The vascular endothelium, a monolayer of endothelial cells (EC), constitutes the inner cellular lining of arteries, veins and capillaries and therefore is in direct contact with the components and cells of blood. The endothelium is not only a mere barrier between blood and tissues but also an endocrine organ. It actively controls the degree of vascular relaxation and constriction, and the extravasation of solutes, fluid, macromolecules and hormones, as well as that of platelets and blood cells. Through control of vascular tone, EC regulate the regional blood flow. They also direct inflammatory cells to foreign materials, areas in need of repair or defense against infections. In addition, EC are important in controlling blood fluidity, platelet adhesion and aggregation, leukocyte activation, adhesion, and transmigration. They also tightly keep the balance between coagulation and fibrinolysis and play a major role in the regulation of immune responses, inflammation and angiogenesis. To fulfill these different tasks, EC are heterogeneous and perform distinctly in the various organs and along the vascular tree. Important morphological, physiological and phenotypic differences between EC in the different parts of the arterial tree as well as between arteries and veins optimally support their specified functions in these vascular areas. This review updates the current knowledge about the morphology and function of endothelial cells, particularly their differences in different localizations around the body paying attention specifically to their different responses to physical, biochemical and environmental stimuli considering the different origins of the EC. Other projections are close to inter-endothelial gap junctions. In these gap junctions, SKCa channels are prevalent. A hyperpolarization of EC occurs by an activation of KCa. An efflux of K+ can have a similar effect as diffusible ‘EDHF’ by the stimulation of vascular smooth muscle Na+-/K+-ATPase and K channels which are inwardly rectifying. Simultaneously, a hyperpolarizing current spreads through myoendothelial gap junctions located on endothelial projections from the endothelium to the smooth muscle. By the spread of ‘conducted’ hyperpolarization along the endothelium of arteries and arterioles to affect conducted vasodilatation the resulting radial EDH is amended. The contribution of EDHF to the endothelium-dependent relaxation is currently actively discussed whether an important feature of a healthy endothelium despite the ongoing debate on its variable nature and mechanisms of action. Experiments have shown that when NO and Prostacyclin are inhibited vasodilation still occurs . The conand IKCa ,79,80. T2, endothelin-1 (ET-1), prostaglandin H2 (PGH2), and reactive oxygen species (ROS) [On the other hand, endothelium derived factors can also induce vasoconstriction. The vasoconstrictors include angiotensin II (Ang II), thromboxane Aes (ROS) ,80,81. Ies (ROS) . The endes (ROS) . In the es (ROS) ,84,85. EIn inflammation, various functions of the endothelium are pivotal. These functions include increasing permeability, vasodilation, increased leukocyte extravasation, and alterations in the control of coagulation and thrombus formation. While resting, intact endothelium is mainly antithrombotic, however with the activation of the EC by stimuli present in pathological conditions, the endothelium may become pro-thrombotic. Stern et al. demonstrated the presence of receptors for coagulation factors on the intact endothelial surface . In vitrThe exchange of fluids, nutrients and hormones that occurs in the capillaries is enabled by the exposure of a relatively small volume of blood to a large endothelial surface. For this trans-endothelial transport, the active vesicular transport system is important, utilizing plasmalemmal structures (caveolae). On the luminal surface of the endothelium numerous receptors for circulating endogenous substances exist, some of them are located in the caveola, offering the essential means for attachment, endocytosis and subsequent movement across the endothelium into the surrounding tissue. In addition, several macromolecules  and enzymatic activities  are also localized on the luminal surface of the endothelium. These components on the endothelial surface play important homeostatic roles and regulate the interaction between blood-borne elements and the interstitium.In the vascular system of a healthy organism, no harmful interactions between platelets and EC take place, although there is close contact between both cell types . PlateleEC secrete various signals and mediators, which are important for the regulation of blood coagulation and platelet functions, in order to prevent platelet adherence and aggregation under normal physiological conditions. Prostacyclin (PGI2) and nitric oxide (NO) are the major antiplatelet agents which are constitutively secreted by EC . Both meBeyond these effects, EC can maintain blood fluidity. This occurs by promoting the activity of numerous anticoagulant pathways, whereas the protein C/protein S pathway is the most important. By the interaction of thrombin with the EC receptor thrombomodulin the protein C/protein S pathway is initiated, facilitating the activation of protein C . FactorsThe formation of a thrombin and thrombomodulin complex also suppresses thrombin to clot fibrinogen or to activate platelets . A rich Upon vascular injury (damage or detachment of EC), blood components like proteins or cells adhere to the denuded subendothelium—e.g., to collagen fibers or to von Willebrand molecules—become activated and aggregate at the site of injury. This evokes the formation of a stable blood clot preventing excessive blood loss. During the development of thrombosis, the significance of an appropriate control of blood coagulation is obvious. Thrombosis, an occlusive clot within a blood vessel, reduces the blood flow to downstream tissues and organs thereby restricting the delivery of nutrients and oxygen. This can induce necrosis in localized tissue and organ necrosis. Large occlusive thrombi can detach and embolize, then occluding distal vessels. The process of thrombosis followed by embolism is collectively termed