Mesenchymal stromal cells (MSCs) are multipotent cells obtained from many tissues including bone marrow, adipose tissue, umbilical cord, amniotic fluid, and placenta. MSCs are the leading cell source for stem cell therapy due to their regenerative and immunomodulatory properties, their low risk of tumorigenesis and lack of ethical constraints. However, clinical applications of MSCs remain limited. MSC therapeutic development continues to pose challenges in terms of preparation, purity, consistency, efficiency, reproducibility, processing time and scalability. Additionally, there are issues with their poor engraftment and survival in sites of disease or damage that limit their capacity to directly replace damaged cells. A key recent development in MSC research, however, is the now widely accepted view that MSCs primarily exert therapeutic effects via paracrine factor secretion. One of the major paracrine effectors are extracellular vesicles (EVs). EVs represent a potential cell-free alternative to stem cell therapy but are also rapidly emerging as a novel therapeutic platform in their own right, particularly in the form of engineered EVs (EEVs) tailored to target a broad range of clinical indications. However, the development of EVs and EEVs for therapeutic application still faces a number of hurdles, including the establishment of a consistent, scalable cell source, and the development of robust GMP-compliant upstream and downstream manufacturing processes. In this review we will highlight the clinical challenges of MSC therapeutic development and discuss how EVs and EEVs can overcome the challenges faced in the clinical application of MSCs. Mesenchymal stromal cells (MSCs) were identified more than 5 decades ago and have captured substantial attention among the scientific community since then due to their potential clinical applications . The abiclinicaltrials.gov.Mesenchymal stromal cells’ particular biological properties, including their self-renewal, proliferation and differentiation potential, make them attractive stem cell resources for pre-clinical and clinical studies that focus on the repair and/or regeneration of damaged tissue . The benThe path from pre-clinical promise into clinical use has not been smooth, however. Multiple MSC therapeutics have failed to demonstrate efficacy in early or late clinical trials, in indications ranging from amyotrophic lateral sclerosis to stroke . DespiteDespite the setbacks the field has experienced, the scale of effort still being applied to MSC-based therapeutics development is testament to the clinical promise that MSCs and their derivatives still hold. MSCs continue to show an excellent safety profile and remain the most studied stem cell population, particularly in the context of therapeutic use.One of the most significant shifts in the MSC research field over the past decade has been the growing recognition that, rather than acting by engrafting and differentiating in sites of disease or injury to directly replace damaged cells, MSCs primarily exert their therapeutic effects via the release of paracrine factors . One of This review aims to highlight the ongoing issues associated with the development of MSC therapeutics and also to discuss how EVs isolated from MSCs overcome these issues to replace or complement cell-based therapies.Most MSC clinical studies have produced disappointing findings despite encouraging results in pre-clinical animal studies. Although 10 MSC therapeutics have received market approval in some jurisdictions , in genein vitro and in vivo studies  per dose varies dramatically, from US$485 to US$111,488 . TechnolCulture medium development is another challenge for MSC production at the clinical level. Culture and expansion of MSCs has traditionally required media enriched with serum, but the complex and variable nature of this mixture of nutrients, growth factors and other constituents poses further challenges for maintaining product consistency. The development of serum-free media or chemically defined media is encouraging but they generally do not perform as well, especially for longer passages and scaling-up. They also put upward pressure on COG .The potential for clinical-scale MSC manufacture has been restricted by limited yield of donated cells, quality variation, and biosafety concerns regarding potential transmission of pathogens. An ideal cell source for industrial-scale MSC product manufacture should offer easy and unrestricted availability, have regulatorily acceptable provenance, present no biological safety risks, and be amenable to unlimited expansion while retaining its original “as harvested” phenotype. In a step toward this ideal, bioengineering approaches to increase both the yield and the homogeneity of MSCs are now being explored. For example, differentiation of induced pluripotent stem cells (iPSCs) to MSCs can be expanded to produce large quantities of cells thereby generating large quantities of highly homogenous MSCs . AlthougDespite the potential of engineered MSC sources to overcome specific limitations in MSC production, additional issues associated with MSC therapeutics persist. Immortalized live cell therapies may retain the issues associated with potential immunogenicity and tumorigenicity, and their engraftment qualities post-administration remain to be elucidated.Although it was previously believed that the therapeutic potential of MSCs was due to cell-to-cell contact or engraftment and differentiation of transplanted MSCs, it has become increasingly clear that MSC therapeutics’ prime mode of action is to release paracrine factors . In one Extracellular vesicles are lipid bilayer-wrapped vesicles approximately 30–200 nanometers in size, released by virtually every cell type in the body. Once thought to be a mechanism for cellular refuse disposal, EVs are now known to be key mediators of cell-to-cell communication, delivering a cargo of lipids, proteins and nucleic acids that reflects their cell of origin. Although significantly simpler than live MSCs, EVs are still highly complex in composition and exert their effects by either transferring bioactive cargo such as functional protein and miRNAs that alter cell fate, or by modulating cell surface receptors and triggering intracellular signaling pathways .In the case of MSC-derived EVs, they have been found to share many attributes such as content, immunophenotype and unique homing ability (tropism) as their parental MSCs . As a rein vivo stability . In one study, MSCs were found to be retained in the lungs , while ain vitro .As EVs can be continually harvested from a MSC population, they can potentially overcome some of the issues of limited supply associated with MSCs themselves. As MSCs are highly amenable to modification, EV production can potentially be boosted to improve scalability. For example, the application of various stimuli to the parent cells, including physical stressors such as hypoxia or mechanical forces, or the addition of various small molecule modulators, can increase EV release . MSCs haFrom the perspective of drug manufacturing, EVs offer several logistical advantages over cell-based therapies. These includes the possible addition of sterile filtration of the drug substance before aseptic filling, unfeasible for whole cells . EVs areThe therapeutic application of MSC-derived EVs is not limited to naïve, i.e., naturally secreted, EVs. The parent cell or its secreted EVs can be bioengineered to generate an EV product with enhanced or altered therapeutic properties. EV engineering strategies can be divided into two broad categories. Firstly, EVs can be engineered to alter their tropism and bias their uptake toward a target cell type. Alternatively, or in addition, EVs can be loaded with a particular therapeutic cargo.A key factor determining the potential therapeutic utility of MSC-EVs is their fate once infused into the body. Several biodistribution studies have used dye- or bioluminescent-labeled naïve EVs to observe EV uptake by different tissues following infusion into animals .In a study on MSC-EVs labeled with a near-infrared lipophilic dye, more than 70% of EVs were found to accumulate in the liver when systemically injected into mice . Other ain vivo tropism may be altered by incorporating selected surface proteins onto their surface. One method for conferring targeting capabilities onto EVs is to manipulate the parent cell. One route is to create fusion proteins, in which the protein tail attaches or inserts into the EV membrane, and the head binds a receptor on the target cell type. In one early example, a fusion protein created between EV membrane protein Lamp2b and the rabies viral glycoprotein (RVG) peptide which binds the acetylcholine receptor on brain cells. These EEVs were used to target neurons, microglia and oligodendrocytes in the brain after systemic injection (Engineered EV (EEV) njection . More renjection . This apnjection .Targeting proteins can also be incorporated onto EVs after their isolation from cell culture. As well as altering EV tropism, proteins can be added to the vesicle surface for a range of other applications. For example, EEVs with protein receptors on their surface have been developed as decoys to