Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1. Instead, these lipids, along with membrane-associated proteins are often organized laterally into domains based on differential physicochemical interactions . One welCTxB associates with the ordered phase of GUVs containing trace amounts of GM1, but this depends on the structure of its ceramide moiety (see further discussion below) C,D 44]..44]. CTxThe intrinsic preference of GM1 itself for raft or non-raft domains also influences the phase in which CTxB preferentially resides. GM1 is classically thought to be a raft-associated glycolipid ,36,43,45GM1 is also known to form nanoclusters in cell membranes and model membranes ,59,60. T5 toxins regulate their association with membrane domains, as well as the general roles that lipid acyl chain structure and protein-mediated lipid clustering events play in membrane organization. They also raise interesting questions about how the structure of CTxB and GM1 controls additional biological activities of the toxin such as its ability to sense and/or induce curvature in cell membranes and influences its endocytic and intracellular trafficking, as discussed further below.Taken together, these findings emphasize the importance of the structure of both CTxB and GM1 in controlling their association with rafts and cellular nanodomains. This has broader implications for our understanding of how ABRegulated membrane shapes are critical to diverse cellular processes such as exocytosis/endocytosis, pathogen vulnerability/protection, therapeutic targeting, and organelle morphology . ProteinTo provide mechanistic insights into how CTxB induces membrane curvature, we recently employed polarized localization microscopy (PLM), a form of super-resolution microscopy that detects membrane curvature ,79. PLM CTxB likely induces membrane curvature by several mechanisms, all of which depend on its ability to cluster multiple GM1s. The first is a direct consequence of the physical shape of CTxB and relative locations of the five GM1 binding pockets. In particular, the GM1 binding pockets on CTxB are located on the perimeter of the homopentamer and elevated above the membrane-binding surface of CTxB. Because of this the membrane must deform to enable binding to more than one GM1 simultaneously . AccordiThe ability of CTxB to induce membrane phase separation in response to GM1 crosslinking also contributes to its membrane bending activity. Local enrichment and crosslinking of GM1 by CTxB creates a local membrane composition enriched in order-preferring gangliosides, which may trigger lipid phase separation. Lipid phase separation can in turn encourage membrane bending by creating a line tension or differential lateral pressure profiles ,87,88,89Crosslinking of GM1 by CTxB may also induce local lipid compression and membrane tension changes, similar to that observed upon crosslinking of Gb3 by STxB . This isCTxB molecules must also act cooperatively to facilitate large-scale membrane shape changes . The mulIn summary, CTxB induces and senses membrane curvature through a complex interplay of physical factors resulting from its molecular shape and lipid crosslinking. Recent results have demonstrated that CTxB locally accumulates and induces membrane bending though the collective result of lipid phase separation, compression, and crosslinking inherent in the multivalent binding of GM1 by CTxB. Ongoing efforts will focus on the precise mechanisms by which local physical changes to the membrane composition and shape recruit the downstream endocytic and intracellular trafficking machinery, for which WT and mutant CTxB are likely to be key tools for research.Endocytosis- a processes by which a plethora of molecules such as nutrients, extracellular cargoes, and activated membrane proteins/receptors are internalized into the cell- is critical for physiological proceedings such as nutrient uptake and intracellular signaling driving cellular homeostasis Mechanistically, endocytosis can be broadly classified into clathrin-dependent and clathrin-independent pathways Studies on the uptake of cholera toxin and in particular its B subunit CTxB have not only enhanced our understanding of the endocytic itinerary and pathophysiology induced by bacterial toxins but also enlightened us about the biophysical modifications that the plasma membrane undergoes in preparation for endocytosis. In this section, we will briefly discuss about different routes of clathrin independent internalization of CTxB. Further, we will also touch upon the utility of CTxB in delineating bacterial toxin internalization pathways that in turn has broadened our knowledge about modalities and machineries driving CIE pathway in general.Early on, it was recognized that CTxB can enter cells via multiple mechanisms, including both canonical clathrin-dependent endocytosis and clathrin-independent mechanisms ,124,125 More recent work has revealed that cholera toxin activates and is internalized by a clathrin-independent pathway dubbed fast-endophilin mediated endocytosis or FEME ,138,139.An important question raised by these findings is how toxin binding is sensed by the cell and translated into a signal that triggers endocytosis. One hypothesis is that clustering of multiple copies of GM1 upon CTxB itself serves as a signal. According to this model, the ability of the toxin to bind multiple copies of GM1, as well as structured clustering of GM1 by CTxB, functionally regulate toxin internalization. In support of this idea, toxin variants engineered to contain as few as one GM1 binding site exhibit strongly attenuated internalization, although they are still capable of completing the intoxification pathway ,145. One5 family toxins such as STx also initiates signaling events [As discussed above, the binding of CTxB to multiple copies of GM1 also regulates its association with ordered domains and the ability of the toxin to stabilize raft domains . Glycolig events . For exag events ,151. It In conclusion, internalization of CTxB occurs via multiple mechanisms and depends on a variety of molecular players operating at the plasma membrane on different time scales. CTxB actively regulates several of these pathways by inducing changes in membrane organization and intracellular signaling in response to toxin binding. These activities are enhanced by CTxB’s ability to cluster multiple GM1s, but glycolipid clustering is not essential for internalization of CTxB or the ability of CTx to cause cellular intoxification. However, many open questions remain about how these events are orchestrated. It is thus clear that CTxB will continue to be a critically important tool to advance our knowledge of bacterial toxin internalization as well as to further illuminate our understanding of mechanisms of CIE.5 toxins from cell surface all the way into the ER requires binding to membrane glycosphingolipids of the host cell, followed by endocytosis, endosomal sorting, and transport of the CTxB-GM1 complex into the retrograde pathway —and for the glycosphingolipids all the way backwards in the secretory pathway to the ER . For exa pathway  [153,1545-toxins have since provided robust tools to study the mechanisms and components responsible for endosomal sorting and membrane trafficking in the retrograde pathway .efore in ,156. The11) [1–10 GFP) fused to TGN or ER targeting sequence by quantifying the evolution of a fluorescence signal upon binding of CTB-mNG211 and mNG21–10 GFP. The assay led to the discovery that perturbations of the sheet and tubular morphology of the ER affects the retrograde trafficking pathway. Moving forward, this approach should be fully amenable to high throughput studies on the underlying biology of membrane trafficking.To address these problems, we modified the new split fluorescent protein technologies to link a small fragment of GFP to CTx  ,157. The11) . Retrogr5 toxins are affected by the structure of their glycolipid receptors [In a second approach, we have directly visualized the trafficking of GM1 itself through the use of fluorescent headgroup-labeled forms of GM1 ,13,158. eceptors . We disceceptors ,13. Ceraeceptors ,163,164.Altogether, these recent advances highlight the continued importance of CTx and GM1 as reporters of retrograde trafficking pathways. The tools described here should ultimately help develop a deeper understanding of mechanisms controlling the intracellular transport of CTx and related toxins, uncover principles that govern both protein and lipid sorting at multiple sites within cells, and design new strategies for delivery of therapeutics.CTxB binds the ganglioside GM1 with high affinity, and GM1 has been shown with the greatest clarity to act as the functional receptor leading to CTx entry into the ER of host cells and the induction of toxicity ,171,172.The evidence for secondary receptors, though in many ways compelling, is largely circumstantial and could have alternative explanations. The glycoprotein receptors do lead to endocytic uptake, for example . HoweverIn this review, we highlighted recent advances in our understanding of membrane biology and biophysics obtained through the use of CTxB. One important conclusion that emerges from these studies is that CTxB is not simply a passive reporter. It can drive phase separation, induce membrane curvature, stabilize rafts, stimulate its own internalization into cells, and re-direct GM1 into different intracellular trafficking pathways. This is not a new message, but it is one worth repeating given CTxB is still sometimes assumed to represent a benign raft and endocytic marker in the literature.It is thus essential to exercise caution when using CTxB as a probe for membrane organization and trafficking mechanisms, especially in poorly defined systems.It is also becoming increasingly clear that not all GM1s are created equal. Depending on its acyl chain structure and degree of saturation, GM1 can associate with ordered or disordered membrane phases in model systems, and form nanoclusters- or not- in cell membranes. CTxB itself also behaves very differently depending on whether it is bound to one or more copies of GM1, as well as their structural features. This can have critical consequences for CTxB’s ability to associate with rafts, bend membranes, and trigger endocytosis. Even changing the GM1/ CTxB ratio is sufficient to evoke some of these changes. Finally, it is important to recognize that lower affinity receptors for CTxB also exist, including other glycolipids and fucosylated secondary receptors. The membrane remodeling activities of CTxB are thus highly context-dependent.5 toxin family and some viruses [The recent pandemic has highlighted the importance of understanding the varied mechanisms by which pathogens gain entry into cells. Many of the membrane remodeling activites of CTxB are shared with other members of the AB viruses ,76,183.  viruses ,183. The viruses ,184,185. viruses . Thus, t


