The current perspectivepresents an outlook on developing gut-likebioreactors with immobilized probiotic bacteria using cellulose hydrogels.The innovative concept of using hydrogels to simulate the human gutenvironment by generating and maintaining pH and oxygen gradientsin the gut-like bioreactors is discussed. Fundamentally, this approachpresents novel methods of production as well as delivery of multiplestrains of probiotics using bioreactors. The relevant existing synthesismethods of cellulose hydrogels are discussed for producing poroushydrogels. Harvesting methods of multiple strains are discussed inthe context of encapsulation of probiotic bacteria immobilized oncellulose hydrogels. Furthermore, we also discuss recent advancesin using cellulose hydrogels for encapsulation of probiotic bacteria.This perspective also highlights the mechanism of probiotic protectionby cellulose hydrogels. Such novel gut-like hydrogel bioreactors willhave the potential to simulatethe human gut ecosystem in the laboratory and stimulate new researchon gut microbiota. The universallyaccepted narrative of probiotics given by the Food and AgricultureOrganization (FAO)2 of the United Nationsstates that “probiotics are live microorganisms, which whenadministered in adequate amounts confer a health benefit on the host”.Earlier research has demonstrated the efficiency of probiotics incuring symptoms related to several bowel disorders, gastrointestinal(GI) inflammations, and diarrhea.3 Microbialcount [colony forming unit (CFU)] and viability are among the essentialconditions for the microbes to have the desired effect on human health.3 Viable microbes that produce useful metabolites to the host areconsidered as probiotics, which are known to exert positive ramificationson host health by offering protective barriers, exclusion of pathogenicmicrobes in the GI tract, and augmentation of the baseline immuneresponse.4The microbial ecosystem present in thehuman gut significantlyaffects the health of the human host.5 However, concerns relatedto their functional viability and delivery to the intended locationof the human gut still persist.6 Evidently,for any probiotic to have beneficial effects, the bacterial suspensionmust pass and survive through the digestive tract and reach the colonin substantial amounts.8 According to a World Health Organization(WHO) report, the minimum number of viable probiotic bacteria in anyfood supplement to be retailed with health claims is 106–107 CFU.9 Moreover,there are other variables, including pH, oxygen, and temperature,that apparently affect the viability of the bacterial suspension administeredas a probiotic.12The application of microbesin food and beverages has a long-celebratedhistory with society initially discovering the benefits of consumingfermented foods. Naturally or commercially produced probiotic bacteriaconsumed by the majority of the population in the form of simple yogurtare directly associated with the early ferments. The apparent healthadvantages of probiotics have led toward their inclusion in a largevariety of food and beverages, including cereals, cheese, ice cream,and milk shakes.16 Carrier systems based on proteins, biopolymers, and lipids are consideredas excellent encapsulation vehicles.17 Protein-basedencapsulated probiotic formulations are composed of several animal-and plant-derived proteins, such as gluten, zein, gelatin, and milkproteins. Probiotic encapsulation in plant-derived vegan proteinsis being considered as an excellent alternative to animal-derivedproteins.18 Lipids, fats, oils, waxes, and resins areanother category of biomaterials considered as a favorable matrixfor encapsulation of probiotics.20Numerous studies have been carried out to investigatethe roleof different encapsulation vehicles in protecting the viability ofcultures throughout processing, delivery, storage, and GI transit.25 Cellulose-based hydrogels on their own or in combination with otherbiopolymers have recently shown great promise to overcome the limitationsof conventional biopolymer-based encapsulation systems.33However,encapsulation systems based on conventional biopolymers,such as alginate, have some limitations in protecting the probioticsfrom gastric fluids.Lactobacillus and Bifidobacterium genera, for health benefits, there is emerging evidence that multiple,difficult to culture, strictly anaerobic, novel probiotic strainsfrom other phyla, such as Firmicutes, Bacteroidetes, and Actinobacteria,may be needed  initiative in Round 22 titled “NewApproaches for Manufacturing Gut Microbial Biotherapeutics”,34 which highlights the importance and significanceof this area of topical research. To tackle this issue and to reducethe cost of production, there is a need to construct hydrogel-basedgut-like bioreactors that can simulate the pH and oxygen (pO2) gradients found across the human gut,51 enabling thegrowth of multiple strains of aerobic and anaerobic bacterial speciessimultaneously to microaerophilic bacteria in the stomach (acidic pH ∼ 1–4and pO2 ∼ 77 mmHg). In the small intestine, thepH becomes increasingly basic (pH ∼ 5–5.5) and the oxygenlevel drops even more (pO2 ∼ 33 mmHg); therefore,facultative anaerobic bacteria grow in this region. Further down thecolon and rectum, pH increases further, reaching values of greaterthan 7, and the oxygen level  drops below 33and 1 mmHg in the colon and near the rectum, respectively. The approximatepH and pO2 values are shown in 51The human GI tract is responsiblefor food digestion, nutrientabsorption, secretion, and motility of undigested parts for excretion.52 Such an idealization of regions makes it easyto design a bioreactor with various compartments interconnected, asin the human gut, that can have crosstalk between them and to simulaterealistic GI conditions in the reactor. The design of the reactorcan be used to control pH and pO2 gradients in the reactorto match human gut conditions. Once the control of the physical gradientsis achieved, the bioreactor can be seeded with various aerobic andanaerobic gut microbes with biotherapeutic potential. The seedingcan be performed with individual strains in different parts of thereactor with a favorable growth environment or multiple strains canbe seeded together as a mixture. The specific microbes will grow intheir comfort zones of a favorable environment, including pH and pO2 values. Bioreactors that can accommodate such a huge variationin the microbial environment are difficult to design. However, itcan potentially be achieved using the optimized design of immobilizationmaterials with varying degrees of spatial structuring and gradients.Because the human gutmicrobiome contains multiple strains of aerobic,microaerophilic, and anaerobic bacteria, it becomes quite essentialto have all of these different types of strains while developing probioticsfor biotherapeutic interventions. However, because these various strainsneed very different nutrient supply, pH, and oxygen levels, they cannotbe grown in a single bioreactor. Multiple strains are generally grownin separate bioreactors, extracted, freeze-dried, and then mixed laterin dry form. This process makes multi-strain probiotic-based healthinterventions expensive. However, from a chemical engineering pointof view, the gut can be treated as a compartmentalized plug flow reactor2.52 Such54 Plant-based cellulosebiomass is abundantly available all over the world. Several inorganicimmobilization materials are not suitable for this application becausethe resultant food products are intended for human consumption. Hence,it is essential to use food-grade biopolymers that are derived fromplant sources.Cellulose hydrogels are promising immobilizing materials with potentialto achieve this goal. They are manufactured from plant- or bacterial-basedcellulose nanofibrils, making them biodegradable, renewable, hydrophilic,and biocompatible.55 These characteristics of cellulose hydrogelsmake it an ideal contender in constructing a gut-like bioreactor,because porosity and cross-linking density of these hydrogels canbe altered to achieve spatial control over pH, oxygen, and nutrientlevels. We also argue that the hydrogels used in the constructionof the gut-like bioreactor must be food-grade, so that the hydrogelscan be used for encapsulation. To our knowledge, this is the firstreport that discusses and develops novel concepts for the growth ofboth aerobic and anaerobic microbial strains of the human gut in asingle bioreactor system.The cellulose-based hydrogels can be chemicallyaltered using functionalgroups or by incorporating with active biomolecules. The cellulosehydrogel structure formation is due to the natural association ofnanofibers in combination with electrostatic stabilization that canbe used to control porosity. The porosity can be varied by concentrationand mixing ratios of various forms of cellulose, such as cellulosenanofibers (CNFs) and cellulose nanocrystals (CNCs). The stabilityof these hydrogels can be further improved by chemical cross-linkingthat leads to flexibility and tenacity to the structure.56 The long aspectratio (>100) imparts flexibility to the fibers, so that it canforman interconnected network when dissolved in water, resulting in hydrogelsof reasonable strength at low concentrations (<1 wt %) withoutexternal chemical cross-linking. On the other hand, CNCs are rigidshort crystals, which typically have low aspect ratios (<30), resultingin rigid morphology. As a result of the rigid crystalline nature,CNCs require high concentrations to form hydrogels of reasonable strength.However, as a result of the completely different characteristics ofCNFs and CNCs, they can be mixed in various concentration ratios toobtain hydrogel of controlled physicochemical characteristics. Forexample, if a highly porous hydrogel network is needed, CNFs can beused. While if rigid and low porosity hydrogel is needed, CNCs canbe used. Therefore, regulation of the concentration and ratio of CNFsand CNCs in a gel system can be used to tune the porosity and rigidityof hydrogels. Surface chemistry modifications and introducing cross-linkingnetworks are the possible methods to achieve mechanically stable CNChydrogels because CNCs alone cannot form stable hydrogels as a resultof the small aspect ratio and rigid structure.57Plant-based cellulose-derived products, such asCNFs and CNCs aremajor classes of this material, which are generally modified or combinedwith other materials to impart desired chemical properties. CNFs generallyhave a diameter of about 5–50 nm, and the fiber length canbe more than 1 μm.58 freeradical polymerization,59 solution casting,60 freezing/thawing cycle,61 three-dimensional (3D) printing,62 andultraviolet (UV)/ion-mediated cross-linking,63 have been extensively studied to incorporate CNCs and CNFs intopolymeric hydrogel networks.Both CNCs and CNFs can be physically and chemicallyentrapped intopolymeric hydrogel matrices to improve their mechanical properties.Rigidity of CNCs allows them to efficiently act as fillers in polymerichydrogel composites. More flexible and soft hydrogels could be obtainedby incorporating CNFs. However, loading ability of CNFs is lower thanthat of CNCs as a result of the tendency for entanglement. Differentmethods, such as homogenization,2 gradients for developing a gut-likebioreactor are controlled porosity and water absorption capacity.Another essential characteristic is that the hydrogels must be food-grade.Below, we discuss a few important studies from recent literature thathave attempted to synthesize cellulose-based hydrogels with controlledporosity and water absorption capacities, albeit using harsh chemicals,which make them unsuitable to be used for human consumption or infood-grade products. We address this issue of chemical-free synthesisof food-grade cellulosic materials later. Although the studies describedbelow used non-food-grade chemicals in the synthesis, they still providethe necessary background information needed to support the hypothesisthat cellulose hydrogels, in theory, can be used to control pH andO2 gradients needed to construct a novel gut-like hydrogelbioreactor.Theimportant characteristics of cellulose hydrogels required tomaintain pH and O65 We present the data obtained in