capture target molecules such as the pro-inflammatory cytokine IL-6, as a potential therapeutic for chronic inflammatory diseases . AnotherMesenchymal stromal cell-EVs are known to possess a cargo that can promote regeneration in damaged tissues, for example by lowering inflammation and inhibiting apoptosis to promote healing. EV engineering allows the possibility of loading EVs with a defined therapeutic cargo that could enhance this capability. MSCs have been engineered to overexpress microRNA-let7c, to generate EEVs which contain elevated levels of this microRNA. Infusion of these MSC-derived EEVs attenuated renal fibrosis in a mouse model of unilateral ureteral obstruction . More reHowever, EV drug loading methodologies also enable the potential development of EEV therapeutics for a wide range of indications beyond tissue regeneration and healing. The key characteristics of EVs–non-immunogenic capsules able to deliver cargo to a target cell type–appear to make them ideal as drug delivery vehicles, such as for targeting tumor cells.Many therapeutics, from small molecule drugs to RNAs, need to be shepherded to their site of action, either to overcome unfavorable pharmacokinetics, as protection from metabolic breakdown, or to reduce off-target effects. Synthetic delivery systems such as liposomes or nanoparticles have been limited by poor stability in storage, toxicity, poor efficacy and a limited capability to deliver cargo to target tissues other than the liver .As knowledge surrounding EV biogenesis and mechanisms of cargo sorting has increased, new strategies have emerged to generate EEVs that contain a defined therapeutic cargo. The techniques associated with the generation of EEVs include incubation with drugs for delivery  or transIn head-to-head testing, EVs were recently shown to be superior to liposomes for RNA transfer, delivering a cargo of RNA several orders of magnitude more efficiently into cells . The bigExtracellular vesicle loading can also take place after EV isolation from cell culture. Techniques including freeze-thawing, sonication, electroporation, osmotic shock and saponin permeabilization have been developed to temporarily disrupt the EV membrane sufficient for uptake of a therapeutic cargo. However, as the cargo loading method itself may alter the biophysical characteristics or biological function of treated EVs, and the loading efficiency can vary between different types of cargo, care must be taken to determine the appropriate cargo-loading methodology in each case .www.ClinicalTrials.gov database to assess the therapeutic effects of EVs in several therapeutic arenas. MSC-EVs have demonstrated acceptable safety and tolerability profiles, and promising signs of therapeutic efficacy ; a Phase I study of MSC-EVs loaded with a siRNA therapeutic for patients with pancreatic cancer (NCT03608631); and a study to assess MSC-EV administration for improvement of disability in patients with acute ischemic stroke (NCT03384433).There are also studies underway to test inhaled MSC-EVs in patients with acute respiratory distress syndrome (ARDS) associated with severe cases of COVID-19 . Extracellular vesicles from other sources than MSCs have also been used in clinical trials. Significantly, in addition to the early phase trials conducted by academic groups, several trials of experimental EV therapeutic products developed by biopharmaceutical companies are now underway, reflecting the increasing maturity of the therapeutic EV field. These trials include platelet derived EVs tested by Exopharm in two Phase I clinical trials for wound healing applications , 2020. CDespite rapid development in the EV field and the commencement of clinical trials, several challenges remain and should be resolved before EVs can achieve widespread clinical utility. These challenges are discussed in the following section.Although regulatory approval and safety of clinical application of EVs appears feasible, and may be simpler than cell-based therapy, important barriers that need to be addressed for clinical GMP-grade production of EVs include selection of the starting cellular material and optimizing cell culture, purification, quantification, and quality control . UnderstAs EV are a secreted product of cells, their manufacture is heavily dependent on the ability to produce large quantities of cells in ways that do not alter their phenotype. However, the opportunities for producing large quantities of stem-cell based conditioned medium with which to undertake meaningful scale-up of EV production are limited.Generally, for stem cell derived EVs, the potential cell sources for EV production are either MSCs, or pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or iPSCs derived lineages. In each case there are advantages and disadvantages associated with their clinical use, which are summarized in in vitro and in vivo, neither promoting nor inhibiting tumor growth  for cell culture and EV collection, which cannot be applied for clinical application . AlthougLarge-scale isolation and purification of EVs is one of the most significant challenges in downstream processing for clinical GMP-grade EV production. Ultracentrifugation (UC) remains the most popular method of EV isolation, despite ongoing concerns over product purity, and that UC protocols are not directly scalable for clinical-scale manufacture . AlternaAs different isolation and purification methods have an impact on EV characteristics and produce different population of EVs, the need for standardized GMP is emphasized . MISEV gQuantification of EVs is another fundamental issue which remains challenging. How to accurately measure and assess EV purity is a critical issue when evaluating EV dosage for both pre-clinical and clinical applications. At present, there is no single method that can accurately measure EVs. One of the most common approaches to measuring EVs is determining the total amount of proteins and particle numbers. There are several methods and instruments measuring EV numbers including nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), and dynamic light scattering (DLS). However, copurification of other proteins by measuring total protein concentration and current particle tracking approaches might be biased toward a designated EV size range which does not discriminate EVs from other nanoparticulate materials. Recent developments in this field include nanoflow cytometry, imaging flow cytometry, and ExoCounter with optic disk technology to quantify EVs . A combiFor GMP compliance, fill and finish of the final product should be performed in a closed system. This might present as a logistical challenge for small biotech companies and academic labs operating at research scale . Overallin vitro and in vivo assays to ensure post-thaw potency of the EVs product is retained (Finally, storage and long-term stability of EVs is another important issue if EVs are to be used as a third party “off the shelf” product. EV quantity decreased in a dose-dependent manner at room temperature, at 4°C, −20°C, but not when frozen in −80°C, therefore most protocols store EVs at −80°C long term without any reported changes in EV profile and structure . Preservretained . Alternaretained . StandarExperimental MSC therapies continue to show promise in pre-clinical research. This promise, however, is still to translate into widespread clinical use. The inherent complexity of a live cell product presents considerable challenges to successful therapeutic translation. Although cell engineering approaches may address some of these challenges, the potential risks associated with cell-based therapeutics, such as tumorigenicity and undesired differentiation, and limitations such as rejection of cells and poor engraftment, will remain.Mesenchymal stromal cell-EVs, as a cell-free product, offer a potential pathway through these challenges. An increasing number of studies have shown that the therapeutic effects of MSC-EVs are equal to those of the MSCs. The use of EVs rather than MSCs protects against issues of tumorigenicity, immunogenicity, and poor engraftment. In addition, EV-based therapeutic manufacture, as well as transport and storage, promises to be more readily scalable at lower cost than live cell MSC therapeutics. However, to achieve their full clinical potential and to avoid some of the pitfalls that have contributed to clinical trial failures of MSC therapeutics, EVs need to be thoroughly investigated in terms of purification, quantification, characterization and potency before their clinical use. Genetic modification of the source cells for large scale manufacture of MSC-EVs may serve as an effective strategy to further improve the therapeutic effect of MSC derived cell-free products. The increasing sophistication with which EVs from a variety of cell sources can be engineered, to enhance their homing capabilities and bolster their therapeutic cargo, further demonstrates the considerable potential of this versatile therapeutic platform.RK, JJ, JMC, and MS designed and wrote the content, and revised the manuscript. All authors contributed to the article and approved the submitted version.MS, JJ, and RK are employed by Exopharm Ltd. JMC is a contractor to Exopharm Ltd.