Amyotrophic lateral sclerosis (ALS) is a fatal, multisystem neurodegenerative disease that causes the death of motoneurons (MNs) progressively and eventually leads to paralysis. In contrast, delayed onset muscle soreness (DOMS) is defined as delayed onset soreness, muscle stiffness, loss of force-generating capacity, reduced joint range of motion, and decreased proprioceptive function. Sensory deficits and impaired proprioception are common symptoms of both ALS and DOMS, as impairment at the proprioceptive sensory terminals in the muscle spindle is theorized to occur in both. The important clinical distinction is that extraocular muscles (EOM) are relatively spared in ALS, in contrast to limb skeletal muscles; however, the blink reflex goes through a gradual impairment in a later stage of disease progression. Noteworthy is, that, the stretch of EOM induces the blink reflex. The current authors suggest that the impairment of proprioceptive sensory nerve terminals in the EOM muscle spindles are partially responsible for lower blink reflex, beyond central origin, and implies the critical role of Piezo2 ion channels and Wnt-PIP2 signaling in this pathomechanism. The proposed microinjury of Piezo2 on muscle spindle proprioceptive terminals could provide an explanation for the painless dying-back noncontact injury mechanism theory of ALS. On th2 and R1 components of the blink reflex? By answering these questions, we could possibly reveal further the difference in this distinct preservation phenomenon and could provide additional clues in reference to the degeneration mechanism of ALS.The important clinical distinction is that extraocular muscles (EOM) are spared from progressive degeneration in ALS, in contrast to limb skeletal muscles ,16,17,182Blink is a burst bilateral eyelid closure associated with concomitant eye movements. It provides a protective barrier for the eyes and facilitates the distribution of the tear film on the corneal surface. Accordingly, harsher tapping of the eyes elicits a bilateral blink reflex with the features of a nociceptive reflex . Blink rThere are six EOM controlling eye muscles. The motor units of EOM are distinguished from other muscles because they have a special set of myosin heavy chains and they also have multiple neuromuscular innervation sites per single myofiber . An inteOur knowledge about the MNs of EOM is fairly well-founded, in contrast to our knowledge pertaining to the sensory innervation of EOM . Muscle 3A recent hypothesis in reference to the cause of DOMS concludes that the primary injury of this dichotomous noncontact injury is a transient microdamage of the Piezo2 ion channel at the proprioceptive terminal in the muscle spindle ,15. PiezThe suggested transient Piezo2 channelopathy mechanism in DOMS is as follows. The peripheral ending of proprioceptive nerves in the muscle spindle is theorized to be exposed to a terminal arbor degeneration (TAD)-like lesion , as coulFurthermore, this stress-related Piezo2 microinjury at the proprioceptive terminals is suggested to compromise the static encoding of the stretch reflex ,15,36. A4It has been observed that familial ALS patients had significantly reduced blink rates than sporadic ALS patients and controls . Even ea2 and 2 subcomponents and R2 is more prone to inhibitory effects of repetitive stimulation and prepulses [2 subcomponents is often challenging due to temporal overlap [2 and R1 and eventually there was no detectable blink reflex [2 subcomponent is devoted to the direct impairment of the stretch pathway or to other indirect lesions affecting the excitability of the reflex [Longitudinal follow-up data showed that blink reflex impairments progressed over time in ALS patients, namely, the latencies of Re of ALS . It has repulses . The dis overlap . The grak reflex . The alte reflex . Howevere reflex ,10,15.Note that the electromyogram (EMG) of stretch reflex comprises three components: short latency (largely Type Ia afferent mediated), medium latency (largely dominantly Type II afferent mediated) and long-latency  responses ,47,48,491 component  of the blink reflex, MLR (polysynaptic) is analogous with the R2 subcomponent (polysynaptic with similar transduction time as 2 and minor differences in the axon length). Furthermore, the temporal overlap of R2 subcomponents of the blink reflex could be due to the minimal difference in the critical axon length difference in terms of transmission as opposed to the stretch reflex responses of extremities. Since the order of impairment in ALS starts with the 2 and R1 components of the blink reflex [Accordingly, the current authors suggest that the SLR (monosynaptic) of the stretch reflex acts analogous with the Rk reflex , this cok reflex ; howevermentclasspt{articlk reflex ,17,18. Fk reflex . Correspk reflex . No wondk reflex ,53 due tk reflex . TherefoA recent research study demonstrated that eye blinks of Piezo2-deficient mice were lower than wild types when the blinks were elicited by von Frey filaments . It is i2 and later R1 components, could eventually initiate the dying-back mechanism propagating toward the MNs of EOM at the very last stage of ALS disease progression but it is a lengthy process, especially under elevated Wnt signaling circumstances, and therefore it could not evolve to this point.Interestingly EOM are relatively spared in ALS ,16,17,185The current authors put forward the late involvement of somatosensory neurons in the unanswered question of whether the lower blink rate in ALS has a peripheral or central origin. At a later stage of ALS disease progression, the impairment of proprioceptive sensory nerves in the EOM muscle spindles could be partially responsible for lower blink reflex, beyond central origin. Moreover, the current authors imply the critical role of Piezo2 ion channels and Wnt-PIP2 signaling in this pathomechanism. The proposed microinjury of Piezo2 on muscle spindle proprioceptive terminals could even provide an explanation for the painless dying-back noncontact injury mechanism theory of ALS.


Obesity is a growing health concern, as it increases risk for heart disease, hypertension, type 2 diabetes, cancer, COVID-19 related hospitalizations and mortality. However, current weight loss therapies are often associated with psychiatric or cardiovascular side effects or poor tolerability that limit their long-term use. The hypothalamic neuropeptide, oxytocin (OT), mediates a wide range of physiologic actions, which include reproductive behavior, formation of prosocial behaviors and control of body weight. We and others have shown that OT circumvents leptin resistance and elicits weight loss in diet-induced obese rodents and non-human primates by reducing both food intake and increasing energy expenditure (EE). Chronic intranasal OT also elicits promising effects on weight loss in obese humans. This review evaluates the potential use of OT as a therapeutic strategy to treat obesity in rodents, non-human primates, and humans, and identifies potential mechanisms that mediate this effect. The obesity epidemic and its associated complications  increaseClinicalTrials.gov registry, National Institutes of Health). Given the current state of the obesity epidemic and lack of highly effective treatment options, this review focuses on OT as an anti-obesity therapy and mechanisms that contribute to these effects in genetically obese  is well recognized for its role in osmoregulation , prosocily obese  and dietly obese  as well ly obese  and obesly obese  and assely obese . The barly obese , the potly obese , impairmly obese , aggressly obese , hyponatly obese , adversely obese  through ly obese , and feely obese   OT is synthesized by magnocellular and parvocellular neurons in the paraventricular nucleus (PVN) as well as the magnocellular neurons in the supraoptic nucleus (SON) in mice , rats S, non-humMature OT and the carrier neurophysin are processed from the OT/neurophysin 1 prepropeptide  with botThe extent to which OT is expressed in outgoing projections from the PVN to hindbrain CNS sites linked to the control of body weight appear to vary based on targets and species. Kirchgessner and Sclafani initially proposed that OT projections from the PVN to hindbrain were important in the control of food intake and body weight based on findings from their lab in which knife cuts that sever PVN-hindbrain projections were found to disrupt OT fibers  and resuP = 0.13) and increased Fos  within ChAT (+) neurons of the thoracic spinal cord  that express OT appear to be involved with modulating cardiovascular function, stress response, thermoregulation and energy expenditure (via BAT thermogenesis) . Data from a combination of pharmacological, microdialysis and/or tract tracing studies suggest that PVN OT neuronal projections to the NAcc are involved with modulating social behavior and feeding reward . HerissoIt is well established that the adiposity signal, leptin, acts, in part, in the ARC to reduce body weight in lean animals by activating anorexigenic [proopiomelanocortin (POMC)] neurons while simultaneously inhibiting orexigenic [neuropeptide Y (NPY)/agouti-related peptide (AGRP)] neurons see . What reExisting data suggest that OTRs within the VMH are important in both the control of food intake and energy expenditure see  in rats in vivo fiber photometry to suggest that OT reduces food cue (sucrose)-elicited activation of dopamine neurons within the VTA suggesting that OT may reduce reward intake, in part, through an inhibitory effect on dopamine neurons and their response to rewarding food cues. Eric Krause and colleagues provided neuroanatomical confirmation that a subpopulation of VTA OTR (+) neurons express dopamine (10%) [in addition to glutamate (≈ 44%)], some of which project to the NAcc  and medial amygdala (MeA) (∼30–59 fibers/side) in rats  to potentially control energy expenditure. There is wide overlap in OTR distribution in mice and rats in areas that include the forebrain hypothalamus /mouse /rat van] and basfatty (fa/fa) rat, Koletsky (fak/fak) rat, ob/ob, db/db and Sim1± mice] , subcutaneous (sc) or intravenous], intraoral (in combination with a proton pump inhibitor)  or intra1± mice] , ovariec1± mice] , a rat m1± mice] , as well1± mice] .Numerous findings suggest that OT reduces body weight or weight gain in rodents and non-human primates, in part, by reducing energy intake see , intranaob/ob mice  indicate that OT-treated animals lose more weight relative to pair-fed control animals . These f/ob mice . Note th/ob mice  in male /ob mice . Further/ob mice .The most direct evidence in support of an important role for OT in the control of energy expenditure stems from pharmacological studies that included measurements of energy expenditure. Acute administration of OT into the 3V or VMH boosted energy expenditure or oxygen consumption as determined by indirect calorimetry in rodents . These eWe know from recent studies that acute forebrain (3V) and hindbrain (4V) injections of OT stimulate IBAT temperature [functional measure of BAT thermogenesis ] in bothOxytocin-ires-Cre mice was associated with a small elevation of locomotor activity, energy expenditure and sc IBAT temperature and a close to significant elevation of sc IBAT temperature (P = 0.13). Another study found that VMH administration of OT stimulated short-term physical activity in rats for 1-h post-injection but these effects failed to coincide with the more prolonged effects of 3V or 4V OT on IBAT temperature that we have found in our studies in rats (The majority of studies indicate that the effects of OT on IBAT temperature appear to contribute to non-shivering thermogenesis (mediated by BAT thermogenesis) rather than shivering thermogenesis . Namely, chronic administration of OT is not associated with elevations in locomotor activity in rats  or mice  in rats  and mice in rats . Yuan etde novo synthesis of brown adipocytes in white adipose tissue (WAT) culminating with increased expression of uncoupling protein-1 (UCP-1) and the production of heat . Further