Through several thousand million years of adaptive evolution, venomous animal species, toxic plants, and microorganisms have developed a large group of high-affinity and stable natural-based biomolecules, which have now been isolated and identified. Some of these biomolecules can specifically bind to human receptors/ligands, which means they have a great potential to be used as novel prototype drug candidates for disease treatments such as hypertension, cancer, diabetes, drug-resistant bacterial infections etc. In the last 2 decades, a lot of studies have been focused on certain types of specific molecular targets on cells, which can be recognized by natural-based biomolecules, to unveil their use in different aspects of clinical prognosis and therapeutic applications. However, many natural-based prototype drugs entering clinical trials are still struggling because their modes of action are not fully identified. Thus, we must do more lab-based and preclinical work toward this research direction.In this Research Topic, we brought together leading researchers to exchange and share their findings on the hot topics of novel identified venom-based and plant-derived natural biomolecules with potent pharmacological activity and their corresponding molecular mechanisms to fulfil the gap which is hindering their potential use as novel prototype drug candidates for disease treatments such as cancer, hypertension, diabetes, anti-bacterial infection, and neurological disorders.Chong et al. identified two cytotoxins from Naja sumatrana (NS-CTX) and Naja kaouthia (NK-CTX), which were highly potent in inhibiting the growth of lung cancer cell lines selectively. Chong’s research team further concluded that these cobra cytotoxins have such potential; however, their highly evolved and diet-adapted cytotoxic nature might have limited the intended application as a safe and effective drug. From their study, the data underscores the need for a comprehensive, fundamental investigation that addresses the selectivity and cell death mechanism of venom proteins in the quest for novel anticancer agents. However, the anticancer potential of these toxins could be further improved through structural modification to deliver a more specific, molecularly targeted cancer therapy in the future. In another research led by de Avelar Júnior et al., their findings confirmed that the peptide named LyeTx I-b is an excellent potential candidate for combined chemotherapy to treat breast cancer. In addition, de Avelar Júnior’s team also indicated that in vivo studies are essential to validate the use of LyeTx I-b as a therapeutic drug candidate, alone or combined with cisplatin. With the concept of using natural biomolecule and FDA-approved drugs as combined cancer therapy, Caballero et al. study provided another example to show two component prototypes named SF3 and SF4 for development as new drugs for glioblastoma treatment. Their findings stimulate studies to use these compounds in combination therapy with a rapamycin-like activity. Caballero’s team concluded that future studies would be conducted to characterize, synthesize the molecules, and evaluate the efficacy and safety in preclinical models.An important aspect of this Research Topic is to unleash the anticancer potentials of the natural biomolecules such as cytotoxins and peptides. Yang et al. carried out extensive metabolomic analysis. They found that 26 biomarkers can be regulated by folic acid via metabolic pathways of amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, citrate cycle, and pyruvate metabolism, which may be the potential therapeutic targets of folic acid against ischemic stroke. Folic acid, as a natural anti-fibrosis agent, has significant activity in protecting against middle cerebral artery occlusion-induced rat ischemic stroke model by delaying pathological development, reversing the metabolic biomarkers, and mainly regulating the perturbation in amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, citrate cycle, and pyruvate metabolism. The data from this study also indicated that this integrated metabolic biomarker screening platform could better understand the therapeutic effect and mechanism of drugs. In another study, Liu et al. identified a novel fibrinolytic protein named EPF3, with strong fibrinolytic activity, which was purified from Pheretima vulgaris. Liu’s research team constructed a three-dimensional structure of EPF3, and further performed a molecular docking analysis. They predicted that EPF3 could directly interact with antithrombotic target proteins, which was further confirmed by further studies. In conclusion, this antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain plasminogen activation. EPF3 possesses the potential to be developed into a promising antithrombotic agent.Cardiovascular disorders such as ischemic stroke and thrombotic disease have been considered major causes of death worldwide. Ogunyemi et al.) employed in vitro, in silico and in vivo studies to provide further insights into the alpha-amylase inhibitory potential of selected pregnane-rich chromatographic fractions and four steroidal pregnanes phytochemicals (SPPs). Their study showed that steroidal pregnane and pregnane glycosides were derived from Gongronema latifolium Benth. Might be exploited as inhibitors of pancreatic alpha-amylase as an oral policy for impeding postprandial blood glucose rise for the treatment of diabetes.Alpha-amylase is widely exploited as a drug target for preventing postprandial hyperglycemia in diabetes and other metabolic diseases. However, inhibition of this enzyme by plant-derived pregnanes is not fully understood. Herein, Ogunyemi and his research team  would reverse RA-induced changes in several cystometric and biochemical parameters characteristic of bladder overactivity and to thus check if this herbal supplement could be a reasonable strategy, as a future pharmacological treatment for patients with OAB. Zapała’s results concluded that Urox® were potent in reversing RA-induced changes in several cystometric and biochemical parameters that are determinants of OAB. Furthermore, they observed no effects on basic cardiovascular parameters and daily urine output, which may promote the initiation of the analysis of its safety in humans.Antimuscarinic drugs are well-known agents for a kind of neurological disorder that have earned their place in the management of overactive bladder (OAB). The study conducted by Amorim-Carmo et al. described some strategies to optimize scorpion AMPs. Amorim-Crmo and his team addressed the primary sequence, biotechnological potential, and characteristics that should be considered when developing an AMP derived from scorpion venoms. In addition, this review contributes toward improving the understanding of rationally designing new molecules, targeting functional AMPs that may have a therapeutic application.Finally, a review article by Overall, the research and review articles included in this Research Topic expand our knowledge of the latest studies in venom-based and plant-derived natural biomolecules. These bioactive molecules with potent pharmacological activities have their corresponding molecular mechanisms to fill the gap hindering the potential use as novel prototype drug candidates for disease treatments. In addition, the state-of-the-art analytic techniques and research platforms that have been widely adopted in the last half decade will continue to contribute to future studies in the field of drug discovery from natural sources.


Internet gaming is acknowledged as a common leisure activity among adolescents yet only a little known about the pscyhodevelopmental roots. Emotion recognition and metacognition which are proved to be determinants in behavioral disorders may be considered salient factors in also internet gaming disorder (IGD).The research to date has focused on psychological comorbidities rather than risk factors , whereas, improved early intervention would be more likely if risk factors were well defined, especially before the onset of the illness. The objective of this study was to investigate these areas by analyzing associations between metacognitive beliefs, emotion recognition, and IGD among late adolescents with tendency for pathological gaming behavior.806 high school students were recruited and instructed to take Internet Gaming Disorder Scale (IGDT), Meta-Cognitions Questionnaire for Children and Adolescents (MCQ-C) and Reading the Mind in the Eyes Test - Children’s Version (RMET).Mean comparisons corresponding to IGD risk potential showed that positive meta-worry and superstitious, punishment, and responsibility beliefs of MCQ-C were significantly higher in the risky group, whereas; RMET neutral subtest was significantly higher in the non-risky group. Additionally, a positive correlation was found in all subtests of MCQ-C, RMET positive emotions and IGD. Regression analysis revealed that RMET positive subtest and positive meta-worry of MCQ-C predict IGD risk.The findings of the study partially corroborated the early results found among early adolescents; however, they also indicated the requirement of distinct therapeutic approach for cognitive interventions of IGD in late adolescence period.No significant relationships.