studies by Chang et al.,42 Geng,47 and Luan et al.26 in 42 have used epichlorohydrin(ECH) to cross-link a blend of cellulose and alginate solution tosynthesize hydrogels with tunable macroporosity and water absorption.It was found that the gel became stiff, porosity decreased, and waterabsorption increased with an increasing concentration of cellulosein the blend  (MBA) as a cross-linking agentto prepare micro- and macroporous hydrogel by increasing the concentrationof MBA. It was found that with increasing MBA concentration, the gelsbecame stiffer, more transparent, and porous and water absorptionincreased (rows II and III of 46 has presenteda schematic model of how the solvent (NaOH/urea) and cross-linkerMBA result in the formation of an entangled cellulose network as aresult of hydrogen bonding in the absence of a cross-linker, resultingin low porosity and transparency, whereas the inclusion of MBA inbetween the cellulose molecules increases transparency, stiffness,porosity, and water absorption with increased MBA concentrations.The reader is referred to studies by Geng47 for further details. Luan et al.26 haveused a combination of native cellulose fiber (CF) and CNF to preparecellulose macrogel particles by extrusion into hydrochloric acid (HCl),as shown in row IV of 66 that synthesizedCNFs from carrot pomace using chemical-free methods offers a hopefor preparing food-grade cellulose hydrogels from either agriculturalor food waste.Many researchers have attempted to combine cellulosicmaterialswith other biodegradable polymers and biopolymers, such as sodiumalginate and other proteins.row I of 3. Geng4667 Most of these physical methods are usedin preparation of cellulose-based foams (aerogels and cryogels), butthey can be used for hydrogels as well. For example, during the preparationof hydrogels, a high shear force can be used to trap air/gas bubblesof controlled size inside the hydrogel solution, which can act asa template for creating pores.68 Similarly,oil-in-hydrogel or hydrogel-in-oil emulsion69 can be prepared with a specified emulsion drop size, which can belater removed by solvent extraction, leaving a specific pore structure.An ice crystal templating method can also be used, where controlledfreezing of water can create pores.70 Theexisting experimental results of porosity tuning of cellulose demonstratethe potential of using them to simulate an intestinal environment.Apart from chemical cross-linking methods to control porosity,there exists an array of physical methods that can be used to regulateporosity.74 The details of the immobilinestructure along with some examples of acidic and basic acrylamidobuffers and their use to generate immobilized pH gradients for isoelectricfocusing of proteins are shown in detail in 74 Our approach of using only cellulose hydrogels along with buffersis presented in the next section.Generation of pH gradients insidethe proposed hydrogel-based bioreactoris essential for simulating the human gut environment and multi-strainprobiotic production. Immobilized pH gradients generated using immobilinescovalently grafted onto polyacrylamide gels with their use in isoelectricfocusing for protein separation is a well-established technology,which is reviewed in detail by many authors.As shown in 2) and low pH buffer with air  and carboxymethyl cellulose (CMC) have beenreported to offer better protection and stabilization against adverseGI conditions.As shown in 31 and Luan et al.,26 where a combinationof alginate/CNF and native CF/CNF were used to control the porosity,water absorption, and swelling of the hydrogel network in variouspH conditions. Hao et al.31 used alginate/CNFin various ratios to prepare hydrogels of various transparencies andporosities, as shown in We describe two important studies by Zhanget al.31 presented a mechanismof protection of probiotic bacteria by SA–CNF composite hydrogels,as schematically shown in 31 explainedthat, in acidic pH, protonation of carboxylic acid groups in sodiumalginate and CNF chains contributes to reduced electrostatic repulsion.31 The increased attraction results in the formationof hydrogen bonds between carboxylic acid and protonated carboxylgroups. This leads to the shrinkage of the SA–CNF compositehydrogel and protection of probiotic bacteria being released in thestomach.31 In acidic pH, the shrinkagewas found to decrease with the increase in cellulose concentrationSA–CNF composite hydrogels, whereas pure CNF hydrogel did notshrink. However, in basic pH, SA–CNF composite hydrogels swelled,whereas CNF hydrogels did not swell. In both acidic and basic pH cases,the CNF backbone imparted stability to the structure of compositehydrogels.31 Such a protonation and deprotonationmechanism in acidic and basic pH conditions could be the reason forprobiotic encapsulation and controlled release properties of cellulosehydrogels.31Zhang et al.26 useda combination of native CF and CNF to prepare controlled porositycellulose macrogel particles, where it was found that, with an increasein the CNF concentration, the average porosity increased from 0.6to 2.1 μm. As shown in 26As described earlier, Luanet al.31 found that there was an ionic interactionandcross-linking between adjacent SA and the carboxyl group of CNF molecules.In this case, both SA and carboxyl contents of CNF provided the requiredpH responsive behavior. The CNF network also provided overall structuralstability required to withstand dissolution in SIF. However, in thecase of the CF–CNF composite hydrogel, Luan et al.26 have found that pH-responsive behavior was importedto the hydrogels by the carboxyl content of CNF. The porosity alsocorrelated well with the carboxyl content of CNF in the compositehydrogel. In acidic pH (SGF), the CF–CNF hydrogel with a higheramount CNF (high porosity) shrank a little but swelled a lot in basicpH (SIF). Hence, the porosity as well as swelling behavior resultingfrom the carboxyl content of CNF contributed to the release of encapsulatedprobiotic bacteria. Highly porous hydrogels swell a lot in SIF andrelease the encapsulated probiotics quickly, whereas low-porosityhydrogels with high amounts CF do not swell much in SIF and, hence,release the bacteria over time.In the case of SA–CNF composite hydrogels, Zhang etal.31 and Luan et al.,26 composite cellulose hydrogels can be used tocontrol the release of probiotic bacteria by tuning either the dissolutionof one component in the hydrogel or the porosity. These studies formthe basis for our proposed approach in using a cellulose hydrogelbioreactor for encapsulation and controlled release of multiple probioticbacteria.As explained in the previous sectionby two example studies ofZhang et al.As explained earlier, probiotic bacteria that favorlow pH andaerobic conditions for growth will be grown in highly porous hydrogels,whereas probiotic bacteria that favor high pH and strictly anaerobicconditions will be grown in low-porous hydrogels. When these individualhydrogels with individual probiotic bacteria are freeze-dried, thebacteria become encapsulated within the respective hydrogel matrix.During the transit through the GI tract, the bacteria encapsulatedin highly porous hydrogels are released first, followed by less poroushydrogels. When the porosity and pH responsiveness of the hydrogelsare tuned, the controlled release of individual bacteria in the regionof the gut favorable for the respective probiotic bacteria can beachieved.2 levels, hydrogel-based bioreactors and immobilizationtechnology appear to be a feasible option.Fermentation strategies, such as immobilized cellulose-hydrogel-basedbioreactors, are highly suitable and promising to produce multipleprobiotic strains. With the ability to maintain pH gradients and diffusedO88 Additionally, there are reports on the applicationof the continuous production strategy for large-scale cultivationof probiotics. Although the strategy has been quite successful forproviding a high cell yield, continuous fermentation always runs arisk of contamination and, even more importantly, altering the characteristicsof probiotics in the process.89 Nonetheless,over the past few years, the concept of immobilization has been appliedfor a great length in probiotic research. The commercial productionof probiotics and especially monoculture probiotics is still largelybased on the free cell culture in batch mode. This is primarily dueto the fact that bacterial cells produced through the process of immobilizationvary greatly in terms of physiology, morphology, and growth characteristics.90 Future research should be directed toward efficientimmobilization support for large-scale production of food items, suchas yogurt, milk solids, and cornflakes.91Additionally, onthe basis of the stability, biosustainability,and biodegradability of cellulose nanofibers, application of cellulosehydrogels in the fermenter can propel the research on designing probioticsin a whole new dimension. Designing such a study where probioticsare propagating in their natural habitat  requires elaborated levels of planningand engineering. However, such studies are crucial considering thepresent scenario because the hydrogels are non-toxic, non-hazardous,recyclable, and biodegradable. Additionally, incorporation of thesehydrogels will also lead the way forward for attaining sustainabledevelopment goals and achieving a circular economy. There are alreadya few studies that implemented the idea of cultivating more than twoprobiotic strains using the co-culture fermentation method in a bioreactor.2 gradients. Essentially, the prospectshint toward emerging engineering aspects along with simulation studiesof the intestinal tract for efficient probiotic production along withgenerating a deeper understanding of the human gut ecosystem.This perspective proposes the concept of designing next-generationbioreactors with the central idea of applying cellulose hydrogelsfor efficient production of multiple probiotic strains using a conventionallaboratory-scale fermenter. As described earlier in the perspective,properties of cellulose hydrogels make it feasible to control andset up pH and pO


Anorexia nervosa is an eating disorder that causes, among the well-known somatic symptoms, brain volume loss which was associated with neuropsychological deficits while the underlying pathophysiology is unknown. In this review article, we summarize the findings of glia cells in anorexia nervosa animal models and try to deduce which role glia cells might play in the pathophysiology of eating disorders, including anorexia nervosa. A better understanding of glia cell function in the regulation of food intake and eating behavior might lead to the identification of new drug targets.Eating behavior is controlled by hypothalamic circuits in which agouti-related peptide-expressing neurons when activated in the arcuate nucleus, promote food intake while pro-opiomelanocortin-producing neurons promote satiety. The respective neurotransmitters signal to other parts of the hypothalamus such as the paraventricular nucleus as well as several extra-hypothalamic brain regions to orchestrate eating behavior. This complex process of food intake may be influenced by glia cells, in particular astrocytes and microglia. Recent studies showed that GFAP In the central nervous system (CNS) exist billions of cells with various functions, molecular structures, and morphologies. These cells build up complex circuits to fulfill a specific task in the human body, i.e., eating behavior. However, the circuits are not limited to neuronal cells, also glia cells play an important element in these pathways. Recent studies showed that glia cells are active and important regulators in food intake behavior and are, thus, potential drug targets for metabolic disorders. Therefore, we summarize in this review the neuronal circuits of eating behavior in the hypothalamus, the role of glia cells in this process, and the potential influence of glia cells for eating disorders with a specific focus on anorexia nervosa (AN).The regulation of food intake is one of the most complex behavioral processes in the CNS and we are still far away from entirely understanding its complexity. While a balance between energy intake and expenditure results in body weight