in vitro studies should also include UCP-1 which was not examined in this study. It will be important to determine if the conflicting data are due, in part, to dose, route of administration, acute vs. chronic application, in vitro vs. in vivo conditions, lack of overlap of brown adipocyte specific markers between studies and if these effects can be blocked by an OTR antagonist. These findings raise the possibility that OT may stimulate energy expenditure through multiple CNS and/or peripheral sites and raise the question as to the extent to which BAT thermogenesis and “browning” of WAT contribute to these effects.Previous studies indicate that OT may help stimulate the transformation of white adipocytes to more metabolically active “brown” adipocytes. The process of “browning”  of WAT m of heat . We rece of heat  but not /db mice  but the /db mice  and EWAT/db mice , it is p/db mice  in eithe/db mice  or EWAT /db mice , 2015. SOxytocin receptor or OT deficient mice are associated with adult-onset obesity  that appThe obesity phenotype observed in older OT and OTR deficient mice does not appear in younger mice. For example, there is no difference in body weight between OTR null mice and wild-type mice that are younger  aWhether the peripheral effects of OT on gastric emptying in rodents is mediated, in part, through activation of peripheral or central OTRs is still not known. CNS administration of OT reduces gastric motility  and thesOxytocin may also reduce gastric emptying through the local release of cholecystokinin-8 (CCK-8) and subsequent activation of vagal afferents that innervate the hindbrain. Systemic administration of OT inhibits both gastric emptying and stimulates the release of CCK-8 , 2008, bThe extent to which circulating OT may inhibit food intake through suppression of the orexigenic signal, ghrelin, is controversial. It has been reported that ghrelin administration centrally can stimulate OT release in rodents  and heteThe extent to which circulating OT may enter the CNS remains controversial . CirculaIntranasal administration enables relatively rapid uptake into the CSF of several neuropeptides and hormones, including insulin, vasopressin, and the melanocyte-stimulating hormone, adrenocorticotrophic hormone (4–10), within 30 min in humans . IntranaOne question is how translatable are metabolic data that are generated in animal models following central or peripheral administration given that OT is largely being administered intranasally in humans. As previously discussed, OT given by peripheral route of administration is likely to act by both peripheral and central OTRs (particularly at higher doses) to inhibit food intake and potentially stimulate thermogenesis and energy expenditure. There is a very limited amount of metabolic data following intranasal administration in rodent models, but it has been found to largely recapitulate the effects of central and peripheral OT to reduce food intake  and/or win vitro and in vivo data suggest that OT reduces dyslipidemia and increases lipolysis in rodent and non-human primate models. OT was found to stimulate glycerol in 3T3-L1 adipocytes , it did stimulate enzymes associated with lipolysis (hormone-sensitive lipase and patatin-like phospholipase domain containing 2)  . Chronicining 2)  and DIO ining 2)  relativeining 2)  or pair-ining 2) . Taken tThe OTR is a G protein-coupled receptor (GPCR) that is coupled to the Gαq alpha subunit (Gαq) or the Gαi alpha subunit . Given tIn addition to ghrelin, the OTR has been found to form homo- and heterodimers with other receptors. OTRs and vasopressin V1a and V2 receptors also form homo- and heterodimers . In addip < 0.001) weight loss at the end of the trial and the weight reduction was significantly larger than the placebo at the end of the trial. No adverse events (AEs) were reported during the trial. In addition, in a single case study, chronic intranasal OT was found to reduce obesity and food intake in an obese 13-year-old adolescent following removal of a craniopharyngioma . Collectively, this may adversely affect cardiac function and hamper its translational potential  given thPre-clinical data in rodents and non-human primates suggest that OT elicits weight loss, in part, by both central and peripheral mechanisms to reduce food intake (homeostatic and hedonic feeding) and impact energy expenditure in the absence of visceral illness or tolerance. While there is much enthusiasm over the potential use of OT as a therapeutic strategy to treat eating disorders and obesity, we await the results of ongoing clinical trials in obese humans for additional confirmation of its feasibility as a long-term weight loss strategy and assessment of adverse side effects. There is an ongoing need to identify an optimal dose, frequency, and duration of administration and to examine the dose-response effects of intranasal OT on body weight and adiposity in individuals with obesity.Combination drug treatment has demonstrated impressive efficacy in inducing weight loss  for re. We thinLong-term efficacy of OT treatment for obesity remains to be established. The effect of OT on thermogenesis, appetite and stress reduction may offer unique properties that current therapies do not have. OT as an adjunct therapy for obesity is also worth exploring. For instance, pre-clinical and clinical studies indicate that OT in combination with the opioid antagonist, naltrexone, is an effective strategy to reduce food intake  as well as body weight (humans). Peripheral (intravenous) administration of OT and naltrexone was recently found to be effective at reducing palatable 10% sucrose solutions as well as intake of a high fat/high sugar diet in rats . IntranaJN helped write and edit the manuscript and she also helped generate JB has a financial interest in OXT Therapeutics, Inc., a company developing highly specific and stable analogs of oxytocin to treat obesity and metabolic disease. The authors’ interests were reviewed and are managed by their local institutions in accordance with their conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ameliorating graft injury induced by ischemia and hypoxia, expanding the donor pool, and improving graft quality and recipient prognosis are still goals pursued by the transplant community. The preservation of organs during this process from donor to recipient is critical to the prognosis of both the graft and the recipient. At present, static cold storage, which is most widely used in clinical practice, not only reduces cell metabolism and oxygen demand through low temperature but also prevents cell edema and resists apoptosis through the application of traditional preservation solutions, but these do not improve hypoxia and increase oxygenation of the donor organ. In recent years, improving the ischemia and hypoxia of grafts during preservation and repairing the quality of marginal donor organs have been of great concern. Hemoglobin-based oxygen carriers (HBOCs) are “made of” natural hemoglobins that were originally developed as blood substitutes but have been extended to a variety of hypoxic clinical situations due to their ability to release oxygen. Compared with traditional preservation protocols, the addition of HBOCs to traditional preservation protocols provides more oxygen to organs to meet their energy metabolic needs, prolong preservation time, reduce ischemia–reperfusion injury to grafts, improve graft quality, and even increase the number of transplantable donors. The focus of the present study was to review the potential applications of HBOCs in solid organ preservation and provide new approaches to understanding the mechanism of the promising strategies for organ preservation. Solid organ transplantation (SOT) is an optimal, lifesaving treatment choice for patients with end-stage organ failure. The number of transplants has increased dramatically in the last decade, but there remains a considerable imbalance between the demand and supply of donor organs worldwide , 2021b. Ischemia and hypoxia are the leading causes of graft damage during the storage of isolated organs and may lead to a variety of destructive effects, such as ion imbalance, uncoupling of mitochondria, activation of endothelial cells, activation of various cell death programs, and proinflammatory immune responses . The curHBOCs are cell-free carriers obtained by processing free hemoglobin via chemical modification, genetic modification, or embedding . The oxy2O2-driven formation of ferryl myoglobin (Mb), which generates radicals and contributes to renal dysfunction  to have the ability to bind and release oxygen. Hb is a tetrameric protein molecule of ∼64 kDa consisting of two noncovalently bound αβ dimers . Free Hbfunction . Moreovefunction . Hb crosfunction . HBOCs wfunction . Howeverfunction . Subsequfunction . Since 2function . HBOCs afunction . Owing tfunction , initiatfunction , tumors function , and othArenicola marina (M101) (Arenicola marina), HBOC-201 (bovine), hemoglobin-vesicles (HbV) (human), polymerized human placenta hemoglobin (PolyPHb) (human), and hemoglobin glutamer (HbG) 200 (bovine). An overview of the different oxygen carriers used in solid organ preservation is provided to understand their properties better  to increase the stability and molecular size  , upregulAlthough there are differences in these vascular parameters, there are few reports on vasoconstriction damage in the preservation of isolated organs. The following reasons may underlie the relative lack of reports: carbon monoxide (CO) production is increased during the degradation of heme ; HBOCs pin vitro  , Celsiorin vitro . The appin vitro .Compared to other solid organs, the clinically isolated heart has a relatively short safe cold storage time (up to 4–6 h). The heart is an aerobic organ that relies on a continuous supply of oxygen to produce high-energy phosphate ATP for mechanical functions. The supply of oxygen via HBOCs supports the resynthesis of ATP in mitochondria of isolated heart tissue, which delays the damage of ischemia and hypoxia and extends the cold storage time . Li et aex vivo heart perfusion (EVHP) to meet the needs of myocardial metabolism, but it did not minimize myocardial injury or provide superior preservation of myocardial function  of the graft in the M101 group were significantly improved, and the graft vascular resistance was reduced after 5 h of reperfusion in the recipient. The study identified a circulating biomarker of ischemia–reperfusion, high mobility group box 1 protein (HMGB1). The expression of HMGB1 in the serum of the M101 group tended to be lower, and the activation of the HMGB1/RAGE (advanced glycation end products) axis in the control group explained the inflammatory response that led to PDG. The early serious events that occurred in the first few hours after transplantation were sufficient to capture the development of PDG. Therefore, the protective effect of M101 on IRI prevented the occurrence of PGD. Another study reported that SCS for up to 36 h after adding M101 reduced the risk of PGD after lung transplantation, which may be related to a significant decrease in circulating interleukin-6 (IL-6) levels (ex vivo lung perfusion (EVLP) for 12 h; the M101 lung showed better lung function after transplantation. M101 has inherent oxidative stress reduction activity, but it produces no significant difference in oxidative injury. The effect of M101 in prolonging the isolated lung’s preservation time may not be as an