Manganese (Mn) plays a multifaceted role in the survival of pathogenic and symbiotic bacteria in eukaryotic hosts, and it is also important for free-living bacteria to grow in stressful environments. Previous research has uncovered components of the bacterial Mn homeostasis systems that control intracellular Mn levels, many of which are important for virulence. Multiple studies have also identified proteins that use Mn once it is inside the cell, including Mn-specific enzymes and enzymes transiently loaded with Mn for protection during oxidative stress. Emerging evidence continues to reveal proteins involved in maintaining Mn homeostasis, as well as enzymes that can bind Mn. For some of these enzymes, Mn serves as an essential cofactor. For other enzymes, mismetallation with Mn can lead to inactivation or poor activity. Some enzymes may even potentially be regulated by differential metallation with Mn or zinc (Zn). This review focuses on new developments in regulatory mechanisms that affect Mn homeostasis and usage, additional players in Mn import that increase bacterial survival during pathogenesis, and the interplay between Mn and other metals during Mn-responsive physiological processes. Lastly, we highlight lessons learned from fundamental research that are now being applied to bacterial interactions within larger microbial communities or eukaryotic hosts. Additiooswitch)  and reguWhile the components of bacterial Mn homeostasis systems were being uncovered, mechanistic models of metal selectivity were developed to account for how proteins avoid mismetallation . In geneBoth Mn deficiency and toxicity are key problems that bacteria must avoid. How Mn deprivation occurs, particularly during pathogenesis, and its consequences for bacterial survival has been reviewed elsewhere . In contFundamental insights about bacterial physiology in pure culture have advanced understanding of the complex interplay in Mn usage and homeostasis in diverse ecosystems, and the field is ripe to examine the contribution of Mn-responsive systems during infection in the host. Here, we summarize recent advances in Mn stress responses and provide an outlook for the near future.Beyond established modes of regulation of Mn homeostasis , new finMn homeostasis in bacteria is maintained by the expression and activities of Mn import and export machinery. Transcriptional regulation of Mn transporters has long been recognized to contribute to virulence . Beyond Streptococcus pneumoniae, CvfD, contributes to virulence through several physiological pathways, including Mn and Zn homeostasis  protein levels. This shifts SOD activity away from Mn-dependence and towards reliance on the cambialistic Fe-using SOD, preserving bacterial resistance to ROS when Mn is scarce.The sRNA RsaC contributes to a Mn-sparing response in importer . Base-patrans-acting RNA regulation, well-established to regulate diverse stress responses  importer MgtE via activation of an intramembrane protease, YqgP , together with an additional outer membrane TonB-dependent Mn transporter (MnoT), has also recently been described  can also contribute to Mn transport. In scarcity . In Vibrlular Mn . Overexpescribed . In Burkransport . The smaresponse . It is uresponse . It willMn influences dynamic and context-dependent processes such as bacterial virulence and biofilm formation. To do so, Mn acts in competition with other metals found within the intracellular metal milieu which is comprised of labile (freely accessible) metal ions and protein-bound (poorly accessible) metal complexes. The size and speciation of the labile metal pool is imprecisely known, especially for Mn which exists predominately in the spectroscopically hard-to-detect +2 oxidation state in cells and binds weakly to biomolecules . NonetheStreptococcus pathogenesis are strongly affected by perturbations in cellular Mn:Zn equilibrium   . These bi (UPEC) .B. subtilis identified targets of Mn intoxication in a highly Mn-sensitive strain (yhdQ), a putative Mg efflux transporter homologous to MpfA in S. aureus and CorC in E. coli, and the Mg-sensing riboswitch preceding the primary Mg importer MgtE. Loss of mpfA increased intracellular Mg levels, which might counteract inhibition by excess Mn, similar to what has been observed in Saccharomyces cerevisiae ppGpp by the RelA-SpoT family protein SAH, which exhibits strong cofactor preference for Mn over Mg in vitro  homeostasis and vice versa . In UPEC with Cu . In UPEC with Cu . In the ase AcnA .Overall, high levels of a single metal can perturb the intracellular amounts of other metals , causingFundamental studies of bacteria in pure culture have defined the strategies microbes use to acquire and remove Mn to combat Mn fluctuations, as well as the intracellular usage of Mn in enzymes and ROS detoxification. It is now commonly accepted that Mn availability, whether limited or in excess, can affect bacterial fitness. However, deciphering the functions of Mn is challenging as there is significant interplay between Mn and other metals. Many studies do not fully mimic the natural habitat where pathogenic bacteria derive Mn in hosts, but are essential for building fundamental understanding of how metals impact bacterial infection. With this knowledge, defined questions can be asked in the more complex multi-organism infection system.S. aureus virulence in the heart by protecting bacteria from ROS, while not affecting other bacterial species in other niches (In mammalian hosts, inflammation leads to rapid readjustment of the metal milieu to disrupt metal homeostasis, exploiting both essentiality and toxicity of metals to control bacterial infection. Host dietary metal uptake further influences the metal landscape . At the r niches .In addition to Mn deficiency imposed by nutritional immunity, it is clear that bacteria are vulnerable to excess Mn, given the ubiquity and numerous types of Mn exporters across bacteria. Pathogenic bacteria lacking Mn exporters also have reduced colonization and virulence . HoweverIn summary, new modes of regulation of Mn homeostasis and intracellular management demonstrate that Mn is critical for bacterial growth and virulence. We anticipate additional post-transcriptional and post-translational regulatory mechanisms affecting Mn levels and usage to be uncovered. We have likely only begun to understand the scope of how metal balances affect physiological responses through enzymes regulated by differential metallation. We look forward to pathogenesis studies which examine how Mn factors are used by bacteria growing in different host niches to counteract host immune defenses. New technologies that can quantify labile metal pools, as well as total Mn levels, in real-time in response to changing conditions would significantly aid the endeavor to characterize the bacteria-host interaction.


Cutaneous chronic wounds are a major global health burden in continuous growth, because of population aging and the higher incidence of chronic diseases, such as diabetes. Different treatments have been proposed: biological, surgical, and physical. However, most of these treatments are palliative and none of them can be considered fully satisfactory. During a spontaneous wound healing, endogenous regeneration mechanisms and resident cell activity are triggered by the released platelet content. Activated stem and progenitor cells are key factors for ulcer healing, and they can be either recruited to the wound site from the tissue itself (resident cells) or from elsewhere. Transplant of skin substitutes, and of stem cells derived from tissues such as bone marrow or adipose tissue, together with platelet-rich plasma (PRP) treatments have been proposed as therapeutic options, and they represent the today most promising tools to promote ulcer healing in diabetes. Although stem cells can directly participate to skin repair, they primarily contribute to the tissue remodeling by releasing biomolecules and microvesicles able to stimulate the endogenous regeneration mechanisms. Stem cells and PRP can be obtained from patients as autologous preparations. However, in the diabetic condition, poor cell number, reduced cell activity or impaired PRP efficacy may limit their use. Administration of allogeneic preparations from healthy and/or younger donors is regarded with increasing interest to overcome such limitation. This review summarizes the results obtained when these innovative treatments were adopted in preclinical animal models of diabetes and in diabetic patients, with a focus on allogeneic preparations. Skin ulcers are open sores often accompanied by the sloughing-off of inflamed tissue. A slow-healing ulcer of the leg  is typically associated with complications of poor blood circulation, such as varicose veins, deep venous insufficiency, arterial and peripheral vascular diseases. Other causes of leg ulceration include trauma, bacterial and/or mycotic infections, and neuropathy related to diabetic disease. Pressure ulcers (bed sores) are a very common complication in elderly hypomobile patients. Cutaneous chronic wounds represent a major global health burden in continuous growth since this pathology is closely linked to the higher incidence in the aging population of chronic diseases, including diabetes. Women experience more pain and have a worse quality of life than men . There iThe prevalence of skin ulcers and the cost of treatments are very high. In industrialized countries, it has been estimated that 1%–2% of the population will experience a chronic wound during their lifetime . Lower lChronic ulcers are difficult to heal because of the diminished blood flow interfering with the healing process. Patient care is concerned with preventing a superimposed infection in the ulcer, increasing blood flow in the deeper veins, and decreasing pressure within the superficial veins. Different treatments have been proposed: biological, surgical, and physical. However, these treatments are mostly palliative and none of them can be considered fully satisfactory. More recently, treatments with allogeneic stem/progenitor cells or platelet-rich plasma (PRP) have been proposed as alternative therapeutic options. This review summarizes the results obtained when these innovative treatments were adopted in diabetic ulcer patients.via interactions with the extracellular matrix (ECM). Platelets entrapped in the provisional fibrin clot release cytokines, primarily the stromal-derived factor-1 , and growth factors that promote immune cell recruitment and resident cell activation. Hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF) are among growth factors released by platelets . Diabetic neuropathy (damage to the foot’s sensory nerves) results in foot deformities and/or ulcers that increase the chance of lower-extremity amputations when no treated. When concomitant peripheral arteriopathy occurs, the risk of DFU development strongly increases .A global prevalence of more than 6% was estimated for DFU with consequences on clinical and social costs . Some amStandard DFU management comprises the removal of necrotic tissue (debridement), interventions on the infection, application of dressings to protect the wound and maintain a moist environment necessary for promoting cell activity, offloading, and strict glycemic control . SurgicaSkin grafts are the most obvious therapeutic approach for replacing the lost tissue of skin