thrombo-embolism and can culminate in a variety of local or chronic disorders . SimilaIn general, EC in different regions of the body fulfill similar demands, however some features and response characteristics are distinct. Aird was the first to describe this the heterogeneity of EC . The str2, and pH, veins and arteries are also different at a cellular and molecular level [Arteries and veins have different functions. Arteries deliver oxygen and nutrients to tissues, while veins remove cellular waste. Due to these distinctions in function, as well as of local differences in blood pressure, blood flow, pOar level . Subsequar level ,106,107.ar level ) sometimar level ).This heterogeneity of EC promotes to their physiological diversity across the vascular tree . The endConsequently, EC in the various vascular beds respond differently to signaling molecules. Comparative studies with freshly isolated EC from coronary micro-vessels, coronary arteries and veins showed regional differences in the capacity of prostaglandin synthesis . ResultsIn addition, the response of cells from various vascular beds to inflammatory cytokines and vasoactive agents differs, where the postcapillary venules response is most pronounced. Snopko et al. reported that microvascular EC, grown as typical monolayers, producing significantly less prostacyclin than arterial or venous EC after stimulation with serum .Moreover, the response of venous and aortic EC to chemical agents can differ, too . FunctioA comparison of cultured EC from arteries, veins, and micro-vessels revealed that not only arteries and veins but in particular, microvascular EC displayed distinct differences showing specific genomic properties and other differences at the tissue level . This isEC in culture may not display the characteristics seen in vivo in the absence of their typical basement membrane , althougAn additional problem with these models was shown to be the difficulty in obtaining and successfully culturing capillary EC from different vascular beds. As of now, this is a limitation for comprehensive comparative studies of the capillary endothelium. There are further limitations when artificial substrata like glass, polymers or metals are used which are foreign to EC. The development of more sophisticated culturing conditions could contribute to the identification of biochemical and functional features, which are unique to the endothelium of specific organs, and may provide insight into interactions between toxins and the vascular bed.The thickness of the glycocalyx varies tenfold from several hundreds of nanometers to several micrometers throughout the vasculature . DiffereUnder normal physiological conditions, EC remain in a quiescent state. However, in response to tissue injury or hypoxic conditions, ECs can more or less rapidly form new vessels by a highly orchestrated process of vessel sprouting, called angiogenesis . AngiogeAngiogenesis is strongly associated with the vascular basement membrane (BM) and the extracellular matrix (ECM). In addition, the interaction between cells and the ECM is required. The first step involves the opening of existing capillaries and partial degradation of surrounding ECM allowing cell infiltration. In initiating this process, EC adopt a proteolytic phenotype and begin to break down the basement membrane by matrix metalloproteases (MMP) . MMP andIn 1990, Jung et al. could document a case of angiogenesis in a human . The in-After fifty-three days a growth of a new capillary after capillary occlusion was observed. Over a period of 47 days, the mean capillary growth velocity was measured with 4.7 µm/day. Between the 85th and 98th day after occlusion a maximum growth rate of 10 µm/day was found. A minimum growth rate of 2 µm/day occurred during the first 11 days.An important question to consider was whether the old capillary channel was used again or a completely new channel was formed. Differences can be seen by superimposition of the picture after growth of the new capillary . The peNumerous genetic and molecular signals were identified over the last decades to regulate this process  and manyA common attempt to repair or replace biological tissues is the implantation of biomedical devices or synthetic matrices. Often, an adverse foreign body reaction occurs in the vicinity of implanted devices , associaMetastasis distant from the primary tumor are created through tumor cell infiltration into the blood circulation and transport to other regions of the organism via the blood flow . Hematog(i) express specific membrane receptors , whose ligands would be found most favorably on EC of the target organ, or(ii) a lump of tumor cells can locally induce blood coagulation, creating an embolus mixed of blood and tumor cells which favors attachment of the embolus to EC (unspecific gatekeeper effect for the emigration of tumor cells from the blood circulation ).The organ-specific heterogeneity of EC favors settling of tumor cells and metastatic spread in specific organs.This review focuses on the morphology and function of endothelial cells, particularly their differences in different localizations around the body paying attention specifically to their different responses to physical, biochemical and environmental stimuli considering the different origins of the EC. It has to be kept in mind, that the results presented here were gathered either from in vitro analyses or from animal studies. Ethical considerations do not allow to study endothelial cells from different vascular regions in humans. This is true for healthy persons and especially for patients with different kinds of diseases. That is why, the transfer of these results to the situation in humans has to be regarded with caution. In general, it is of utmost importance for the conduct of in vitro studies to generate culture conditions, e.g., cell culture media, cell substrates and cell environments, which create growing similarity to the situation in situ/in vivo.A further deepening of the understanding of the endothelial cell biology is essential for the elucidation of specific mechanisms in tissue engineering and regenerative medicine or for novel approaches in the treatment of cardiovascular diseases.