antioxidant, but it may optimize tissue oxygenation in the area of atelectasis. Briefly, adding M101 preservation solution prolongs the preservation time of isolated lungs up to 48 h, which will help overcome long-distance obstacles and expand the donor pool.Lung transplantation has been the most effective treatment for most end-stage lung diseases since 1983 . IRI is ) levels . CompareLiver graft quality depends on the donation status of the donor and the preservation effect during transportation from the donor to the recipient. During the preservation of the isolated liver, insufficient oxygen supply followed by reperfusion leads to an inflammatory network, and oxidative stress in the tissue leads to IRI. HBOCs induce the production of heme oxygenase 1 (HO-1) . Topp anNMP is generally performed using perfusion solutions based on RBCs to maintain physiological osmolarity, oncotic pressure, and adequate metabolic support. Laing et al. presented the first experience using an HBOC-based perfusion fluid to perfuse five discarded high-risk human livers in a human model of NMP . Livers 2) ± M101 (respectively or combined)] with HMP on the perfusion of isolated kidneys in a pig kidney auto-transplantation model  and maintained a high level of kidney injury molecule-1 (KIM-1) in the circulation. The study showed that supplementation with M101 with or without 100% O2 improves the HMP effect on kidney recovery and late graft results.Kidney transplantation is the therapy of choice for end-stage renal diseases. IRI is a complex process that involves oxidative stress, mitochondrial uncoupling, and the coagulation cascade, which affect early graft function after organ transplantation and have an adverse effect on the long-term survival of grafts . Thuillion model . The resGradual rewarming is a new perfusion modality in which HBOC maintains the preservation of perfusion of organs from hypothermia to normothermia. A transient increase in vascular resistance and methemoglobin was observed after preservation of the kidney using a preservation solution supplemented with HBOC-201 in a model of gradual rewarming but improved ultrafiltrate production, glomerular filtration rate, and sodium reabsorption during the reperfusion phase . In the Pancreas or islet transplantation is a treatment for type 1 diabetes and pancreatitis . During Muscle is a promising alternative site for islet transplantation, facilitating the rapid restoration of islet vasculature; however, it is susceptible to hypoxic limitation in the early posttransplant period. By co-transplanting islets with an oxygen carrier-PolyHb in mice, Daniel et al. found a dose-dependent reduction in β-cell hypoxia . Yet, thDue to the global shortage of organ resources, the imbalance of donor–recipient needs, and the increasing number of high-risk donors, these issues have led to considering what should be done to preserve grafts better. Although standard preservation solutions are currently used for static cold preservation, the repair and evaluation of marginal organs may rely on multiple combinations of MP, oxygenation strategies, and temperature regulation. Many efforts have been made by clinical practitioners and researchers to explore ways to mitigate graft ischemia–reperfusion injury, improve graft quality, and increase recipient and graft survival rates. Originally developed and utilized as a blood substitute, HBOCs are an ideal class of oxygen carriers with the ability to bind oxygen and release oxygen. HBOCs have now been extended to apply to tissues or organs in hypoxic conditions, where they act as an oxygen bridge to improve tissue oxygenation. The addition of HBOC to the preservation solution to improve the quality of the graft is a new promising strategy because they offer several advantages compared with traditional preservation solutions. First, HBOCs improve the oxygenation of isolated organs during SCS and enhance the quality of grafts. Second, the combination of HBOCs and MP provides a more targeted method, which can replace RBC perfusion to improve and evaluate the quality of high-risk grafts. In addition, preservation solutions containing HBOCs may be easily used in all solid organ transplants without further improving the existing preservation solution or perfusion platform protocols. Recent clinical trials have demonstrated the safety and efficacy of HBOCs in combination with mechanical perfusion platforms for liver and kidney preservation and improved prognosis for recipients and grafts.Future prospects address the need for translational medicine research to determine the exact therapeutic dose and organ preservation time for human solid organs. Coordinated multicenter clinical trials are also needed in the subsequent clinical studies to accumulate larger, homogeneous cohorts. Additionally, some clinical trials with a randomized controlled design would be performed. This review summarizes the latest and most important research on HBOCs in solid organ preservation. Therefore, based on scientific evidence, HBOCs, one of the oxygen carriers for organ preservation, are ideal candidates for organ preservation additives. The mechanism of organ preservation by HBOCs has not been fully understood; the relevant mechanism would be investigated in depth in future studies to determine a more optimal preservation method. In addition, the vascular resistance, methemoglobin content, and oxidative damage index of grafts and recipients would also be closely monitored when using HBOCs. The future development of HBOCs may give a fascinating insight into the understanding of the safety and effectiveness of new strategies for solid organ preservation.


Vascular endothelial cells produce and release compounds regulating vascular tone, blood vessel growth and differentiation, plasma composition, coagulation and fibrinolysis, and also engage in interactions with blood cells thereby controlling hemostasis and acute inflammatory reactions. These interactions have to be tightly regulated to guarantee smooth blood flow in normal physiology, but also allow specific and often local responses to blood vessel injury and infectious or inflammatory insults. To cope with these challenges, endothelial cells have the remarkable capability of rapidly changing their surface properties from non-adhesive (supporting unrestricted blood flow) to adhesive (capturing circulating blood cells). This is brought about by the evoked secretion of major adhesion receptors for platelets  and leukocytes (P-selectin) which are stored in a ready-to-be-used form in specialized secretory granules, the Weibel-Palade bodies (WPB). WPB are unique, lysosome related organelles that form at the trans-Golgi network and further mature by receiving material from the endolysosomal system. Failure to produce correctly matured VWF and release it through regulated WPB exocytosis results in pathologies, most importantly von-Willebrand disease, the most common inherited blood clotting disorder. The biogenesis of WPB, their intracellular motility and their fusion with the plasma membrane are regulated by a complex interplay of proteins and lipids, involving Rab proteins and their effectors, cytoskeletal components as well as membrane tethering and fusion machineries. This review will discuss aspects of WPB biogenesis, trafficking and exocytosis focussing on recent findings describing factors contributing to WPB maturation, WPB-actin interactions and WPB-plasma membrane tethering and fusion. Endothelial cells comprise the inner lining of blood vessels and thus the first cellular barrier separating blood and tissue. They form single-layered epithelia that differ in morphology, molecular characteristics, physiology and function depending on the type of vascular bed. As such they seal blood vessels and control traffic of nutrients, hormones, growth and differentiation factors, particles and cells  to and from the vasculature. Moreover, through selective secretion and uptake as well as production and decoding of signaling molecules they regulate blood vessel homeostasis including clotting and coagulation, fibrinolysis and thrombosis as well as vascular tone and local inflammatory reactions.One striking characteristic of endothelial cells relates to the adhesive properties of their apical cell surface that faces the blood vessel lumen. In the normal physiological state this surface does not interact firmly with leukocytes, erythrocytes and platelets thereby permitting an unrestricted blood flow and blood cell circulation. However, upon insult and endothelial cell activation surface properties change rapidly allowing leukocytes and platelets to adhere to the vessel wall. These cell interactions are vital to ensure proper responses to blood vessel injury (platelet plug formation and initiation of coagulation) and inflammatory or infectious insult (recruitment of leukocytes to sites of tissue damage or infection). Endothelial cells can actively control these surface properties by the regulated presentation of specific adhesion molecules. To do so, vascular endothelial cells are equipped with unique secretory organelles that store among other things leukocyte and platelet adhesion receptors to be released on demand. In honor of their initial discovery by Ewald Weibel and George Palade in electron microscopic analyses of rat and human pulmonary arteries these organelles were termed Weibel-Palade bodies (WPB) . Only laWPB are born at the trans-Golgi network (TGN) where they bud off in the form of discernible structures. Their dimensions and unique morphology are dictated by the main cargo VWF, a large glycoprotein synthesized and first processed in the ER  GBF1 (a GEF for Arf1 and 4) as a factor promoting ER/Golgi trafficking of VWF. GBF1 can be activated by phosphorylation by AMP-activated protein kinase (AMPK), a key enzyme coupling metabolic changes to cellular signaling, and it was shown that low glucose levels and subsequent AMPK activation lead to GBF1 phosphorylation and a resulting upregulation of anterograde VWF trafficking. This in turn produces smaller WPB and reduces VWF secretion  Figure . Arf GTP2+/phospholipid-binding protein annexin A8 as a LEL-localized component of the machinery facilitating LEL-to-WPB delivery of CD63 (2+ mobilization). Moreover, the exocyst complex was identified as a target of BLOC-2 in endothelial cells and exocyst depletion or inhibition phenocopied the WPB maturation defects seen in BLOC-2 deficient cells. In this study exocyst was also found to serve a second function in impeding WPB exocytosis at the PM  in the process of maturation that is accompanied by a microtubule-dependent movement to the cell periphery . The detailed molecular mechanisms responsible for releasing WPB from the cortical anchorage and enabling their PM contact are largely unknown, but they are likely to involve WPB associated RabGTPases. A central role for Rab27a in this event has been shown by Bierings and coworkers  as a negative regulator of the constitutive pathway . The majoworkers  who repooworkers . Rab46 wreleased  P2] via its AnxA2 subunit and to WPB-bound Munc13-4 via its S100A10 subunit P2 producing PI4P 5-kinase are required for efficient histamine-evoked WPB exocytosis , has been shown to promote WPB exocytosis most likely by providing a link or tether between the organelle surface and a PM-bound complex consisting of annexin A2 (AnxA2) and S100A10  Figure . In this subunit . A speciocytosis . In the ocytosis   levels in pathophysiological scenarios .