Idiopathic generalized epilepsy (IGE) represents a common form of epilepsy in both adult and pediatric epilepsy units. Although IGE has been long considered a relatively benign epilepsy syndrome, a remarkable proportion of patients could be refractory to treatment. While some clinical prognostic factors have been largely validated among IGE patients, the impact of routine electroencephalography (EEG) findings in predicting drug resistance is still controversial and a growing number of authors highlighted the potential importance of capturing the sleep state in this setting. In addition, the development of advanced computational techniques to analyze EEG data has opened new opportunities in the identification of reliable and reproducible biomarkers of drug resistance in IGE patients. In this manuscript, we summarize the EEG findings associated with treatment resistance in IGE by reviewing the results of studies considering standard EEGs, 24-h EEG recordings, and resting-state protocols. We discuss the role of 24-h EEG recordings in assessing seizure recurrence in light of the potential prognostic relevance of generalized fast discharges occurring during sleep. In addition, we highlight new and promising biomarkers as identified by advanced EEG analysis, including hypothesis-driven functional connectivity measures of background activity and data-driven quantitative findings revealed by machine learning approaches. Finally, we thoroughly discuss the methodological limitations observed in existing studies and briefly outline future directions to identify reliable and replicable EEG biomarkers in IGE patients. Idiopathic generalized epilepsy (IGE) accounts for almost 20% of all epilepsies in adults and children and is associated with a strong genetic background . IGE hasIGE has traditionally been considered to have a favorable prognosis, though increasing evidence has shown that up to 40% of patients are refractory to treatment and a proportion of them may experience unremitting seizures throughout their clinical history . Drug reIn this manuscript, we briefly review the EEG features commonly observed in IGE patients and analyze those associated with drug resistance by examining the findings observed during both routine EEG and prolonged ambulatory EEG recordings (paEEG). We also provide an overview of the recent discoveries in the field of advanced EEG analysis. All studies were evaluated with special attention to the methodological limitations. Finally, we discuss future directions to identify reliable and replicable EEG biomarkers in IGE patients.A literature search for relevant articles was carried out on online databases  covering a period from 1980 to March 2022 using the following medical subject headings: IGE , EEG , drug resistance , biomarkers , and prognosis. The EEG hallmark of IGE syndromes is the occurrence of interictal symmetric, bisynchronous, and regular generalized spike-wave discharges (GSWDs) and generalized polyspike-wave discharges (GPSWDs) in the context of normal EEG background activity. Interictal discharges are usually more frequent during non-rapid eye movement sleep (NREM) and in the first hour after awakening  7]. The. The7]. While ictal and interictal EEG patterns are almost indistinguishable in absence seizures, different ictal EEG patterns are usually observed in myoclonic and generalized tonic-clonic seizures. The ictal EEG pattern in the latter is typically characterized by a diffuse attenuation in EEG amplitude followed by generalized rhythmic activity that increases in amplitude and decreases in frequency (tonic phase), eventually followed by repetitive polyspike-wave complexes (clonic phase) . In myocAtypical interictal EEG abnormalities can also be frequently encountered, such as focal slowing or focal epileptiform discharges (EDs), asymmetric or asynchronous discharges, irregular morphology of GSWDs/GPSWDs, and generalized fast discharges . The reported frequency of these atypical EEG findings is highly variable across different studies (ranging from 13% to 66.4%) due to the heterogeneous criteria used to define them and classify IGE syndromes, as well as the methodological discrepancies among studies  ,11. The When considering activation procedures, hyperventilation and intermittent photic stimulation are the most widely used during routine EEG recordings. Hyperventilation may facilitate EDs and typically triggers absence seizures in CAE/juvenile absence epilepsy patients . IntermiAn overview of the frequency of the above-mentioned EEG patterns is illustrated in During the previous decades, several authors have tried to identify possible EEG findings that predict treatment response. Special attention has been paid to the prognostic significance of atypical EEG findings, particularly focal EEG abnormalities, during routine exams. Matsuoka first reported the association between focal EEG abnormalities and poor treatment response in JME patients , and hisAnother debated EEG variable is represented by PPR. The most solid evidence comes from studies evaluating PPR in CAE patients prior to initiating antiseizure medications (ASM). In a retrospective study, Incecik et al. reviewed the EEGs of 52 drug-naïve CAE patients and evaluated the baseline variables predicting failure to the first prescribed ASM . They foWhen considering a manual assessment of the background activity, the presence of EEG slowing was found to be associated with a lack of medication response in CAE patients in two well-known studies in which baseline EEG was reviewed and patient outcome was assessed prospectively and/or retrospectively ,37. HoweEye-closure sensitivity consists of GSWDs/GPSWDs that typically appear within 1–3 s after eye closure and last 1–4 s, which may occur across different IGE and non-IGE genetic generalized syndromes  ,40. AmonFollowing the study by Matsuoka et al. that described frequent activation of GSWDs/GPSWDs during demanding mental activities , a few aFinally, the occurrence of early cessation of interictal EDs after ASM initiation with respect to baseline EEG was found to be a reliable indicator of good seizure outcome in IGE in a study performed by Steinhoff et al. . SeveralThe bidirectional relationship between sleep and epilepsy has long been investigated. While NREM sleep promotes the hypersynchrony of neuronal networks, thus facilitating the occurrence of EDs , poor seHowever, Sun et al. recently explored GPTs (defined as bursts of at least five spikes lasting less than 1 s) and GPFA  occurring during sleep in a relatively large cohort of IGE patients : in fact, GPFA was actually observed in 21.1% of drug-resistant patients and in no drug-responsive one. The prognostic significance of GPT and GPFA was later replicated in other single-center retrospective studies ,60. MoreUnfortunately, all these studies were burdened by remarkable methodological limitations, particularly regarding their retrospective design and the timing of sleep EEG exams. Since only post-treatment paEEG recordings were evaluated in most patients, the predictive role of GPFA and GPT cannot be definitely confirmed, and these patterns might also represent an epiphenomenon of prolonged unremitting seizures in refractory patients. This hypothesis has been partially corroborated by the results of Jensen and colleagues, who tried to overcome the methodological limitations regarding the timing of EEG recording . The autAnother EEG finding that