Achieving multi-level devices is crucial to efficiently emulate key bio-plausible functionalities such as synaptic plasticity and neuronal activity, and has become an important aspect of neuromorphic hardware development. In this review article, we focus on various ferromagnetic (FM) and ferroelectric (FE) devices capable of representing multiple states, and discuss the usage of such multi-level devices for implementing neuromorphic functionalities. We will elaborate that the analog-like resistive states in ferromagnetic or ferroelectric thin films are due to the non-coherent multi-domain switching dynamics, which is fundamentally different from most memristive materials involving electroforming processes or significant ion motion. Both device fundamentals related to the mechanism of introducing multilevel states and exemplary implementations of neural functionalities built on various device structures are highlighted. In light of the non-destructive nature and the relatively simple physical process of multi-domain switching, we envision that ferroic-based multi-state devices provide an alternative pathway toward energy efficient implementation of neuro-inspired computing hardware with potential advantages of high endurance and controllability. The recent advancements of data-driven learning paradigm such as artificial deep neural networks (DNN) have achieved superhuman performance in various applications including image/pattern recognition, natural language processing, and developing autonomous intelligence  based on novel physical mechanisms can lead to significant reduction of leakage power and achieve high on-chip density compared to CMOS, while mimicking key neuro-synaptic functionalities for neuromorphic computing  materials such as magnetic RAM (MRAM)  motion in ferromagnets and multi-domains induced in exchange-coupled heterostructures can be leveraged to achieve multi-level nanometer-scale devices. Section 3 focuses on ferroelectric devices, including FeFET and ferroelectric tunnel junctions (FTJ) as the basic configurations for generating ferroelectric multi-level states. Both fundamental material physics of multi-level devices as well as representative demonstrations of neural functionalities are covered. Section 4 reiterates the opportunities with ferroic multi-level devices toward developing neuromorphic hardware in comparison with other technologies. We will highlight key advantages and challenges for each of these technologies, followed by discussions and proposals regarding the potential pathways for addressing the challenges. We conclude this review with the proposal that through the co-design of device, circuit, and algorithm, multi-level ferroic devices could provide exciting opportunities of constructing large-scale and energy efficient cognitive computing systems.Spintronic materials have shown clear advantages for developing next generation non-volatile memory with potential combination of high speed, low power, and unparalleled endurance. In particular, magnetic tunnel junctions (MTJ) have been extensively investigated and demonstrated reliable memory read and write schemes in device dimension down to tens of nanometers  at the FM/HM interface and the broken inversion symmetry in the heterostructure stack , SOT-DWM devices in a crossbar array can have their DW position (and thus conductance states) accurately programmed to map a synaptic weight matrix, as is illustrated in In addition to synaptic plasticity, neuronal behaviors can also be realized based on the domain dynamics involving SOT-driven DWM. While a mono-domain SOT-MTJ or STT-MTJ is fully capable of making a stochastic spiking neuron based on the sigmoidal switching probability function of excitation currents , and thus hindering the deployment of the DWM devices beyond prototype demos  ordering, and particularly its interaction with ferromagnetic ordering to introduce non-coherent response to external excitation such as spin currents. The idea of using AF materials to modify FM in bilayer F/AF blocks has already been used such as exchange bias in MTJ stack used in magnetic sensor and MRAM , can facilitate multi-domains due to the absence of long range exchange interaction and dipole fields. In polycrystalline AF metallic thin films such as PtMn or IrMn, AF grains are formed with a dispersion of crytalline orientation and grain size, leading to variation in the AF ordering orientation and distribution of switching energy barrier among AF grains. Therefore, an inhomogeneous exchange bias is expected at a interface of FM/AF bilayer heterostructure. In presence of external fields or spin currents, the nucleation of the different regions in the FM layer may be impacted differently by the adjacent AF domains underneath, leading to multiple domains with non-coherent nucleation or gradual switching of the whole device area characterized by sloped hysteresis curves , leading to a magnetic cluster size similar to the physical grain size. The grains with a finite switching field distribution will respond to external excitation non-coherently, generating multi-domain states. It has been recently revealed that intermediate magnetic states can be retained under gradually increased external magnetic field, demonstrating a possibility of making memristors based on the multi-domain switching dynamics  in most of recent device-level demonstrations are not feasible for CMOS circuits to sense and process in a integrated chip. Combined efforts of microstructure segregation and antiferromagnetic order on MTJ-based platform could potentially pave the way for providing scalable multi-level spintronic integrated devices for neuro-inspired computing.Ferroelectric materials can maintain electric polarization states that can be switched by applying voltages (electric fields), and therefore have been under continuous investigations for non-volatile memory applications. With multi-domain switching dynamics taken into consideration, ferroelectric materials are capable of realizing not only as binary synapse but also as multi-level synapse. FeFET and FTJ are the most studied device structures for implementing ferroelectric neuromorphic hardware, and will be discussed in details in the following subsections.3 (BTO) and PbZrxTi1−xO3 (PZT) are used as ferroelectric materials. In addition to perovskites, novel 2-dimensional (2-D) materials such as transition metal dichalcogenides are being explored in ferroelectric devices  offers distinctive advantages such as improved retention, scalability, and high CMOS compatibility in comparison to previously investigated materials such as PZT  among the FE grains  of channel conductance with an maximum on/off conductance ratio (Gon/Goff) of 45  and drain current IDS. And leaky behavior due to reduction of the remnant polarization retention has also been observed. Such leak in ferroelectric can be resulted from gate leakage current, effects from interface charges, oxide breakdown, intrinsic depolarizing field in a ferroelectric capacitor, or a reversed inhibitory electric field. It was also observed that abrupt change in channel conductance can take place under scaled devices which have just few grains so that each grain switching effect are largely reflected into the conductance change, effectively emulating a step-like integrate-fire neuron. The sharp transition from OFF to ON state is attributed to formation of conductance pathway in the channel when a sufficient number of subdomains have switched.Moreover, neuron behaviors such as input accumulation, leaking in membrane potential, and spike firing can also be emulated utilizing the field-time dependence of domain nucleation kinetics in ferroelectric materials, as shown in 2-based thin films has shown great potential toward the development of non-volatile neuromorphic devices. The capability of emulating both synapse and neuron functionalities with the same device structure could provide an advantage toward hardware integration. More importantly, the ferroelectric material has high CMOS compatibility, while the possibility of realizing symmetric conductance modulation, as well as demonstrated high on/off ratio makes it competitive for implementing both training and inference machine learning tasks at large scales. Meanwhile, some critical issues still remains. In general, HfO2-based FeFETs still have endurance and retention issues associated with the interfacial layer deposited between ferroelectric and channel. This is because the degradation in remnant polarization due to the impact of charge traps, defects, and oxide breakdown. Currently the endurance of HfO2-based FeFET is about 109 cycles , BiFeO3 (BFO), and Hf0.5Zr0.5O2 (HZO) have demonstrated large range of modulation in the tunneling electroresistance  . FTJs are two terminal devices with a layer of ferroelectric material sandwiched between top and bottom metallic layers of electrodes, where sizeable tunneling currents can occur at room temperature if the ferroelectric layer is as thin as few nanometers. Although the concept of FTJ has been proposed in 1971  applied over the tunnel barrier to switch the polarization, making it challenging for scalable circuit integration. One of the approach to reduce the operation voltage is thinning ferroelectric tunnel barriers, however, the retention and stability of polarization is deteriorated, limiting retention of current FTJs on the order of 10–100 h , it is worthwhile to note that high precision weight matrices can be mapped to multiple crossbars in large scale implementation of in-memory computing. Moreover, multi-level cells having large number of states would typically require a higher precision readout circuitry to interface between digital and analog domains, dominating the power and area costs  with numerous intermediate states. Hence, multi-level spintronic devices can be well suited for implementing frequently updated components such as neurons, while ferroelectric devices are considered to be more suitable for implementing analog synapses, given the device characteristics of large memory windows between states, low read/write energy, fast switching, and superior