Defences to pathogens such as SarCoV2 in mammals involves interactions between immune functions and metabolic pathways to eradicate infection while preventing hyperinflammation. Amino acid metabolic pathways represent with other antimicrobial agent potential targets for therapeutic strategies. iNOS-mediated production of NO from Arg is involved in the innate inflammatory response to pathogens and NO overproduction can induce hyperinflammation. The two Arg- and Trp-catabolising enzymes Arg1 and IDO1 reduce the hyperinflammation by an immunosuppressive effect via either Arg starvation (for Arg1) or via the immunoregulatory activity of the Trp-derived metabolites Kyn (for IDO1). In response to amino acid abundance mTOR activates the host protein translation and Coronaviruses use this machinery for their own protein synthesis and replication. In contrast GCN2, the sensor of amino acid starvation, activates pathways that restrict inflammation and viral replication. Gln depletion alters the immune response that become more suppressive, by favouring a regulatory T phenotype rather than a Th1 phenotype. Proliferating activated immune cells are highly dependent on Ser, activation and differentiation of T cells need enough Ser and dietary Ser restriction can inhibit their proliferation. Cys is strictly required for T-cell proliferation because they cannot convert Met to Cys. Restricting Met inhibits both viral RNA cap methylation and replication, and the proliferation of infected cells with an increased requirement for Met. Phe catabolism produces antimicrobial metabolites resulting in the inhibition of microbial growth and an immunosuppressive activity towards T lymphocytes. Host defences to pathogen invasion, including bacteria or virus such as SarCoV2, involves in mammals a combination of initial innate immune and protective responses to eradicate infection that proceeds by directly acting on the pathogens and with locally induced inflammation, subsequently balanced by different mechanisms of immunomodulation and disease tolerance to prevent secondary harmful hyperinflammation. These processes include for a part a series of complex interactions between immune functions and metabolic pathways resulting from a convergent evolution of the two systems , 2. ThisArginine (Arg) is a non-essential amino acid for healthy humans involved in the synthesis of proteins and in the urea cycle and acting as precursor for different molecules including glutamate (Glu), citrulline (Cit), and nitric oxide (NO), an important bioactive molecule with both cardiovascular, immunological, and neurological signalling functions, and antimicrobial cytotoxic activity . In humaArg catabolism involves in mammals four enzymes including nitric oxide synthase , arginase , arginine decarboxylase (ADC), and arginine glycine amidinotransferase (AGAT) . The thrThe relative expression of the two Arg catabolizing enzymes iNOS and Arg1, and the production of associated Arg-derived metabolites, are related to the intrinsic regulation of macrophage polarisation, function, and activation state, and to the profile and time course of the immune response against viruses and bacteria pathogens  is a precursor for the synthesis of proteins and for several molecules involved in the regulation of diverse biological processes, including the amino acid kynurenine (Kyn), NAD, serotonin and melatonin . In mammThe enzyme IDO1, mainly expressed by DCs, is involved in the control of acute inflammation by an immunoregulatory immunosuppressive effects linked to its catabolic activity that induce a rapid change of Trp and Kyn levels that causes Trp deprivation in the microenvironment and the generation of immunoactive Kyn metabolites . Kyn is Identification of mechanisms that control the functions and fate of IDO1 opens new strategy for the pharmacologic control of IDO1 functioning and its immunoregulatory effect and modulating IDO1 catalytic activity is a promising therapeutic approach to modulate the immune response during the different steps of the immune response to pathogens infection. For some viral infections high IDO1 activity represents a main cause of immune unresponsiveness during the initial immune responses that aims to eradicate pathogens and downregulation of the short-term immunosuppressive regulatory effects of IDO1 should improve this short-term immune response. In contrast, during the second phase of the response, upregulating and increasing the activity of IDO1 could participate to prevent harmful hyperinflammation.At the crossroad between Arg and Trp metabolism and their regulatory effects, the two enzymes Arg1 and IDO1 are involved in the secondary control of acute hyperinflammation associated to pathogens infection by an immunoregulatory immunosuppressive effects via either amino acid depletion (as for Arg1) or via the combined effects of immunoregulatory Kyn and signalling activity (as for IDO1) , 32. HowRapidly proliferating cells, including several immune cells, require a supply of some amino acids such as Gln and upregulate metabolic pathways providing these amino acids, and these processes can be used as therapeutic targets against infection . The ami3 and aspartate and to the synthesis of the nucleotides purines and pyrimidines involved in the synthesis of DNA and RNA. Gln acts as a nitrogen donor for rapidly dividing cells, such as lymphocytes, in which it is critical for nucleotide synthesis and energy production [3 and in mammals glutaminase is highly expressed in lymphocytes and macrophages of lymphoid organs and increase in response to inflammatory stimuli [The degradation of Gln leads to the formation of NHoduction  and thisoduction . Glutami stimuli . Gln act stimuli . The TCANaive T lymphocytes are rapidly activated following pathogens invasion and Gln is essential for their proliferation and biosynthetic activities requiring high energy supply met by increased absorption and use of Gln –51. DuriGln depletion alters the balance of the immune response that become more suppressive, by favouring a regulatory T phenotype rather than a Th1 phenotype, even in a cytokine environment inducing the Th1 phenotype, associated with an inhibition of IFN‑γ secretion . Gln comAmino acid sufficiency and deficiency and amino acid and protein metabolism are controlled by the amino acid sensing pathways mTOR and GCN2 with a crosstalk between the two pathways . In respAnother example relies to serine (Ser) that is also required in large amount for rapidly growing cells –69. Ser In the same way, the two sulphur-containing amino acids, the essential amino acid methionine (Met) and the semi-essential amino cysteine (Cys), are also involved in immunoregulation. Cys is strictly required for T-cell proliferation because T cells lack the enzyme converting Met to Cys and must import Cys via their transport system . Cys avaMoreover, the interleukin 4-inducible 1 enzyme, secreted or localised in lysosomes, catalyses Phe catabolism leading Phe depletion and to the production of the a-ketoacid phenylpyruvate, and H2O2 with antibacterial toxic effects potentiated by the associated production of NH3 and basification of the medium , 80. TheAmino acid metabolism and signalling processes participate to the control of pathogen infection and to the regulation of the inflammation induced by activation of innate, adaptive, and regulatory immune responses. The crosstalk between catabolism of amino acids and the immune system represents an important process for tuning immune reactivity and reducing hyperinflammation associated to immune responses to infections. Among amino acids, Arg availability and the catabolic enzyme iNOS are involved in the inflammatory innate response through the production of NO, while Arg and Trp availability, associated to the two catabolic enzymes Arg1 and IDO1, represent catabolic pathways involved in modulating immune reactivity and exerting regulatory effect on inflammation and on the adaptive immune responses. Moreover, the amino acids Gln, Ser, Cys, Met, and Phe are also being considered as amino acids involved in pathogen development and in the balance of the immune reactivity. Overall, excess amino acid and mTOR hyperactivation appear as an environment favouring virus invasion while amino acid starvation and GCN2 activation restrict viral invasion. These different mechanisms represent potential therapeutic targets to control infection and associated hyperinflammatory response causally related to morbidity and mortality induced by pathogens, including coronavirus.