ulcers. Cadaveric cryopreserved skin allografts can be used to stimulate healing, particularly for deep wounds with exposed bones and tendons . BesidesSince the pioneering works of Autologous keratinocytes and dermal fibroblasts were found to promote diabetic ulcer healing in few clinical studies. In a small group of patients with deep diabetic ulcers (Wagner degree 3), autologous fibroblast grafts induced complete re-epithelialization, with no recurrence for at least 2 years . A very Allogeneic skin cells from non-diabetic donors were also used to promote healing of chronic DFU without major safety problems being encountered . KeratinSeveral clinical studies proved the ability of keratinocytes from healthy donors to safely reactivate the healing response in DFU non-responding to conventional treatments. Epidermal substitutes were administered on a hyaluronic acid support , or loadAllogeneic dermal substitutes with normal fibroblasts plated in a spongy matrix of hyaluronic acid and atelo-collagen were used as dressings to promote granulation tissue formation prior to autologous skin grafting . The allBetter performances were achieved with allogeneic implants containing both keratinocytes and fibroblasts. Apligraf , approved by FDA for diabetic ulcer therapy, is a cryopreserved, bi-layered allograft, formed by foreskin-derived fibroblasts seeded within a bovine collagen I matrix overlaid by a stratified epithelium of neonatal keratinocytes . A pivotProgenitor cells of mesodermal origin, also named mesenchymal stem cells (MSCs), reside in many tissues, including dermis and skin annexes  Figure . It is sMSCs from different tissues share the expression of specific cell surface markers and lack those of hematopoietic and endothelial cells . They maA great number of studies in animal models of diabetes proved MSC efficacy in promoting skin repair . Stem ceA critical issue with MSC therapeutic potential is the poor cell survival and engraftment . To reinex vivo expansion represent limiting factors in their use. Adipose tissue-derived MSCs (AD-MSCs) manifest properties similar to BM-MSCs . BM-MSCs provided promising results in both preclinical and clinical studies . However BM-MSCs , but, un BM-MSCs . Additio BM-MSCs . However BM-MSCs . MSCs fr BM-MSCs . WJ-MSCs BM-MSCs . Other M BM-MSCs   and by increasing both anti-inflammatory cytokines , and prostaglandin E2 (PGE2)  . A recen2 (PGE2) . This coFollowing encouraging preclinical results, few preliminary clinical studies with allogeneic MSCs in chronic diabetic ulcers have been published . A studyA phase 1 dose-escalation study aimed at evaluating safety of placental MSCs (Cenplacel) in patients with DFU and PAD showed no adverse effects at all doses tested . After 3www.clinicaltrials.gov). Of those, eight test the effect of a product containing allogeneic AD-MSCs from healthy donors in a hydrogel sheet (ALLO-ASC-Sheet). A randomized, single-blind, phase 2 clinical trial testing efficacy and safety of this commercial product versus standard therapy has been published  in neuropathic DFU.Twelve studies using allogeneic MSCs for diabetic ulcer treatment are presently recorded in the National Institutes of Health (NIH) clinical trial registry (ublished . DespiteInduced pluripotent stem cells (iPSCs) are obtained from differentiated adult cells following ectopic co-expression of genes coding for transcription factors conferring a pluripotent stem cell phenotype . Under pInduced PSCs obtained from dermal fibroblasts and subsequently differentiated into smooth muscle cells showed an increased ability to release growth factors and to promote wound healing in diabetic mice when transplanted in a tridimensional scaffold . They imDermal fibroblasts from non-healing DFU were found to dedifferentiate to iPSCs as efficiently as fibroblast from non-lesional diabetic skin or from non-diabetic skin . KashpurSince iPSCs from dermal fibroblasts release a richer ECM compared to original fibroblasts, they have been used to develop tissue engineering scaffolds. These scaffolds enhance the activity of DFU fibroblasts that release higher amount of glucosaminoglycans, VEGF and anti-inflammatory cytokines, and reduce the secretion of proinflammatory cytokines .Based on these findings, iPSCs derived from dermal fibroblasts could be considered for a non-invasive cell therapy in DFU. However, strong regulatory constraints exist to their use, the methodology to obtain the cells is highly sophisticated, and, at present, iPSCs are produced only in very specialized laboratories.Skin substitutes and stem/progenitor cells mainly act not by engrafting , but ratwww.clinicaltrials.gov; NCT04134676). This study was aimed at testing the therapeutic potential of umbilical cord MSC on healing of chronic ulcers.In particular, MSC-secreted exosomes have been quite extensively tested at preclinical level as potential tools for cell-free therapy in cutaneous wound healing . These nAmong allogeneic approaches for diabetic foot ulcers, it has to be mentioned the amniotic membrane. This natural dressing is being used for decades to promote tissue regeneration and in skin repair as it releases factors promoting cell function, it is non-immunogenic, reduces pain, inflammation and fibrosis, among other properties . AmniotiActivated platelets release high amounts of growth factors and active molecules capable to trigger cell proliferation, matrix remodeling, angiogenesis and to modulate inflammation at the wound site . MoreoveIn vitro experiments from our group showed an initial transient enhancement of the inflammatory response , followed by the establishment of an anti-inflammatory microenvironment due to prostaglandin E2 (PGE2) production  can present a significant variability in platelet and other blood component concentrations Moreover, patients with specific health status  or individuals with age-related problems  are not retained ideal candidates for the treatment with autologous platelet derivatives . A standAn allogeneic PRP or platelet-derived product could reduce the risk of poor standardization. However, the possibility of an immune reaction should be considered. More recently, two articles investigated the effects exerted by an allogeneic non-homologous PRP. In these studies, performed in a mouse model of diabetes, the xenogeneic platelet-rich gel enhanced the healing process without adverse reaction . These oThe aforementioned meta-analysis on selected clinical trials evaluating PRP for DFU treatment  includedClinical evidence, including ours, confirms that the treatment with allogeneic platelet derivatives allows healing also of ulcers hard to treat and slow to heal, which would not benefit of a “standard traditional” treatment. If the efficacy of quality controlled allogeneic PRP is confirmed by additional studies, with this relatively low-cost treatment no longer would patients need to undergo more sophisticated invasive procedures. This would be particularly important for patients who are severely disabled . Prompt and effective long-term treatment of ulcers and chronic wounds will reduce disease complications  and mortality rate.Cutaneous chronic wounds, and in particular chronic ulcers in diabetes, represent a major clinical and societal problem in continuous growth because of population aging. Different treatments have been proposed: biological, surgical, and physical. However, most of these treatments are palliative and none of them can be considered fully satisfactory. Cell-based therapies, including stem cell conditioned medium and cell released microvesicles, together with platelet-rich plasma (PRP) treatments, are today innovative therapeutic approaches that share as common goal the reactivation of silent endogenous regeneration processes. In most cases, cells and PRP are obtained from patients as autologous preparations.Positive preclinical data suggested the adoption of cell therapy on diabetic patients to promote healing of chronic ulcers unresponsive to standard care. Since preclinical data were mainly obtained in rodent models, that only partly recapitulate features of human diabetic wounds, and because preclinical studies often suffer from non-rigorous setting and analysis, so far, the translation to the clinic was often disappointing. Well-structured, large, highly controlled clinical trials are therefore needed to test cell therapy efficacy in diabetic ulcers.Ex vivo expanded Mesenchymal Stem Cells from different body sites, either of the patient or of a donor individual, manifest high potentialities in promoting healing of diabetic wounds as compared to skin grafts. However, despite the positive results already obtained, due to rules imposed by the regulatory agencies , the need of highly sophisticated cell culture facilities, and the consequent high cost of the procedures, the MSC therapeutic approach cannot be easily applied to patients of largely diffuse pathologies, such as chronic diabetic ulcers. Moreover, for autologous MSCs, the logistics to obtain a biopsy from the patient, expanding the cells in the culture lab, and returning to the patient the final product as a preparation to be immediately used, makes the approach even more difficult to be adopted. It is also to note that in metabolic disorders, such as diabetes or in elderly individuals, cell number reduction and defective paracrine activity could hamper the effectiveness of autologous cell-based therapy.As for allogeneic therapy, a safety issue related to allo-recognition and rejection may arise as a problem. Preliminary evidence on MSC therapy indicates no major problems following allogeneic cell topical administration. However, a careful evaluation is needed before translation of allogeneic cells to the clinical practice. It is now widely recognized that the therapeutic activity go to head of the transplanted cells strongly relies on their secretome and on released microvesicles. Indeed, secretome and released microvesicles hold great promise as a therapeutic option in regenerative medicine. However, the same regulatory restrictions applying to pharmaceuticals apply to allogeneic stem cells, cell secretome or cell-released microvesicles.Great promises rely on the use of PRP that could be easily prepared with low invasive techniques and at a relatively low cost. Moreover, at variance with cell and cell-derived products, in most countries PRP is not classified as a pharmaceutical, and it is considered a blood transfusion product. A critical issue with PRP therapy is the lack of standardization in the preparation procedures. This problem is at the root of the discrepancies in results reported in literature. Most of reported PRP treatments have utilized a product obtained from the blood patients (autologous PRP). An allogeneic PRP obtained from a pool of healthy subjects could be a better alternative to the use of autologous PRP. Allogeneic PRP preparations can be quality controlled before their use and can be stored in a freeze-dried form as “off the shelf” product for a future use.It is expected that allogeneic PRP will have the same, if not better, performance, than autologous PRP. The few published reports seem to confirm this expectation. However, large randomized clinical trials comparing allogeneic PRP to other treatments are still missing. This is mandatory for a reliable, efficient application on a large scale of allogeneic PRP in the clinical practice.