maintenance, insufficient food intake for a longer period of time results in body weight loss, which can be severe (reviewed in ). AffereSeveral peripheral mediators exist which influence neuronal network activities with leptin, ghrelin, insulin, and glucose being the most widely studied. Leptin often referred to as the “satiety hormone”, is produced by adipocytes, proportional to fat levels (reviewed in ). HigherAnother peptide hormone regulating food intake is ghrelin synthesized in the hypothalamus and endocrine cells of the gastric mucosa. Its effects are antagonistic to those of leptin . GhrelinA subset of neurons located in the ARC of the hypothalamus synthesize NPY and AgRP. The central function of these neurons is to stimulate food intake. Accordingly, NPY/AgRP neuronal activity is elevated when the body is in a state of energy deficit . Acute aWhile the food intake regulating the function of NPY/AgRP neurons is well documented, there is evidence that eating behavior is also regulated by these neurons. Eating behavior includes feeding practices, food choice and motives, dieting, and eating-related problems. Activating only AgRP neurons substantially increased motivation for eating and drove intense food-seeking behavior, demonstrating that these neurons also orchestrate other complex behaviors in adult mice . When foFos expression as well as gliosis in the ARC and postsynaptic brain regions [A receptor agonist) suppresses Fos activation and maintains food intake  led to a lean phenotype and a resistance to diet-induced obesity in mice [A receptor subunits are increased in an animal model for AN [It is a matter of debate which factors mediate the orexigenic function of the NPY/AgRP neurons. The inactivation of the genes encoding NPY, AgRP, or both had little effect on body weight regulation, suggesting that something else produced by these neurons does regulate their orexigenic function . Since A regions ,18. Beyod intake . The dir in mice . An addil for AN . Thus, tAnother main population in the ARC of the hypothalamus are POMC neurons, in which the precursor protein pro-opiomelanocortin (POMC) produces many biologically active peptides via a series of enzymatic steps in a tissue-specific manner, including melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH), and β-endorphin. These peptides play a role in an array of biological activities in the central and peripheral nervous systems (reviewed in ), howeveIn terms of eating behavior, the sensory detection of food is sufficient to rapidly reverse the activation state of these neurons induced by energy deficit  suggestiGlia cells have long been considered passive cells which support the mechanical stability of neuronal networks. These cells are currently recognized as active regulators of neuronal function and are therefore able to shape different types of behavior such as learning and memory . In In pathological conditions e.g., after CNS injury, reactive astrogliosis triggered by cytokines  is induIn conclusion, knowledge about the physiology of astrocytes creates opportunities to identify new targets to treat diseases including eating disorders. We now address the influence of astrocytes in food intake processes because these cells are subject to change in the ABA animal model ,36.2+-dependent exocytosis of neurotransmitters [2+ code and other signal-transduction pathways (reviewed in [2+-dependent transporters L-glutamate/L-aspartate transporter (GLAST) [It has been shown that astroglia in vitro directly signals to neurons and other astroglia through Casmitters ,38. Subssmitters ). Today iewed in ). Theoreiewed in  as well iewed in  from theiewed in ). These  (GLAST)  and glut (GLAST) , glutami−/− mice ate significantly less than mGluR5+/+ controls when both groups were refed [Following the hypothesis that a dysfunctional glutamatergic gliotransmission system may influence behavior, it has been shown that some extrasynaptic N-methyl-D-aspartate (NMDA) receptors antagonists drive antidepressive effects in animal models of depression . Furtherre refed . Howeverre refed , it remare refed . A recenre refed . In summAstrocytes are strategically located around neurons and synapses and can, due to the expression of neurotransmitter receptors on their membranes, sense neuronal activity. In response, astrocytes release neurotransmitters or co-transmitters such as glutamate, GABA, D-serine, or adenosine triphosphate (ATP) which can, as a result, influence neuronal activity. Assuming there is reciprocal communication between astrocytes and neurons, scientists started examining the possible role astrocytes may have in regulating behavior. Studies in animals have shown that astrocyte interaction with synapses occurs under physiological and pathological conditions, and this indeed shapes behavioral responses ,53,54. FIn the next section, we will discuss to what extent peripheral mediators of feeding behaviors, in particular leptin, exert their effects via modulation of astrocyte function.As already outlined above, the “satiety hormone” crosses the blood-brain barrier (BBB) through a selective transport system  and bindIn comparison to leptin-mediated peripheral food intake signals, astrocyte function appears to interfere with the appetite-stimulating hormone ghrelin (reviewed in ). Astroc+ cell number increased, and these cells developed a more complex morphology indicating that astrocytes respond to nutritional changes. A mechanism due to an additional specialized astrocytic subtype in the hypothalamus, the Gomori-positive astrocyte, is also possible . This siewed in . In concBesides astrocytes, microglia (another type of glia cell) have been shown to influence food intake by regulating signaling cascades, and this cell type will be discussed in the following Section.Microglia cells were recognized as a specific cell entity more than a century ago . Since tFollowing inflammation, the morphology of microglia changes dramatically into an amoeboid morphology with retracted processes, and they are also capable of regulating plasticity by modifying synaptic connections . Once acIn terms of glia cell interaction, it is known that astrocytes and microglia communicate with one another and, as a result, astrocytes influence microglia function and morphology, and vice versa (reviewed in ). The coThe involvement of inflammation and microglia cells in the complex processes of food intake regulation is not completely clarified, however, our understanding is constantly growing. The results of several studies suggest that excessive food intake results in microgliosis ,81,82. ATo sum up, there is good evidence that both glia cell populations, astocytes as well as microglia, are important entities to mediate food intake pathways, potentially also in nutrient-deficient conditions as in eating disorders like AN.AN is characterized by body image disturbances and significant weight loss . AN has In terms of neuropathology, AN is associated with extensive brain volume changes. Two meta-analyses have demonstrated in detail that gray matter volume is decreased by 6% and white matter volume by 4% in patients with acute AN (reviewed in ). These The cellular changes that underlie this brain volume reduction in patients with AN are mostly unclear. Dehydration as the sole reason for brain volume loss seems unlikely due to normal serum and normal to low urine osmolarity . Therefoad libitum food intake not longer than 1–3 h per day, combined with free access to a running wheel [Animal studies have given first insights into the underlying pathophysiology of AN. The ABA animal model mimics the most important pathological parameters of patients with AN such as amenorrhea, bodyweight reduction, hypothermia, hypoleptinemia, and hyperactivity. The first ABA model consequently called the original model, combines a period of ng wheel . Paradoxng wheel . Interesng wheel . In thisng wheel ,110. In ng wheel .Some studies addressed the important question of whether the brain atrophy observed in patients with AN can be mimicked in the ABA model and which cellular alterations underlie this brain atrophy under conditions of food restriction. To analyze chronic food restriction, our group established a slightly modified ABA model that avoids the high mortality rate observed in the original model, whilst including a fixed level of body weight loss maintained over a longer period of time. The start of the modified ABA model is characterized by an acute starvation phase, in which the rats received 40% of their baseline food intake until a 25% weight reduction is reached. Afterward, a period of chronic starvation is maintained by holding the animals’ body weight at 75% of their initial body weight for two weeks by giving them just enough food to maintain this weight . Thus, t+ astrocyte cell density in the white matter corpus callosum (CC) and the cerebral cortex (CX) of ABA rats in comparison to controls , the starvation-induced effects were mainly reversible in the CX but not CC in ABA animals  model also exists, in which the animals receive 2.5% NaCl solution to drink leading to dehydration and reduced body weight . In line reduced . MoreoveAnother consequence of starvation appears to be inflammation shown by an increase of IL-1β and IL-1R1 protein expression in the hypothalamus of ABA animals . FollowiThe described changes on the cellular level are paralleled with behavioral deficits shown in ABA rodents, e.g., impaired memory performance in the novel object recognition (NOR) task  and reduCell culture studies additionally gave interesting insights into glia cell pathology. An in vitro semi-starvation model in primary rat astrocytes has been established in our group by Kogel et al. . The celNpy and Agrp mRNA expression was upregulated in ABA animals while Pomc expression was reduced [Pomc mRNA level; this may be dependent on the length of starvation [Consequently, another important question is how food intake-related pathways are changed due to starvation processes in the ABA model. A few studies have shown first insights into this topic: Increased Fos expression in the hypothalamus (ARC and supraoptic nucleus) indicates neuronal activation in ABA animals when compared with normally fed animals . Further reduced . Howeverarvation . Furtherarvation . Surprisarvation . The admarvation . Chronicarvation . To conc+ cell density. We suggest that these cellular changes also occur in the ARC of the hypothalamus associated with astrocytic dysfunction and an inflammatory environment (+ cell density loss could be reduced proliferation of astrocytic precursor cells, apoptosis/necrosis of astrocytes, decreased Gfap mRNA expression, and/or potentially due to UPR induction which might be linked to an inhibition of GFAP protein synthesis. Our observation of reduced overall cell proliferation suggests that disturbed astrocyte turnover plays an important role, a hypothesis that will be clarified in the future with immunofluorescence double staining experiments. Additionally, reduced astrocyte density can lead to disturbed astrocytic function which, in turn, could change behavior potentially explaining neuropsychological deficits in psychiatric disorders. Causal interactions between astrocytes and behavioral alteration in starvation need further exploration.In summary, the brain volume reduction in the ABA model is linked to a reduction in GFAPironment . The reaAs previously described, also neurons play an important role in food intake control and behavior. For example, neuronal impairment can worsen the energy-deficient condition by changes in synaptic plasticity which can be investigated in animals with neuronal and/or astrocytic specific fluorescent protein expression.In conclusion, experimental animal models for AN demonstrate the potential influence of glia cells in the neuropsychological progression of eating disorders. Astrocytes and perhaps microglia are therefore an important research area in the context of eating behavior to find potential new drug targets for interventions in patients with eating disorders, especially AN. Further research should focus on the interaction between astrocytes and neurons in the hypothalamus to analyze the complex interplay in food intake-related pathways.