The complex ecosystem in which tumor cells reside and interact, termed the tumor microenvironment (TME), encompasses all cells and components associated with a neoplasm that are not transformed cells. Interactions between tumor cells and the TME are complex and fluid, with each facet coercing the other, largely, into promoting tumor progression. While the TME in humans is relatively well-described, a compilation and comparison of the TME in our canine counterparts has not yet been described. As is the case in humans, dog tumors exhibit greater heterogeneity than what is appreciated in laboratory animal models, although the current level of knowledge on similarities and differences in the TME between dogs and humans, and the practical implications of that information, require further investigation. This review summarizes some of the complexities of the human and mouse TME and interjects with what is known in the dog, relaying the information in the context of the temporo-spatial organization of the TME. To the authors' knowledge, the development of the TME over space and time has not been widely discussed, and a comprehensive review of the canine TME has not been done. The specific topics covered in this review include cellular invasion and interactions within the TME, metabolic derangements in the TME and vascular invasion, and the involvement of the TME in tumor spread and metastasis. Cancer, the uncontrolled proliferation of cells, is a significant cause of morbidity and mortality in humans and their canine companions worldwide , 2. The The interplay between the TME and tumor cells is paramount in the progression and response to neoplastic growth. While our understanding of the TME in dogs is rudimentary, there are the similarities in tumor heterogeneity between dogs and humans , which aWhile the definition of “cancer stem cells”  is mostly semantic, the importance of cells that retain or acquire stem-like features in the tumor cannot be underestimated. Whether they be few, as in traditional hierarchically-organized tumors, or many, as in stochastically organized tumors, these cells contribute to remodeling the TME .Recent work characterized genome-wide gene expression signatures in canine tumor models  that were grouped according to their hierarchical organization . Cell liIntriguingly, the CSC condition appears to be at least partly under extrinsic control. Depletion of CSCs in cultured canine and human cell lines leads to reprogramming of differentiated cells to become CSCs, maintaining the population in a steady state . The sigThe role of adipocytes in the TME has received more attention as evidence mounts for a link between obesity and cancer risk in dogs and humans , 18. AdiAdipocytes promote neoplastic development through a variety of mechanisms, including supporting angiogenesis (described later in this review), manipulating tumor cell metabolism, and encouraging a pro-inflammatory state, leading to the recruitment of macrophages. Adipocytes play an important role in reprogramming tumor cell metabolism. For example, ovarian cancer cells co-cultured with abdominal adipose cells were shown to coerce neighboring adipocytes into supplying free fatty acids, thereby providing substrates for sustained tumor cell replication . The rolRecently, attention has been given to the influence of adipose-derived mesenchymal stem cells (ad-MSCs) in tumor progression. The secretome of ad-MSCs is incompletely understood but is thought to have overarching immunomodulatory and pro-angiogenic properties . The immIn vitro studies using cell lines from various species, although to the authors' knowledge not from dogs, have demonstrated that normal, non-cancer associated fibroblasts and the matrix they produce are capable of inhibiting the spread of tumor cells, a phenomenon termed neighbor suppression  including fibrillar collagen, elastin, laminin, fibronectin, and glycosaminoglycans . Fibroblpression –31. Sincpression , many thpression , 33. Neipression .Cancer-associated fibroblasts (CAFs) are corrupted by the neoplastic cells in their proximity and have drastically different functions than their non-transformed counterparts. The origin of CAFs is not entirely clear; many theories on their origin claim CAFs originate from resident mesodermal precursors –38. An iMesenchymal stem cells (MSCs), also known as undifferentiated fibroblasts or mesenchymal stromal cells, are another important component of the TME. These cells are phenotypically plastic cells that originate from the mesoderm . MSCs hoTumor-associated ECM is markedly different from ECM in a non-pathologic milieu. As an active driver of tumor progression, tumor-associated ECM is reorganized, directing tumor cell migration and promoting local invasion along collagen fibers , 51. FurInteractions between tumor cells, stromal cells, and the ECM are heterogeneous and tumor-specific. However, fragmentation of hyaluronic acid, which is pro-inflammatory, and deposition of tenascin-C seem to occur in most tumor types –58. The The extensive heterogeneity and adaptation of the tumor niche is partly dependent on intercellular communication. Malignant cells co-opt developmental programs of intercellular communication to create and maintain a niche with unique properties that promote growth and survival . InterceSoluble mediators of communication have been relatively well-described in humans, although there is little information available as to the impact of the stroma and soluble factors in dogs. Kim et al.  showed tIntercellular interactions are also critical to establish and maintain the tumor immunosuppressive barrier, by excluding or incapacitating host immune cells. For example, expression of pro-apoptotic molecules, such as FasL can target infiltrating effector T cells in the tumor environment, while sparing apoptosis resistant tumor cells, CAFs, and cancer-associated endothelial cells. For more information on immune cells within the TME, please see the next section on metabolism, vascular