has been investigated during the last decade as a potential biomarker of drug resistance is GSWD/GPSWD duration. Seneviratne et al. retrospectively reviewed the 24-h EEGs of 105 IGE patients and investigated the association between the duration of seizure remission  and several EEG features . They anHowever, in the aforementioned study conducted by Jensen and colleagues,  the progNeurophysiology practice is currently based mainly on the visual inspection of EEG recordings, which may provide only a limited amount of information, especially in the context of partially stereotyped EEG patterns, as in IGE . In receThe prognostic significance of EEG background slowing in IGE patients has been assessed through quantitative EEG power analysis by Abela and colleagues, who found a correlation between a shift to slower alpha rhythms, especially over posterior regions of the brain, and poor seizure control during the 12 months prior to study, suggesting a more pronounced cortical dysfunction in drug-resistant patients . HoweverOther authors explored EEG markers of treatment resistance by using current source density measures in ED-free resting-state EEGs to compare differences in pre and posttreatment EEG between responders and non-responders . The autIn addition, Yeom et al. explored the prognostic significance of the localization of the GSWD generator in CAE patients , followiResting-state interictal background EEG  can be used to analyze functional connectivity between different cortical areas by measuring the statistical dependence between their signals. Several EEG-based functional connectivity measures have been developed based on amplitude, frequency, and phase ,76. AmonHowever, since IGE pathophysiology has been consistently attributed to the involvement of widespread brain regions and extensive networks, complex brain network investigation based on graph theory might be more valuable to identify markers of treatment resistance ,80,81,82Additionally, microstate analysis is an increasingly applied method to assess EEG-based brain network dynamics in the millisecond range . Past stFinally, other authors developed a new machine learning method to simultaneously assess several quantitative EEG features in drug-naïve IGE patients in order to automatically discriminate between responders and non-responders with artificial intelligence algorithms. Despite the low number of included patients, one study found an excellent discrimination ability of their support vector machine model based on 10 different EEG features . Future After an extensive literature review, we could identify some EEG findings associated with treatment resistance in IGE patients, which are summarized in However, despite some interesting results, it is worth to mention that the previously highlighted limitations prevent us from recommending the use of these EEG patterns/features as prognostic biomarkers in routine clinical practice: in fact, prospective studies with systematic use of 24-h EEG recordings are warranted to draw more solid conclusions. Recently validated automated methods could be used to reduce interrater differences and the time needed to manually review paEEGs, thus allowing to conduct large-scale studies ,94.Regarding advanced EEG analysis, some of the reviewed computational techniques (including machine learning approaches) yielded excellent results and prompted future multicenter studies including large cohorts of patients to validate these findings. Apart from its prognostic impact, a better understanding of the quantitative EEG features and connectivity measures associated with treatment resistance may also provide crucial insights into the pathophysiological mechanisms underlying drug resistance in IGE, which may help the development of new treatments for refractory cases, including targeted neuromodulation approaches.


The formation of amyloid fibers is associated with a diverse range of disease and phenotypic states. These amyloid fibers often assemble into multi-protofibril, high-order architectures in vivo and in vitro. Prion propagation in yeast, an amyloid-based process, represents an attractive model to explore the link between these aggregation states and the biological consequences of amyloid dynamics. Here, we integrate the current state of knowledge, highlight opportunities for further insight, and draw parallels to more complex systems in vitro. Evidence suggests that high-order fibril architectures are present ex vivo from disease relevant environments and under permissive conditions in vivo in yeast, including but not limited to those leading to prion formation or instability. The biological significance of these latter amyloid architectures or how they may be regulated is, however, complicated by inconsistent experimental conditions and analytical methods, although the Hsp70 chaperone Ssa1/2 is likely involved. Transition between assembly states could form a mechanistic basis to explain some confounding observations surrounding prion regulation but is limited by a lack of unified methodology to biophysically compare these assembly states. Future exciting experimental entryways may offer opportunities for further insight. Normal cellular homeostasis requires maintenance of a correctly folded proteome , which is enabled by a network of tightly regulated protein-quality control factors including chaperones and proteases . When thAmyloidogenic proteins provide an excellent model to elucidate this interplay. These proteins can access alternative conformations under physiological conditions and assemble into aggregates that are associated with either new functional or disease states. Each of these phenotypic states can often exist within a range of distinct severities and stabilities dictated by the underlying fold of the protein determining them . The perSc leads to progressive and infectious neurodegenerative disease for which there is currently no treatment or cure  [PSI+] prion is determined by the amyloid state of the translation termination factor Sup35, which results in a partial loss of function and the associated read-through of stop codons [psi−] state [PSI+] state in growing yeast cultures exists within a balanced cycle of Sup35 expression, conversion into amyloid aggregates, fragmentation of these aggregates by chaperones, and transmission of these aggregates to daughter cells at cell division to maintain both the amyloid form and sufficient Sup35 activity to support viability  alone can lack any detectable infectious activity in either cell culture or rodent bioassays . HoweverSc . Nonethein vitro ,44, thouin vitro . Subjectl prions  through l prions  and orall prions  routes oOne possible explanation is structural differences between fibrils. Indeed, structural analysis of rec-PrP subjected to PMCA with or without the addition of co-factors found infectious preparations to contain smaller oligomeric species, while non-infectious material contained longer fibers , althougPSI+] strain [psi−] cells to the [PSI+] state  strain . Most de+] state , suggestPSI+] variants when transfected into yeast cells [psi−] strain  [PSI+] variants by expressing Sup35 mutants  prion, Sup35 exists as a heterogeneous mixture of aggregate sizes in vivo as determined by fractionation of [PSI+] lysates on sucrose gradients and by migration by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) [PSI+] lysates, in contrast to detergent-sensitivity of aggregates