDSCs, regulatory T cells  and proteoglycans, is characterized by regions suitable for redox-regulation by ROS . For insHereby, we reviewed features and mechanisms harnessing cell detachment from the primary tumor able to circulate into the bloodstream and ultimately colonize a distant organ establishing a metastatic niche, which could remain quiescent for years and become manifest and clinically relevant all at once by unpredictable signals. All the aspects related to the metastatic program have been discussed focusing on how ROS can influence specific features of MCCs, which favor migratory and stem phenotype acquisition allowing MCC circulation into the blood and seeding at the metastatic site of distant organs, where metastatic niche develops. Specifically, ROS might influence the development of MCCs within the primary tumors with intermediate features between epithelial and mesenchymal phenotype and gain of stemness and migratory skills favoring circulation into the vasculature. The journey of MCCs into the blood is closely followed by ECs, PMNs, and platelets, whose functions depends on ROS availability. Once reached the site of metastasis, either homotypic or heterotypic clusters might give rise to a metastatic niche with specific structural and metabolic characteristics, able to sustain metastasis survival and protect from immune system surveillance.Supplementary file1 (PDF 99 kb)Supplementary file2 (DOCX 89 kb)Below is the link to the electronic supplementary material.