invasion, and immune cells within the TME.ex vivo and ~100–500 microns in vivo , which is involved in the formation of some types of prostaglandin production, has been shown to increase expression of VEGF-A mRNA in tumors, thus having pro-angiogenic properties . The mecThe clinical implications of HIF-1 and VEGF expression and regulation have been the subjects of recent investigation. Moeller et al.  were theHypoxia affects innate and adaptive immune function in multiple and complex ways . MacrophIn the absence of oxygen, cells are obligated to use glycolysis to produce ATP. Reprogramming energy metabolism is regarded as a hallmark of cancer, as described by Hananhan and Weinberg . The “WaLactic acid is a major component that fuels metabolic symbiosis between the aerobic and hypoxic tumor compartments . LactateUtilization of lactate has also been described in tumor-associated fibroblasts, which have low expression of CAV-1, an inhibitor of myofibroblast differentiation . An infoOnce tumor-associated blood vessels are formed, they are structurally and epigenetically abnormal, which facilitates metastatic spread. These vessels tend to be irregularly dilated and tortuous with increased permeability, decreased pericyte numbers, and abnormal deposition of collagen type IV in the basement membrane. Endothelial cell adhesion molecules, such as selectins and integrins are required for leukocyte transmigration into tissues , 138. ItLike neoangiogenesis, lymphangiogenesis can act as an important gateway to tumor metastasis. The density of lymphatic vessels within a tumor has been correlated with tumor growth and metastasis in both dogs and humans [In their landmark update of the Hallmarks of Cancer in 2011 , HanahanThousands of studies have examined the composition of the TME in humans and animals. Most studies focused on one or a few features in isolation, such as infiltration by immunosuppressive elements like Tregs or by tumoricidal NK cells or cytolytic T cells. For example, increased CD4+:CD8+ T cell ratios were correlated with decreased survival in dogs with mammary carcinomas , and enrRecent advances in next generation sequencing and bioinformatics, as well as the availability of high-quality samples that comprise The Cancer Genome Atlas (TCGA), made it possible to divide the immune landscape of human tumors into six distinct steady states or subtypes . ThorssoRecent advances in sequencing technology and bioinformatics  are beinGorden et al.  showed tIt is widely accepted that macrophages play a major role in molding the TME, making them attractive targets for tumor therapy. Myeloid antigen presenting cells (APCs), and especially dendritic cells, control the initial steps in the cancer-immunity cycle, engulfing tumor cells and tumor debris and presenting it to T cells in the draining lymph nodes . Since tImmune recognition of the tumor intensifies the selective pressures that drive tumor evolution through the process of immunoediting . T cellsThe elimination phase of immunoediting gives way to an equilibrium phase where the immune system appears to control the tumor. However, editing is not static, and the process eventually favors outgrowth of resistant tumor cells that “edit” the epitopes recognized by the immune system. Editing can occur through downregulation of major histocompatibility complex (MHC) proteins, upregulation of proteins that resist T cell activation and killing, acquisition of the ability to kill activated T cells or resist T-cell induced apoptosis, or alteration of target epitopes by further mutation or epigenetic regulation . The hosClinicalTrials.gov Identifier: NCT01583686).Some common steps in the evolution of the immune microenvironment of cancer has led to the development of highly effective immunotherapies. Immune checkpoint blockade using antibodies that interfere with binding of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to CD80 and CD86, or with binding of PD-1 to PD-L1 and PD-L2, are the first therapies directed against the TME that have been effective at achieving meaningful cancer control. Durable remissions in as many as 50% of patients with advanced cutaneous melanoma, various types of tobacco-related malignancies, gastrointestinal tumors, and certain blood cancers have been achieved via these therapies , 191–193Spontaneous canine cancers provide a rich resource to understand both conserved and species-specific mechanisms that create and maintain the tumor immune landscape. Dogs can teach us much about therapeutic immune system manipulation in the context of cancer, including the potential to alter the TME to enhance immune responses. There are numerous published studies on the subject, including using pharmacologic intratumoral delivery of vectors encoding FasL , a varieThe components of the TME work in concert through epigenetic and functional means to promote tumor cell invasion and metastasis. Although metastasis can occur at any point in space and time during the course of tumor evolution, the cellular, structural, and molecular components of the TME are able to enhance numerous pro-tumorigenic activities, which in turn facilitate invasion and enhance the metastatic potential of tumor cells. The organization of the primary tumor niche requires significant alterations to the normal extracellular environment. Molecular interactions are highly specific to different tumors and can vary substantially even within tumor types; but the general features include “loosening” of stable cell-cell adhesion, loss of cell polarity, and reorganization of the cytoskeleton, as well as stiffening of the extracellular matrix, which enhances motility, facilitates invasion, and enables the metastatic phenotype , 71, 73.Changes in cell-to-cell contacts in sarcomas have been less well-studied, almost certainly due to the rarity of these tumors in humans. The concept of EMT in sarcomas presents a paradox: some, but not all sarcomas exhibit aggressive, rapidly metastatic phenotypes, and cells in