Compartmentalisation by bioenergetic membranes is a universal feature of life. The eventual compartmentalisation of prebiotic systems is therefore often argued to comprise a key step during the origin of life. Compartments may have been active participants in prebiotic chemistry, concentrating and spatially organising key reactants. However, most prebiotically plausible compartments are leaky or unstable, limiting their utility. Here, we develop a new hypothesis for an origin of life environment that capitalises upon, and mitigates the limitations of, prebiotic compartments: multi-compartmentalised layers in the near surface environment—a ’scum’. Scum-type environments benefit from many of the same ensemble-based advantages as microbial biofilms. In particular, scum layers mediate diffusion with the wider environments, favouring preservation and sharing of early informational molecules, along with the selective concentration of compatible prebiotic compounds. Biofilms are among the earliest traces imprinted by life in the rock record: we contend that prebiotic equivalents of these environments deserve future experimental investigation. Bulk geochemical environments have several deleterious properties as possible hosts for chemistry that may have given rise to life. Such environments are largely dilute, water-rich, and overly complex—all characteristics that can limit the efficacy of prebiotic reaction pathways. Take the so-called water paradox: the contrast of water as the universal solvent for life with its thermodynamic inhibition of similarly universal condensation reactions involved in, for example, nucleic acid formation . CondensExtant life elegantly overcomes the water paradox, concentration problem, and kinetic barriers by harnessing enzyme-driven reactions to sustain electrochemical gradients across membranes. The cell membrane is built around an amphiphile bilayer that regulates diffusion, with inward facing hydrophobic groups and outward facing hydrophylic groups. This fundamental relationship between spatial structure, energy flow, and chemical behaviour in extant life draws attention to the possible role of structured environments in fostering prebiotic chemistry, which may be more analogous to extant cells than dilute bulk environments, for example, mineral/rock pores, microdroplets, amphiphile-based vesicles, and so on ,4,5,6. HThe key distinction of our hypothesis from previous protocell-centric scenarios for prebiotic chemistry is environmental in nature. The prebiotic utility of electrochemical gradients across, and low water activity . We suggest that scum layers may mediate molecular diffusion and water activity to help overcome the concentration problem, arithmetic demon, water paradox, and kinetic barriers to prebiotic chemistry.Many near-surface environments on Earth, including a large fraction of all lakes and oceans, are characterised by a surface microlayer of distinct physical chemistry and composition to an underlying bulk solution . AmphiphFinally, scum layers, the focus of this work, are insoluble organic-rich environments of macroscopic thickness at the water surface f 19]. H. H19]. HThere are several early Earth environments where scum layers could have accumulated via these mechanisms. Abiotic amphiphile sources include exogenous fatty acids  as well A body of theoretical, experimental, and field observations suggest that subaerial restricted basins could have accumulated high amphiphile concentrations, which then self-assembled into prebiotic compartments a-iii 8,,24,25,26Although experimentation and modelling will be needed to test these possibilities, the formation of scum layers on the prebiotic Earth does appear plausible in several environmental settings. Indeed, biologically active multi-compartmentalised scum layers, or biofilms, have been abundant on Earth throughout its history . Such biLongevity: The scum hypothesis posits that multi-compartmentalised and buoyant organic-rich environments may have been stronger candidates than free-floating individuals for driving certain reaction pathways in prebiotic chemistry. Much like a modern biofilm, built from constituent cells, a scum layer would have a longer lifespan in aggregate than that of its compartments—and therefore much longer than that of free-floating individuals [ividuals . Ensemblividuals . Long-liConservation of genetic material: Conservation of functional/informational polymers would plausibly occur, owing to slower overall diffusion between scum multi-compartments and their bulk environment, than would be observed for isolated compartments -irradiated regions of an aqueous environment, which increasingly appears to be a strict requirement for performing prebiotic chemistry on near-biological time-spans [me-spans . Conversme-spans ,36. The me-spans . Water ime-spans . Howeverme-spans . MeanwhiOpportunities for environmental cycling: A viable environmental setting for prebiotic chemistry also requires a mechanism of selective cycling, that is, the earliest stages of evolution by natural selection, wherein individual compartments in the scum represent a form of progenote [rogenote . This coCaveats: Standing in the way of these apparent advantages are some important caveats. Modern scum layers are apparently largely unstructured  [uctures) ,40. It tWe explore whether scum-type environments have any inherent utility in comparison to isolated compartments for driving prebiotically relevant reactions. Condensation reactions provide a useful case study for these purposes, being ubiquitous in prebiotic chemistry . Take, fThe equilibrium position of this condensation reaction is driven to the right by removal of water, where further forward reaction must occur in order to re-attain equilibrium. Several mechanisms may reduce water activity in naturally occurring prebiotic environments. Molecular crowding in a water-poor solution can result in confining cavities of especially low ensation . Secondaensation ,44,45. Tensation .We have previously defined multi-compartmentalised scum layers as buoyant accumulations of structured organic molecules, for example, vesicles and protocells. In addition to further opportunities for the formation of confining spaces, such structured amphiphiles have additional promising properties for driving prebiotic condensation chemistry ,50,51,52It is not the intention of this article to prove that scum-type media are specifically more advantageous for driving prebiotic condensation reactions than free-floating prebiotic compartments. However, there are indications that film, foam, and scum layers all have some thermodynamic potential for hosting (at least) prebiotic condensation reactions. With this basic level of prebiotic utility justified, we next explore whether the macroscopic nature of a scum layer may be particularly advantageous for initiating and sustaining dynamic equilibrium—a defining attribute of living cells.Cells must protect biomolecules  against degradation by hydrolysis. This protection is ultimately achieved by cycling between the cellular environment and dehydrating microenvironments formed during enzyme-substrate binding . Such cod), and total moles (n) of all species (i) undergoing reaction,The total chemical energy available to do work  that the compartment is a closed system, which lacks diffusive exchange with dilute surroundings, or 2) the surroundings are non-dilute: product can build up and diffuse back into the compartment at a rate such that an equilibrium position for the reaction is achieved. As the compartment becomes selectively leaky, maintaining the ability to induce forward reaction but more rapidly losing any product generated, a dynamic equilibrium is achieved. Compartments will approach a dilute limit once diffusion removes product as fast as it is generated in. At this dilute limit, the compartment is furthest from being at local thermochemical equilibrium: reactant and product concentrations will reflect those in the wider environment, and Equation  and a second multi-compartmentalised environment of prebiotic interest . The change in concentration of products in environment s (We construct a simple kinetic model of condensation chemistry and molecular diffusion taking place in a scum layer . We solvEquation ).(3)Δ[CProduct source and sink fluxes can be written as the product of environmental volumes (4)0=rfd, there may be diffusion of product from environment s into environment d. Given that the dilute environment is of much greater volume than the scum layer, we assume that d is approximately zero at steady state. We can simplify our equation for product sources and sinks in the scum layer accordingly,Even though there is no spontaneous forward reaction in environment s in our model scenario cannot be greater than the rate of forward reaction, that is, every product molecule produced is balanced via diffusion of a product molecule to environment d. In this case we can express product diffusion as some scaled value of the forward reaction rate ,(6)fdis). Finally, to obtain the forward and reverse reaction rates, for the general condensation reaction ))Kdiff caSubstituting for unknowns, we can then rearrange Equation  and solvs—our measure of whether the equilibrium achieved is more dynamic or thermochemical in nature—as,Using this information in conjunction with Equation , we can We use favorable values for rate constants and values for free energies of reaction that characterise at least some condensation reactions in desolvating  and a scum layer (environment s). The results of this calculation are shown in Using these assumptions, we calculate s is at its maximum. As the rate of diffusion between environment s and d is set to lower values  facilitation of forward reaction, may allow prebiotic reactions to proceed with constant supply—potentially helping to beat universal problems in prebiotic chemistry, such as the arithmetic demon [If diffusion is rapid enough to remove the majority of product  under a range of conditions . The formation of unstructured scum layers seems prebiotically plausible on current evidence—the requirements for multi-compartmentalisation, meanwhile, are less obvious. It could be argued that surface-charge interactions may act to repel close-packed prebiotic compartments that are chemically similar to one another. It is therefore possible that distinct membrane types may be required to overcome such electrostatic instabilities, or perhaps mixed systems will also be unstable, leading to unstructured systems of questionable prebiotic utility. We recommend, and intend to pursue, an experimental effort focused on the critical issue of scum formation pathways. If positive results are obtained for prebiotic plausibility of scum layers, future experiments can then begin to consider questions about prebiotic utility.Scum-type media should be tested for their ability to concentrate reactants from a dilute bulk environment, promote forward reaction, and selectively concentrate product molecules of interest. Results should be compared to those obtained using a mixture of free-floating prebiotic compartments. Important tests include the ability of scum layers to (1) concentrate phosphate and drive phosphorylation reactions, (2) harness a combination of surface scum UV irradiation and juxtaposed UV shielding to drive prebiotic chemistry, (3) drive general condensation polymerisation chemistry, as well as provide selection mechanisms that favour accumulation of longer/more useful products , and (4)Finally, the ability of scum layers to selectively concentrate molecules of increasing size should be considered. Longer molecules should diffuse more slowly across semi-permeable membranes , implyinWhilst we have pointed to the probable compatibility of scum layers with broadly important condensation reactions, there are many outstanding issues about the diversity, efficiency, and selectivity of prebiotic chemistry taking place in scum layers, as well as the overall placement of these environments in testable scenarios for the origin of life. Despite these outstanding issues, we note that buoyant bacterial communities have a deep ancestry on our planet—representing the earliest known biological structures in the rock record . We cont