Inhibitory control (IC) is a fundamental cognitive function showing age-related change across the healthy lifespan. Since different cognitive resources are needed in the two subcomponents of IC (cognitive inhibition and response inhibition), regions of the brain are differentially activated. In this study, we aimed to determine whether there is a distinct age-related activation pattern in these two subcomponents. A total of 278 fMRI articles were included in the current analysis. Multilevel kernel density analysis was used to provide data on brain activation under each subcomponent of IC. Contrast analyses were conducted to capture the distinct activated brain regions for the two subcomponents, whereas meta-regression analyses were performed to identify brain regions with distinct age-related activation patterns in the two subcomponents of IC. The results showed that the right inferior frontal gyrus and the bilateral insula were activated during the two IC subcomponents. Contrast analyses revealed stronger activation in the superior parietal lobule during cognitive inhibition, whereas stronger activation during response inhibition was observed primarily in the right inferior frontal gyrus, bilateral insula, and angular gyrus. Furthermore, regression analyses showed that activation of the left anterior cingulate cortex, left inferior frontal gyrus, bilateral insula, and left superior parietal lobule increased and decreased with age during cognitive inhibition and response inhibition, respectively. The results showed distinct activation patterns of aging for the two subcomponents of IC, which may be related to the differential cognitive resources recruited. These findings may help to enhance knowledge of age-related changes in the activation patterns of IC. Inhibitory control refers to the ability to suppress unwanted actions that are not appropriate for the current situation or to resist distractions and adapt to conflicting situations . By doinIn the recent years, there has been an increase in the number of studies examining the behavioral and neural developmental trajectory of inhibitory control across the lifespan refer to . HoweverIn neuroimaging research examining the neural developmental trajectory of inhibitory control, the results are also not consistent. Increased frontal activation in adults compared with children has been observed in some fMRI studies . Rubia ePrevious meta-analyses have also compared brain activation across different age groups to explore the age effect of brain activation during the process of inhibitory control . For exaThe lack of agreement in the existing studies on the behavioral and neural developmental trajectory of inhibitory control across the lifespan may be due to studies including samples with limited age ranges or the fact that few existing studies or theories have systematically differentiated age-related changes in different inhibitory control tasks. For example, Most current studies on the behavioral and neural developmental trajectory of inhibitory control across the lifespan include only a single type of inhibitory control task . For exaIn the recent times, an increasing number of studies have not only focused on explaining the diversity, scope, and range of inhibitory control functions, but have explored the intrinsic variability of different components of inhibitory control. These studies have suggested that inhibitory control is not a unitary construct, but is the one which can be further differentiated into different subcomponents . For exaTo differentiate the cognitive process during inhibitory control, inhibitory control can also be classified into two subcomponents—cognitive and response inhibitions—depending on the cognitive process of different inhibition targets in inhibitory control tasks . CognitiDifferences between inhibition difficulties and complexity of the two types of inhibition tasks might originate from the differences in cognitive resources recruited in these subcomponents of inhibitory control tasks . Stahl eCognitive inhibition can be captured by paradigms including the Stroop, Flanker, and Simon and stimulus response compatibility (SRC) . In the In line with the findings of behavioral studies regarding cognitive inhibition and response inhibition, studies characterizing the neural correlates of inhibitory control have also found a significant difference in neural correlates between the two components. For example, In this study, we investigated how neural activation during the two subcomponents of inhibitory control changes across the healthy lifespan with aging using meta-analytic technology. The study had two aims: that first was to characterize the common or distinct neural correlates in the two subcomponents of inhibitory control and the second was to identify the distinct age-related activation pattern in the two subcomponents of inhibitory control. Since the cognitive resources recruited in the two subcomponents are different, we hypothesized that the activation patterns of inhibitory control may show distinct age-related trajectories in the two subcomponents of inhibitory control, or differential activation patterns may be shown across different age groups in cognitive and response inhibitions.First, two online citation indexing services—PubMed and Web of Science—were searched. This search used the keywords “fMRI” with “interference resolution,” “action withholding,” “action cancellation,” “response inhibition,” “cognitive inhibition,” “inhibitory control,” “stop signal,” “stopping,” “go nogo,” “action restraint,” or “countermanding,” including articles published prior to April 2020, yielding a total of 9,419 articles. After removing duplicates, a total of 7,985 articles were screened. We then compiled 39 eligible articles identified in a previous meta-analysis . The folWe extracted the following information from each study: authors, year of publication, sample size, experimental design, paradigms, mean age with the age range, task contrasts, stimulation types, and cluster coordinates in the MNI or Talairach space.Cognitive inhibition is the inhibitory process of suppression of competing cognitive processing to solve relevant problems . For theResponse inhibition is the process of suppression of a prepotent response to perform a different, more context-appropriate response . To meas1 to identify the brain regions activated during inhibitory control. Peak effect coordinates from each study were convolved with a spherical kernel (r = 16 mm)   toolbox1= 16 mm)  to generp < 0.05, FWE-corrected across the entire brain). Specifically, to characterize brain activation patterns, first, we identified brain regions that showed significant convergence across 278 studies comprising 4,393 foci from 290 contrasts. Then, contrast analyses were conducted to verify the differences in cognitive demand for the two subcomponents of inhibitory control and capture the selectively or preferentially activated brain regions for two subcomponents of inhibitory control: cognitive inhibition vs. response inhibition. All cluster coordinates were analyzed in Montreal Neurological Institute (MNI) standard stereotaxic spaces.Previously used meta-analyses, such as the activation likelihood estimate (ALE), count how many peak coordinates are within each voxel divided by the brain regions and compare this to the number expected by chance if the peak coordinates were randomly distributed in the brain. This method is limited by the consequence that the peak coordinates in any single study may overly influence the results from analyses . Using Mp < 0.05 to control for family-wise error rate. We performed two meta-regression analyses in ES-SDM. Data involved in the meta-regression analyses were derived from response inhibition contrasts and cognitive inhibition contrasts separately. Given that different age ranges were reported in the original articles, the age computed in the meta-regression analysis as a continuous variable was determined by the mean age of each sample in the original articles. The results from the two regression analyses were then compared to identify brain regions with distinct age-related activation patterns in the two subcomponents of inhibitory control.To further assess age-related change in activation patterns in the subcomponents of inhibitory control, the effect-size seed-based d mapping (ES-SDM) toolbox (SdmPsiGui-v6.21 from the Seed-based d Mapping project) was used to perform meta-regression analyses because the ES-SDM software can provide accurate results of regression analyses incorporating meta-regression methods. This is achieved by first using peak coordinates and their statistical values to recreate statistical parametric maps and then conducting an image-based meta-analysis . The fulFurther, given that age was computed in the meta-regression analysis as a continuous variable determined by the mean age of each sample in the original articles, the mean age was affected by extreme values, which cannot well represent the age distribution of all subjects in each original study. As mentioned above, to more completely explore the age-related changes in the two subcomponents of inhibitory control within individuals of different ages, we performed additional MKDA analyses as validation analyses. We divided the datasets from all articles included in the current meta-analysis into four age groups: under 18 years for under-aged children, 18–35 years for young adults, 35–55 years for middle-aged adults, and 55–80 years of age for older adults. Then, we performed contrast analyses and computed the differences among all age groups in the two subcomponents of inhibitory control: under-aged children vs. young adults, young adults vs. middle-aged adults, middle-aged adults vs. older adults, under-aged children vs. middle-aged adults, young adults vs. older adults, and under-aged children vs. older adults.Additional validation analyses were performed to reduce the impact of the number of experiments, the different types of inhibition tasks, and the behavioral performance reported in the studies included in the results of the current meta-analysis.First, studies on response inhibition  included a much larger number of experiments compared to those on cognitive inhibition (54 experiments). To test the effect of the number of experiments, we randomly selected 54 contrasts from the response inhibition data and repeated the MKDA and meta-regression analysis with age as a variable using the same settings.Second, leave-one-out analysis was performed to test homogeneity in the cognitive inhibition tasks and response inhibition tasks separately. For the two subcomponents of inhibitory control, we removed each type of inhibition task one at a time: including Flanker, Simon, Stroop, WCST, and other tasks for cognitive inhibition, and tasks including antisaccade, Go/NoGo, and stop signal tasks for response inhibition. MKDA analyses were separately performed on the remaining studies with a total of five activation maps for cognitive inhibition and three activation maps for response inhibition. Then, we pooled the activation maps to obtain the overlapping rate maps for cognitive and response inhibitions, respectively. The overlap rate maps obtained from leave-one-out analysis and the results from MKDA on cognitive or response inhibition were then contrasted to test whether different tasks employed in studies influenced the activation patterns in the subcomponents of inhibitory control. Furthermore, since the meta-analysis results of cognitive inhibition and response inhibition identified different major regions , we conducted contrast analyses on the regions identified in the leave-one-out meta-analysis results for cognitive inhibition and response inhibitions separately.Third, to investigate whether older adults that performed a task similarly to younger adults may show different patterns of activity to older adults who underperformed relative to younger adults, we selected a total of 81 studies which reported task performance with successful inhibition in response inhibition tasks. Then, we performed a meta-regression analysis and several contrast analyses separately.The MKDA of the 278 studies showed significant activation of clusters in both hemispheres, including the frontal cortex, the angular gyrus, and the supplementary motor area . The resIn both hemispheres, activated areas during the cognitive inhibition tasks included the inferior frontal gyrus, precentral gyrus, anterior insula, inferior parietal lobule, supplementary motor cortex, superior parietal lobule, superior frontal gyrus, middle cingulate gyrus, inferior frontal gyrus, and angular gyrus . UnilateData from response inhibition experiments revealed activations in the right middle frontal gyrus, the right angular gyrus that extended to the middle temporal gyrus and superior temporal gyrus, the right inferior temporal gyrus, and the left middle cingulate gyrus. In addition, activation areas in both hemispheres were observed in the supplementary motor cortex, middle cingulate gyrus, superior frontal gyrus, precentral gyrus, inferior frontal gyrus, anterior insula, inferior parietal lobule, and supramarginal gyrus .Activation patterns common to the two subcomponents of inhibitory control were derived by conjunction analysis . RegionsContrast analyses of cognitive inhibition and response inhibition revealed that significantly different regions were activated in the two subcomponents of inhibitory control. Specifically, compared to response inhibition, a higher level of activation was found during cognitive inhibition in the left superior parietal lobule . On the In the cognitive inhibition tasks, results from the meta-regression analysis with age as a continuous variable across all studies showed a positive association with clusters in the followings: (1) the middle cingulate cortex, anterior cingulate cortex, and insula in both hemispheres; (2) the angular gyrus, superior parietal lobule, inferior frontal gyrus, and supplementary motor cortex in the left hemisphere. In addition, a negative association between age and clusters was found in the bilateral middle frontal gyrus, left inferior parietal lobule, right angular gyrus, and right inferior frontal gyrus .Activation in the response inhibition tasks showed significant positive correlations with age in the right angular gyrus, right middle frontal gyrus, bilateral inferior parietal lobule, and bilateral middle cingulate cortex, whereas a negative correlation with age was found in the followings: (1) the anterior cingulate cortex, inferior frontal gyrus, insula, hippocampus, and superior parietal lobule