Brain metastasis (BrM) is an area of unmet medical need that poses unique therapeutic challenges and heralds a dismal prognosis. The intracranial tumor microenvironment (TME) presents several challenges, including the therapy-resistant blood–brain barrier, a unique immune milieu, distinct intercellular interactions, and specific metabolic conditions, that are responsible for treatment failures and poor clinical outcomes. There is a complex interplay between malignant cells that metastasize to the central nervous system (CNS) and the native TME. Cancer cells take advantage of vascular, neuronal, immune, and anatomical vulnerabilities to proliferate with mechanisms specific to the CNS. In this review, we discuss unique aspects of the TME in the context of brain metastases and pathways through which the TME may hold the key to the discovery of new and effective therapies for patients with BrM. In all cases, the intricate web of interactions between metastatic cells and the microenvironment of the central nervous system (CNS) begins before any actual seeding and evolves in a temporally and spatially dependent manner. Eluding to the complexity of the brain’s microenvironment, we now have a better understanding from patient spatial modeling that there is a nonuniform spatial distribution of metastasis to preferential brain regions according to primary cancer subtype. Use of predictive spatial modeling to reveal that primary cancers have distinct CNS topography patterns of BrM.5 These cancer-specific brain topography patterns may underlie the ability of tumor cells to adapt to regional neural microenvironments in order to facilitate colonization and establishment of metastasis. While the full range of interactions might be heterogeneous across tumor origins and mechanisms, there are key shared elements and pathways that can be considered to better understand the current state of the field, its future, and the implications for clinical advancements in the treatment of this particularly irascible diseaseThe importance of dynamic interactions between the native tissue components and metastatic tumor cells has become increasingly apparent over the past few decades of cancer research. When metastatic cells invade, they integrate into and modify the environment in complex pathways with important implications to tumor establishment, aggressiveness, and response to treatment. Understanding these relationships within the tumor microenvironment (TME) is essential to the exploration of brain metastasis (BrM) research and clinical progress. Today, BrMs comprise over 50% of all intracranial tumors and occur in up to 40% of all patients suffering from metastatic cancer.Even before consideration of BrM, the CNS microenvironment is a unique compartment within the body. The resident cells create a complex and dynamic microenvironment in their own rights, with interactions between neurons, astrocytes, oligodendrocytes, microglia, pericytes, immune cells, and the extracellular matrix essential to normal development and function. This ecosystem is separated from the peripheral vasculature by the blood–brain barrier (BBB), a selective filter composed of tightly connected endothelial cells, pericytes, and astrocyte projections within a dense basement membrane. The invasion of metastatic cells into this closely regulated environment results in an evolving TME distinct from any other seen in systemic metastases. In this review, we will cover how this distinct environment governs metastatic seeding and growth in the brain, and how this knowledge can be used to develop improved clinical concepts to treat or to prevent BrM. We start with the various microenvironmental factors of the brain metastatic cascade, then focus on 4 microenvironmental aspects that we consider key for a better understanding of fundamental BrM biology, and that at the same time are also instructive for clinical translation. These are the perivascular niche, angiogenesis and blood vessel interactions, tumor–immune interactions, and neuron–tumor crosstalk.11 One unique element of the process to the CNS is the initial attack on the BBB. Feng et al.12 utilized a murine model of acute monocytic leukemia to show that BBB permeability was increased by secreted matrix metalloproteinases 2 and 9, through disruption of the tight junctions (TJs) between endothelial cells. This functional change is the result of the downregulation of essential TJ proteins ZO-1, claudin-5, and occludin.12 The same group further characterized the role of secreted VEGF in compromising the BBB through similar mechanisms, and subsequent work has shown an ameliorating effect with VEGF inhibition via monoclonal antibody.14 The work by Li et al.15 in small cell lung cancer found that BrM is more common in patients with high levels of placental growth factor (PLGF). Their study of the effect in vitro revealed that the secreted factor also acts through a VEGFR-mediated pathway to break down TJs, exposing the underlying CNS to invasion by circulating tumor cells. Furthermore, Lyle et al.16 identified alterations in the pericyte population of high-permeability BBB metastases, showing an enriched population of desmin + pericytes. Beyond undermining the structural integrity of the BBB, expression of tumor-specific cellular adhesion molecules is also increased prior to metastatic colonization. In a murine breast cancer model, Soto et al.17 found that the brain endothelial vascular cells upregulate multiple cell adhesion molecules (CAMs), in particular, E-selectin, vascular cell adhesion molecule (VCAM-1), ALCAM, ICAM-1, VLA-4, and β4-integrin, in response to circulating tumor cells. At the same time, these molecules were upregulated on the vascular surface, their ligands were similarly highly expressed on the circulating tumor cells both, highlighting a potential mechanism for increased susceptibility to CNS colonization.Prior to extravasation of metastatic cells through the BBB into the CNS environment , it is p18 highlighted the influence of tumor-derived exosomes, finding that such complexes containing proteins, nucleic acids, and lipids in a breast cancer model can enter the CNS parenchyma through the BBB by compromising the endothelial cell endocytic pathway. Fong et al.19 identified a role for these exosomes with their study on complexes containing the miR-122 miRNA, which suppress glucose uptake in astrocytes and create a more hospitable environment for nascent metastatic cells. In their in vivo model of breast cancer BrM, the group showed that inhibition of miR-122 recapitulated normal glucose uptake in the brain and decreased the incidence of metastasis. Another implicated miRNA is miR-181c, which has been also demonstrated to disrupt the BBB and increase the incidence of BrM in an in vivo breast cancer model, in this case through the disruption of an actin localization pathway via downregulation of the PDPK1 gene.20 Rodrigues et al.21 identified an exosome-contained factor, CEMIP, that promotes metastatic invasion of the CNS. This protein was found to be specifically upregulated in brain-tropic metastatic cells and induces pro-metastatic changes in both brain endothelial cells and microglia by supporting a pro-inflammatory milieu. The group added further evidence to their results with the finding that high CEMIP expression in patients’ primary tumor samples correlated to a shorter time to BrM and worse overall survival. Altogether, the development of a premetastatic niche by secreted factors from the primary tumor is an important step in establishing a pro-metastatic microenvironment at the future site of metastasis.The CNS microenvironment beyond the BBB is also modulated by circulating factors prior to metastasis. Morad et al.22 This confirms earlier studies that demonstrated a slow-cycling phenotype of breast cancer circulating tumor cells (CTCs) in the clinical setting of brain metastatic disease and a particular brain metastatic potential of cancer cells with a more stem-like phenotype.23–25 It is unknown whether we can molecularly better characterize brain metastasis-initiating cancer cells in the future and develop more effective approaches for their specific targeting for the prevention of BrM.Tumor cells are continuously circulating in the bloodstream over the course of cancer growth, but only a small fraction will eventually grow to a clinically relevant brain macrometastasis. This is certainly partly due to the inefficiency of the brain metastatic cascade after tumor cell arrest and extravasation in the brain. In light of extensive tumor cell heterogeneity in all adult cancer entities studied so far, it is likely that only a tiny subfraction of tumor cells can “seed.” Recent work has shown that BrM-initiating breast cancer cells are particularly slow-cycling and express various stemness pathways.26 However, the finding that leukemic cells from the bone marrow use small bridging veins (emissary vessels) with laminin-rich microenvironments to reach the CNS raises the question whether similar mechanisms can also be in place for CNS colonization of solid malignancies, which frequently disseminate to the bone marrow, too.28 Malignant cells may travel along the abluminal surface of vessels that are topologically contiguous with the subarachnoid space, and these cells can migrate directly to the CNS, bypassing the need to enter and exit the vasculature.29 This anatomic trafficking pathway has also recently been shown to play a role in inflammatory processes, suggesting the possibility of an important function in immune surveillance and CNS tumor responses.It is widely accepted that BrM is hematogenous, which means that CTCs in the bloodstream arrive in brain (micro)vessels, arrest, and extravasate. This is supported by extensive intravital imaging studies and also the fact that no anatomical system exists in humans that would explain a lymphogenic arrival in the brain.31 These clinical observations may suggest that distinct molecular programs underlie brain parenchymal versus meningeal metastasis.Intriguingly, breast cancer patients who develop leptomeningeal metastases do not always have parenchymal metastases, but they all have vertebral bone metastases.32 The exact cellular process of extravasation in the brain is not well understood so far, but appears to be a dynamic process of tumor cell–blood vessel wall interactions.26 After tumor cell extravasation that is already mastered by a minority of brain-arrested cancer cells only, the following metastatic cascade into the CNS occurs across a harsh selective pressure, with fewer and fewer cells progressing to the next step of the sequence. Kienast et al.26 characterized the first foray of metastatic cells beyond the BBB with real-time in vivo multiphoton laser-scanning microscopy study of BrM formation. The vast majority of even the extravasated cancer cells fail to establish a foothold within the tissue, and the surviving cells remain near the vessel wall in the critical perivascular niche. Whenever they leave that niche in the first 2 weeks after brain colonization, they die. Studies in primary brain tumors have revealed the lasting importance of this location within the tumor, as a key site for the cancer stem cells that drive tumor growth and angiogenesis through a range of mechanisms including differentiation into vascular endothelial cells.33–35 In the initial stages, the perivascular niche is the crow’s nest from which the proliferating tumor cells can redirect vascular remodeling to meet their own needs. In glioblastomas, the perivascular niche also serves as a cancer stem cell reservoir that is critical to tumor progression and treatment resistance.36–39 Translationally, the German consortium (prevent_BM) has focused on key biological processes in this crucial perivascular niche for survival and resistance in the brain to develop novel molecular therapies against the earlier steps of BrM for improved BrM prevention.The initial intravascular arrest, extravasation, and