Pancreatic cancer is a disease which is known to be highly deadly, with only 8% of people diagnosed surviving for more than 5 years. Multiple factors contribute to this poor survival rate, including a lack of early symptoms leading to late diagnosis and a high tendency for the cancer to rapidly spread throughout the body. Although there has been a lot of work in this area, we still lack an effective cure for pancreatic cancer. In this review, we aim to outline the latest research on possible treatments, including those which disrupt signalling pathways in the cell, disturb the cancers’ fuel sources, overload cancer cells with mutations, and re-direct the body’s immune system to attack the tumour. We also look at how we could eliminate a particularly aggressive group of cells within the tumour (‘cancer stem cells’) which are thought to be important to pancreatic cancer growth and spread.G12C mutant inhibitors. Moreover, new research, as outlined in this review, has highlighted promising new approaches including the targeting of the tumour microenvironment, enhancement of immunotherapies, epigenetic modulation, and destruction of CSCs.Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer that is strongly associated with poor prognosis and short median survival times. In stark contrast to the progress seen in other cancer types in recent decades, discoveries of new treatments in PDAC have been few and far between and there has been little improvement in overall survival (OS). The difficulty in treating this disease is multifactorial, contributed to by late presentation, difficult access to primary tumour sites, an ‘immunologically cold’ phenotype, and a strong tendency of recurrence likely driven by cancer stem cell (CSC) populations. Furthermore, apparently contrasting roles of tumour components (such as fibrotic stroma) and intracellular pathways (such as autophagy and TGFβ) have made it difficult to distinguish beneficial from detrimental drug targets. Despite this, progress has been made in the field, including the determination of mFOLFIRINOX as the standard-of-care adjuvant therapy and the discovery of KRAS Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer that arises in the exocrine glands of the pancreas  and compCytotoxic chemotherapy remains the mainstay of treatment in PDAC , althougOther strategies currently being investigated include pathway inhibition, metabolic targeting, DNA damage, immunotherapeutic agents, epigenetic targeting, tumour microenvironment (TME) modification, and destruction of cancer stem cells (CSCs). Pre-clinical successes have been seen in many of these fields, although, as of yet, they have not translated into clinical practice. Outside of therapeutics themselves, the determination of biomarkers to guide drug selection and improvements in pancreatic cancer screening and detection are areas that will be key in improving patient outcomes.It is now well established within the field that PDACs can be stratified into different molecular subtypes, which impact OS and therapeutic response ,17. ThisWork over recent years has attempted to target multiple pathways vital to growth and development in PDAC. This has been extensively reviewed by Stoica (2020)  and NevaAs with other cancer types, cellular metabolism in PDAC cells differs from non-cancer cells  55] and and55] aMore recently, mitochondrial metabolism has been of interest, with high levels of oxidative phosphorylation (OXPHOS) occurring in a subset of patients  despite Autophagy has been another area of interest in PDAC, being an important energy source that enables cancer cells to survive in the nutrient-deprived environment driven by desmoplasia and poor vascularity. Interestingly, this pathway performs opposing functions throughout the course of tumourigenesis. Initially, autophagy impairs tumourigenesis via the maintenance of cellular homeostasis, but later supports tumour growth by promoting fuel generation via intracellular organelle catabolism . This laReactive oxygen species (ROS) and antioxidants have similar contrasting roles over the course of PDAC development, with lower levels of ROS promoting tumour initiation at the pre-malignant phase and higher levels then supporting metastatic progression . AttemptEliciting DNA damage in order to promote cellular senescence has proved to be a successful approach in multiple cancer types. Most notably, the use of PARP inhibitors in BRCA mutated breast cancers has been a major step forward . PARP enATR and ATM, serine/threonine kinases involved in DNA damage response (DDR) signalling , are othDespite being widely regarded as an immunologically ‘cold’ cancer type there has been increasing interest in the potential of immunotherapies to treat PDAC . DisappoFor example, agonists to CD40, a member of the TNF receptor superfamily commonly expressed in immune cells, enabled antigen-presenting cells to increase levels of T-cell activation in tumour explants from a mouse KPC model . A phaseOutside of ICIs, multiple immunotherapies have been trialled with varying results. Mesothelin-targeting CAR-T cells have been trialled in six patients and were well tolerated, with one patient experiencing a 69% decrease in metabolically active tumour volume . HoweverVaccine therapy has also been tested, with GVAX, an allogeneic whole-cell cancer vaccine, expressing a granulocyte macrophage colony stimulating factor, being used in combination with chemotherapy during the ECLISPE trial . DisappoNew research has also aimed to target immune cell types outside of T cells. Neutrophils are abundant within the PDAC TME and are associated with poor clinical prognoses. They are known to promote tumour growth through ECM modulation and cytokine production and are also thought to aid in metastasis by supporting the survival of circulating cancer cells, enhancing stem cell characteristics of metastasis initiating cells, and establishing a hospitable niche for metastasis growth ,92,93. TThe abundance of fibrotic stroma, known as desmoplasia and derived mainly from pancreatic stellate cells, is a feature typical of PDAC. This dense extracellular matrix acts as a physical barrier preventing delivery of therapeutic agents to tumours and barring entry of immune cells to contribute to the ‘immunologically cold’ TME ,96,97. HThis is illustrated in the case of targeting hyaluronic acid (HA), a glycosaminoglycan (GAG) overexpressed in PDAC stroma and associated with low immune response and poor prognosis ,104. HA Cancer stem cells (CSCs) refer to a specific subset of cells within tumours, which display self-renewal and the ability to produce differentiated progeny, characteristics typically associated with normal stem cells. First described in 1997 in acute myeloid leukaemia , CSCs haMuch work has been carried out in this area aiming to characterise and selectively target pancreatic CSCs. JAK-STAT and Hedgehog pathways, known to be involved in CSC maintenance ,117, havSpecific metabolic targeting of CSCs is also a promising approach, with PCSCs highlighted as being dependent on oxidative phosphorylation in (OXPHOS) in order to survive. Inhibition of this pathway by metformin was able to produce rapid apoptosis specifically within the PDAC CSC population while non-CSC cells were less dramatically affected, only undergoing cell cycle arrest . FurtherHowever, it is important to note that increasing bodies of work suggest that PCSCs, and CSCs as a whole, are not a defined subpopulation but rather a state that non-CSC tumour cells are also able to enter . This meAlteration to epigenetic pathways within cells is known to be vital in cancer onset and progression and is thought to stimulate stem cell-like characteristics within tumour cells. Targeting epigenetic mechanisms in order to abolish this cellular plasticity and trap cells in a more restricted state, thus re-sensitising tumours to chemotherapy, is a promising approach to ablate PCSCs . HistoneThis could take the form of bromodomain and extra-terminal domain (BET) proteins, which are able to bind to acetylated lysine residues on histones and subsequently modulate gene expression. Promisingly, in PDAC, the bromodomain inhibitor I-BET 672 showed anti-tumour activity and cytotoxic synergy with gemcitabine in vitro and in vivo in pre-clinical research ,129. FurA further positive step forward has arisen from new models, which will enable clinicians to better tailor epigenetic therapy regimes to specific patients. Organoids, small 3D multicellular in vitro constructs able to mimic in vivo structures, have proven to be capable of recapitulating primary tumour characteristics in PDAC and modelling unique patient cancers ,137. ImpMore recently, Bian et al. were able to define MYC-high and MYC-low PDAC subtypes using a set of 16 transcriptional targets of c-MYC in patient-derived tumour xenograft (PDTX) models, with the MYC-high subgroup demonstrating increased sensitivity to the BET inhibitor JQ1 . This inDespite only modest improvements in patient outcomes, there have been multiple steps forward in PDAC treatment over the past few years. Importantly, mFOLFIRINOX has been highlighted as the standard-of-care adjuvant therapy, and combined chemotherapies such as paclitaxel and gemcitabine or FOLFIRINOX have been developed for advanced-stage disease. Furthermore, Olaparib maintenance therapy has been shown to improve outcomes in those with BRCA mutations and ICIs could enable long-term survival in patients with MMR deficient PDAC. As with oncology as a whole, in recent years, PDAC literature has demonstrated a clear shift from classical chemotherapy to more targeted approaches. This has led to a greater emphasis on strategies such as pathway inhibition, metabolic targeting, and attack of PCSCs. Unfortunately, there are multiple challenges affecting the development and application of precision medicine in PDAC, including rapid disease progression requiring a short timeframe between diagnosis and commencing treatment, the difficulty of tissue acquisition for tumour samples, and tumour heterogeneity, which is likely to limit specific pathway inhibitors to small subsets of patients . DespiteOutside of pathway inhibition, immunotherapeutic, stromal, and epigenetic approaches have demonstrated encouraging results and are set to become more prominent. Methods aiming to convert PDAC from an immune ‘cold’ to an immune ‘hot’ tumour could open up the disease to whole new areas of medicine through enabling immunotherapeutic targeting. Meanwhile, targeted therapies and epigenetic approaches show promise in the annihilation of PCSCs, known to be a prerequisite for the successful treatment of PDAC. These promising results over several areas of research will hopefully translate to improved patient outcomes in the coming years.