CMOS compatibility , suffers resistance drift due to relaxation of the amorphous state (high resistance states). Such drift in device conductance significantly degrades the desirable data retention and thus requires additional circuit-level compensation scheme in real applications, leading to additional energy consumption and delay  can be a major hindrance  will become more severe at larger crossbars, leading to non-ideal output current values compared to the ideal crossbar output current I = G · V. Note that using devices with low ON-state resistance will be more susceptible to the non-ideal IR drop, and thus a high ON-state resistance is desirable for accurately performing crossbar-based inference task . The chalcogenide alloy in PCM experiences frequent expansion and contraction under heating during writing process, and the high probability of physical detachment at the interface between the alloy and the heating elements can lead to permanent defects in devices, leading to hard errors such as stuck at fault when device states are no longer changeable. As for the ReRAM, both intrinsic device variation originated from fabrication process and the writing/resetting of ReRAM devices can contribute to severe device defects that can significantly degrade the accuracy of the crossbar in-memory computing. In contrast, the endurance of ferromagnetic devices is much better in comparison with ReRAM and PCM, since switching in spin-based devices do not need electro-forming of conduction filaments . The endurance of production-ready STT-MRAM can be as high as 1014–1015 cycles with write latency of 10 ns, making MRAM much more competitive for data memory requiring frequent updates. And newly emerging SOT-MTJ equipped with separated read and write current paths could provide even more flexibility in design fast and high-endurance devices. The leverage of MRAM technology for neuromorphic computing can provide a viable path toward making crossbar arrays with high endurance for training and learning. Moreover, writing in PCM and ReRAM requires high energy consumption and latency due to the device characteristics. The phase transition in PCM needs substantial write current  compared to the highest possible bits per cell in synaptic devices (about 1–5). Therefore, the mapping of input vectors and weight matrices into hardware may involve multiple arrays or multiple columns (Shafiee et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intercellular communication is a critical process that ensures cooperation between distinct cell types and maintains homeostasis. EVs, which were initially described as cellular debris and devoid of biological function, are now recognized as key components in cell–cell communication. EVs are known to carry multiple factors derived from their cell of origin, including cytokines and chemokines, active enzymes, metabolites, nucleic acids, and surface molecules, that can alter the behavior of recipient cells. Since the cargo of EVs reflects their parental cells, EVs from damaged and dysfunctional tissue environments offer an abundance of information toward elucidating the molecular mechanisms of various diseases and pathological conditions. In this review, we discuss the most recent findings regarding the role of EVs in the progression of cancer, metabolic disorders, and inflammatory lung diseases given the high prevalence of these conditions worldwide and the important role that intercellular communication between immune, parenchymal, and stromal cells plays in the development of these pathological states. We also consider the clinical applications of EVs, including the possibilities for their use as novel therapeutics.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00787-y. Exosomes are formed through the inward budding of the multivesicular body, an intracellular component of the endocytic pathway, and are typically 30–150 nm in diameter. In contrast, MVs are formed through outward budding of the plasma membrane and are generally 100–1000 nm in diameter. Exosomes and MVs overlap in size and density and share many of the same biosynthesis components, making them experimentally difficult to distinguish. Therefore, throughout this review, we will refer to exosomes and MVs as EVs. While EVs were initially believed to be cellular waste products, their status as key players in intercellular communication is now broadly recognized [Studying the biological function of extracellular vesicles (EVs) is an emerging research area, and the body of work supporting the transfer of communicating components through secreted EVs has greatly expanded. EVs represent a novel axis of intercellular communication, contributing not only to tissue homeostasis but also to the pathogenesis of immune-mediated diseases. EVs are spherical, lipid-bilayer delimited structures that are universally secreted by nearly all cell types and organisms. While EVs are subcategorized into several groups based on their biogenesis pathway, the term cognized , 3. Addicognized , 5. The cognized . These dWhile much evidence is accumulating regarding the importance of EVs as effective communicators, much less has been studied regarding the role of EVs in the development of pathological conditions. Recent insights gained from the experimental study of EVs derived from human subjects, tissue culture, and animal models are shaping our understanding of the cellular communication patterns that unfold during disease progression. As tissue damage develops, the information that is transmitted between stromal, immune, and dysfunctional cells dictates pathogenesis and outcome. This review serves to highlight recent work that uncovers factors and signaling pathways that are involved in EV-mediated communication within injured and diseased environments. It should be noted that EVs have been implicated in many diverse pathologies, including cardiovascular, renal, and musculoskeletal diseases –9. HowevTumor cells interact with the immune system throughout all stages of disease development, and crosstalk between tumor cells and immune cells is critical to cancer progression . Several2 (PGE2) in macrophages, leading to the recruitment of additional macrophages and the progression of gastric cancer [Within the TME, EV-mediated crosstalk is a multi-directional process. Tumor cells may reprogram and influence the behavior of immune cells, which send new information back to the tumor Fig. . In the c cancer . Due to c cancer . UltimatAdditionally, tumors often adapt to exploit the immune system’s intrinsic regulatory mechanisms in order to avoid immune surveillance. One of the key immune checkpoints involves programmed cell death protein 1 (PD-1) and its ligand PD-L1 . In normEVs clearly play a central role in intercellular communication between tumor and immune cells. Cancer cell-derived EVs participate in driving immune cells to adopt pro-tumor phenotypes, allowing the tumor to systemically evade immune surveillance and establish favorable microenvironments. Additionally, EVs secreted by dysregulated immune cells in the tumor microenvironment can induce changes in cancer cells, such as increased migration and proliferation, that contribute to the progression of cancer. These studies highlight the importance of EVs in mediating tumor–immune cell crosstalk in the TME.2 to induce intrinsic ROS generation was also shown to result in enhanced EV secretion [Chemotherapy is a widely implemented systemic therapy to treat cancer patients. Chemotherapeutic drugs generally affect rapidly proliferating cells in many parts of the body in addition to the tumor . Most checretion .Recently, Shen et al. revealed an EV-mediated mechanism of chemotherapy resistance in breast cancer . Cancer Chemotherapy-induced alterations in the relationship between EVs and cholesterol transfer was recently studied in acute myeloid leukemia (AML) . AML celThe majority of cancer patients will undergo surgery as part of their therapy. Patients with locally controlled solid tumors are more likely to receive surgery as their sole or primary form of treatment . Evidenc., whose work has suggested that granulocyte colony-stimulating factor (G-CSF) from tumor cells induces activation and release of neutrophils into the circulation, where interaction with tumor-derived EVs promotes the release of NETs [Complications arising from CTC dissemination are further compounded when considering the role of surgery-induced immune activation. Surgical injury results in the activation of immune cells, such as monocytes, lymphocytes, and granulocytes, and enhances the postoperative inflammatory environment . While t of NETs . To our  of NETs . In a mo of NETs . While cRadiation therapy is an integral component of palliative and curative care for cancer patients, with estimates that over 50% of patients will receive radiation as part of their treatment . IonizinFurthermore, the factors transferred by EVs in this context have recently been highlighted as multiple studies have reported changes in the composition of EVs following IR. Several groups have analyzed changes in vesicles secreted from head and neck cancer cells following moderate doses of in vitro radiation. Mutschelknaus et al. found nearly 80 differentially regulated proteins in EVs isolated from cells treated with 6 Gy of radiation . ProteinRadiation-induced alterations in EV signaling are believed to be influenced by oxygen levels in the environment, but this relationship is poorly understood. As portions of the TME are often hypoxic, it is important to understand how IR further compounds changes in cellular crosstalk. Jung and colleagues investigated EV dynamics under normoxic and hypoxic conditions and with or without IR treatment in a breast cancer model . They reWhile studies regarding radiation-induced modifications of EV crosstalk have focused primarily on the response of cancer cells, the EV-mediated radiation response in normal tissue cells has yet to be significantly explored. As normal tissue irradiation has been implicated in cancer recurrence under immunocompromised conditions, EV-mediated tumor–stromal interactions following IR will be