initial colonization of the CNS by circulating tumor cells mark the next step in the evolution of the brain TME. Even the intravascular arrest is associated with relevant microenvironmental changes in the brain: microthrombosis and reduced microvascular perfusion, which likely impact the microenvironment. Platelet clotting and fibrin formation at the very site of arrested tumor cells were important for their long-term persistence and extravasation in the brain, with multiple interesting avenues for BrM prevention particularly by anticoagulatory therapies.26 The degree to which each pathway is present varies by tumor type.41 In vascular co-option .47–50Angiogenesis can provide a modified niche for perivascular cancer cells, but its implications extend beyond that aspect. Regarding tumor-directed angiogenesis, the primary driving mechanism appears through the VEGF pathway. On its own, VEGF stimulates angiogenesis and increased vascular permeability.51 Current research indicates that tumor cell entry into the brain occurs mainly via crossing of the BBB. However, recent data suggest the involvement of BCSFB as a route of entry for circulating tumor cells that reside within the CSF. Although these barriers tightly regulate neural homeostasis and immune privilege in the CNS, their restrictive nature also contributes to obstacles for effective drug delivery into the brain.52 Thus, tumor cells that successfully cross into the brain parenchyma use the brain as a sanctuary from chemotherapeutic insult and immune clearance.The brain is structurally and functionally isolated from the peripheral circulation, to limit exposure of the CNS to external influences at interfaces where the blood and the cerebrospinal fluid (CSF) interact with the neural milieu. To this effect, the brain has evolved to have 2 main barriers of restriction, namely, the BBB and the blood cerebrospinal fluid barrier (BCSFB).53The BBB consists of a non-fenestrated capillary endothelium, ensheathed by a network of pericytes, astrocytic foot processes, and microglia that together constitute this neurovascular unit. Central to its barrier functions are tight and adherens junctions that regulate the selective permissiveness of the BBB and restrict paracellular diffusion between plasma and brain interstitium. TJs connect adjacent endothelial cells and consist of 3 membrane proteins, namely, claudin, occludin, and junction adhesion molecules. These are further associated with cytoplasmic accessory proteins like Zona occludens  and cingulins which are anchored to the actin cytoskeleton. Adherens junctions comprise of cadherin–catenin complex and associated proteins.54 Similarly, brain-seeking tumor cells that arrest within the BBB capillaries can alter the barrier properties, enabling entry into the brain parenchyma. Extravasation into the brain takes longer than in other organs, and studies show it takes lung cancer (LC) cells 48 hours, breast cancer 2–7 days, and melanoma up to 14 days to invade the BBB.55 Enhanced migratory capacity of tumor cells within the BBB vasculature and interference in normal endothelial structure and function contribute to CNS metastasis. BrM from triple-negative and basal breast cancer is associated with BBB disruption based on GLUT1 and BRCP expression profiles, while Her2+ BrM tends to preserve BBB function.56 Melanoma cells in vitro induce endothelial apoptosis and reduce trans-endothelial resistance by proteolytic disruption of TJs claudin-5 and ZO-1 during migration, after which the endothelium repairs itself.57 These cells also interact with VCAM-1 found on the surface of endothelial cells activated by stimuli like TNF-α and interferons, indicating that tumor cells extravasate the BBB preferentially at sites of inflammation. PDX models show that the BBB is disrupted in BrM from breast, lung, and prostate cancers due to downregulation of Msfd2a, a fatty acid transporter expressed in the endothelium.58 This is accompanied by impaired TGFb and FGF signaling in the endothelium and loss of normal astrocyte function within the BBB.59 The expression of alpha2,6-sialyltransferase ST6GALNAC5, usually restricted to the brain, was found to be enhanced in BrM breast cancer cells, enhancing their adhesion to the endothelial cells and passage through the BBB.60 In LC, tumor-derived PLGF and enhanced expression of proteases MMP9 and ADAM10 facilitate passage through the BBB through disassembly of TJs in VEGFR1 expressing endothelial cells, and degradation of BBB ECM.61 Breast cancer cells also secrete exosomes containing miRNAs 105 and 181c and transfer these miRNAs to endothelial cells, disrupting BBB TJ functionality.20 Thus, although there is heterogeneity in the mechanisms of BBB disruption, brain-seeking tumor cells collectively modulate the brain endothelium to attempt successful BrM colonization.Although burdened with the critical function of protecting the brain against injury, inflammation, and pathogens, the integrity of the BBB is compromised in conditions like stroke, brain trauma, and neurodegenerative diseases like multiple sclerosis and Alzheimer’s disease.23 The CSF comes into close contact with blood in 2 areas, which formally make up the BCSFB: (1) where the arachnoid membrane envelopes the subarachnoid space and (2) where choroid plexus (CP) projects into the ventricular system.62 Because CP microvasculature lacks TJs, it is porous to large molecules, unlike the BBB endothelium. This is due to the absence of capillary ensheathment by astrocytic foot processes and the expression of “pore-forming” claudin-1 in choroid plexus, rather than “barrier-forming” claudins 3, 5, and 12 evident in the brain. The functional BCSFB is therefore dependent on TJs within choroid epithelium, rather than their “leaky” endothelium. Correspondingly, the BCSF is more permeable with a TEER of 150 ohm cm2 in vitro, compared to 1500 ohm cm2 measured in the BBB in vivo.63CTCs in the CSF and leptomeninges are therapeutic roadblocks and potential “seeds” of metastasis in the brain and spine.64 Increase in tumor-derived complement C3 within the CSF was shown to disrupt the BCSFB by activating the C3a receptor in the choroid epithelium. This leads to unrestricted inflow of growth factors and nutrients across the BCSFB, facilitating the growth of leptomeningeal metastases.65 Recent in vitro studies show that while LC cells migrate through the BBB and BCSFB at comparable rates, breast cancer cells preferentially migrate through the BCSFB by degrading TJs in the choroid epithelium.66 Neuroblastoma cells were able to migrate across an intact BCSFB in a paracellular fashion within 24 hours in vitro, without affecting the integrity of choroid epithelium.67Despite its contact with the CSF and its high permeability, the BCSFB is an understudied route of tumor cell entry into the CNS. Rare cases of intraventricular metastases have been reported from renal, lung, GI, breast, and bladder cancers, some found to be juxtaposed right alongside CP cells that line the lateral ventricles.68 Studies have demonstrated that the CNS microenvironment imposes specific and distinct metabolic pressures on metastatic tumors, as similar energy source flexibility has been found in brain metastases relative to matched primary tumors.70 Fischer et al.71 identified in their study of melanoma brain metastases an increased utilization of oxidative phosphorylation pathways compared to paired extracranial metastases. These findings offer direct translational opportunities, as they demonstrated in vivo efficacy of an oxidative phosphorylation inhibitor on murine survival. In a separate study, the same group showed that greater enrichment of oxidative phosphorylation expression is clinically relevant and associated with shorter survival after resection in humans, which could be targeted in their mouse model through metformin treatment.72 Fukumura et al.73 supported these findings with their 2021 study that found enhanced oxidative phosphorylation across lung, breast, and renal cell carcinomas again compared to matched primary or extracranial metastatic tumors, mediated through a separate mechanism from the PGC-1α implicated in melanoma.74 In breast cancer, Ebright et al.75 identified elevated HIF1A expression compared to matched primaries, a mediator of hypoxic signaling associated with glycolytic pathways. Ngo et al.76 highlighted a similar effect with their findings of the importance of PHGDH, a catalyst in the serine synthesis pathway, that provides metastatic cells with an essential amino acid of limited availability within the CNS. Additionally, specific cell populations within the CNS have been implicated in driving the metabolic profile of brain metastases. Zou et al. demonstrated that interactions between astrocytes and melanoma brain metastases activate PPARγ within the metastatic tumor, which is critical to modulation of glucose homeostasis and fat metabolism, with a resultant sensitivity to PPARγ inhibitors specific to CNS metastases. The authors implicated the lipid-enriched brain microenvironment and specifically polyunsaturated fatty acids within astrocytes in the mechanism of this effect.7 These results emphasize a theme of distinct but parallel pathways leading to common endpoints within the CNS microenvironment. The ability of metastatic cells to overcome the resource limitations of the brain is an essential predictor of their success, and this condition presents an additional dimension for specific targeting of CNS metastases.Metabolomics is a rapidly developing field within the study of oncology broadly with significant relevance to the specific and general cases of the metastatic microenvironment. The brain itself is a hub of metabolism within the body, consuming 20% of available glucose-derived energy along with its ability to rapidly adapt to various metabolic states and alternative energy sources, including acetate, glutamine, and branched-chain amino acids.77 Owing to their diverse functionality, they also play important roles in the disease progression of CNS malignancies. In BrM, astrocytes exhibit both pro- and antitumor functions.78 Early after tumor cell infiltration, astrocytes are activated by gliosis and contribute to neuro-protection by inducing tumor cell death through nitric oxide production and plasmin secretion, and forming reactive astrocyte (RA) boundaries delineating the metastatic lesion from the adjacent normal brain.80 However, they also promote BrM formation and colonization at various stages. They protect tumor cells from chemotherapy, and support BrM growth through the formation of tumor–astrocyte gap junctions that inhibit intratumoral calcium uptake, and stimulate release of tumor-supportive cytokines through the innate STING pathway.82 In vitro studies show enhanced tumor cell growth on co-culture with astrocytes. These effects are attributed to the release of soluble factors like heparanase, IL6, TNF, and IL1 by astrocytes, which stimulate metastatic determinants like endothelin-1 receptor expression, ERK phosphorylation, and induction of survival genes like BCL2L1 and TWIST1 in tumor cells.83–86 Astrocytes also regulate immune response within the brain during BrM progression. Priego et al.87 identified the presence of STAT3+ RAs in established BrM. The secretome from these RAs was sufficient to suppress T-cell activation and limit any antitumor effects on BrM cells through immunosuppressive molecules like VEGF-A, TIMP-1. Furthermore, they demonstrated that in response to macrophage migration inhibitory factor secretion from these RAs, CD74+ macrophages in the BM vicinity express midkine, a factor that promotes tumor cell growth.Astrocytes are glial cells involved in tissue homeostasis, maintenance of the BBB, regulation of neuronal synaptic responses, and immune signaling.88 RAs and microglia also contribute to neuronal death through persistent inflammation in response to tumor cell exposure. Furthermore, unlike normal breast epithelium, metastatic