formed by Sup35 overexpression in [psi−] strains in the absence of prion conversion [PSI+] when transfected into a [psi−] strain also increases  lysates and analysis by SDS-PAGE or RT-PCR reveals co-captured proteins and ribosomal RNA [Podospora anserina [PSI+] yeast lysates are consistent with the bundling of fibrils even in the presence of SDS [PSI+] yeast lysates in the presence of Tween-20 [PSI+] aggregates purified in the absence of detergent are present as short, single filaments and ring-like oligomers [PSI+] aggregates as a template  aggregates under endogenous expression conditions, the direct comparison of the species observed under native and detergent conditions, and the correlation of these observations to biological outcomes. With many experimental conditions to induce transitions between the [psi−] and [PSI+] states having been identified, such approaches have the potential to directly correlate higher-order structure to biological impact.While there is clear evidence from EM analysis of yeast cells that these kinds of higher-order architectures can be present in vivo, they have only been observed with the expression of the isolated PrD to levels well beyond that of the endogenous protein ,12,14,97psi−] cell expressing Sup35-GFP and a fluorescent reporter of the [PSI+] phenotype to a [PSI+] cell expressing untagged Sup35. Upon cell fusion, the prion phenotype emerges concomitantly with Sup35 incorporation into foci at the single-cell level  induction. In these analyses, Sup35-GFP or PrD-GFP fusions are overexpressed at high levels, and the emergent aggregation patterns observed by fluorescence microscopy in live cells. Induction of [PSI+] by this method is associated with the presence of Sup35 aggregates detected as large foci and/or ring-like structures, which have been interpreted as filament-containing complexes , [PIN+] and HET-s prions [PSI+] strain with LatA makes PrD-GFP adopt a diffuse pattern  induction [PSI+] is induced, and gene mutations that prevent [PSI+] induction also do not support the formation of ring structures [PSI+] without continued high levels of Sup35-PrD  prion, the complexity of these dynamics cannot be currently reduced to a molecular mechanism. Indeed, conditions that lead to [PSI+] appearance, such as Sup35 overexpression and Hsp104 inhibition, correlate with transition into laterally associated multi-fibrillar structures as observed by cryo-EM [PSI+] appearance coupled with parallel analyses of structural dynamics may offer experimental entryways to gain this insight.While the appearance and evolution of these structures clearly correlate with the emergence of the  particles adopt in vivo during this time of stable equilibrium, and how does it change during the process of prion loss? At a minimum, a stable prion state requires the formation of an aggregate capable of templating its continual growth by interaction with and conversion of native-state protein, being fragmented to create multiple complexes, and being transmitted to other cells . The minPSI+] inheritance. The presence of visible foci alone may phenotypically indicate the prion state, but these structures do not necessarily represent the propagon. Fluorescence microscopy methods and their limit of detection have improved over time, so the interpretations derived from the presence or absence of visible foci should be taken in context of the probable limits of detection at the time of publication. In addition, previous studies have visualized different fusion proteins expressed to different levels and in different metabolic contexts, complicating interpretations. Nonetheless, multiple studies combining genetic and microscopic analyses indicate that changes in the sizes of visible Sup35 foci impacts [PSI+] becomes unstable upon Sup35 overexpression [PSI+] induction, suggesting that these structures arise when chaperone activity becomes limited. As was true during induction, the rod-shaped foci formed during GdnHCl treatment are composed of longer filaments laterally associated in parallel to each other  loss [PSI+] loss during Hsp104 overexpression: fragmentation inhibition  loss , decouplI+] loss , the latI+] loss . Three mhibition , transmihibition , and dishibition . Howeverhibition ,134,135 PSI+] stability under certain conditions [PSI+] lysates on sucrose gradients has not been assessed, structural spacing between fibrils in vivo as determined by cryoEM does indeed accommodate the possibility of these kinds of interactions [SSA1-21 mutant background under native conditions, although this effect appears to be independent of the size of detergent-resistant species observed by SDD-AGE which remain unchanged [SSA1-21 mutation. While the effects are complex and complicated by variations in experimental conditions, including fusion proteins, expression levels, and metabolic states, these observations are intriguing and could represent a true measurable and regulated transition between higher-order architectural states with biological implications. Indeed, if titration of Ssa1/2 induces Sup35 complexes to adopt a larger, less mobile form, these species would rapidly lose transmissibility, potentially explaining the faster kinetics of curing by Hsp104 overexpression in comparison with Hsp104 inhibition which requires continued fibril growth to exceed the size threshold for transmission [Despite the limitations on currently available observations, transition between states of higher-order architecture during Hsp104 overexpression could help explain these data. For example, increasing Hsp104 levels has been shown to titrate Ssa1/2 from fluorescent foci formed during Sup35 PrD overexpression , and Ssanditions ,137. Theractions , and sevractions , and Supsmission ,101. ComEvidence is compelling that prion complexes can adopt high-order assemblies in vivo and that these assemblies appear structurally ordered. The biophysical nature of these higher-order architectures or their role in amyloid biology is, however, still unclear. To resolve this unknown, a concerted effort to isolate, quantify, and assess the biological impact of distinct protein assemblies in parallel is crucial. As noted throughout this review, an ensemble of approaches and methodologies have been developed, but these are most frequently used in isolation of one another, raising questions as to how the methods relate to one another and the true nature of the aggregation states that they have described.Integrating current studies to reveal the correlation between amyloid assemblies and their biologic impact may, however, provide pathways to resolving the existing ambiguity. Specifically, unique conditions are known to promote the assembly of distinct structures in vitro. The biologic activity of these structures should be assessed in parallel using existing cell-based and/or animal models, provided that the abundance of each can be determined by quantifying particle number. Similarly, in vivo models can be subjected to conditions and/or treatments that change assembly state. By using common protocols to extract these species in parallel, direct comparison of their structures using existing methods becomes possible. Carefully controlled application of existing methodologies in parallel and under the same experimental treatment regimens will undoubtably help relate these observations and define the true nature and biological significance of these unique higher-order assemblies.