these tumors undergo phenotypic changes that resemble EMT. Yet, all sarcoma cells possess a mesenchymal phenotype. This has given rise to a more nuanced vision of EMT, and the reverse process called mesenchymal to epithelial transition (MET), where the transcriptional and epigenetic mechanisms that regulate these transitions give rise to metastable phenotypes that are adaptive . In otheEmerging evidence suggests that exosomes are critically important mediators that mold the distant or metastatic tumor niche in blood-derived and solid tumors. Exosomes are formed by inward budding of early endosome membranes by the endosomal sorting complex required for the transport (ESCRT) complex. Mature endosomes, also known as multi-vesicular bodies (MVBs) fuse with plasma membranes releasing exosomes vesicles to the extracellular space. Exosomes circulate systemically and can bind to and merge with other cells, creating a mechanism for horizontal transfer of activated oncoproteins, oncogenic DNA, and oncogenic and regulatory microRNAs . For exaCancers are complex and dynamic multicellular tissues; multiple distinct events contribute to initiation, promotion, and progression. Ultimately, these events converge into more rigid molecular programs and create recognizable histological tumor phenotypes that are widely conserved across species. Tumor formation is a tightly orchestrated process, molded by selection to support growth and survival of a clonal population of immortalized cells. This review has demonstrated the complexity and intricacies of the TME in the human and mouse, and established, to the best of the author's abilities, the same complexities within the dog. While there is much room for growth in the understanding of the TME in the dog, the current knowledge base in conjunction with the information known about the TME in humans and mice, provides a solid foothold for the development of further basic and clinical endeavors.KL, JK, MD, and JM contributed to the writing of this manuscript. Figures were rendered by KL and AS. Editing was performed by all authors of this paper.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.


The complement system is integral to innate immunity, and it is an essential deterrent against infections. The complement apparatus comprises of >30 fluid-phase and surface-bound elements that also engage with the adaptive immune system, clear harmful immune complexes, and orchestrates several salutary physiological processes. An imbalance in the complement system's tightly regulated machinery and the consequent unrestrained complement activation underpins the pathogenesis of a wide array of inflammatory, autoimmune, neoplastic and degenerative disorders. Antibody-mediated rejection is a leading cause of graft failure in kidney transplantation. Complement-induced inflammation and endothelial injury have emerged as the primary mechanisms in the pathogenesis of this form of rejection. Researchers in the field of transplantation are now trying to define the role and efficacy of complement targeting agents in the prevention and treatment of rejection and other complement related conditions that lead to graft injury. Here, we detail the current clinical indications for complement therapeutics and the scope of existing and emerging therapies that target the complement system, focusing on kidney transplantation. Complement proteins account for 3 g/l of plasma and make up ~15% of the globulin fraction  (a) the The approval of the complement-targeting anti-C5 antibody, eculizumab, by the United States Food and Drug Administration (FDA) for the rare disease paroxysmal nocturnal hemoglobinuria (PNH) in 2007 has led to a renaissance in complement therapeutics . GPI serIn atypical hemolytic uremic syndrome (aHUS), complement overactivity leads to systemic thrombotic microangiopathy involving the kidney, central nervous system, heart, and gastrointestinal tract. In 2011, the FDA approved eculizumab for use in aHUS following a clinical trial demonstrating significant improvement in renal function with its use . HereditKidney transplantation is the therapy of choice for eligible patients with end-stage renal (kidney) failure. However, mismatches in human leukocyte antigens (HLA) between the donor and the recipient can cause the recipient's immune system to reject the transplanted allograft. The mechanism of immune injury in rejection has been the subject of intense research over the last few decades. This has led to a detailed understanding of the molecular processes involved in rejection, and it is now clear that the complement system plays a central role, especially in the pathogenesis of antibody mediated rejection (AMR). Several groups have recently published results of clinical trials exploring the role of complement blockade in AMR of transplant kidneys.Kidney transplantation is the treatment of choice for end-stage renal disease (ESRD) due to the superior long-term survival, cost savings to the health care system, and the greater quality of life it offers relative to dialysis , 13. HowPreformed circulating anti-HLA antibodies are present in up to 30% of patients awaiting kidney transplantation as a result of previous exposure to allo-HLA antigens from blood transfusions, pregnancies, and previous transplants and may cause immediate graft failure due to hyperacute rejection . The incThe advent of sensitive solid-phase Luminex platform bead-based assays for the detection of HLA antibodies has enhanced our ability to detect DSAs and diagnose AMR , 23. In The presence of preexisting DSA increases the risk of AMR and may adversely impact the graft survival even in recipients of negative lymphocytotoxic cross-match kidney transplants . C1q-binEnhanced understanding of the etiology of AMR and the prominent role played by the complement system implies a possible role for pharmacologic complement inhibition in clinical transplantation. The terminal complement inhibitor, Eculizumab, and C1 esterase inhibitor (C1-INH) have