breast cancer cells express low levels of the serpin pigment epithelium-derived factor (PEDF) contributing to reduced neuronal health during BrM progression. Restoration of PEDF in xenografted mice results in increased tumor suppression.89Neurons mediate the propagation of electro-chemical signals within the brain and are critical for the normal functioning of the CNS milieu. However, our understanding of their role in BrM progression is limited. BrM lesions cause solid stress on surrounding brain tissue resulting in neuronal loss and neurological dysfunction.90 showed that BrM breast cancer cells shift their metabolic requirements and adapt GABAergic properties usually attributed to neurons. Breast cancer cells also show enhanced expression of receptors for BDNF and NGF, 2 major neurotrophic factors indicating the ability to respond to these stimuli within the neuronal niche.91 Neurons, heretofore assumed to be bystanders in BrM, are now shown to be directly involved in tumor cell growth. Aggressive breast cancer cells upregulate GluN2B, a metabotropic glutamate receptor, that primes them for successful BrM. Within the brain, these cells form pseudo-tripartite synapses with surrounding astrocytes and neurons and utilize glutamate secreted by presynaptic neurons, augmenting tumor growth and BrM colonization.92The seed and soil hypothesis states that successful BrM can be established by tumor cells that can adapt to the neural niche. Recent studies have highlighted the gain of neuro-adaptive attributes in brain-seeking tumor cells, particularly in breast cancer. Neman et al.93 Our knowledge of the heterogeneity and function of brain macrophages in the progression of CNS metastasis is limited. CD68+ or Iba1+ brain macrophages were detected within and around BrM lesions from breast and lung cancer and melanoma. Although these tumor-associated macrophages (TAMs) showed markers of phagocytosis, they did not seem to activate inflammation or adaptive immunity, indicating adaptation of a pro-tumorigenic phenotype.94 In vivo studies show accumulation of activated and reactive brain-resident microglia and infiltrating macrophages within BrM lesions, leaning toward a pro-tumorigenic subtype particularly in parenchymal BrM compared to those in the dura.95 In vitro, microglia promote breast cancer cell growth and invasion in co-culture, as well in living brain tissue slices. These properties could be blocked by inhibition of microglia function with bisphosphonate clodronate or by Wnt antagonist DKK-2.96 Currently, our knowledge of TAM involvement in leptomeningeal metastases is limited; however, perivascular and meningeal macrophages may play a role at these sites due to their role in immune cell recruitment to the CNS.97The CNS has parenchyma-associated resident innate immune cells called microglia and nonparenchymal macrophages in the perivascular, meningeal, and choroid plexus regions.98 demonstrated through their single-cell mass cytometry study of resected tumors that BrM has a greater proportion of invading immune cells relative to primary brain tumors. The effector CD8+ T cells were found to express high levels of both co-stimulatory and co-inhibitory molecules. Furthermore, they showed a preferential accumulation of regulatory T cells (Tregs) in BrM relative to gliomas. Typically these cells serve an essential role in tempering auto-immunity; however, in the setting of the metastatic brain TME, their anti-inflammatory activity creates a more permissive environment for tumor progression.99 The immunosuppressive environment of brain tumors has been highlighted in both primary and metastatic tumors.100 Analysis of BrM samples from a range of primary tumor types revealed distinct trends across histologies regarding distribution and phenotyping. Harter et al. identified melanomas and renal cell carcinomas as the most immunologically active tumors, with diffuse infiltration throughout melanoma samples, though infiltration has been shown in a range of histological origins.102 In this study as well, a high degree of inhibitory markers was identified, highlighting the problem of tumor-induced T-cell dysfunction.102 Ogiya et al.103 conducted a study of matched tumor samples from primary and metastatic brain tumors in breast cancer patients and found significantly lower infiltration of immune cells in the BrM. Completion of similar studies across other primary tumor types will be important to characterizing heterogeneity within the metastatic microenvironment and its implication for future treatment.The presence of infiltrating immune cells into the TME of BrM presents both an additional challenge and a well of potential therapeutic targets. Friebel et al.104 observed that peripheral T cells may not require access to the brain, because they can experience the parenchymal antigenic repertoire within the deep cervical lymph nodes (dCLNs). In contrast to glioblastoma, combined anti-PD1 and anti-CTLA4 have intra- and extracranial activity in melanoma. Mice with either intracranial or flank tumors benefit from checkpoint inhibitor therapy while survival benefit in mice with only intracranial tumors treated with the VEGF-C (a growth factor for lymphatic vessels) with an anti-PD1 combination therapy were similar to mice with both intracranial and flank tumors treated with checkpoint inhibitor therapy alone. Ligation of the dCLNs removed the VEGF-C benefit in mice with intracranial tumors, but not mice with both intracranial and flank implants. T-cell priming through expression of VEGF-C in the CSF, or through a flank tumor, enables checkpoint inhibition in the CNS. However, in the case of a tumor that is confined to the CNS at a steady state , immune checkpoint inhibitors alone do not confer notable benefits. Educating immune cells outside the CNS may hold the key to a new strategy to increase lymphatic drainage, thereby enhancing immunosurveillance and overcoming the immune ignorance of CNS tumors.Song et al.105 Additionally, these cells exhibit a phenotype that actively inhibits local T-cell proliferation, further emphasizing the co-option of immune effector cells into a pro-tumorigenic microenvironment by BrM.106 The prognostic implications for the degree of immune infiltration are currently unclear, with some studies indicating no correlation and others linking infiltrating immune cell density to both edema and overall survival.107 Beyond local immunosuppression, brain tumors are also linked to systemic lymphopenia and exhaustion.108 With these populations of cells both present within the TME and systemically available, identifying therapeutics to functionally activate them is an important pursuit going forward.Beyond infiltrating lymphocytes, neutrophils have also been found to be highly abundant within BrM. These cells have been shown to be similarly influenced by the TME, with upregulation of anti-inflammatory markers including ADORA2A.Measures taken in clinical practice for prevention of BrM include prophylactic whole-brain radiotherapy (WBRT) in tumors with a high risk of CNS relapse like small cell lung cancer, although it is only offered to a minority of patients because of potential neurotoxicities. New more targeted and well-tolerated approaches are needed.109 In addition to monoclonal antibodies targeting VEGF-A, tyrosine kinase inhibitor, nanobody targeting VEGF-A and Angiopoietin-2, have also shown activity in BrM, which indicates a class effect of these drugs.109 Clinically, the anti-VEGF-A antibody bevacizumab can exert even single-agent effects in BrM of breast, lung, and colorectal cancers, as shown in a recent case series and earlier studies.110–112 However, response assessment using MRI can in principle be misleading in this setting as antiangiogenic therapies can reduce gadolinium uptake, although this seems much less the case than in glioma.48 However, these anti-VEGF-A treatment effects in BrM patients appear clinically meaningful, particularly as a salvage therapy for heavily pretreated patients, and may also mitigate the need for immunosuppressive corticosteroids.The preventive efficacy of antiangiogenic drugs appears to be restricted to a limited number of tumor types  and the brain, which provides an example of how the brain TME can differ from the rest of the body.114 Unfortunately, historically BrMs are known to be particularly treatment-resistant to both systemic and radiotherapeutic approaches. SRS has been shown to extend survival in these cases, though melanoma and renal cell carcinoma are known to be particularly radioresistant.115 Tumor resistance to systemic therapy is often attributed to obstruction and active exclusion by the BBB, in particular, p-glycoprotein (ABCB1) mediated efflux.116–118 However, interactions within the TME have also been shown to play important roles in BrM chemoresistance. RAs within the tumor drive upregulation of survival genes in tumor cells, in particular, GSTA5, BCL2L1, and TWIST1. Kim et al.86 demonstrated that upregulation of these genes was directly correlated with tumor resistance, and that their regulation was directly dependent on gap junction communication between astrocytes and tumor cells, with disruption increasing therapeutic efficacy.Standard treatment of BrM is typically centered on radiotherapy, with surgical resection indicated in cases with significant mass effect or hydrocephalus. Today, stereotactic radiosurgery (SRS) has largely replaced WBRT, particularly in cases with limited metastatic loads.119 Interactions within the microenvironment have been shown to increase treatment resistance through the PI3K–AKT–mTOR pathway and PTEN loss within metastatic cells.119–122 Importantly for consideration of future therapeutics, inhibition across these pathways increased the susceptibility of tumor cells to traditionally ineffective chemotherapy agents.120 Various small molecular inhibitors targeting driver mutations including HER2, EGFR, ABL, and BRAF have shown efficacy in the treatment of appropriately selected BrM; however, broad success has not been found as most tumors do not have targetable oncogenes.123–125Additional mechanisms of chemotherapy resistance have been shown to be mediated by signals within the brain TME, along with calcium sequestration by RAs.127 Kluger et al.128 administered pembrolizumab, an inhibitor of the PD1 pathway, in 23 patients with asymptomatic melanoma BrM and identified a promising overall survival rate of 48% at 2 years. Another study targeting the CTLA4 pathway in 72 patients showed activity against melanoma BrM along with systemic responses, also with evidence of long-term survivors .129 Knisely et al.130 found clinical benefits with the addition of ipilimumab, an anti-CTLA4 agent, to SRS in their study of 77 patients. In a subsequent study by Amaral et al.127 in 380 patients, a corresponding benefit was found for the combination of immunotherapy, using both PD-L1 and CTLA4 inhibition, with surgery and radiosurgery upfront. These findings highlight the concept of combinatorial therapies and the likelihood that utilizing several methods to broadly and specifically target tumors is a compelling avenue forward in the treatment of BrM. Clinical trials of immunotherapeutics in BrM are ongoing, which will provide important evidence of their role and applications in future multimodal treatment paradigms.Attempts to reverse the immunosuppressive environment of BrM to harness the circulating and infiltrating immune cells have been an active area of research. Essential key questions to future investigations are highlighted in It is clear today that the TME in BrM is of high and specific pathobiological importance and at the same time provides ample avenues for more effective therapies. The emerging data from neuron–cancer cell interactions, angiogenesis, brain’s barrier, specific immunological features, and other aspects that are covered in this review hold the promise to extend our armamentarium to treat or even prevent this challenging disease.