The antitumor potential of personalized immunotherapy, including adoptive T-cell therapy, has been shown in both preclinical and clinical studies. Combining cell therapy with targeted metabolic interventions can further enhance therapeutic outcomes in terms of magnitude and durability. The ability of a T cell receptor to recognize peptides derived from tumor neoantigens allows for a robust yet specific response against cancer cells while sparing healthy tissue. However, there exist challenges to adoptive T cell therapy such as a suppressive tumor milieu, the fitness and survival of transferred cells, and tumor escape, all of which can be targeted to further enhance the antitumor potential of T cell receptor-engineered T cell (TCR-T) therapy. Here, we explore current strategies involving metabolic reprogramming of both the tumor microenvironment and the cell product, which can lead to increased T cell proliferation, survival, and anti-tumor cytotoxicity. In addition, we highlight potential metabolic pathways and targets which can be leveraged to improve engraftment of transferred cells and obviate the need for lymphodepletion, while minimizing off-target effects. Metabolic signaling is delicately balanced, and we demonstrate the need for thoughtful and precise interventions that are tailored for the unique characteristics of each tumor. Through improved understanding of the interplay between immunometabolism, tumor resistance, and T cell signaling, we can improve current treatment regimens and open the door to potential synergistic combinations. Success of immunotherapy directed against cancer is predicated on the fact that tumor cells can be specifically targeted by various populations of immune cells, minimizing damage to healthy tissue while exclusively eliminating cancerous growth. Adoptive cellular therapy (ACT) for solid tumors has gained increased attention in recent years following its initial success in metastatic melanoma  and moreTumor cells arise from transformations that fundamentally alter a cell’s proliferative and survival capacities. As such, the metabolic demands for cancer cells are different than those of a nonmalignant cell. In 1956, Warburg observed increased rates of glycolysis and lactate production in tumors, indicative of a preference for aerobic glycolysis over the more efficient oxidative phosphorylation pathway for generating ATP , a term In addition to metabolite synthesis, cells normally integrate information regarding nutrient availability and stress to regulate survival and proliferation; unfortunately, tumor cells have co-opted many aspects of these signaling cascades, including ones involving Mammalian Target of Rapamycin (mTOR), in order to sustain rapid division . mTOR haThe high level of glucose consumption by tumor cells provides not only fuel for glycolysis, but also increases flux through the Pentose Phosphate Pathway (PPP), which is consequently also elevated in malignant cells compared to normal cells . This paEvidence of plasticity in T cell metabolism has been demonstrated in the context of multiple metabolic pathways. Glutamine provides the fuel needed for the tricarboxylic acid (TCA) cycle, which in turn generates the intermediates needed for lipid, protein, and nucleic acid synthesis crucial for rapidly proliferating cells . PredictAs part of Warburg physiology, large amounts of lactic acid are generated as byproducts of aerobic glycolysis, which is subsequently secreted from the tumor cells and contributes to the acidification of the tumor compartment . This inin vivo with an enhanced kynureninase to degrade kynurenine increased infiltration and proliferation of cytotoxic CD8s, resulting in improved tumor control in a variety of tumor types. Interestingly, the treatment had no effect on tumor growth in immunocompromised mice, suggesting that the antitumor mechanism of kynureninase is contingent upon the presence of immune cells , the first and rate-limiting step of tryptophan degradation as part of the kynurenine pathway, which fuels many essential biological processes including energy metabolism and generation of the co-factor Nicotinamide adenine dinucleotide (NAD+) . IDO is ne cells . The exaEffective antitumor immune therapy is predicated on the function of dendritic cells (DCs), which uptake and present tumor antigen to activate naive T cells and induce their proliferation . DC ablaA similar tale of functional balance can be told for the role of adenosine monophosphate-activated protein Kinase (AMPK) pathway in DC activation and T cell effector capabilities. AMPK is crucial to regulating energy balance within the cell, turning on ATP-producing catabolic pathways when cellular stress reduces the amount of ATP available . AMPK acIn TCR-T, human T cells are engineered with a TCR of interest and then infused into the patient, where the hope is that those cells will traffic to the tumor and surrounding lymph nodes, setting up long-lived cytotoxic populations which will attack the tumor. In order for this therapy to be successful, the T cells must be able to survive in the TME, and thus are subject to many of the same pitfalls that plague immunotherapy in general – namely, exhaustion of effector cells, poor viability, and loss of effector function . The commem) (EM) phenotype. The cells also increased their expression of glucose-transporter 1 (GLUT1) to increase glucose uptake  to glycolysis to meet energy requirements , 86, Fimem) . Inhibitmem)   . Glucosee uptake , which ie uptake   in activated cells can help enhance antitumor function. In a mouse model of lymphoma, adoptively transferred effector CD8s preconditioned in low glucose conditions were able to mediate complete clearance of tumor, with an enhanced effector phenotype and increased number in circulation. TGR T cells redirected their utilization of glucose carbons to the TCA cycle rather than glycolysis, resulting in a drastic increase in ATP generation, and were better poised to handle oxidative stress due to the sustained synthesis of the reactive oxidative species (ROS) scavenger glutathione – two characteristics which allow these T cells to better survive in the TME compared to cells which have not undergone preconditioning. Metabolomic interrogation of these TGR effector cells showed that these cells were able to enhance their glucose uptake following re-exposure to glucose and increase flux through the PPP , both ofIn a similar vein, glutamine restriction can also yield comparable results to glucose restriction. T cells cultured in glutamine depleted conditions or treated with inhibitors of glutamine metabolism were shown to increase survival and promote tumor clearance. Phenotyping of the tumor infiltrate showed that glutamine restricted T cells displayed lower levels of exhaustion markers and increased proliferation, cytokine production, and markers associated with long-lived memory populations. Metabolically, these cells showed increased mitochondrial spare respiratory capacity, reduced mitochondrial ROS, and increased glycolytic flux, indicating that glutamine restriction can skew cells metabolically towards a memory-like phenotype with greater self-renewal and antitumor capabilities . Just asDuring glycolysis, the generation of pyruvate from phosphoenolpyruvate (PEP) by pyruvate kinase generates one molecule of ATP . Knockdode novo pyrimidine synthesis pathway. Inhibition of CAD decreases available pre-rRNA, which limits the ribosomal biogenesis required for synthesizing new proteins to support proliferation and cytokine production. Overexpression of CAD was shown to enhance proliferation and improve cytokine production upon rechallenge , a binding partner of mTORC2 crucial to its activation, was able to generate robust T cell responses similar to wild type controls upon vaccination, indicating that deficiencies in mTORC2 did not affect effector capabilities. Interestingly, upon rechallenge, these cells displayed increased markers of memory, robust cytokine production, and were present at a higher frequency in the spleen. This is possibly due to the fact that mTORC2 prevents activation and nuclear translocation of Forkhead Box O1 (FOXO1), a transcription factor which is responsible for upregulation of memory-associated transcripts like L-selectin (CD62L) and C-C Motif Chemokine Receptor 7 (CCR7) . Additioin vitro pharmacological or genetic screens to determine optimal treatment regimens for personalized metabolic medicine. A recent study described insights gained from such a screening process, where T-cell and tumor cells were placed in coculture in the presence of 240 different pharmacological perturbations to assess which compounds influenced T cell activation. Not only were they able to gain understandings about vulnerabilities of T cells and their underlying mechanisms, but they were also able to identify compounds in which T cells were differentially affected compared to tumor cells. For example, in the context of a B16-OVA tumor with OT-1 T cells, glutathione peroxidase 4 (GPX4) inhibition induced ferroptosis in CD8+ T cells while leaving tumor cells mostly unaffected, and this sensitivity was attributed to acyl-CoA synthetase long-chain family member 4 (ACSL4)  , one of  (ACSL4) . One couThe fundamental principle behind TCR-T is that tumor-specific TCRs can be engineered onto a bulk population of T cells, and this population can then be redirected to target the tumor while sparing healthy tissue . HoweverAnother challenge in TCR-T has been optimizing the phenotype of the TCR-engineered T cell population throughout the expansion process to yield long-lived cells which will persist and carry out effector functions in their new niche. It has been found that a central memory phenotype of adoptively transferred T cells was optimal for obtaining the best antitumor responses in patients ; howeverT cell sensitivity to their cognate peptide/major histocompatibility molecule (MHC) appear to be governed by both intrinsic and induced factors – there is an intrinsic affinity of a TCR for its ligand, but also induced dynamic TCR clustering , both ofUpon activation, naïve T cells typically switch their metabolism from primarily oxidative phosphorylation to glycolysis . Howeverin vitro have a different metabolic profile than cells activated in physiologic conditions, with the former acquiring more of a Warburg-like metabolism and the latter displaying higher rates of oxidative metabolism  G12D mutation-targeting TCR-T to mediate regression of metastatic pancreatic cancer, with the response ongoing 6 months post-treatment . To ensuFuture TCR-T approaches will seek to shorten engineering time, improve cell function and phenotype, and broaden the applicability of therapy to more patients. Although initial trials with TCR-T utilized a patient’s own cells for engineering (autologous cell therapy), this requires that engineering and expansion be completed for each individual, which is resource and time intensive. There has recently been a push towards “off the shelf” allogeneic products, whereby healthy donor cells can be engineered to form a bank of cells which can then be infused into any HLA-matched patient, reducing time and engineering costs; additionally, the availability of large numbers of these cells allows for repeat dosing , 132. HoWM, conceptualization, writing‐original draft, and figure design.The author would like to acknowledge the research team at BioNTech for their support of this work.The author is an employee of BioNTech USA.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.