been the subjects of recent clinical trials exploring the potential of targeting the complement cascade in the prevention and treatment of AMR , 27, 28.DSA depletion by plasmapheresis combined with intravenous immunoglobulin (IVIg) is currently the mainstay of treatment for AMR . HoweverIn AMR, activation of the classical pathway is triggered by ligation of the C1 complex to HLA-antigens that are bound by DSA. Therefore, complement inhibitors targeting C1 can be leveraged to treat AMR while sparing the alternative pathway, thereby potentially averting infectious complications . AdditioC1 complex is a multi-molecular cluster constituted by the PRP, C1q, and two serine proteases, C1r, and C1s . Upon acSensitization to HLA is often a significant challenge to kidney transplantation . The calEculizumab's role in the prevention of AMR in HLA-incompatible kidney transplants was tested in an observational study of 26 highly sensitized patients by Stegall et al. . The ratP = 0.76)  .Post-hoc analysis demonstrated that the failure to show benefits in the treatment arm was likely related to trial design, inasmuch as there was discordance between the central and local pathology assignment of the diagnosis of AMR. When the central pathologists re-analyzed the biopsies with the clinical data that had been available to the local pathologists, the discordance went away, and the difference between the treatment and control groups became significant. Likewise, if all categories of AMR severity were assigned the diagnosis of AMR, the results were also significant. Most clinicians believe that the diagnosis of AMR is binary, and the grading system used in the study was not clinically relevant, again reinforcing a positive result to the study  to enter the trial. This had the effect of lowering rates of AMR in both study arms below expected based on prior reports in HLA incompatible transplant recipients /CRISPR-associated 9 (Cas9) technology, various groups have created genetically altered pigs to facilitate xenotransplantation . In addiMore recently, progress has been made by combining multiple glycan knockouts with human complement and coagulation regulatory gene knock ins . In 2016While currently available, transgenic knockout pigs may provide a solution to the problem of potent and immediate humoral responses due to pre-formed anti-Gal antibodies and allow transplantation. However, agents utilized in most of the successful preclinical primate studies do not have a track record of safety in humans. Therefore, to realize the goal of clinical xenotransplantation with sustained graft function in humans, tolerance induction is seen as a key element.Neisseria meningitidis and other encapsulated bacteria like Streptococcus pneumoniae have been well-documented in people with inherited deficiencies in complement components  and Shire ViroPharma; served as a paid consultant for Alexion, Shire ViroPharma and CSL, Behring; and received travel honoraria from Alexion and Shire VioPharma during the conduct of this study. He served on advisory boards for Genentech Scientific/ROCHE, True North/iPerian, Novartis, and Hansa Medical; received consulting fees from OrbidMed, GuidePoint Global, Sucampo, Astellas, and Shire; and received research grants from Immune Tolerance Network, ViroPharma, Hansa and Alexion. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiac disease causes 33% of deaths worldwide but our knowledge of disease progression is still very limited. In vitro models utilising and combining multiple, differentiated cell types have been used to recapitulate the range of myocardial microenvironments in an effort to delineate the mechanical, humoral, and electrical interactions that modulate the cardiac contractile function in health and the pathogenesis of human disease. However, due to limitations in isolating these cell types and changes in their structure and function in vitro, the field is now focused on the development and use of stem cell-derived cell types, most notably, human-induced pluripotent stem cell-derived CMs (hiPSC-CMs), in modelling the CM function in health and patient-specific diseases, allowing us to build on the findings from studies using animal and adult human CMs. It is becoming increasingly appreciated that communications between cardiomyocytes (CMs), the contractile cell of the heart, and the non-myocyte components of the heart not only regulate cardiac development and maintenance of health and adult CM functions, including the contractile state, but they also regulate remodelling in diseases, which may cause the chronic impairment of the contractile function of the myocardium, ultimately leading to heart failure. Within the myocardium, each CM is surrounded by an intricate network of cell types including endothelial cells, fibroblasts, vascular smooth muscle cells, sympathetic neurons, and resident macrophages, and the extracellular matrix (ECM), forming complex interactions, and models utilizing hiPSC-derived cell types offer a great opportunity to investigate these interactions further. In this review, we outline the historical and current state of disease modelling, focusing on the major milestones in the development of stem cell-derived cell types, and how this technology has contributed to our knowledge about the interactions between CMs and key non-myocyte components of the heart in health and disease, in particular, heart failure. Understanding where we stand in the field will be critical for stem cell-based applications, including the modelling of diseases that have complex multicellular dysfunctions. Heart failure is a global pandemic affecting over 26 million people worldwide and is becoming increasingly prevalent with an ageing population . DespiteDespite our increasing knowledge about factors influencing the initiation and progression of heart failure, historical and current study designs are unable to map the intricate interactions between cardiomyocytes (CMs) and their surrounding environment in an accurate