in the left hemisphere and (2) the superior frontal gyrus, cerebellum, insula, and inferior frontal gyrus in the right hemisphere .To characterize distinct brain regions with age-related changes in activation patterns in the two subcomponents of inhibitory control, we overlapped the results from regression analyses in the two subcomponents with age and found different age-related activation patterns in the subcomponents . ActivatContrast analyses among different age groups for cognitive inhibition showed that regions with significantly higher levels of activation in adults  than under-aged children were located in the bilateral inferior frontal gyrus, right insula, and left supplementary motor area . CompareContrast analyses among different age groups for response inhibition showed that regions with significantly higher levels of activation in adults than under-aged children were located in the bilateral angular gyrus, insula, and right middle frontal gyrus . CompareThe evaluation of the number of experiments contrasting the two subcomponents showed no significant differences between the real contrasts and the randomly selected 54 experiments for response inhibition. The activated brain areas are reported in The results from leave-one-out analysis  were highly consistent with the results of brain activation patterns in each component of inhibitory control derived from MKDA analyses. Further details are reported in Meta-regression and contrast analyses on successful inhibition showed a similar activation pattern of brain regions including the inferior frontal gyrus, supplementary motor area, inferior parietal lobule, insula, angular gyrus, middle cingulate cortex, and occipital gyrus as compared to current MKDA analyses on response inhibition. The activated brain areas and activation maps are reported in Applying MKDA and ES-SDM allowed the current meta-analysis to characterize the neural correlates and age-related effects in different subcomponents of inhibitory control. We observed brain areas including the inferior frontal gyrus, insula, middle cingulate cortex, and inferior parietal gyrus being activated in the two subcomponents. Contrast analyses conducted to elucidate the distinct neural substrates for each subcomponent revealed that relative to response inhibition, cognitive inhibition produced stronger activation in the left superior parietal lobule, whereas response inhibition primarily recruited the right inferior frontal gyrus, insula, middle temporal gyrus, and angular gyrus. Importantly, by performing a meta-regression analysis with age as a continuous variable, we found distinct age-related activation patterns in different subcomponents of inhibitory control in brain regions including the left anterior cingulate cortex, left inferior frontal gyrus, left superior parietal lobule, and bilateral insula. Overall, our results indicated common and distinct neural correlates and distinct age-related activation patterns in the two subcomponents of inhibitory control.The MKDA results showed that brain regions including the inferior frontal gyrus, insula, middle cingulate cortex, and superior parietal lobule were activated by both inhibition subcomponents. Therefore, this suggests that the inferior frontal gyrus, insula, middle cingulate cortex, and inferior parietal lobule played the core roles in inhibitory control , which iThe main clusters of activation in the two subcomponents of inhibitory control were observed in the followings: (1) the inferior frontal gyrus extending to the insula and (2) the middle cingulate cortex and the superior parietal lobule. The inferior frontal gyrus is known to engage in the process of inhibiting automatic but irrelevant actions while activating task-relevant responses at the same time . MoreoveThe anterior insula has been considered as the center that controls brain activity across different tasks and stimulus modalities and regulates inhibitory control mechanisms W. . Wager eAdditionally, our research found that the right superior occipital gyrus and left middle occipital gyrus were commonly activated in the two subcomponents of inhibitory control, which is in line with the results from previous studies and meta-analyses . InteresIn this meta-analysis, the inhibitory control paradigms classified as cognitive inhibition required conflict resolution and inhibition of response tendencies for successful responding . When peIn contrast, for response inhibition, the Go/NoGo and stop signal tasks encompass future action selection and inhibition of a predominant response tendency or an ongoing response, respectively. As mentioned above, the inferior frontal gyrus plays an inhibitory role in resolving conflicts during response execution, and the anterior insula is involved in the regulating process of response inhibition. Thus, there were more activated regions in response inhibition than cognitive inhibition, primarily located in the inferior frontal gyrus and anterior insula. The distinctiveness of response and cognitive inhibition, we suggest, may partly be due to the different cognitive demands in these inhibitory control tasks. Participants are required to resolve conflicts and to involve more sensory or stimulus-related neural activity in cognitive inhibition tasks, whereas the demand for inhibitory control may further increase in response inhibition tasks, which require inhibiting a predominant tendency or stopping of already-initiated actions. Furthermore, these tasks differ in terms of task-related complexity. Suppressing a response tendency or canceling an ongoing action might increase the inhibitory demand as compared to suppressing interference due to irrelevant information or resolving conflicts, as is the case in cognitive inhibition tasks . As the Interestingly, results from contrast analyses of cognitive inhibition and response inhibition showed a predominantly lateralized differential activation between response inhibition and cognitive inhibition. We hypothesized that the underlying reason may be the different types of stimuli  materials used in inhibitory control tasks. By compiling a list of all types of stimulation used in the included literature refer to , we founIn this meta-analysis, we observed neither a completely coherent increase nor a decrease in the inhibitory network with increasing age in the two subcomponents. In the cognitive inhibition tasks, activation showed a positive association with age in the anterior cingulate cortex, insula, superior parietal lobule, and inferior frontal gyrus. The results from contrast analyses among different age groups also showed that compared to under-aged children, activation in the inferior frontal gyrus and insula was stronger in adult groups. These age-related changes fit with the existing literature showing that prefrontal regions, including the inferior frontal gyrus and the middle frontal gyrus, become more active with aging . A previA different pattern of functional age-related changes was found in the response inhibition tasks. We found the activation of the response inhibitory network, including the left anterior cingulate cortex, bilateral inferior frontal gyrus, insula, left superior parietal lobule, and right superior frontal gyrus, was negatively correlated with age. Contrast analyses also showed a linear decline with increasing age in the activation of brain regions including the bilateral inferior frontal gyrus, insula, middle cingulate cortex, and inferior parietal lobule when performing response inhibition tasks. These seemingly differential results might also be explained by the differences in the inhibition processes involved.In line with a previous study , the curSeveral neuroimaging studies have contributed greatly to enhancing our knowledge of the neural correlates of subcomponents of inhibitory control or age-related change in the activation in the two subcomponents . HoweverThrough synthesizing data from different subcomponents, we found that brain regions including the inferior frontal gyrus and anterior insula, as well as regions including the middle cingulate cortex and supplementary motor cortex, were consistently activated across all inhibition tasks. This finding may suggest that these brain areas are core inhibitory control regions. Meanwhile, different age-related changes in the activation between the subcomponents of inhibitory control could be observed. Functional reorganization of the aging brain in different inhibitory control tasks showed a complex pattern of increase and decline: the corresponding cognitive inhibition tasks required older adults to increasingly recruit the core inhibitory network and additional inhibitory regions, such as the frontal regions and bilateral insula. However, a contrary pattern of age-related decline in the inhibitory network including prefrontal areas and middle cingulate cortex was shown during the process of response inhibition. The current results suggest that these differences might result from the increasing demands on inhibitory function from cognitive inhibition to response inhibition. Furthermore, age-related increased activation of additional inhibitory networks was limited. When the task demand exceeded the older adults’ capacity, activation in the inhibitory network decreased clearly. These findings are of significance for the understanding of the neuro-developmental mechanisms of inhibitory control and may provide insights into inhibitory control deficits in clinical settings.As mentioned above, common and distinct activation patterns are involved in the processes of proactive and reactive inhibition, and age-related changes in activation patterns in proactive and reactive inhibition have been investigated. Experimental evidence from neuroimaging studies supports a right-lateralized frontoparietal circuit being widely recruited through the reactive inhibition process . In addiWe acknowledge that the current study still has some limitations. First, we note that a potential limitation in meta-analysis methods in general is that any meta-analysis method is prone to publication bias; in this study, we only considered the results available in the published literature and original studies which reported coordinates. Moreover, we could not control the statistical methods used in original articles for thresholding the data. There is an emerging trend to store unthresholded statistical maps, which will allow image-based meta-analyses to be performed in the future studies .The second limitation relates to the bias of employing different tasks for measuring the components of inhibitory control and including studies using a variety of stimulation types. Specifically, our meta-analysis adopted a mixture of inhibitory control tasks as different subcomponents of inhibitory control, such as the Stroop, Simon, and Flanker tasks used to capture cognitive inhibition and the GNG, stop signal task, and antisaccade tasks to capture response inhibition, which consequently increased the heterogeneity of the study designs. Leave-one-out analysis was performed as validation analysis to test homogeneity in cognitive inhibition and response inhibition separately. The results from leave-one-out analysis were consistent with the results of brain activation patterns in each component of inhibitory control from the MKDA analyses and the details are reported in Third, considering that task difficulty and behavioral performance in different inhibition tasks may influence age-related brain activity in different individuals, and not all studies included in the current meta-analysis reported behavioral performance for each participant or task difficulty, we selected a total of 81 studies which reported task performance with successful inhibition in response inhibition tasks. Then, we performed a meta-regression analysis and several contrast analyses separately. The results refer , 5 were Fourth, the findings from this study are of significance for the understanding of the neuro-developmental mechanisms of inhibitory control and may provide insights into cognitive health in older adults. However, cognitive health is affected not only by physiological factors such as age, but also by socio-demographic factors; particularly significant is educational attainment. Understanding the links between education and cognitive health or cognitive aging could improve the cognitive prognoses and offer clues about the mechanisms underlying cognitive decline that can be targeted by interventions. Previous studies found that a better level of education might affect cognitive ability in older adults and attenuate aging-related decline in cognition functions . This maIn this meta-analysis, we examined the neural correlates of subcomponents of inhibitory control and the difference in age-related changes in activation between the subcomponents. Activation of the middle cingulate cortex, supplementary motor area, inferior frontal gyrus, inferior parietal lobule, and anterior insula was common across the different inhibition processes, which revealed that these regions are the core neural system engaged in inhibitory control. On the other hand, differences in the activation patterns of subcomponents of inhibitory control with aging showed a complex pattern in the functional reorganization of the aging brain. Specifically, when performing cognitive inhibition tasks, stronger activation of the core inhibition regions was observed in older adults, whereas activation in prefrontal areas in older adults declined during response inhibition tasks. We summarize that these differences may be driven by the different demands between inhibitory control tasks. Aging individuals recruit additional inhibition-related brain regions when performing an inhibitory control task. However, with an increasing demand of inhibition tasks, limited reallocation of cognitive resources in older adults eventually results in a lower level of the activation of inhibitory brain regions in older adults during inhibitory control processes. These results may further enhance our knowledge of age-related changes in the activation patterns of inhibitory control and may provide insights into inhibitory control deficits in clinical settings.The original contributions presented in the study are included in the article/The study was reviewed and approved by the Institutional Review Board (IRB) of Southern Medical University.JL: conceptualization, data curation, data analysis, writing the manuscript, reviewing, and editing. XS, YW, CW, and RH: writing—reviewing and editing. RZ: conceptualization, supervision, funding acquisition, and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.