Pancreatic cancer is one of the most aggressive diseases among solid tumors. Most patients are diagnosed with advanced or metastatic disease and are characterized by poor chemosensitivity. Therefore, earlier diagnosis and novel therapeutic possibilities for pancreatic cancer patients are urgently needed. Liquid biopsy is an emerging technology that allows the noninvasive sampling of tumor material. Nowadays, liquid biopsy has shown promising results as diagnostic, prognostic and predictive biomarkers, but it has not yet been universally adopted into regular use by clinicians. In this review, we describe different components of liquid biopsy, especially circulating tumor cells, circulating tumor DNA and exosomes and their potential clinical utility for pancreatic cancer patients. It is one of the most lethal types of human cancer with 5-year survival rate less than 10%-7. Radical surgical resection remains the only potential curative option for PDAC patients; however, the probability of relapse is high. The average survival of resected patients ranges from 12 to 20 months, and the 5-year survival rate after surgery and adjuvant systemic therapy is approximately 20%-7. Around half of PDAC patients are diagnosed with distant metastasis. Unfortunately, progress in the management advanced-stage disease has been very modest in the last decades. Actually, chemotherapy regimens (gemcitabine and abraxane or FOLFIRINOX) are gold standard in the treatment of locally advanced or metastatic PDAC, although providing only slight improvements in OS, reaching at best few months-10. No target therapies and no immunotherapy approaches are nowadays clearly effective in PDAC. Only the implementation of genetic testing can change a very narrow treatment landscape for small subsets of patients with actionable aberrations: in particular, in BRCA1/2 mutated setting, olaparib (PARP inhibitor) might be proposed as maintenance strategy. Therefore, only few different treatment combinations are currently available to metastatic PDAC patients, and it is important to switch between them wisely.The most common pancreatic neoplasm is pancreatic ductal adenocarcinoma (PDAC), occurring in more than 85% of pancreatic tumor cases. Blood is undoubtedly the most common fluid employed as source of tumor components, and “liquid biopsy” is a novel technology that allows the noninvasive sampling of tumor material. In particular, tumor components released in body fluids such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), vesicles and exosome (EVs) have been studied as predictors of chemoresistance in PDAC . All together, these components represent TME.In conclusion, today, a universally accepted subtyping of pancreatic tumors  has not been followed by success in increased effectiveness of available therapies or identifying tumor biomarkers for early diagnosis and therapeutic response monitoring in PDAC patients. On the one side, some epithelial tumor cells undergo passive intravasation, escaping from the primary tumor. On the other side, some tumor cells undergo EMT, secrete digestive enzymes, lose their polarity and cell-cell/matrix adhesion and gain migratory properties. There are two models of CTCs intravasation: single CTCs detachment from tumor site or collective CTCs invasion. This means that CTCs may travel as single cells or clusters,35. CTCs which access the intravascular space have to overcome selection processes exerted by the hemodynamic stress of bloodstream and programmed cell death (termed “anoikis”) occurring when cells detach from the surrounding extracellular matrix and immune cell attacks,30. There is evidence that malignant cells possess specific characteristics that enable them to survive mechanical stresses. These properties allow CTCs to survive during their passage through the circulation. Moreover, CTCs develop anoikis resistance by the EMT process, promoting proteins linked to anoikis resistance and cell-remodeling, thus modifying energy metabolism, growth factor receptors expression, membrane microdomains, reactive oxygen species and lipid rafts. In the bloodstream, CTCs must evade immune system after leaving the immune privileged site of the primary tumor. CTCs have developed strategies to escape immune response, such as intracellular transcriptional changes leading to the upregulation of PD-L1 inhibiting T cell-mediated immunity; production of inhibitor cytokines that negatively modulate NK cell anti-tumor activity; EMT-related processes promoting epithelial cell plasticity leading to less immunogenicity; and promotion of platelets number and activity that modulate NK activity, reduce CTCs shear stress in the bloodstream and increase CTCs extravasation. CTCs traveling in the bloodstream are not a monolithic entity, but a heterogeneous one. The CTCs population’s heterogeneity is induced by acquired genetic and epigenetic alterations, EMT events, clustering phenomenon and microenvironment-associated cues. There is evidence of epithelial CTCs expressing epithelial markers such as epithelial cell adhesion molecule (EpCAM); mesenchymal-like CTCs expressing traditional mesenchymal markers such as vimentin; CTCs co-expressing epithelial and mesenchymal markers; and CTCs expressing positive stem cells (CSC) markers such as CD4 and CD133 [which are called circulating tumor stem cells (CTSCs)]. EMT promotes acquisition of mesenchymal and CSC markers, inducing so-called EMT-CTCs. CTSCs and mesenchymal-like CTCs, derived from the EMT event, have higher invasion and migratory potentials. CTSCs demonstrate self-renewal and differentiation into multiple cell types, capacities that make them more capable of reaching and colonizing distant organs. Further, CTSCs and mesenchymal-like CTCs contribute to promote resistance to conventional anticancer treatments by many mechanisms such as increasing drug efflux, promoting cell dormancy and increasing genomic DNA repair. A fraction of CTCs, called disseminated tumor cells (DTCs), has the capacity of extravasation, entering distant sites (seeding process) and then interacting with new a microenvironment and progressing toward metastases. DTCs undergo mesenchymal-to-epithelial transition (MET) with subsequent proliferation and dissemination, or, alternatively, they might enter in dormancy and generate metastasis a long time after their establishment in the distant site.CTCs are tumor cells which enter the bloodstream by breaching the basement membrane of the pancreatic acinus and ducts, stroma and vessel walls. This transition is driven by two principal mechanisms: forces of interaction created internally to the tumor mass and the EMT program8 hematologic cells per mL of blood or even less in early-stage cancer. In pancreatic tumors, CTCs entering the bloodstream are conducted to the liver through the portal vein. Thus, thoss in the portal vein might provide a better representation of the CTC population.CTCs in pancreatic cancer are rare, around one CTC per 10. A meta-analysis analyzing 623 PDAC patients demonstrated that the patients with positive CTCs had poorer progression free survival (PFS) and overall survival (OS) than the group of PDAC patients without CTCs. This might suggest that CTCs could be a promising diagnostic and prognostic biomarker in PDAC