model of the disease. Major limitations when modelling heart failure include species mismatch when using CMs isolated from animals , in vitrIn this review, we discuss the disease models of the physiological and pathological composition of the myocardium, paying a particular focus on the potential that stem-cell derived cell types present in developing an accurate in vitro model of heart failure, and also to the key myocyte-non-myocyte interactions that have been delineated thus far—these findings must be considered in future models. There is clear clinical relevance in being able to accurately model human cardiac diseases in vitro. The withdrawal of drugs from the market due to unobserved toxic effects is unfortunately common. A systematic review identified that in the US, 14% of post-marketing drug withdrawals between 1953 and 2014 occurred due to cardiac toxicity ,7. HistoIn our search for an accurate disease model for human cardiac failure, a huge asset has been the development of human stem cell-derived cells. Human embryonic stem cells were first isolated from human embryos in 1998 ,28,29. TA new approach for producing stem cell-derived cells is through human induced pluripotent stem cells (hiPSCs) ,30,31. UInitial progress has been made in using hiPSCs to better understand cardiomyopathies [opathies , rhythm opathies , valvulaopathies , and metopathies . In the opathies ,37. In dopathies .However, despite the benefits that hiPSCs have over other sources of CMs in disease modelling, it is important to note the immaturity in structure and electrophysiology of hiPSC-CMs, reviewed extensively by our group . These, The CMs are the contractile cells of the myocardium, but it is important to consider the adult human heart as a multicellular organism. The healthy adult human myocardium is composed of many cell types, with the most abundant cell types thought to be CMs, fibroblasts, endothelial cells, and perivascular cells. CMs occupy most of the myocardium volume; between 70–80% in the adult and constitutes 30–40% of the cells by number . AlthougCardiac remodelling is defined as a group of molecular, cellular and interstitial changes that manifest clinically as changes in size, mass, geometry, and function of the heart after stimulation and stimuli at disease onset such as cardiac ischemia, inflammation, pressure overload, and pharmacological toxicity ,97,98,99As we previously described, CMs and non-myocyte intercellular interactions are central in the initiation and progression of cardiac dysfunction. Therefore, CM-specific analyses cannot model all cardiac diseases. Future models should use various cell types including CMs, fibroblasts, endothelial cells, vascular smooth muscle cells, lymphocytes, and neurons and the ECM , as wellThe complex tissue and organ architecture of the heart is maintained by extensive ECM networks, composed of fibrous proteins , adhesive glycoproteins , and proteoglycans. These guide the anisotropic alignment of CMs, form the mechanical environment for cells and contribute to the stress-strain relationships of the heart. In health, the fibres of the ECM use the energy produced in systole for the re-lengthening of CMs during diastole. It is important to note that in heart failure there is a CM hypertrophy as well as collagen deposition in the ECM ,111,117.Cardiac fibroblasts account for 20% of the non-myocyte components of the heart . The numThe vasculature is critical in the delivery of oxygen and nutrients to CMs and, thus, momentum has gathered in the modelling of vascularized microenvironments ,135. In SMCs display plasticity in switching expression between contractile and proliferative (synthetic) phenotypes in the regulation of vessel tone and blood pressure. The challenges to studying diseases of the vasculature include the limited proliferation capacity and rapid senescence of adult human SMCs and hiPSC-SMC. Many studies have shown that the in vitro modulation of hiPSCs can direct them to become specific subtypes of SMCs; displaying a proliferative, synthetic or contractile phenotype. These SMCs closely resemble native SMCs at both the transcriptional and functional levels, with their pluripotency effectively silenced. There is promising evidence to suggest that SMCs have a significant role in the construction of the tunica media in the maintenance of vessel tension and contraction-relaxation capabilities and can be reproducibly created using SMC. However, less reliable is the evidence for the formation of functional vessels using hiPSC-ECs and SMCs in the co-culture. It is believed that the pattern of gene expression varies between in vivo and in vitro studies, as well as in the somatic tissue from which the stem cells are sourced .The human body’s adaptive immune system is designed to protect the body from injury. The central cellular components of this immunity are T- and B-cells that arise from lymphoid progenitor cells in the bone marrow. The cellular components of innate immunity are myeloid cells, including monocytes, macrophages, dendritic cells, natural killer cells, as well as neutrophilic, basophilic, and eosinophilic granulocytes. Heart failure is a state of chronic inflammation , with a Studies in which coronary ligation in adult C57BL/6 mice has been used to mimic chronic ischemic heart failure showed that the initial injury causes a global expansion and activation of CD4+ T-lymphocytes and the expansion of memory T-cells in the spleen. The key findings are that the cardiac and splenic T-cells in heart failure are primed to induce cardiac injury and remodelling , and retain this memory upon the adoptive transfer from donor mice to naïve recipient mice . There iThe dynamic interconnections between the heart and the neuronal system that controls cardiac function are bidirectional; dysfunction in either system can trigger a cascade that alters the functioning of the other. Peripheral neural networks are composed of all the necessary neural machinery for the reflex control of