imperative to study to more comprehensively understand the effects of IR-associated changes in EV composition on intercellular communication and disease progression .Metabolic disorders, generally characterized by hyperglycemia, hyperlipidemia, hypertension, and high body-fat, are estimated to affect about one-fourth of world’s population . MoreoveFreeman and colleagues recently revealed that patients with diabetes had higher levels of circulating EVs compared to euglycemic individuals . FurtherThe liver plays an essential role in metabolism and the immune system. Within the liver, hepatocytes can send metabolically active enzymes via EVs. Angulo et al. recently reviewed the current status of active enzyme-carrying EVs in liver conditions . NotablyThe worldwide prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing, with recent estimates claiming roughly 1 in 4 individuals worldwide are afflicted with NAFLD . These rEmerging evidence has shown that immune cell-derived EVs play a critical role in the pathology of inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Airway lining fluid, which is sampled by bronchoalveolar lavage, is highly populated by EVs . EVs fouIn addition to being increased in biofluids in response to inflammatory stimuli, EVs carry critical communicating cargoes that implicate them in immune-mediated pathology. In fact, EVs in the lung have been demonstrated to carry cytokines that play critical roles in the communication between immune cells. For example, in mouse models of infection-exacerbated asthma and COPD, EVs secreted into the airway carry the pro-inflammatory cytokines IL-18 and IL-1β, resulting in airway lining fluid and lung tissue neutrophilia . In mousApart from cytokines, reports also show that immune cell-derived EVs carry active enzyme cargoes. Macrophages, dendritic cells, and neutrophils secrete EVs carrying leukotriene biosynthesis enzymes while EVs secreted by activated T cells have been shown to carry the TCR-associated kinase Lck –110. ThiImmune cell-derived EVs in the lung have also been shown to carry nucleic acids, where the miRNA content of EVs has been the subject of extensive study. Extracellular miRNAs derived from immune cells are increased in the airway lining fluid of mice with induced allergic airway inflammation, and these miRNAs are likely packaged into EVs as they are protected from degradation by RNases . This inOwing to the difficulty of identifying the active EV components that exert a biological function on target cells, many studies have identified possible roles for EVs in inflammatory lung diseases without identifying a molecular mechanism. For example, Cañas and colleagues found that eosinophils isolated from the peripheral blood of asthmatic patients, but not healthy controls, secrete EVs that induce apoptosis in epithelial cells and inhibit wound healing in vitro . This suIn addition to their pro-inflammatory effects, immune cell-derived EVs can also play anti-inflammatory and regulatory roles in pathologic lung inflammation. Alveolar macrophages secrete EVs containing suppressor of cytokine signaling 1 (SOCS1) and SOCS3, which are proteins that inhibit inflammatory cytokines involved in the JAK/STAT3 pathway . These sEVs serve as vital mediators of intercellular communication between immune, parenchymal, and stromal cells in tissues. As such, it is intriguing to consider how the information provided by EVs may be harnessed to diagnose and treat a wide range of diseases. Many studies have investigated the use of specific cargo components in EVs derived from biological fluids to use as biomarkers for disease detection. For example, EVs with particular lipid and miRNA cargoes, such as ceramides and miRNAs with target transcripts involved in cytokine and MAPK signaling, are altered in airway lining fluid collected from asthmatics compared to healthy patients , 125. MaBeyond use as biomarkers, knowledge of EV-mediated communication may be directly applied to the treatment of tissue injuries and immune-mediated diseases. Targeting the interactions between parenchymal cells and immune cells may result in favorable outcomes that attenuate disease progression. In liver diseases, EV-mediated transfer of miR-122 has been implicated in the development of an inflammatory state that is critical to steatohepatitis development , 91, 96.Processes involving polarization of naïve or pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype are appealing targets for novel immunotherapies. Tumor cells in the TME rely heavily on polarization of macrophages to create a pro-tumor and immunosuppressive environment , 26. FurRegarding cancer therapies, several strategies may be explored to target EV-mediated communication in an effort to maximize the efficacy of traditional therapies and eliminate therapy-induced recurrence. Improvements to oncological surgeries through endoscopic techniques may allow fewer CTCs to enter the circulation, which may reduce the likelihood of interaction with NETs and primary tumor-derived EVs in circulation and limit the formation of metastatic niches. Another key focus should be restricting interactions with primary tumor-derived EVs and neutrophils. Targeting EV–neutrophil interactions is a challenge, but blocking TF-positive EVs through mAb-based therapies is one such option. Much work remains to be done to elucidate the connection between radiotherapy-injured tissues and EV-mediated communication that results in cancer recurrence. Radiotherapy could be improved by enhancing normal tissue radioprotection such that EV release and dysregulated communication do not result from IR-induced DNA damage. Protecting normal tissue cells from ROS damage induced by chemotherapy may also abrogate abnormal EV-mediated communication.h2) cells and may be able to attenuate allergic airway inflammation [h2 cells are thought to be drivers of the pathology seen in allergic asthma. Taking advantage of immune cell-derived EVs has also been applied to the treatment of ALI. One study showed that neutrophil membranes can be disrupted by nitrogen cavitation to produce EV-like particles, which can be loaded with drugs and administered to mice with ALI to attenuate inflammation [In inflammatory lung diseases, EVs derived from immune cells may be manipulated to serve as couriers of molecules that have therapeutic effects when delivered to their target cells. This has been explored through the development of EVs decorated with chimeric antigen receptors (CARsomes). CARsomes derived from dendritic cells were shown to induce apoptosis in antigen-specific T helper 2 (Tammation . Notablyammation . AlthougOutside of the pathologies discussed in this review, EV-based therapeutics for clinical wound healing applications have recently been explored. Particularly, mesenchymal stem cell-derived EV therapies are quickly moving toward clinical applications . Stem ceThe work discussed in this review underscores the importance of EVs in intercellular communication networks that govern tissue injury and disease progression. In the diseases and conditions explored here, healthy cells are exposed to stress- and damage-inducing conditions upon progression of the disease state. These conditions amplify EV secretion, where the ensuing EV-mediated communication results in ongoing crosstalk between immune cells and stromal and parenchymal cells. Ultimately, these interactions can reshape the tissue microenvironment to promote the survival, proliferation, and resistance of dysfunctional cells. While our understanding of EV-mediated communication is expanding rapidly, there are clear gaps in our knowledge of the role of EVs in the progression of pathological conditions. As such, much of the research highlighted here requires comprehensive follow-up studies to further solidify therapeutic targets. Nonetheless, the insights gained from these studies provide a framework to establish alternative and improved therapies for patients suffering from complex diseases.


Glucocorticoids are essential endogenous steroid hormones secreted in response to stress, and treatment with synthetic forms is widespread for numerous inflammatory conditions including asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, and arthritis . EmerginAs such, the recent prospective cohort study by Cairat et al.  is an imUnlike prior studies, Cairat et al. were able assess whether the associations of glucocorticoids and breast cancer differed according to hormone receptor status, histological subtype, tumor grade, and stage at diagnosis. This is particularly important given the various pharmacological properties of glucocorticoids and evidence for cross-talk between estrogen receptors and glucocorticoid receptors in mammary epithelial cells , which sThis study is informative in teasing apart heterogeneity in glucocorticoid-breast cancer associations by hormone receptor status to isolate the potential impact of estrogen pathways. As authors of this study note, estrogen inhibition induced by glucocorticoids may explain the lower risk of ER-positive tumors observed in this study. Moreover, given that ER-positive tumors tend to be less aggressive than ER-negative tumors, the inverse associations observed for early stage disease may reflect the preponderance of ER-positive tumors in this group. Systemic glucocorticoid use also affects immune system function and insulin sensitivity, which could induce adipose aromatase activity and estrogen production and also directly stimulate breast cancer cell growth and invasion , therebyThe potential impact of these findings are substantial, given the widespread and increasing use of glucocorticoids over the past several decades , 9, and In conclusion, this study’s findings are intriguing and merit follow-up to further characterize the potential role of glucocorticoids and other anti-inflammatory therapies in breast cancer etiology. Specifically, future studies recognizing the complex interplay between inflammation and adaptive immune response in breast cancer are needed to inform our understanding of etiology and the development of personalized medicine approaches in breast cancer.