patients. In another study, Ren et al. showed the presence of CTCs in 80.5% of stage III and IV PDAC patients before any therapy. Examining the CTCs at different time-points (prior to initiation of 5-fluorouracil-based chemotherapy and after seven days of treatment), the presence of CTCs decreased to 29.3%, suggesting a potential role of CTCs in early response to anticancer prediction. Another recently published systematic review and meta-analysis of 19 studies assessing 1320 PDAC patients showed that CTC positive patients had significantly shorter OS and PFS than CTC-negative patients. This again suggests that CTCs may have a prognostic role in PDAC patients.In pancreatic cancer, CTC detection was identified as a possible diagnostic biomarker and their enumeration correlates with staging, prognosis and tumor resectability,56. In small-cell lung cancer (SCLC), genomic analysis of CTCs with relative molecular classifier has allowed assigning patients into chemosensitive or chemorefractory group. The possible prediction of therapy response based on CTCs is currently under evaluation in different cancer types, such as prostate, breast, colorectal, SCLC and gastric cancer, in both preclinical and animal models. To our knowledge, the only study that evaluated CTCs as predictor biomarker of treatment response in PDAC was published by Yu et al. The authors analyzed gene expression profile of CTCs in 50 patients with metastatic or locally advanced PDAC. They created and validated Pharmacogenomic (PGx) modeling of tumor tissue to predict efficacy of chemotherapeutic agents in preclinical cancer models. CTCs isolated from patients’ blood were profiled and the PGx model was used to predict effective and ineffective chemotherapeutic regimens. The authors concluded that, using previously created PGx models to predict chemotherapy efficacy, the clinical benefit was seen for PDAC patients treated with chemotherapy regimens predicted to be effective vs. chemotherapy regimens predicted to be ineffective with regard to PFS and OS.Referring to chemoresistance, in prostate and breast cancer, characterization of CTCs have shown capacity of early prediction of treatment response. Generally, CTCs represent blood available cells originating from primary tumor or metastatic lesions giving phenotypic and molecular tumor characterization in real time. Therefore, they might have importance in tumor diagnosis, staging and prognosis, as well as prediction of response to cancer treatment.The confirmation that CTCs might function as biomarkers of chemoresponse is essential to identify “non-responders” and therefore to avoid ineffective treatments. The portion of cfDNA which refers specifically to the tumor-derived portion is called circulating tumor DNA (ctDNA). Many studies have found a correlation between tumor aggressiveness and the quantity of the absolute amount of ctDNA. As PDAC exhibits the highest frequency of KRAS mutations, KRAS is likely to be the best-characterized tumor-related gene that also occurs at an early stage of PDAC. Therefore, several studies have focused on mutKRAS ctDNA detection  and its potential role in PDAC development, extrapolating prognostic and predictive data. Kruger et al. found KRAS mutated ctDNA in 75% of pancreatic tumors, slightly higher (79%) if only metastatic patients were considered. They also noticed that the quantity of mutKRAS ctDNA significantly differed between patients with non-progressive disease and progressive disease at time of first re-staging. They observed an early increase in mutKRAS ctDNA during the first two days after chemotherapy followed by a decrease in mutKRAS ctDNA, usually after the first or second week of chemotherapy. This trend was seen above all in PDAC patients with good response or disease stabilization at the time of radiological re-staging. An initial increase of mutKRAS ctDNA is observed in most patients, although an increase after two weeks might correlate with disease progression. Therefore, the authors suggested Day 14 might be a crucial time point to evaluate early kinetics. Hadano et al. evaluated 105 patients who underwent surgery for resectable PDAC and identified KRAS mutation in both tumor and plasma samples before resection in 33 patients. KRAS status concordance between tumor specimens and matched plasma samples was 100%. Moreover, the presence of ctDNA, and in particular mutKRAS ctDNA, in plasma samples was significantly associated with poorer outcome in both disease-free survival (DFS) (6.1 months vs. 16.1 months) and OS (13.6 month vs. 27.6 months) analyses. This study suggests a potential prognostic role of ctDNA at early stage PDAC: patients with high ctDNA levels at diagnosis may benefit from neoadjuvant chemotherapy before undergoing surgery. The changes in cDNA levels in pre-surgery and post-surgery liquid biopsy might provide a proof of therapeutic efficacy and management strategies of PDAC. Wei et al. explored application of cfDNA and ctDNA profiling in monitoring tumor burden change following FOLFIRINOX treatment in 38 PDAC patients. Their results show a high degree of concordance between radiologic therapy response and dynamics of ctDNA allele fraction. Cheng et al. collected plasma samples from 13 metastatic PDAC patients. They observed that the presence of mutKRAS ctDNA correlated with radiologic tumor responses. Watanabe et al. analyzed the importance of one-point determination of mutKRAS ctDNA levels before chemotherapy or surgery treatments, in order to predict treatment outcome. They did not prove any association between the presence of mutKRAS ctDNA before surgery and relapse-free survival. Nevertheless, the presence of mutKRAS ctDNA before chemotherapy start was correlated with worse prognosis . Furthermore, the emergence of mutKRAS ctDNA in PDAC patients was associated with poor prognosis. The emergence of mutKRAS ctDNA within six months of chemotherapy termination correlated with worse PFS. mutKRAS ctDNA in patients disappeared in response to drug treatment. Similar results were presented by Del Re et al., who noticed that PDAC patients undergoing chemotherapy treatment and displaying mutKRAS ctDNA increase on the 15th day of therapy had disease progression; conversely, the early mutKRAS ctDNA modulation was not associated with tumor response. Therefore, mutKRAS ctDNA is a potential biomarker of chemosensitivity/chemoresistance to anticancer treatment in PDAC patients. Monitoring the levels or the emergence of tumor molecular alterations in mutKRAS ctDNA during anticancer treatment is a potential biomarker to monitor treatment resistance or response.Both benign and malignant cells undergo physiological processes such as apoptosis or necrosis with subsequent release of genetic material into circulation which might be detected peripherally as cell-free DNA (cfDNA), serum, saliva. In comparison to ctDNA, exosomes have a longer circulating half-life. As tumor cells produce exosomes without pause, their detection in peripheral blood is independent of certain events such as apoptosis or cell necrosis-73. The components of exosomes were studied as diagnostic, prognostic and prognostic biomarkers in PDAC, as well as for novel therapeutic approaches. In one study, expression of exosomes containing miRNA-483-3p was higher in