The pre-metastatic niche is a recently established concept that could lead to targeted therapies that prevent metastasis before ever occurring. Considering that 90% of cancer mortality results from metastasis, the PMN is thus a salient opportunity for intervention. The purpose of the current review is to cover what is known specifically about the hepatic pre-metastatic niche, a topic that has garnered increasing research focus within the last decade. We discuss the methods of communication between primary tumors and the liver, the involved cell populations, the key changes within liver tissue, and perspectives on the future of the field.Primary tumors can communicate with the liver to establish a microenvironment that favors metastatic colonization prior to dissemination, forming what is termed the “pre-metastatic niche” (PMN). Through diverse signaling mechanisms, distant malignancies can both influence hepatic cells directly as well as recruit immune cells into the PMN. The result is a set of changes within the hepatic tissue that increase susceptibility of tumor cell invasion and outgrowth upon dissemination. Thus, the PMN offers a novel step in the traditional metastatic cascade that could offer opportunities for clinical intervention. The involved signaling molecules also offer promise as biomarkers. Ultimately, while the existence of the hepatic PMN is well-established, continued research effort and use of innovative models are required to reach a functional knowledge of PMN mechanisms that can be further targeted. The liver is a major hub of metastasis for primary tumors from around the body. Gastrointestinal malignancies such as colon, pancreatic, and gastric carcinomas have a high tendency for hepatic spread and for breast carcinomas, sarcomas, and melanoma as well . ConsideRecent evidence has begun to shed light on a new aspect of cancer biology termed the “pre-metastatic niche” (PMN). The concept was first posed by Stephen Paget’s 1889 “seed and soil” theory, which speculated that circulating tumor cells of distinct cancer origin (the seeds) had a higher predilection to spread to organs with compatible characteristics (the soil) . Work wiAlthough research on the PMN has primarily focused on the lung since this time, a significant body of work now exists regarding the pre-metastatic liver as well. While clinical recognition of the PMN is not yet feasible, it is likely that gastrointestinal malignancies that predominate liver metastases are the same cancers which favor hepatic PMN formation. In line with this notion, the bulk of primary literature that has begun to elucidate the hepatic PMN has used colorectal carcinoma and pancreatic ductal adenocarcinoma models. The current dynamic can be thought of as a game among three players: the primary tumor of interest, the liver, and the systemic immune population of bone-marrow-derived cells (BMDCs). Signaling initiated by the primary tumor causes varying interactions among these three entities that ultimately result in principle changes that define the PMN: ECM remodeling, inflammation, immune suppression, and increased vascular permeability. In this review, we will discuss the signaling mechanisms, cellular components, and the characteristic changes involved in the hepatic PMN. Thereafter, we will present perspectives on the PMN targets and biomarkers that may one day offer clinical utility as well as emerging models that might lead to further discovery on the mechanisms underlying the PMN. A schematic of the salient components to the hepatic PMN can be seen in To influence the distant hepatic PMN, primary tumors must be able to communicate remotely through the blood stream. This is accomplished through the production and secretion of molecular components that either influence the PMN directly or instigate the recruitment of BMDCs to the niche, where they then have further effects. Thus, primary tumor signaling serves as the first step in PMN formation. The following sections will describe the known signaling mechanisms utilized and their downstream targets, and the molecular components have been summarized in One secreted molecule that has been studied extensively is the tissue inhibitor of metalloproteinases (TIMP)-1. Seubert et al. demonstrated that TIMP-1 released by lymphoma cells could recruit CXCR4+ neutrophils to the liver by upregulating SDF-1 in the PMN . SubsequBeyond TIMP-1, other molecular factors have been implicated in the development of the PMN. In a colorectal carcinoma (CRC) model, Wang et al. demonstrated that tumor-derived VEGFA stimulated tumor-associated macrophages (TAMs) in the surrounding stroma to produce CXCL1. In the blood stream, CXCL1 then recruited CXCR2+ myeloid-derived suppressor cells (MDSCs) from the bone marrow to the hepatic PMN, where they facilitated formation of liver metastases (mechanisms of MDSC involvement to be reviewed in a later section) . Further− immature myeloid cells to the pre-metastatic liver through an interaction with CCR1 [Primary tumor cells can also independently secrete chemokines to further develop the PMN. Human CRC cell lines injected into mouse spleens were shown to secrete CCL15, which then recruited CD34+ Gr-1ith CCR1 . Antagonith CCR1 . CCL2 isith CCR1 . HoweverExosomes are extra-cellular vesicles (EVs) that contain varying components of proteins, lipids, RNA, and/or DNA that can be horizontally transferred to other cells. These EVs are surrounded by a lipid bilayer that gives them protection and enables their travel throughout the circulatory system. Their diameter ranges from 30–150 nm, and they can be actively shed by cells to communicate with others both locally and distally. Depending on the integrins expressed on their surface, they can also deliver their cargo in a cell-specific manner . Due to their unique composition and characteristics, exomes engage in cell communication throughout a broad range of physiologic functions and pathologies. Of particular interest is their involvement within the metastatic cascade. Ranging from promoting an inflammatory vascular niche in brain metastases to facilitating breast cancer dormancy, they have been implicated in both the development of the primary tumors along with progression of established metastases. Their function within these contexts has been reviewed extensively ; in thisBy secreting exosomes into the bloodstream for hepatic uptake, primary tumors can manipulate the liver from afar before ever seeding it. Non-small cell lung cancer can secrete exosomes containing regulatory microRNA-122-5p that increases the expression of mesenchymal markers N-cadherin and Vimentin in native hepatocytes upon uptake while decreasing the epithelial marker E-cadherin. The ensuing migratory phenotype with less cellular adhesion proteins may ultimately make the liver more amenable to tumor cell invasion . CRC exoExosomes can also be utilized to modulate stromal cells directly. In a study by Li et al., gastric tumor cells could upregulate TGF-β1 transcription in Kupffer cells via cell-specific exosome signaling. Consequently, TGF-β1 expression in the hepatic niche activated the SMAD2/3 pathway in invading tumor cells, which increased their stem cell-like properties and led to a higher metastatic burden . In addiWhile their further roles in the characteristic changes of the PMN will be reviewed in a later section, their involvement can already be appreciated. From directly altering hepatocytes to engaging distant immune cells, exosomes offer several opportunities for therapeutic targeting. Special attention in future study should be paid to this form of pre-metastatic communication.Bone-marrow-derived cells (BMDCs) are integral to the development of the hepatic PMN. They are mobilized to the liver via primary tumor stimulation, where they coordinate a metastasis-friendly microenvironment through a variety of mechanisms. Each subpopulation has corresponding capabilities and has been implicated in unique ways, but collectively, BMDCs comprise a third cellular component outside of the primary tumor or liver that is vital to PMN priming. Myeloid-derived suppressor cells (MDSCs) are one of the most influential populations of cells to the development of the PMN and have been implicated in the metastatic progression of many primary cancer types. They comprise a heterogeneous group of cells derived from myeloid progenitors that have powerful immunosuppressive properties along with roles in inflammation, angiogenesis, and ECM remodeling. The two primary sub-populations are split between monocytic MDSCs and granulocytic MDSCs. While they are part of the normal-functioning immune system, pathologic activation via primary cancers can recruit these cells to distant microenvironments and co-opt their function for cancer progression .Previous research in contexts outside of the PMN have uncovered a number of different mechanisms by which MDSCs can stifle the natural immune response. Through production of nitric oxide (NO) and reactive oxygen species (ROS), they can suppress cytotoxic T-cell function by increasing antigen-specific tolerance and disrupting the T-cell receptor . They alBeyond their direct roles, MDSCs also influence other components of the immune system. They are able to induce NK cell anergy, thereby reducing clearance of migrating cancer cells. Additionally, they can augment the function of T-regulatory cells, which are known immunosuppressors ,48. AnotIn terms of spatiotemporal localization, MDSCs could already be found in the blood of mice with pre-malignant pancreatic neoplasms, with their concentration only increasing with further oncogenesis . This suUltimately, MDSCs are critical players in the development of the hepatic PMN. While there has been much study on the function of these cells and their roles in liver metastasis, still there is a dearth of evidence that specifically investigates their mechanisms of action during the elusive pre-metastatic timeframe. Not only does this present an opportunity for further study, but the ensuing knowledge might offer therapeutic targets early in the metastatic cascade, which could halt the development of clinical liver masses before detectable colonization.Another population of BMDCs mobilized to the liver are macrophages. While resident macrophages known as Kupffer cells exist in healthy liver stroma, during metastasis, a similar yet distinct population of leukocytes of monocytic origin travel into the hepatic niche from the circulatory system. Like MDSCs, they can generally promote metastasis via several different mechanisms including angiogenesis, immunosuppression, and ECM remodeling although whether they behave similarly in the PMN is unknown. What is known, however, is that they do accumulate in the liver prior to metastasis and promote subsequent colonization . This reOne point of interest about tumor-associated macrophages (TAMs) is their inherent plasticity. They can differentiate into subpopulations with vastly different consequences on inflammation and immune regulation. Depending on their incoming cytokine signaling, they