Sinonasal squamous cell carcinomas are a group of diverse tumors affecting the nasal cavity and paranasal sinuses. As a direct consequence of their rarity and heterogeneity, diagnosis is challenging, and treatment does not follow universally accepted protocols. Though surgery represents the mainstay of treatment, neoadjuvant and adjuvant therapies have pivotal roles in improving outcomes of patients treated with curative intent. Indications to endoscopic surgery have been expanding over the last three decades, but a considerable number of patients affected by sinonasal squamous cell carcinoma still need open surgical procedures. Management of the neck in patients affected by sinonasal squamous cell carcinoma is controversial. Curative-intended treatment of recurrent and/or metastatic tumors, alongside palliation of uncurable cases, represent poorly explored aspects of this disease.Sinonasal squamous cell carcinoma is a rare tumor affecting the nasal cavity and paranasal sinuses. Several aspects of this disease, ranging from epidemiology to biology, pathology, diagnosis, staging, treatment, and post-treatment surveillance are controversial, and consensus on how to manage this sinonasal cancer is lacking. A narrative literature review was performed to summarize the current evidence and provide the reader with available data supporting the decision-making process in patients affected by sinonasal squamous cell carcinoma, alongside the authors’ personal opinion on the unsolved issues of this tumor. The review has highlighted several advances in molecular definition of epithelial cancers of the sinonasal tract. Surgery represents the pivot of treatment and is performed through an endoscopic transnasal approach whenever feasible. Open surgery is required for a large proportion of cases. Reconstruction of the defect follows principles of skull base and cranio-maxillo-facial reconstruction. Chemotherapy is given as neoadjuvant treatment or concomitantly to radiotherapy. Photon-based radiation therapy has a crucial role in the adjuvant setting. Particle therapy is providing promising results. Management of the neck should be planned based on the presence of clinically appreciable metastases, primary tumor extension, and need for recipient vessels. Biotherapy and immunotherapy are still underexplored therapeutical modalities. Squamous cell carcinoma (SCC) of the sinonasal tract (SNSCC) is “a malignant epithelial neoplasm arising from the surface epithelium lining the nasal cavity and paranasal sinuses and exhibiting squamous differentiation” . DespiteA recent study on 4994 SNSCCs registered at the United States National Cancer Institute’s Surveillance, Epidemiology, and End Results revealed an incidence of 0.32 new cases/100,000 habitants/year, with a steadily declining trend of −2.6%/year over the last 3 decades . The malMost relevant up-to-date evidence on pathological, diagnostic, and therapeutical characteristics of SNSCC are hereby summarized in a narrative fashion. For those aspects lacking in evidence and consensus, the authors will report their experience-based opinion, yet recognizing it represents the lowest level of evidence in the scientific literature.SCC is the most common malignant neoplasm of the nasal cavity and paranasal sinuses. Approximately 50–60% of all sinonasal cancers are SCC . SNSCC iThe maxillary sinus is the most common site affected by SCC of the sinonasal tract (approximately 60% of the cases), followed by the nasal cavity (25%), and ethmoidal complex (15%) . SCCs arMalignant transformation of Schneiderian papillomas is a known phenomenon, particularly for an inverted papilloma, whose transformation rate to SCC is reported as being as high as 10% . ApproxiInterestingly, approximately 20–25% of SNSCCs test positive for HPV. The majority of these are in the NKSCC cohort, which is histomorphologically similar to HPV-positive SCC of the oropharynx. The implications of this association are not clear yet. However, recent studies suggested a more favorable outcome in HPV-positive SNSCCs ,25,26,27Another pathological entity to be considered is NUT carcinoma ,30. HistThe differential diagnosis of poorly differentiated SNSCC is challenging and includes: SNUC , NECs, and adenoid cystic carcinoma ,34,35,36Preoperative histologic diagnosis of a given sinonasal lesion is essential to correctly plan a proper treatment strategy. Even to an experienced subspecialty pathologist in referral centers, this could be challenging because of non-specific presentation, overlapping appearance of different neoplasms, and the large miscellany of different possible histologies. Preoperative diagnosis has been reported to be non-concordant to the final histopathological diagnosis in 7–10% of HN cancers ,38. Not Another crucial step in treatment planning for sinonasal tumors is the analysis of local extension, with special reference to skull base, orbit, and infracranial spaces , which allows a “T category” to be attributed to a given sinonasal cancer according to the latest TNM classification criteria . In thisSinonasal tumors can extend to the adjacent regions by transgressing the bony walls of the nasal cavity and paranasal sinuses. Structural changes and reabsorption of these thin bone structures are well depicted by CT by virtue of its high spatial resolution and capability of demonstrating bone density alterations ,43,44. O18F-fluorodeoxyglucose-positron emission tomography (FDG-PET)/CT, are associated with higher probability of malignancy .,62.61,62The main goals of such therapy were reported to be a decreased risk of local recurrence and distant metastases, which are the main patterns of treatment failure of locally advanced sinonasal cancers . In addiClinicalTrials.gov (accessed on 1 June 2021) Identifiers: NCT03493425, NCT02099175, NCT02099188, NCT00707473).In 2011, Hanna et al. reported a series of 46 patients undergoing an induction ChT-including treatment for an SNSCC . ChT regIntra-arterial ChT aims at maximizing cytotoxicity while minimizing side effects related to systemic distribution of chemotherapeutic agents. The intra-arterial route permits the delivery of a high concentration of a cytotoxic agent directly to the primary tumor, in an effort to achieve local tumor response, thus omitting mutilating surgery and enabling organ-sparing treatment, while ensuring adequate survival outcomes. The maxillary artery is the preferred vessel to reach the sinonasal tract, thus the intra-arterial infusion finds its natural application for advanced stage SCCs of the maxillary sinus. Nakatani et al. employed neoadjuvant intra-arterial ChT combined with radiotherapy (RT), followed by surgery in 8 patients with advanced maxillary sinus cancer; they reported a response rate of 100% (complete response in 60% of cases), and none of the patients experienced grade 3 to 4 toxicity . InuyamaDespite the scarcity of data on this treatment modality, some groups advocated the use of neoadjuvant chemoradiation to optimize treatment morbidity while maintaining adequate oncologic results . RadiatiSurgery has an acknowledged role in the treatment of SNSCC ,81. As fThe current literature is uniform in highlighting the prognostic significance of achieving negative margins, as opposed to leaving microscopic or macroscopic residual disease. Jafari et al. published a large NCD study on 7808 patients affected by SNSCC, demonstrating a stepwise OS decrease ranging from complete resection to microscopically involved margins and macroscopic residual disease . InteresThe surgical approach should be selected based on tumor extension, which dictates the extent of the ablation .The surgical approach should be selected based on tumor extension, which dictates the extent of the ablation. The debate on whether endoscopic or open surgery should be selected has been slowly progressing towards consensus. There is evidence that endoscopic transnasal surgery provides a shorter hospital stay  and surgIn case of an endoscopic resection without transnasal craniectomy, reconstruction is often unneeded. When available, local pedicled mucoperiosteal flaps  ,101 can A complex reconstruction is required after a large surgical resection involving the maxillary and/or frontal bone; the maxillary and frontal bones are centerpiece of the middle and upper face, respectively, and their resection has aesthetical, functional , and vital  implications. Moreover, sinonasal malignancies most often require a multimodal management, with a high rate of patients sent for adjuvant treatments, ,113,114 Owing to the rarity of SCC arising from the frontal sinus,  principlAs a final remark, tissue bioengineering is expected to provide game-changing possibilities in the field of craniomaxillofacial reconstruction, but, to date, most reported experiences are anecdotal and applied to nononcologic scenarios .Involvement of cervical lymph nodes is considered one of the most significant prognostic factors in HNSCC. Clinical evidence of nodal metastases was reported to be around 13% in the largest series of nasal cavity and paranasal sinus SCCs . The perSNSCC patients with clinically positive neck should receive a neck dissection as a part of surgery. There are no data to support one type of neck dissection over the others, therefore selective, comprehensive, or radical neck dissection have been alternatively advocated ,128. To On the other