Nuclear imaging is a powerful non-invasive imaging technique that is rapidly developing in medical theranostics. Nuclear imaging requires radiolabeling isotopes for non-invasive imaging through the radioactive decay emission of the radionuclide. Nuclear imaging probes, commonly known as radiotracers, are radioisotope-labeled small molecules. Nanomaterials have shown potential as nuclear imaging probes for theranostic applications. By modifying the surface of nanomaterials, multifunctional radio-labeled nanomaterials can be obtained for in vivo biodistribution and targeting in initial animal imaging studies. Various surface modification strategies have been developed, and targeting moieties have been attached to the nanomaterials to render biocompatibility and enable specific targeting. Through integration of complementary imaging probes to a single nanoparticulate, multimodal molecular imaging can be performed as images with high sensitivity, resolution, and specificity. In this review, nanomaterial nuclear imaging probes including inorganic nanomaterials such as quantum dots (QDs), organic nanomaterials such as liposomes, and exosomes are summarized. These new developments in nanomaterials are expected to introduce a paradigm shift in nuclear imaging, thereby creating new opportunities for theranostic medical imaging tools. Molecular imaging is a non-invasive medical imaging technique capable of providing detailed images and information at molecular and cellular levels. Molecular imaging enables the visualization of cellular function through the different imaging modalities such as Positron Emission Tomography (PET) and Single-photon Emission Computed Tomography (SPECT). Imaging probes or biomarkers are used for tracking specific molecular pathways in particular targets in a living system ,2. The mBionanomaterials including organic and inorganic materials can be easily assimilated in living systems. These small-sized nanomaterials can penetrate into tiny capillaries and propagate across biological barriers, enabling detection of changes occurring at molecular levels. Combining these unique properties with being biocompatible has accelerated the application of nano-biomaterials for molecular imaging . Nanomat18F), carbon-11 (11C), gallium-68 (68Ga), iodine-124 (124I), and copper-64 (64Cu) for PET and technetium-99m (99mTc) for SPECT. 18Fluorinated 2-deoxy glucose (18F-FDG) is one of the most extensively used PET tracers. The design of 18F-FDG is for detecting the higher expression of glucose transporters in cancer cells Dalargin to poly(butylcyanoacrylate) nanoparticles injected systematically showed accumulations in the brain, indicating that the nanoparticle complex had crossed the BBB. The development of nanomaterials for specific targeting of the Central Nervous System (CNS) still remains extremely challenging due to the safety and efficacy concerns. However, there has been great advancement in the design of nanoparticle as carriers, drugs or probes to efficiently and safely propagate across the BBB. With more knowledge and better understanding of nanomaterials and their mechanisms, more success in the design of a nanomaterial probe may be achieved in targeting neurological diseases in the near future, creating more opportunities in therapeutics and diagnostic clinical applications.The limitations of crossing biological barriers such as the blood–brain barrier (BBB) and small capillaries can also be overcome using nanomaterials. These nanosized vessels are small enough to propagate across the barriers, delivering drugs to targeted sites . For exaBionanomaterials in particular have been widely investigated for theranostic applications due their natural ability to assimilate easily in body systems. These natural nanoparticles are equipped with unique properties such as biocompatibility, biodegradability, and stability in blood circulation, thereby overcoming some of the limitations that need to be considered in the design and development of a novel imaging probe . For exaTo improve their biocompatibility, targeting specificity, and easily controllable properties, nanomaterial-based nuclear imaging probes require necessary surface modifications such as coating and targeting moiety attachments, which include conjugation with peptides, antibodies, and many other targeting molecules . The modThe nanomaterial probe administered intravenously into the living system is assimilated either by the hepatobiliary system or by the renal system. To improve the biocompatibility, the nanoparticles are coated with hydrophilic polymers that include linear chain polymers of polyethylene glycol (PEG) and branched copolymers ,51. The Apart from PEG, several other coating polymers including polyvinyl pyrrollidone (PVP) , polyvinD. Dissociation constants determine the affinity with which the molecules can bind to each other. Melamide, succinimide, and other carbodiimide reactions are exploited to form covalent ester bond to link typically an alcohol group–carboxylic group to create an ester bond. Many such reactions are useful in molecular imaging for functionalizing the surface of the nanoparticles FBEM) for in vivo biodistribution and PET imaging studies Zr(oxinate)4 neutral complex. The radiometal complex was encapsulated into the exosomes and ensured that the surfaces of the exosome were not affected. The lipophilic complex [89Zr]Zr(oxinate)4 was able to pass through the lipid bilayer of the vesicles where 89Zr dissociated from the oxine ligands; dissociated free 89Zr then bound to intravesicular metal-chelating ligands, such as proteins and nucleic acids within the exosomes. Biodistribution studies using exosomes derived from pancreatic cancer cells (PANC1) via PET-CT displayed 89Zr-PANC1 exosomes accumulated in the liver, spleen, bladder, lymph nodes, and brain within 1 h of intravenous (iv.) injection [Intraluminal labeling of radionuclides to exosomes was demonstrated through the use of a metastable lipophilic [njection . As exosWith the development of nuclear imaging probes using nanomaterials, pathological processes at molecular levels can be revealed that could potentially help in early detection and interventions of various diseases. Enhancement of nanomaterial targeting capabilities as imaging probes has been explored through various approaches such as surface modifications and targeting moieties attachment. In this review, we summarized developments of nanomaterials as nuclear imaging probes through various modifications. The applications of nanomaterials have been extensively exploited for biocompatibility, in vitro stability, and in vivo biodistribution and animal imaging. Despite promising results from in vivo studies, there are still challenges that need to be overcome before nanomaterials can be applied as diagnostic and/or therapeutic nuclear medicine and imaging tools in clinical applications. For example, the reproducibility of various nanomaterials needs to be systematically determined, and the synthetic methods need to be standardized to ensure their biocompatibility so that theoretical modelling of nanomaterial integration can be guided for cell tracking and cell internalization. The review provides some future research directions to those with expertise in radiochemistry, nuclear imaging engineering, and other bio-imaging research. It is envisaged that the research thrust area combining these nanomaterials including exosomes can lead to a new generation of nuclear imaging probes and introducing a paradigm shift in theranostics.