can either become an M1-type with anti-microbial, anti-tumor cytotoxic properties, or they can become an M2-type that promotes immunosuppression and tissue remodeling, which favor tumorigenesis . While tTakano et al. demonstrated this plasticity within the context of hepatic metastases using a CRC model. Tumor-derived EVs containing microRNA-203 caused monocyte polarization into the M2 phenotype. In vivo, this led to increased liver metastasis although the question remains whether this similarly happens in the PMN . ClinicaNeutrophils are another key element of the immune system under investigation for their involvement in liver metastasis. Traditionally, neutrophils are seen as first responders of the innate immune system that clear pathogens and inflammatory debris through induction of phagocytosis, production of ROS, and release of lytic enzymes . HoweverAs mentioned previously, CXCR4+ neutrophils were shown to accumulate in hepatic PMN following primary tumor TIMP-1 secretion . AlternaAs was the case with tumor-associated macrophages, the further mechanisms by which neutrophils facilitate the PMN within the liver need to be elucidated. One unique avenue to explore relates to their capacity to release neutrophil extracellular traps (NETs), which are extracellular DNA webs that have previously been shown to trap circulating lung carcinoma cells within hepatic sinusoids and increase micro-metastases . BecauseIn addition to the populations mentioned above, NK  cells are another immune cell type with probable involvement in the PMN. They are highly abundant in the liver and play salient roles in mitigating cancer progression in several malignancies including hepatocellular carcinoma . This isIn addition to recruited BMDCs, native liver cell populations are the downstream effectors of primary tumor signaling. This is primarily mediated by stromal cells , but parenchymal hepatocytes have been shown to be involved as well. Moreover, the extracellular matrix (ECM) both serves as the physical bedrock for cellular activity and is also directly involved in pre-metastatic changes.Hepatic stellate cells (HSCs) are liver-specific pericytes located in the space of Disse that differentiate into highly proliferative and mobile myofibroblasts with diverse pro-inflammatory effects. While they are physiologically activated by liver injury and inflammatory signaling, tumor cells can also induce this change to promote metastatic colonization of the liver . Upon paThe extent of their involvement in metastatic progression has motivated increasing attention to their influence at the pre-metastatic phase. Nielson et al. showed that murine PDAC tumors could recruit macrophages to the liver, which then influenced HSCs to produce a fibrotic microenvironment through periostin secretion . PeriostHSCs also promote the PMN by recruiting MDSCs to the liver. Under the influence of EVs derived from BMDCs in lung-cancer-bearing mice, they can remodel the ECM to be more susceptible to incoming MDSC migration . In lineRecently, Zhao et al. demonstrated that HSCs could communicate back with the primary tumor following activation by miR-181a-5p released from CRC cells. Following EV uptake, HSCs facilitated CRC cell migration, invasion, and metastasis formation by increasing expression of α-SMA and fibronectin in the liver ECM while reducing vitronectin and tenascin C. Moreover, they also secreted CCL20 into circulation, which upregulated further miR-181a-5p production by primary tumor cells in a positive feedback loop . This crS100 gene expression has also been shown to correlate with clinical development of metastasis in patients [In contrast to the recruited macrophages described previously, Kupffer cells are the liver’s endogenous macrophage population that reside within the lumen of the sinusoids and act as the first line of defense against incoming insults. During metastasis, they serve a myriad of functions from circulating tumor cell elimination to promoting cell adhesion, invasion, and angiogenesis . Ultimatpatients . In a sepatients .Ultimately, this incongruence underscores the complexity of Kupffer cell involvement in the PMN. It is likely that signaling from the primary tumor dictates the function they serve, but further study is necessary to elucidate this notion. Much like recruited macrophages, determining how to elicit the anti-metastatic phenotype of Kupffer cells could offer an intervention that promotes tumor cell clearance.Liver sinusoidal endothelial cells (LSECs) are a specialized population of discontinuous, fenestrated endothelial cells that form a barrier between the hepatic circulation and the underlying space of Disse and parenchyma. They are highly permeable, create low shear stress, and have a minimalistic basement membrane. Their unique nature allows them to play important roles in liver physiology, immunology, and pathology . During In one study investigating LSEC pre-metastatic involvement, intestinal tumors led to significantly increased expression of apical fibronectin prior to tumor cell dissemination . AdditioMoreover, VEGFA (known promoter of angiogenesis) was found to be upregulated in the pre-metastatic liver tissue of melanoma-bearing mice . This coThe extracellular matrix (ECM) is a collection of proteins collectively referred to as the “matrisome” that serve several physiologic functions. The core matrisome comprises fibrillar proteins such as collagen, glycoproteins such as laminin, and the proteoglycans heparin sulfate and versican. Matrisome-associated proteins, on the other hand, are composed of proteins that process, remodel, and regulate the more traditional ECM components . CollectSignificant evidence has implicated pre-metastatic tissue fibrosis as a promoter of metastatic colonization . Within Interestingly, the ECM changes induced by any given cancer line is specific to the organ of metastasis. Quantitative mass spectrometry analysis of metastases from the same breast cancer cell line to murine liver, lungs, brain, and bone marrow demonstrated distinct ECM changes depending on the context. In the hepatic niche, certain proteoglycans were uniquely upregulated compared to healthy tissue or other sites of metastasis . While tHepatocytes are the parenchymal cells of the liver and comprise the bulk of its mass. Despite their wide array of physiologic roles, their involvement in metastasis is relatively less understood compared to their stromal neighbors. In a murine model of breast cancer metastasis, they directly interacted with invading tumor cells via claudin-2 functioning as an adhesion molecule, and this was necessary for successful colonization . AdditioThe former sections reviewed signaling molecules and cell populations involved in formation of the hepatic PMN, but the crux is ultimately the specific changes within the liver that facilitate ensuing metastasis. The following discussion will review what is known about the liver PMN with regard to four principle PMN changes: ECM remodeling, inflammation, immunosuppression, and angiogenesis and increased vascular permeability. Many of the paramount studies from the last decade that have begun to elucidate the mechanisms behind pre-metastatic priming have directly investigated ECM remodeling. A general theme has been the induction of liver fibrosis leading to immune cell recruitment, with the primary mediators being exosomes, as discussed in previous sections. In addition, they have been shown to cargo CCL2 and recruit macrophages to the liver, stimulating their differentiation into the immunosuppressive M2 phenotype and again increasing the downstream metastatic load . AnotherCollectively, these studies demonstrate a multitude of mechanisms by which primary tumors induce fibrosis within the pre-metastatic liver and link this change to immune cell recruitment and metastatic development. What remains to be explained is precisely how pre-metastatic fibrosis causes these downstream effects. The capabilities of specific immune populations were earlier reviewed, and their roles in primary tumor development and metastatic colonization have been studied, yet the methods by which they prime the pre-metastatic liver prior to dissemination are still unknown. Nonetheless, fibrosis is a known promoter of metastasis, and this is not exclusively contingent on primary tumor signaling. Patients with fibrotic livers according to the NAFLD fibrosis score (NFS) were shown to have increased hepatic recurrence of CRC following primary tumor resection . MoreoveIt has long been known that inflammation is necessary for oncogenesis. Over a century ago, it was shown that chronic inflammation in mouse models could induce spontaneous tumors, and we now accredit this is to the milieu of cytokines, chemokines, and growth factors that support cancer development . The mecMultiple studies investigating the PMN have implicated interleukin 6 (IL-6), a pro-inflammatory cytokine known to have pathologic effects in the dysregulated processes of chronic inflammation and autoimmunity . Shao etSaa 1/2/3/4, Tnfa, Ccl2, and Mmp15) and mobilized distant macrophages and neutrophils to help establish a pre-metastatic inflammatory niche [More unorthodox methods have also been shown to be involved. In a mouse model of CRC, increased primary tumor disruption of the gut vascular barrier (GVB) allowed for bacterial translocation into circulation and eventually the liver. There, they increased transcription of several proinflammatory genes  have also been under investigation for PMN targeting. Park et al. showed that nanoparticles coated with NET-digesting DNase I significantly reduced lung metastasis in mouse models of breast cancer . MoreoveA clinical trial currently under way is investigating the safety profile of dual administration of nivolumab (anti-PD-1 inhibitor) with galunisertib (TGFβ receptor I inhibitor) for advanced refractory tumors (NCT02423343). As previously discussed, TGFβ promotes inflammation, ECM remodeling, and immunosuppression within the hepatic PMN ,29,55. IDespite the growing list of molecular players involved in the hepatic pre-metastatic niche, the field is young and has yet to produce a clinical biomarker amenable to assay for prognostic or predictive information. In theory, the secreted factors or circulating immune populations involved in PMN development could indicate looming metastatic progression, yet still even identifying the PMN with confidence in murine in vivo models is difficult given its spatiotemporal specificity. Exosomes containing unique microRNA signatures have shown promise as prognostic biomarkers for both gastric cancer  and CRC Interestingly, an emerging field called “radiomics” has also offered new avenues of prognostic PMN biomarkers. This entails the use of medical imaging to mine quantitative data that provide clinical insight. In a recent study by Creasy et al., pre-operative CT imaging of stage II/III CRC patients before initial colon resection demonstrated significant differences in liver image qualities between patients who remained disease-free at 5 years