hand, there is no consensus on how to manage the neck in patients affected by cN0 SNSCCs. In a large series of patients with maxillary sinus SCCs published by Cantù et al., the rate of neck metastases, either at presentation or after treatment, was much higher in patients with T2 tumors than in those with T3, T4a, or T4b tumors, differently from any other HN cancer . Based oBerger et al.  recentlyBased on personal experience and data available in the literature, it is the authors’ opinion that neck dissection should be reasonably indicated in case of clinically appreciable nodal metastasis  or when harvesting of recipient vessels is required for reconstruction purposes  .Complete tumor removal followed by adjuvant intensity-modulated RT (IMRT) is the current mainstay of treatment for advanced SNSCCs (pT3-4) and in most intermediate tumors (pT2). SNSCCs classified as pT1 are anecdotal and, therefore, there are no data to support the use of unimodal vs. bimodal treatment. Some histological subtypes of SNSCC  might have pronounced radiosensitivity [66); areas at high risk for microscopic tumor invasion based on initial gross tumor extension and surgical violation are included within the CTV receiving 60 Gy . A 0–5-mm margin is added to both CTV66 and CTV60 to delineate the planning target volumes . Dose per fraction is commonly kept at 2 Gy. Areas that were both spared by surgery and deemed at low risk for microscopic disease should receive 54 Gy in 1.8 Gy-fractions . In cN0 tumors for which elective neck irradiation is indicated, PTV54 includes also lymph nodal levels I to IV; level V should be included in case of nasoethmoidal or nasopharyngeal-involving SNSCC; retropharyngeal nodes are included in case of nasopharyngeal-involving SNSCC, or in case of nodal metastases from a nasoethmoidal SCC. The contralateral nodal basin is included in the radiation field based on a case-by-case evaluation.IMRT is an advanced external-beam, photon-based RT that is most beneficial for complex and irregularly shaped target lesions located adjacent to critical organs at risk . The rel12C-carbon ions) provides several advantages compared to photons-based RT, including the ability to design a sharp dose gradient owing to the so-called “Bragg peak”, increased radiobiological effectiveness thanks to high linear energy transfer, and relative independence of tissue oxygenation [Intensity-modulated particle therapy (IMPT)  with RT alone [Although surgery followed by adjuvant RT(-ChT) has been associated with increased survival outcomes in SNSCCs,  definitiRT alone . They deOverall, sending a patient affected by SNSCC for definitive photon-based (chemo)radiation seems a less-than-optimal treatment path, which should be currently reserved to cases for which complete surgical resection is deemed unfeasible or unacceptably invasive. However, the marked and often unpredictable radiosensitivity displayed by some sinonasal cancers suggests that identification of reliable biomarkers to select SNSCCs which will respond to RT(-ChT) should be one among the several unmet aims in the field of sinonasal oncological research.In view of their physical and biophysical properties, charged particle beams are ideally suited for dose escalation in complex anatomical sites located close to critical and radiosensitive organs . The dosClinicalTrials.gov (accessed on 1 June 2021) Identifier: NCT01586767), whereas another trial is investigating the role of R2 endoscopic surgery followed by IMPT in patients affected by unresectable sinonasal cancer  Identifier: NCT03274414).A registered clinical trial is currently comparing definitive IMPT with IMRT for sinonasal malignancies  is another resource of the therapeutic armamentarium for recurrent sinonasal cancers. ReRT can be indicated as either standalone or adjuvant modality following surgery. Currently, benefits of reRT of recurrent sinonasal cancer in terms of survival and locoregional control is unclear, whereas the potential sequelae, such as osteoradionecrosis and vascular blow-out, are well known and particularly feared ,182. A rStereotactic RT should also be considered in the palliative setting. In particular, stereotactic RT of recurrent, unresectable tumors determining trigeminal neuralgia has proved to be effective in decreasing patient-reported pain and opioid requirement .ChT can be indicated for locoregional recurrences deemed ineligible to locoregional treatment and/or in case of distant metastasis. Drugs used in the palliative setting are similar to those mentioned for neoadjuvant ChT; the choice of combination therapy vs. monotherapy and dose depend on clinical conditions of the patient . The rolSystemic immunotherapy with nivolumab or pembrolizumab is in the process of being possibly entitled as alternative “first line treatment” for recurrent/metastatic HNSCC. However, very little information on the potential effect of immunotherapy on sinonasal tumors is currently available. Riobello et al. demonstrated that membranous expression of PD-L1 is observed in 34% of the tumor cells and in 45% of immune infiltrating cells of SNSCC, thus alluding to a potential benefit from immunotherapy in selected cases . OverallMetastasectomy should also be considered in well selected patients. Location and number of distant metastases, and distant metastasis-free interval following presentation are the main factors to take into account. In sinonasal histologies with well-known quick kinetics, such as SNSCC, the threshold of the distant metastasis-free interval to stratify prognosis was identified earlier than for notoriously slower progressing tumors, such as adenoid cystic carcinoma ; Lu et aAdequate palliation may require different modalities and, therefore, the same actors playing a major role in curative treatments are likely to be somehow involved also in noncurative care. Given the different aim of palliation as opposed to curative treatment, complications and toxicity are, by far, less acceptable, and any treatment modality should be tailored accordingly.Rarity, biological diversity, and heterogeneity of management are the main factors limiting the understanding of SNSCC. These aspects resulted in a limited level of evidence, which does not overcome the threshold of 2–3 ; given the majority of current thoughts on SNSCC are based on level 3-to-5 evidence, recommendations resulting from scientific data should be interpreted cautiously. The present review has highlighted a number of recent refinements in the molecular definition of sinonasal epithelial cancers. Multimodal imaging is essential to reliably stage the local, regional, and systemic extension of the tumor. Surgical ablation is the mainstay of treatment. Neoadjuvant ChT is currently indicated with different aims, including orbit preservation, stratification of prognosis, chemoselection of locoregional treatment, and reduction of distant metastases. Neoadjuvant ChT-RT has been advocated to increase the chances of sparing the orbit and improving the margin status in patients receiving surgery. IMRT, possibly combined with concomitant ChT, plays an essential role in the curative-intended treatment of the large majority of SNSCCs. The advent of IMPT has been providing promising results in the field of sinonasal and skull base cancer treatment and is expected to also be beneficial for SNSCCs. Although the management of the neck is far from being standardized, an algorithm is hereby presented based on available evidence and authors’ personal experience . Managem


Sex differences are essential factors in disease etiology and manifestation in many diseases such as cardiovascular disease, cancer, and neurodegeneration . The bio Genetic, epigenetic, hormonal, immunological, metabolic, and environmental factors affect sex-biased drug responses.Drug repurposing approaches provide a significant advantage over novel drug development by reducing lengthy and costly clinical trials.Advances in compute processing power and optimized algorithms for computational systems have increased the efficiency and feasibility of computational drug repurposing.Multiple challenges still need to be addressed for sex-aware drug repurposing, including the insufficient understanding of the cause of variation of drug responses due to sex differences, better performing sex-aware repurposing methods, and the lack of large and balanced datasets to develop improved methods.Future low-dimensional representations of molecular association and network approaches could significantly impact the field of sex-aware drug repurposing. Attempting to isolate novel therapeutic drug candidates can cost one to two billion dollars and 12–16 years of research . Add. Add1]. Data mining drug repurposing approaches retrospectively analyze information from and across clinical trials, biomedical literature, and other resources with drug outcome or drug target information to identify novel drug indications . These ahttp://www.nlm.nih.gov/research/umls/), ontology sources often remain incongruent and may lead to misconceptions and error [Additionally, researchers should be aware of data mining challenges with regards to variation in data structure and nomenclature. While the NIH is implementing a new Policy for Data Management and Sharing (DMS Policy) effective January 2023 for NIH-funded research to ensure stricter standards for data sharing and the availability of raw data, this