Cardiovascular disease (CVD) is the leading cause of mortality, resulting in approximately one-third of deaths worldwide. Among CVD, acute myocardial infarctions (MI) is the leading cause of death. Current treatment modalities for treating CVD have improved over the years, but the demand for new and innovative therapies has been on the rise. The field of nanomedicine and nanotechnology has opened a new paradigm for treating damaged hearts by providing improved drug delivery methods, specifically targeting injured areas of the myocardium. With the advent of innovative biomaterials, newer therapeutics such as growth factors, stem cells, and exosomes have been successfully delivered to the injured myocardial tissue, promoting improvement in cardiac function. This review focuses on three major drug delivery modalities: nanoparticles, microspheres, and hydrogels, and their potential for treating damaged hearts following an MI. Cardiovascular disease (CVD) affects approximately 30 million adults in the United States of America each year, resulting on average in 647,000 deaths . MyocardIn certain cases of significant coronary artery disease, revascularization may be required by percutaneous coronary intervention and/or coronary artery bypass grafting (CABG) surgery  to restoGrowth factors are bioactive molecules that exhibit a paracrine effect on efferent cells, like endothelial and fibroblasts regulating the extracellular matrix (ECM) deposition . There hNanoparticles can be fabricated by various methods depending on the type of polymer and drug used. Most NPs used for drug delivery purposes are based on lipids or polymeric nanosystems such as DSPC, HSPC, PLA, and PLGA. These nanoparticles can be prepared by thin-film hydration techniques, solvent evaporation, and co-precipitation, respectively. However, more extensive fabrication techniques have been reviewed elsewhere –19. A vaThe microspheres can be fabricated by following similar techniques and using similar polymers; however, factors like homogenization speed, sonication time, the molecular weight of the polymers can significantly influence the size and homogeneity of the microspheres –35. It hHydrogels are extensively employed for the purpose of drug delivery. In general, hydrogels are fabricated by crosslinking hydrophilic polymers forming microporous three-dimensional structures. The porosity of hydrogels can be controlled by changing the degree of crosslinking, thereby influencing the rate of drug release. More importantly, hydrogels can entrap hydrophilic therapeutic proteins with high efficiency and exhibit sustained release as needed. A variety of hydrogels have been reported in the literature for various applications ranging from drug delivery, stem cell delivery, scaffolds to implants. Fabrication techniques for these hydrogels have been discussed elsewhere with extensive details. However, this review highlights the fabrication of a few smart hydrogels employed for cardiovascular applications.2 groups present on gelatin, forming self-crosslinked hydrogels to deliver growth factors and stem cells. Similarly, gellan gum, a naturally occurring polysaccharide, has been used to fabricate biomimetic scaffolds for cardiac cells. The hydrogel scaffold was first fabricated by oxidizing –OH groups present on gellan by sodium periodate, leading to aldehyde functionalized gellan. Later, the modified gellan was crosslinked with gelatin forming hydrogel scaffold . These hydrogels can further be modified by conjugating molecules on the activated functional groups present on the polymer by click-chemistry approaches. Functionalization of these polymers imparts newer functionalities to the hydrogel, like pH or temperature responsiveness, thus rendering them smart hydrogels. Researchers have used naturally occurring alginate that exhibits properties like shear thinning, excellent biocompatibility, and the ability to encapsulate living cells for cardiovascular and wound healing applications. The –OH group present on the alginate is oxidized by using oxidizing agents like sodium peroxide leading to the formation of alginate dialdehyde , 40. Thescaffold , 42. In scaffold . Biocompscaffold , 45. Furscaffold , 47.Synthetic polymer-based hydrogels display versatile properties with a high degree of tunability. One of the most commonly used polymers for tissue engineering and drug delivery applications is polyethylene glycol (PEG) . The funTo overcome the challenges faced by conventional drug delivery, nanoparticle-based formulations have been evolved. Nanoparticle formulations can encapsulate the therapeutic cargo and deliver it to the specific tissue, which gives NPs an edge over the conventional formulation. In addition, recent advancements in nanotechnology have led to developing newer materials for tissue-specific delivery ranging from organic  to inorganic  materials , 53. TheFor the delivery of bioactive molecules to damaged cardiac tissue, a substantial amount of research has focused on developing nanoparticles that can precisely deliver the therapeutics to the targeted tissue, as shown in in vivo study showed inhibition of scar formation and ventricular remodeling, thus displaying the cardioprotective nature of these NPs  (PLGA) NP's size on payload retention. Insulin-like growth factor (IGF-1) was employed for its cardioprotective effect. The results demonstrated that 60 nm PLGA-NPs exhibited a higher loading efficiency and were successfully retained at the injected site for 24 h (h) post-intramyocardial delivery. The hese NPs .Following an acute MI, the inflammatory response leads to the recruitment of pro-inflammatory macrophages and myofibroblasts at the infarcted site; this results in scar formation, leading to LV dysfunction. Nanoparticle-based therapies targeting and modulating these inflammatory cells' responses can present a promising approach for preventing scar formation. Tokutome et al. have reported PLGA-NPs entrapping pioglitazone for tuning the localized immune response by IV administration following a MI. This study's rationale was to inhibit the activation of macrophages to the M1 phenotype (pro-inflammatory). When entrapped in PLGA-NPs, pioglitazone was selectively delivered to the macrophages  and prevented the activation of macrophages, thus providing an anti-inflammatory response. The results demonstrated enhanced LV functioning and minimized scar formation. Furthermore, PLGA and pioglitazone's use imparts a more significant advantage as they are Food and Drug Administration approved and holds immense potential for clinical translation .in vitro. These reports demonstrated the feasibility of using NPs for collateral angiogenesis in the damaged myocardium  along with hypoxia/normoxia responsive VEGF plasmid DNA  resulting from reperfusion surpasses the normal cell's antioxidant mechanism, thus causing cell death. To prevent ischemic injury, attempts have been made to fabricate enzymes mimicking NPs as a possible route to myocardial salvage. Zhang et al. fabricated a biomimetic artificial nanozyme that mimics the activities of enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). These nanozymes target mitochondria by triphenylphosphine (TPP) and result in a significant quenching of ROS in the ischemic region following IV administration with subsequent improvement in LV functioning .Microspheres bring a whole different paradigm to the world of drug delivery due to their relatively small size and other unique properties such as improved drug release kinetics and stability. As microspheres hold a larger volume than NPs, they can encapsulate various drugs and exhibit sustained release. Different polymers have been explored for the fabrication of MPs, including natural  and synthetic , PLA, among others) . They cain vivo results indicated specific localization of the microparticles within 8 h of injection, which lasted for 72 h. It showed a sustained targeting ability of the microsystem essential to inhibit the acute inflammatory response following an MI. Moreover, the same microparticles showed no accumulation in cardiac tissue when injected in normal mice, further proving its targeting efficacy during MI , and hepatocyte growth factor (HGF) that were released in a sustained manner . The samThe inflammatory response following MI is induced by infiltrating pro-inflammatory macrophages within the infarcted region. It has been reported that macrophage polarization toward a reparative phenotype could prevent inflammation and repair the damaged tissues. Pascual-Gil et al. used polymer-based MPs as a way to achieve sustained and local delivery, specifically to the damaged heart focusing on the relationship between neuregulin-1(NRG-1) loaded PLGA-MPs and inflammation. They discovered that the intramyocardial delivery of NRG-1 PLGA-MPs improved the immune response post-MI. Interestingly, when these MPs were administered at various time points, they were shown to improve the anti-inflammatory response post-MI .NRG-1 and fibroblast growth factors (FGF) typically have a shorter half-life and do not have a specified target. However, the pharmacokinetics of both of these growth factors can be significantly improved when used as MPs leading to enhanced bioavailability. Therefore, researchers have increasingly used MPs to deliver these growth factors to treat the infarcted regions post-MI. Similarly, Formiga et al. have used the fibroblast growth factor-1 (FGF1) and NRG-1 growth factors encapsulated in PLGA-MPs and administered to the heart after an acute MI. Additionally, they were able to achieve a sustained release of the growth factors for a more extended period. Three months after intramyocardial administration of MPs, a significant reduction in infarct size was seen with a lower rate of fibrosis than control  and imprMicroparticles have also been explored for the localized delivery of stem cells and engraftment, as reported by Savi et al. The MPs encapsulating hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) were fabricated and complexed with adipose-derived stem cells. They successfully formed a substrate for the cell attachment and, in addition to it, acted as a reservoir for sustained release of growth factors. When these cells and MPs complexes were administered into mice models, they exhibited localized stem cell retention that stimulated the healing of the infarcted myocardium and restored hemodynamics within the region. However, there is an unmet need to address the arrhythmias arising from cell engraftments . AdditioMadonna et al. have employed fibronectin-coated PLGA-MPs encapsulating VEGF as a carrier for adipose tissue-mesenchymal stromal cells. These microcarriers were injected into the infarcted region of the heart. Similarly, treatment with these MPs demonstrated improvement in LV function . In a siFollowing an MI, a drastic increase in the ROS levels is observed in both intracellular and extracellular environments. Thus, enriching the environment with antioxidant enzymes could be used as a therapeutic strategy. Seshadri et al. reported polyketal MPs encapsulating SOD1 quenching ROS both in the intracellular and extracellular environment. On the contrary, only extracellular ROS was quenched when the free enzyme was administered. Furthermore, once injected intramyocardially, the MPs were retained for 3 days in the myocardium and prevented myocardial cell death .Hydrogels are one of the most extensively researched therapeutics for drug delivery and tissue engineering. They are composed of a crosslinked polymer network by forming covalent bonds, hydrogen bonds, and hydrophobic interactions between the polymer chains, thereby forming a depot . The useThe remodeling of the LV following an MI is the consequence of a cascade of inflammatory responses that activates ECM degrading enzymes. Attempts have been made to inhibit the activity of such enzymes from preventing cardiac remodeling after MI. Awada et al. has fabricated fibrin-based hydrogel entrapping polyelectrolyte