and those who developed liver metastases . Of noteThe hepatic PMN is a particularly difficult phenomenon to study due to its temporospatial specificity. It marks a period of tissue change prior to the arrival of disseminated tumor cells, but demonstrating this experimentally through in vivo models is difficult. Present studies typically start by identifying primary tumor signaling molecules of interest, conditioning mice with these molecules, investigating pre-metastatic liver changes, and then challenging with tumor cell injection to determine increased susceptibility to metastasis . While sThree-dimensional tissue models present an area of promise. Carpenter et al. implanted hydrogel scaffolds seeded with human stromal cells into prostate cancer mouse models to serve as proxy PMNs. By imaging microenvironments within the scaffolds, they were able to monitor interactions among disseminated tumor cells, leukocytes, stromal cells, and endothelial cells . MoreoveAdditionally, scaffolds can be prepared from native patient samples through decellularization, which removes the cellular components from tissue and leaves behind the ECM. In a study by Persson et al., ECM scaffolds made from patient breast cancer samples were recellularized with cell lines to assess how established tumor-microenvironments affect cancer progression . When coSpheroid models of CRC have also been used to study the in vitro upregulation of certain signaling pathways during tumor proliferation . This miAlthough Paget originally proposed his “seed and soil” theory in 1889, research supporting the pre-metastatic niche has only truly materialized within the last two decades. In large part, this body of work has focused on the lung PMN, but the hepatic PMN has garnered increasing interest as of late. We now know that dynamic interactions between primary tumor signaling, recruited BMDCs, and native liver characteristics lead to a set of defined changes that foster metastatic growth. Inhibiting the metastatic cascade at this point, prior to the onset of colonization, presents an attractive yet elusive opportunity. Ultimately, this would require both a PMN-specific target and a well-tolerated therapeutic that could be administered long-term in early-stage patients prior to the onset of metastasis. Nonetheless, the need for such treatment is large. Metastasis is the true killer of cancer, and we currently have few strategies to stop its development rather than treat its existence. Hopefully the emergence of the PMN will offer an opportunity to change this paradigm and alleviate the burden of patients who often need it the most.


Induced pluripotent stem cells (iPSCs) are crucial for disease modeling and cell-based therapy because they serve as an infinite source of specific human cell types. The use of iPSCs in cancer immunotherapy and cell transplantation therapy has garnered much attention in personalized medicine. To improve the efficacy and specificity of brain cancer treatment, iPSCs can be used to derive human brain tumor models for therapeutic evaluation. This review summarizes the utilization of human iPSCs to generate brain-specific cells, organoids, and tumor models for brain cancer modeling and drug testing. In addition, current challenges, limitations, and future prospects to find a more efficacious approach for treating human brain cancers are discussed.Brain cancer is a group of diverse and rapidly growing malignancies that originate in the central nervous system (CNS) and have a poor prognosis. The complexity of brain structure and function makes brain cancer modeling extremely difficult, limiting pathological studies and therapeutic developments. Advancements in human pluripotent stem cell technology have opened a window of opportunity for brain cancer modeling, providing a wealth of customizable methods to simulate the disease in vitro. This is achieved with the advent of genome editing and genetic engineering technologies that can simulate germline and somatic mutations found in human brain tumors. This review investigates induced pluripotent stem cell (iPSC)-based approaches to model human brain cancer. The applications of iPSCs as renewable sources of individual brain cell types, brain organoids, blood–brain barrier (BBB), and brain tumor models are discussed. The brain tumor models reviewed are glioblastoma and medulloblastoma. The iPSC-derived isogenic cells and three-dimensional (3D) brain cancer organoids combined with patient-derived xenografts will enhance future compound screening and drug development for these deadly human brain cancers. Early embryonic development marks the division and transformation of a totipotent zygote into pluripotent cells that can give rise to all cell types of the body . The indModels using iPSCs are compelling alternatives to animal models for studying neural disorders and brain tumors because iPSC-derived neurons possess similar genetic information, cell structure, and neuronal connections as those found in the human brain. Pathological models are now available for major neurological disorders that have been used to elucidate pathogenic mechanisms and to evaluate drugs preclinically ,20,21. HDespite appreciable improvement in cancer survivorship, brain cancers continue to show poor prognosis, largely attributable to the absence of suitable brain tumor models that limit the study of complex brain tumor biology and drug development. Glioma and medulloblastoma are two primary brain tumors with the highest incidence in adults and children, respectively . The mosThe past decade has seen a gradual emergence of iPSC-derived human brain tumor platforms ,27,28,29In this review, we discuss strategies for obtaining specific brain cells and brain organoids from iPSCs with a special focus on brain tumor models—their construction and usefulness in shaping brain cancer research.Since their discovery, iPSCs have enabled unprecedented advancements in studying human brain physiology and pathobiology. Researchers have successfully used iPSCs to generate various types of brain cells in 2D cultures to model specific brain regions in 3D organoid systems and to restructure the blood–brain barrier (BBB) in vitro. Collectively, these models contributed to the generation of a brain-like organ that may recapitulate the architecture of a developing human brain.The human brain consists of two major cell types—the neurons and the glial cells—that work in concert to ensure the proper execution of its functions. While the neurons are the primary cells responsible for signal transmission and processing, the glial cells, composed of astrocytes, oligodendrocytes, and microglia, support various neuronal functions and play active roles in synaptogenesis and brain development . These bNeural progenitor cells (NPCs) serve as precursor cells that can differentiate into neurons, astrocytes, and oligodendrocytes, so the generation of NPCs is considered substantial for the study of neurodevelopment and neurological diseases. With the advent of iPSCs, various protocols have been developed to differentiate iPSCs into NPCs . HistoriIt is important to note that NPCs can be generated from iPSCs as region-specific subtypes based on the available morphogens. While wingless/integrated (Wnt), FGF, and retinoic acid (RA) morphogens generate progenitors along the anterior–posterior axis, exposure to Wnt, BMP, and sonic hedgehog (SHH) results in cells with dorsal–ventral identities  . In addiDifferentiation of iPSCs into astrocytes takes a prolonged time and occurs in three steps: (1) neural induction of iPSCs into NPCs through EB formation, (2) expansion of NPCs until they acquire a glial phenotype, and (3) differentiation of glial progenitors into astrocytes driven by several factors, such as BMP and ciliary neurotrophic factor (CNTF), which activate the signal transducer and activator of transcription (STAT) pathway ,38. NeurInvolved in axonal myelination, oligodendrocytes emerge late during normal development and follow an intricate spatiotemporal specification. Similar to astrocytes, oligodendrocytic differentiation from iPSCs begins with NPC generation followed by gliogenesis ,42. The Microglia are the resident macrophages of the central nervous system (CNS) and the primary gatekeepers against infections. Aside from their role in innate immunity, microglia are critical players in neurological diseases, highlighting the need for the development of in vitro human-like microglia models that can recapitulate their in vivo counterparts. Recent protocols for iPSC-derived microglia rely on the latest developmental studies that trace microglia lineage to erythromyeloid progenitors (EMPs) in the yolk sac . In mostMatrigel, an extracellular matrix derived from a mouse sarcoma cell line, into the culture [Matrigel, EBs spontaneously developed into optical cup characteristics of retinal tissue [Matrigel and then culturing the complex in differentiation media with RA supplement [To more accurately model the human brain, a 3D culture system is required. These 3D structures, also termed organoids, consist of organ-specific progenitor cells capable of spontaneous self-organization and differentiation into various tissue-specific cells that can mimic some of the organ’s structural and functional features . We revi culture . When stl tissue . Later ipplement . This prAlternatively, other methods have been described to restrict the differentiation of the organoids into specific brain regions. By adding defined signaling molecules, organoids can be patterned into forebrain, midbrain, or hindbrain. For example, the addition of SMAD inhibitors can guide the organoids into dorsal forebrain fate, and if followed by SHH agonists, can promote ventral forebrain identities . FurtherEven though the addition of external factors has patterned the brain organoids into specific regional identities, these regions lack proper morphogen spatiotemporal gradients, functional connectivity among diverse brain regions, and non-ectodermal cell lineages, such as microglia and endothelial cells (ECs). This can substantially affect brain organoid development, resulting in an unfaithful replica of the human brain .A multitude of studies has emerged to address the aforementioned shortcomings of the patterning protocols. Cederquist et al. engineered a pseudo-signaling center by embedding inducible SHH-expressing iPSCs at one pole of a developing organoid generating forebrain topography reminiscent of in vivo neurodevelopment . AdditioCollectively, despite various pitfalls, these breakthroughs in brain organoid development from human iPSCs have provided unprecedented opportunities to understand and manage brain development and neurological disorders.The blood–brain barrier (BBB) is an exceptionally selective barrier with rich composition of unique brain microvascular endothelial cells (BMECs) and other supporting cells  of the neurovascular unit (NVU). Through its dynamic cellular complexes and tight junctions, the BBB is involved in the maintenance of brain homeostasis and the regulation of any interchange