PDAC patients compared with patients with non-malignant IPMN. Goto et al. studied serum miRNAs enclosed in exosomes in PDAC patients and patients with IPMN. They concluded that in particular three miRNAs  showed elevated expression levels in patients with PDAC and IPMN and therefore might be used as biomarkers for early PDAC detection. Further, expression of exosomal miR-191/21/451 was significantly elevated in patients with PDAC and IPMN compared to healthy controls. In another study, the plasma levels of exosomes containing miR-196a and miR-1246 levels were significantly higher in PDAC patients as compared to the healthy population. Su et al. identified five miRNAs  that have diagnostic potential of early PDAC diagnosis. Further, Zhu et al. showed how different levels of microRNA expression might distinguish patients with PDAC and healthy volunteers, appearing to be diagnostic markers. The potential use of miRNA as predictors of response to chemotherapy has been widely studied in PDAC. For example, miR-21 increases cell proliferation and the expression of factors involved in metastasis formation. The reduction of miR-21 levels has been shown to be predictive of response to gemcitabine therapy, while the reduction of miR-17-5p expression is predictive of response to nab-paclitaxel in PDAC patients. Miyamae et al. showed that upregulation of plasma miR-744 contributed to worse PFS of non-resectable PDAC patients who underwent gemcitabine-based chemotherapy and therefore might be a useful tool to monitor chemoresistance in PDAC. In addition, the reduced tissue expression of MiR-10b represents a potential marker of response to neoadjuvant chemotherapy in PDAC patients. In another study, it has been shown that reduction of miR-181a-5p plasma levels correlated with response to therapy with FOLFIRINOX. Moreover, the reduction was associated with better PFS and OS. Interestingly, it has been shown that chemotherapy can induce the increase of not previously present miRNAs. In the study of Xia et al., chemotherapy with gemcitabine caused an increased expression of miR-155, a hypothesized acquired chemoresistance biomarker. In addition, several studies showed the diagnostic potential of microRNAs in PDAC. Unfortunately, even if numerous studies have been published, microRNAs have not been confirmed as universal biomarkers in PDAC patients. The use of different samples and studies involving different populations, as well as the attempt to use experimental elements, has been a limitation. The potential solution to this issue could be to carry out studies on as homogeneous as possible populations with defined tools, in order to reach a conclusive value.Exosomes are extracellular vesicles that contain proteins, microRNAs (miRNAs) or messenger RNA (mRNA). They act as an important mediator of intercellular communication. They can regulate or even modify their surrounding microenvironment. The components of exosomes can therefore serve as cancer biomarkers. It is possible to detect exosomes by isolating them from various cell fluids including blood. Moreover, levels of GPC1+ crExos correlated with outcome in PDAC patients after radical surgery. The authors concluded that GPC1+ crExos might be considered as a diagnostic biomarker to detect early PDAC stages. However, the role of GPC1+ crExos is controversial. Frampton et al. adopted ELISA to quantify GPC1 levels in PDAC tissues and crExos; they concluded that GPC1+ crExos levels may correlate with tumor burden and response to surgical resection, but they are not useful to distinguish between benign and malignant PDAC lesions preoperatively or to rate the aggressiveness of the tumor. The authors of the two cited papers used different techniques: Melo et al. adopted techniques such as anti-GPC1 antibody labeled beads and subsequently flow cytometry, while Frampton et al. used ELISA, which is more reproducible and clinically amenable in a standard hospital laboratory. Interestingly, Buscail et al. combined quantification of GPC1+ crExos with CTC detection in order to increase their sensitivity and negative predictive value. They observed a negative predictive value of 100% and an overall diagnostic accuracy of 91%. This result could be useful in early stages of PDAC when the tumor is likely releasing fewer circulating biomarkers such as CTCs and exosomes. It could be crucial to make more rapid and effective decisions about the treatment in the early setting of such an aggressive disease.Recently, the cell surface proteoglycan glypican-1 (GPC1) on tumor exosomes was identified. GPC1+ circulating exosomes (crExos) were analyzed in the serum of PDAC patients. The authors demonstrated that GPC1+ crExos were able to distinguish between healthy subjects and patients with a benign pancreas disease from patients with early- and late-stage PDACet al. used exosomes for surviving delivery to pancreatic cancer cell line (MiaPaCa-2) with subsequent restoration of gemcitabine sensitivity in pancreatic cancer cell. Bernard et al. investigated PDAC patients with potentially resectable tumors. They demonstrated that an exosomal DNA KRAS mutant allele fraction (MAF) peak of 1% was associated with PDAC progression, almost two months earlier than CT scan progression. Moreover, in borderline resectable PDAC patients who underwent systemic therapy with neoadjuvant intent, the exosomes DNA KRAS MAF kinetics before and after the completion of therapy correlated with disease progression and therefore an indication for no further surgical resection. All these findings make exosomes a sensitive tool for early PDAC diagnosis, as well as possible prognostic and predictive biomarkers.Some authors have provided evidence on the possible use of exosomes for therapeutic approaches. Aspe . Arginase, cathepsin-B and -E, MMP-1 and -3 and UpA have been established as suitable serum markers of PDAC patients. Therefore, analysis of this panel of enzymes by means of a liquid biopsy is promising for early PDAC detection,93.Liquid biopsy offers the possibility to analyze other cancer biomarkers: serum proteases comprising matrix metalloproteinase (MMPs) 1, 3 and 9; urokinase plasminogen activator (UpA); cathepsin-B and -E; and arginase. These proteases are over- or under-expressed in solid tumors and have a role in malignant progression  and immune dysregulation in cancer. These proteases take part in a proteolysis signal network of proteolysis interaction which in turn interacts with other signal networks such as chemokines, cytokines and kinases co-opted to promote tumor progression. In this review, we give an overview of the current literature on CTCs, ctDNA and exosomes in pancreatic cancer (see also KRAS, might be a signal of microscopic metastases that could spare the futile major surgery. Another field where liquid biopsy might be helpful is their utility for monitoring response to different treatments. In conclusion, the implementation of liquid biopsies in clinical context represents a new hope for precision medicine and personalized treatments for PDAC patients.Nowadays, pancreatic cancer is among the deadliest solid tumors. There is also a lack of personalized therapeutic approaches because pancreatic cancer biopsies are often inadequate for molecular characterizationsee also . Unfortu