complexes for myocardial repair. The rationale of the study was to avoid remodeling and to remuscularize the injured myocardium. To achieve this, metalloproteinases-3 inhibitor (TIMP-3) was entrapped in the fibrin matrix for rapid release and inhibition of ECM degradation. Secondly, for remuscularization, FGF-2 and stromal cell-derived factor 1 (SDF-1α) were formed into polyelectrolyte complexes for sustained release. Taken together, the timely release of these proteins from hydrogel showed significant improvement in cardiac functionality up to 8 weeks post-treatment .in vivo were found to improve cardiac function and boosted the generation of new blood vessels  based nanogels encapsulating cardiac stem cells for localized delivery to the myocardium. The stem cell embedded nanogels were injected in the immune-competent mice that showed no inflammatory immune response prolonging its retention, which otherwise was not observed with only stem cell injections. Results demonstrated a significant improvement in cardiac functionality and remuscularization when compared with only stem cell injections. More interestingly, the group treated with the only nanogel also showed considerable improvement, including scar inhibition, which could have been due to the formation of a physical barrier for immune cell infiltration suggesting the beneficial role of hydrogel . Among s vessels .in vivo. The use of small molecules like BIO that promotes cell cycles in adult cardiomyocytes can aid in remuscularization. Both the BIO and IGF-1 were encapsulated within gelatin NPs. The results demonstrated that the hybrid gel material, when administered to MI-induced rats, showed proliferation of cardiac cells, enhanced revascularization around the infarcted area, and improved overall LV function  and IGF-1 in a hybrid hydrogel system, which could help induce cardiac repair function .The miRNA has shown promise in treating various diseases, including CVD. The limiting factor for miRNA-based therapies is the lack of cell internalization and delivery vehicles. Along similar lines, Yang et al. reported a poly (PFBT) based miRNA delivery system. These NPs were loaded within a hydrogel matrix and administered intramyocardially. The miRNA was seen to trigger the proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs), and promote angiogenesis during hypoxia with lower toxicity than other treatments. Following hydrogel injection, LV function was improved by increasing the ejection fraction from 45–64% and reducing scar size from 20–10% while doubling the capillary density. This introduced a mechanism to deliver miRNA to restore LV function .Cardioprotective hydrogel based on crosslinked polyvinyl alcohol (PVA) has been reported by Li et al. The gel undergoes degradation when exposed to ROS. The advantage of using ROS-responsive hydrogels is that they can prevent ischemic reperfusion injury, where a burst of ROS is produced in response to reperfusion. More interestingly, the hydrogel can also release encapsulated cargo, like bFGF, in response to ROS. Furthermore, the authors have also demonstrated the feasibility of administering the hydrogel to the pericardial cavity in large animals and humans , resultiThe peptide-based hydrogels have opened up a newer array of biocompatible, self-assembled, and biodegradable materials. The primary advantage of a peptide hydrogel over others is the ability to tune the mechanical and chemical properties by altering the amino acid sequences. For example, Lin et al. reported a peptide hydrogel and employed it as a cell delivery material for cardiac repair. When the gel was injected into the myocardium of pigs following an acute MI resulting in heart failure, LV function was improved. More interestingly, the peptide gel alone was also able to show improvement in cardiac function. These findings agree with earlier reports suggesting that blank hydrogels themselves can impart therapeutic benefit and aid in repairing injured myocardium .in vitro and in vivo  is of great clinical importance. Therefore, magnetic NPs surface-functionalized with bio-probes was developed for rapid detection of early ACS diagnostics markers (NCT02226523). The principle behind the technology is to assess the magnetic oscillation of the NPs when exposed to alternating current, as these NPs interact with biomarker protein from the blood samples retrieved from patients; it undergoes aggregation resulting in the reduction of magnetic oscillation events known as immunomagnetic reduction . Thus, t3O4 magnetic NPs have been used for photothermal ablation of atherosclerotic plaque both non-invasively and in a minimally invasive manner (NCT01270139). The NIR light used exhibits deep tissue penetration making it possible for irradiation from outside the body resulting in a remarkable reduction in atherosclerotic plaque compared with controls without adverse events. This has emerged as a newer paradigm in treating atherosclerotic plaque . The autologous cardiac stem cells were implanted in bFGF gelatin hydrogel sheets in patients with severe LV dysfunction. The results demonstrated a decrease in scarring, and a significant enhancement in LV functioning within 6 months of implantation, and improved exercise tolerance. Moreover, the clinical data suggest that this biotherapy can be safely administered to patients .in situ crosslinked hydrogel  is under investigation for its applicability to prevent tissue inflammatory response caused by left ventricular assist device (LVAD). This hydrogel application is anticipated to avoid the inflammatory response and enable surgeons to remove VADs with minimal surgical interventions (NCT01244321) more easily.Similarly, alginate-based hydrogels are used as a support material and investigated to treat ischemic and non-ischemic cardiomyopathy (NCT04781660). Lastly, a polyethylene glycol-based The above-discussed clinical studies have demonstrated the efficacy of biomaterials. Their use as NPs, MPs, or hydrogels shows promise in treating CVD and can be employed in cardiac repair; however, there is a need for further investigation, innovation, and exploration of emerging smart biomaterials to impart therapeutic benefits.Overall, the discovery of newer functionalized polymers provides an excellent potential for achieving sustained delivery of bioactive growth factors in treating the damaged heart. The availability of advanced techniques at our disposal for fabricating nanoparticles and microparticles has facilitated the ease of upscaling at the industrial level. These have also made it possible to control the size from nano to micro range with a great degree of spatio-temporal control, directly affecting drug release rate. In addition, the ever-growing field of biomaterials has demonstrated the possibility that smart hydrogels with sophisticated properties like electroconductivity have further expanded the spectrum of their application in the cardiac field. Another potential future application of these innovative biocompatible polymers could be localized immune-modulation, which can offer therapeutic potential in alleviating adverse immune reactions and aid in healing the damaged heart. The localized availability of therapeutic bioactive molecules has shown promising results in preclinical studies. Therefore, these therapies can further be explored in a clinical setting to facilitate the translation from bench to bedside.SBA and MK: conceptualization and design. SBA, SA, DS, ZN, NP, HW, KB, WZ, NS, and MK: wrote and reviewed the manuscript. The input from all the authors was incorporated in finalizing the manuscript draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Angiosarcomas are a group of aggressive tumors of vascular origin. Although thought to be a rare cancer constituting just 1–2% of all soft tissue sarcomas, recent observations suggest that angiosarcomas are more common amongst Asian populations as compared to the West, suggesting the possibility of distinct genetic or environmental triggers influencing its pathogenesis. Advances in genomic sequencing efforts have led to the discovery of ultraviolet mutation signatures and high tumor mutation burden as common features of angiosarcoma of the head and neck. In addition, multi-omic analyses integrated with clinical data identified 3 subtypes characterized by distinctive etiological and biological phenotypes, with potential implications on precision therapy. The systemic and local immune milieu, as well as the presence of “giant” tumor cells, was also recently demonstrated to influence clinical behavior and patient outcomes, further highlighting complexities of this disease. Improvements in next generation “omic”-based technologies are expected to improve our understanding of angiosarcoma and guide the development of precision oncology in this rare cancer. Although rare, the clinical features and predisposing factors have been fairly well-described in the literature. Notably, the biological heterogeneity of this disease entity is manifested through its diverse forms of clinical presentation and behavior. At the anatomical level, angiosarcoma most often presents as a cutaneous malignancy in the head and neck region, but may also originate from other sites including the breast, viscera, trunk and extremity. In terms of etiology and risk factors, angiosarcoma arises either as primary nditions . CuriousUsing next generation sequencing, The Angiosarcoma Project - a large patient-partnered US-Canadian research initiative, presented evidence for ultraviolet mutagenesis as a common feature of angiosarcomas of the head and neck. This characteristic molecular signature underlies high tumor mutation burden (TMB), a potential biomarker for deriving benefit from checkpoint immunotherapy . This waThe prognosis of patients with angiosarcoma remains dismal, and patients with advanced disease usually do not survive beyond a year despite treatment with conventional chemotherapy or targeted agents. Determinants of survival typically include traditional clinical parameters such as age, tumor size, and stage of disease , while pConventional cytotoxic chemotherapy with anthracycline or taxane-based regimens are currently the most widely used in angiosarcomas, and while treatment response has been shown to improve survival, predictive biomarkers to guide treatment decisions are lacking. By examining the intra-tumoral immune responses and oncogenic signals, it was shown that the dysregulation of several immune-oncology pathways, including angiogenesis, matrix remodeling and metastasis, cytokine and chemokine signaling, as well as an accumulation of neutrophils and macrophages, resulted in primary chemoresistance .in vitro chemoresistance in comparison to regular angiosarcoma cells. The detailed analysis of single cell populations of giant cells and regular angiosarcoma cells revealed similar genomic landscapes, reaffirming their status as tumor-derived rather than infiltrating immune or stromal cells. Interestingly, when examined in the context of intra-tumoral immune signals, the presence of giant cells also correlated with immune pathway dysregulation including matrix remodeling and metastasis, cytokine and chemokine signaling, hinting at a complex interplay between tumor heterogeneity, dysregulated tumor microenvironment and chemoresistance which warrants further investigation.Apart from tumor microenvironment, tumor heterogeneity is also thought to contribute to primary chemoresistance and relapse. We examined resected angiosarcoma specimens and angiosarcoma cell-lines for giant cells, a rare (< 1%), bizarre subpopulation of malignant cells found across various tumor types and thought to contribute to treatment resistance . Giant cHow do we progress research in a rare cancer like angiosarcoma from here? Emerging next generation “omic” technologies beyond bulk whole genomic or transcriptomic sequencing are expected to dissect the composition of the tumor microenvironment of angiosarcoma at higher resolution . Single