between the bloodstream and the CNS . BBB comSpecialized endothelial cells known as BMECs compose the innermost layer of the BBB’s physiological barrier. Alteration of these cells on a structural and functional level is linked to neurological disorders such as traumatic brain injury, Alzheimer’s disease, and Parkinson’s disease ,76. PeriIn a pioneering approach to generate BMECs from iPSCs, Lippmann et al. employed endothelial and neural codifferentiation of iPSCs in an unconditioned medium followed by expansion of the cells in an endothelial cell medium and selective matrix-aided cell purification. By coculturing these iPSC-derived BMECs (iBMECs) with astrocytes, the group was able to induce the expression of endothelial transporters and receptors, in addition to the recreation of BBB attributes such as well-organized tight junctions and polarized efflux transporter activity . The keyPericytes have two distinct origins and can be generated from iPSCs through either mesoderm or neural crest induction . MesoderCoculture of iBMECs with pericytes, astrocytes, and neurons is an attractive approach to enhance BBB properties in models, including transendothelial electrical resistance (TEER) and low passive permeability ,98,99. TIn vitro models derived from human iPSCs proved to be important tools for research considering interspecies variations in BBB receptor expression and the difficulty of using fresh tissue from human biopsies ,106. WhiPowered by their proclivity to self-renew and differentiate into specific cell lineages, iPSCs offer a valuable and easily manipulable model system that can be used to understand brain cancer progression, screen for anticancer drugs, and develop personalized therapeutic approaches. Primary brain tumors arising within the brain mainly consist of gliomas and medulloblastomas, which occur in adults and children, respectively. Diffuse gliomas, the most common primary tumors of the brain, include low-grade gliomas, astrocytomas and oligodendrogliomas, and high-grade glioblastomas that are distinguished from one another by the mutational status of isocitrate dehydrogenase (IDH) genes. IDH mutations are enriched in low-grade gliomas compared to glioblastomas, and thus infer a strong positive prognostic marker . While tThe first human iPSC-derived gliomagenesis model was reported by Sancho-Martinez et al. in 2016 . The modTP53 gene and activation of the G12VHRas oncogene  or neurospheres proved to be a better approach to model glioblastoma with complex genotypic and phenotypic heterogeneity B. The fiA glioma cerebral organoid model called GLICO  was introduced by Linkous et al. This model was generated to replicate patient-specific glioblastomas in an attempt to customize treatment strategies to fit patients’ unique tumors. The authors cocultured different GFP-labeled GSCs derived from patients’ tumors with mature cerebral brain organoids. The resulting tumor cells proliferated, migrated, and integrated themselves within the brain organoid using different routes of microtubules based on the intrinsic properties of the invading GSC line. The GLICO model proved to be advantageous, as it recapitulated many features of in situ glioblastoma. It invaded and subsequently destroyed the organoid tissue, formed microtubules for communication and invasion as seen in in situ glioblastoma, and expressed key genetic features and signaling pathways . This waLately, Goranci-Buzhala et al. compared the protocols described by Linkous and Ogawa with another method that generated a glioma model by simultaneous coculturing of GSCs and iPSCs, in an attempt to characterize GSC invasion patterns. They showed that GSCs tend to home to brain organoids of different maturity levels. This propensity is enhanced for mature brain organoids and varies between primary and recurrent GSCs. They also found that cocultures of GSCs and iPSCs do not reflect the in vivo process . AlthougGlioblastoma avatars derived from genome-edited human iPSCs have been shown to recapitulate the pathobiology of the disease. The study involved the introduction of GBM-associated genetic driver mutations into iPSCs, followed by their differentiation into NPCs. Finally, the edited NPCs were engrafted orthotopically in immunocompromised mice, forming GBM-like tumors. Moreover, cultures of dissociated tumor cells were found to possess cancerous phenotypes and to form, upon re-engraftment, secondary tumors with features similar to the patient samples .Another glioblastoma modeling paradigm that used patient-derived iPSCs was recently reported. The authors deployed iPSCs from a patient with c-met mutation, a hallmark of glioblastoma, to generate a neuronal organoid simulating the disease. The c-met-mutated iPSCs developed neuron-like organoids that lasted up to one year with features characteristics of glioblastoma .Medulloblastoma (MB) is the most common malignant embryonal tumor accounting for 20% of all pediatric brain tumors . AccordiThe molecular classification of sonic hedgehog MB (SHH-MB) has identified constitutive activation of the SHH signaling pathway in patients. SHH-MB accounts for ~30% of all MB cases, with a majority being infants (<3 years of age) and adults (>16 years of age) ,125. DesHuang et al. used iPSCs to generate neuroepithelial stem (NES) cells with genetic tumor predisposition to study medulloblastoma . TransduIn a similar approach, Čančer et al. developed humanized models of SHH-MB utilizing iPSC reprogramming and primary NES culturing. Ectopic overexpression of MYCN in NESCs derived from either iPSC or primary human hindbrain generated in vivo tumors with varying degrees of tumorigenicity. Although both models developed tumors with clinically relevant signatures of infant SHH-MB, the iPSC-derived tumors presented with a more aggressive phenotype. The authors attributed this increased malignancy to Oct4 upregulation and subsequent mTOR activation. This was validated by the fact that mTOR inhibitors effectively suppress viability, migration, and SHH-MB growth .Another iPSC-derived medulloblastoma model was generated by Ikemoto et al. using fibroblasts from four patients with Gorlin syndrome to study the pathogenesis of the disease. One of the models exhibited somatic loss of heterozygosity of the PTCH1 wild-type allele, recapitulating the development of the SHH subgroup of medulloblastoma seen in patients with Gorlin syndrome carrying PTCH1 germline mutations .Analogous to the previous studies, Susanto et al. described the formation of SHH-MB in mouse cerebellum following transplantation of NES cells reprogrammed from iPSCs of Gorlin syndrome patient skin cells with PTCH1 familial mutation. The model showed a progressive upregulation of inflammatory and epithelial-to-mesenchymal transition genes during primary and secondary tumor development, offering the possibility of a stepwise follow-up of tumor progression . The autGroup 3 medulloblastoma occurs in ~25% of all MB patients. It is characterized by poor clinical outcomes with a high incidence of metastasis at diagnosis and 5-year overall survival of <60% . While MIn a recent study, cerebellar organoids were exploited to create a medulloblastoma model simulating the Group 3 subtype. The authors reproduced in vivo the genetic aberrations identified in patients with Group 3 tumors. While mice overexpressing the gene combinations GFI1 + c-MYC (GM) and OTX2 + c-MYC (OM) induced brain tumors, only the latter combination developed medulloblastoma clustered with Group 3 subtype B, highliLately, a synthetic mRNA-driven strategy was used to develop the MYC-driven medulloblastoma model mimicking the Group 3 subtype D. The syParisian et al. developed an inducible SMARCB1 loss-of-function system inside iPSC-derived cerebral organoids. SMARCB1 inactivation is strongly associated with aggressive atypical teratoid rhabdoid tumors (ATRT) in children. They showed the effect of SMARCB1 loss is dependent on neural differentiation stages, with a potential to drive cellular transformation during early stages of neural differentiation . In termKey studies modeling iPSC-derived malignant brain tumors are summarized in Induced PSCs have revolutionized biological and biomedical research for their remarkable renewal and differentiation potential. In neurodegenerative disease research, iPSCs are particularly important since they can be differentiated into brain-specific cell types and used in cell replacement therapy to restore brain function. Production of brain-specific cells is of great interest for generating isogenic brain organoids, BBB, and brain tumor models. At the same time, brain organoid and BBB models have widespread use in developmental studies, brain functional characterization, disease modeling, and drug screening. Concerning brain tumor modeling, iPSCs have attracted a great deal of research interest as they may be engineered to harbor specific mutations and be differentiated into different tumor types.Brain tumor modeling from human iPSCs is superior for disease pathogenesis study because it possesses important species-specific attributes of human brain that are absent in animal models . With thTwo major strategies are employed to obtain iPSC-derived brain tumor organoids: using iPSCs via genome editing technology to introduce driver mutations  or using patient-derived cells . Fully iPSC-derived models such as neoCOR are excellent platforms to study neoplastic transformation mechanisms and tumor origin. They share similar transcriptomic profiles and invasive properties with the original tumor. However, their molecular features may be different from the natural tumor, and they may not retain intratumor heterogeneity . Fully iBrain tumor organoids such as GLICO use patient-derived tumor cells to retain the heterogeneity, histopathology, and genetic makeup of tumor cells, but they still lack a native tumor microenvironment . These mAn important consideration on brain tumor organoid models is keeping them viable for a prolonged time, irrespective of their types . Brain oDespite considerable success in glioma and medulloblastoma modeling, meningioma and ependymoma, two main types of brain tumors, have not been modeled using the pluripotent stem cell approach. Meningioma originates from the meninges, while ependymoma involves ventricle-lining ependymal cells. A potential use of iPSCs involves organoid formation for these tumors. Apart from disease modeling, iPSC-derived brain organoid models can be exploited for neurotoxicological studies, such as understanding the neurotoxic mechanism of antineoplastic drugs or carcinogenicity potency of environmental agents .Brain organoid models could also be useful for faster clinical decision-making at a single-patient level via biobanking, where the genetic features of a patient’s tumor could be matched against a collection of drug-sensitive organoids. Primary tumor-derived organoids are the most relevant mimetics in this direction, as they retain heterogeneous cancer stem cell composition to ensure their simultaneous growth and desired cellular hierarchy ,138. Co-