has been a pervasive issue from past data sharing up until this point . Due to nd error . In summAnother drug repurposing approach, ligand-target binding prediction, identifies drugs predicted to bind to a disease target  based on their binding affinity . MoleculMolecular association methods identify targets or patterns from molecular data  that correlate with disease, therapeutic outcomes, and/or drug candidates . MoleculNetwork approaches evaluate mathematical graphs (nodes joined together by edges) representing relationships as edges between different nodes like genes, proteins, diseases, and drugs to identify drug repurposing candidates . One of However, a promising approach is to use a combination of computational and experimental approaches to identify and validate drug repurposing candidates Table . For exaLastly, with some complex diseases such as cancer, the use of combinational drug therapy increases the rate of success because different combinations of drugs can have synergistic effects on the same target or multiple targets . For exaIn this section and Table Currently, there are limited methods available to either select sex-specific drug repurposing candidates that will be effective against a disease of interest or prioritize candidates to avoid SBAEs. While these methods fall under the same drug repurposing categories, the following sex-aware methods are variations that adjusts input data, parameters, and/or algorithms for sex differences to identify sex-specific drug candidates or SBAEs. We did not consider drug repurposing methods that used sex as a covariate as sex-aware because using sex as a covariate reduces the impact of sex in computational models. These models will not identify sex-specific drug candidates and adverse events. Here we summarize the currently available sex-aware approaches or studies for sex-aware drug repurposing Table .Table 2SThe first sex-aware drug repurposing approaches use data mining to identify SBAEs based on case information from patient adverse events databases such as FAERS or VigiBase. A study from Yu et al. calculated the reporting odds ratio for a sex-bias adverse event based on FAERS’ case reports . They foFurthermore, there are some additional limitations with data mining studies. For example, adverse events are often voluntarily reported by healthcare professionals, consumers, and drug manufacturers . Even wiNext, we identified two sex-aware molecular association studies that used molecular biomarkers to identify sex-specific drug candidates or SBAEs. The first, a study by Kfoury et al., identified drug repositioning candidates not currently FDA approved for any condition that might benefit GBM patients . This grOne sex-aware experimental approach is the manipulation of sex hormones as a therapy for a disease. Such manipulation is used when one sex tends to respond better to current therapies and/or have better outcomes than the other. For example, in AD, which is more prevalent in females compared to males, leuprolide acetate, an androgen deprivation therapy used for the treatment of prostate cancer, has been investigated for the treatment of AD as it might slow the progression of the disease , 157. ThWhile most of these methods performed poorly due to limited, sex-balanced datasets. Future development of strategies to more accurately model sex-bias from these unbalanced datasets via methods similar to transfer learning approaches done by Gao and Cui study or leverage new and more balanced datasets is required. Furthermore, sex impacts biological systems in multiple ways  , 33. ThiMolecular association and network approaches are promising candidates for future sex-aware drug repurposing methodologies. Molecular association studies provide molecular biomarkers that might be causal for different responses to drugs . These mAs an emerging computational approach for sex-aware drug repurposing, network techniques developed by the Network Zoo have been used to build gene regulatory networks to identify regulatory pattern differences between males and females from GTEx tissue samples . The autDue to several limitations, data mining and ligand-target binding prediction approaches are challenging to adapt for sex-aware drug repurposing. First, data mining approaches typically require large amounts of balanced data, equal male and female data points. However, several databases are not balanced. For example, the UK biobank is more female-biased  while GTEx is more male-biased  , 161. InLigand-target binding prediction methods could also be difficult to adjust for sex differences. Several studies have identified that sex differences can be due to gene regulatory and hormone signaling , 151. ThSeveral challenges exist across basic, translational, and clinical research in assessing sex as a biological variable (SABV) in in vitro, in vivo, and in silico model systems Fig. . These cCTPS2 and DLG3 X chromosome genes are known to cause differences in drug response  and reactivate expression of silenced genes on the inactive X chromosome, a process known as inactive X erosion [In vitro cell line models are important to drug repurposing because many drug screens and validation experiments use these models for testing the efficacy and toxicity of drug repurposing candidates. However, some cell lines become “de-sexualized” after losing a Y chromosome, in the case of male cell lines, or the loss of an X chromosome, in the case of female cell lines . Additio erosion . ResearcDrosophila genus of flies, XX, XXY, and XXYY flies are female; while XY and XO flies are male [C. elegans, has XX hermaphrodites and XO males [Additionally, genetic and hormonal sex differences are also difficult to account for in vivo animal models as these can vary across organisms . While tare male . In flieare male . AnotherXO males . In addiXO males . OverallXO males , 177.Another challenge for in vivo models is sex differences in human phenotypes might not be present in a given model organism. For example, the longevity between males and females varies significantly between species and human females, similar to yellow baboons, tend to live longer . Mice, aSf1 knockout mouse [Sry gene is moved from the Y chromosome to an autosome to generate four genotypes: XXF (XX mice with ovaries), XXM (XX mice with testes), XYF (XY mice with ovaries), and XYM (XY mice with testes) [In vivo animal studies have historically excluded female animals from many study designs, resulting in less female data . For exaut mouse , 191, seut mouse , 193, orut mouse , 195. In testes) , 195. No testes) .The biological impact of sex extends also to impacts on in silico modeling of biological systems. The exclusion of female animals in preclinical studies  and the We envision sex-aware drug repurposing as a standard analysis used in drug repurposing research due to the overwhelming evidence that sex is important for drug response. Even if a disease does not show a known sex difference, the variation of drug responses between the sexes warrants investigations of SBAEs and drugs that might have sex-specific therapeutic effects. While several drug repurposing strategies attempt to find drug candidates without the influence of sex, sex-aware drug repurposing identifies drug candidates that will have differential effects between the sexes by either having variations in therapeutic effects between the sexes or cause an adverse event in one sex . In this review, we highlight several FDA-approved drugs and drug candidates that have different therapeutic effects, such as the BET inhibitor drug candidate for GBM . A potenHere we described sex-aware drug repurposing and discussed the challenges and future of sex-aware drug repurposing. Drug repurposing is a valuable method for identifying drug candidates for FDA approval because of its ability to prioritize efficacious drug candidates at a reduced cost compared to traditional drug discovery , 83. HowThere is an urgent need to address the following: (1) the lack of balanced data to develop accurate models for sex-aware drug repurposing, (2) the need for a variety of improved sex-aware drug repurposing methods, and (3) the scarcity of studies relating to sex differences and variation in drug response between the sexes. Increased representation of females in biomedical research and clinical trials through balanced sex studies or female-only studies is needed to improve drug repurposing approaches. While some methods are available to overcome limited datasets , 155, ulUpon reflection, one promising sign that sex-aware methods will improve the field of drug repurposing is how tissue-aware drug repurposing has improved the field. While sex is an essential factor in drug response, other factors such as tissue differences, aging, development, race, social, and environmental factors are also important to consider for drug repurposing methods. For example, a study investigated tissue-specific genetic features of drug target genes  . They diIn conclusion, the development of sex-aware drug repurposing methods is essential but challenging due to the understudied complexity of sex differences. We recognize these challenges for sex-aware drug repurposing, but its potential for biomedical research and patient care outweighs the difficulties. In the future, sex-aware drug repurposing will identify safer and more efficacious drug candidates for both males and females.