the heart . The intCurrent disease modelling simplifies the complexity of the heart by using a simple co-culture; whereas cardiac organoids have not yet been reported . RecentlLooking ahead, beyond modelling the heart as a closed system, we must also consider its place as an organ within the human circulatory system. A number of clinical and animal studies have implicated inflammation as a key contributor to myocardial remodelling. Prolonged exposure to inflammatory cytokines not only triggers signalling cascades within the heart but also other organs ,146. TheOur knowledge of human diseases has improved enormously over the last few decades, owing to advancements in the in vitro and animal models that allow us to investigate the intercellular interactions between CMs and non-CM cell types, demonstrating their importance in simple 2D co-cultures, as well as within a 3D scaffold. Increasingly, it has become standard to use hiPSCs, especially hiPSC-CMs, in models for human diseases due to their patient’s origin. However, with many of the models of human diseases dependent on the ability of the CMs to recapitulate native adult human CM physiology, differences between CMs in vitro and in the native myocardium pose challenges to fully realizing the potential of human disease models. The advancements in stem cell biology and tissue engineering have spearheaded the development of in vitro cardiac models that can employ patient- or disease-specific CMs and non-myocytes in culture. As it becomes increasingly clear that CMs are intricately modulated by the extensive extracellular interactions in the native myocardium, more accurate future in vitro myocardial models will be more physiologically relevant with the ability to provide multiple biochemical, mechanical and electrical readouts, for real-time monitoring of disease progression and functional endpoints. The development on new biomaterials will not only provide support to patient-specific stem cell-derived cells but will also contribute to their maturation and their guiding alignment, providing the relevant stiffness and be dynamically replaced over time by ECM components secreted from the cells ,154,155.Overall, a more solid picture of the myocyte-non-myocyte interactions that occur in the native myocardium using relevant models will enable us to develop a greater understanding of how the adult myocardium functions. This will provide the unique opportunity to study strategies for disease intervention in human in vitro disease models, spanning the gap between 2D culture and in vivo testing, thus reducing the cost, time, and ethical burden of the current approaches.


Organic electrochemical transistors (OECTs) are recently developed high-efficient transducers not only for electrochemical biosensor but also for cell electrophysiological recording due to the separation of gate electrode from the transistor device. The efficient integration of OECTs with electrochemical gate electrode makes the as-prepared sensors with improved performance, such as sensitivity, limit of detection, and selectivity. We herein reviewed the recent progress of OECTs-based biosensors and cell electrophysiology recording, mainly focusing on the principle and chemical design of gate electrode and the channel. First, the configuration, work principle, semiconductor of OECT are briefly introduced. Then different kinds of sensing modes are reviewed, especially for the biosensing and electrophysiological recording. Finally, the challenges and opportunities of this research field are discussed. Transistors were invented by William Shockley and his coagent in 1947 for the precluded amplification efficiency and reliability of vacuum tubes. With the discovery of conducting conjugated polymers in the late 1970s, organic conducting polymer-based transistors have been widely investigated. Among of them, organic thin-film transistors have attracted much attention due to their broad range of applications, especially in biological system, which includes two types of transistors, i.e., organic field effect transistors (OFETs) and organic electrochemical transistors (OECTs). OFETs always use small organic molecules and organic conjugated macromolecules as semiconductor, in which the gate voltage is applied across the gate insulator and through field effect doping the gate electrode modulate the channel current. Differently, OECTs use the electrolyte medium between the channel and the gate electrode rather an insulator layer. When applying gate voltages, electrochemical doping or de-doping from the electrolyte modulate the channel current. In this case, the OECTs is easier to be used in biological system since most of biological reaction or species are occurred in an electrolyte medium. More importantly, compared with other kinds of OFETs, OECTs always bear low working voltages (below 1 V), essentially evaluating its potential application in biological system.Based on these unique properties of OECTs, various applications have been explored during the last two decades , includiSome of previous reviews have summarized the physics and principles of OECTs  and the drain  are controlled by applying constant voltages, which are referenced to the source electrode (D), reveals the channel's doping state, which is proportionable to the quantity of mobile holes or electrons in the channel.Bernards and Malliaras  systematlectrode . The potTo qualitatively describe the working principle of OECT, The Bernards et al. postulates that there are two circuits of the OECTs: an ionic circuit and an electronic circuit, which separately describe the ions flow in the gate/electrolyte/channel system and the charges flow in the source/channel/drain system according to Ohm's law . In the m = ∂ID/∂VG). High transconductance values reflects the effective gating but leads to the slow operation of OECTs  the channel/gate area ratio is irrelevant to the detection linear  and n-type (commonly based on copolymers of thiophene and fluorene) has been used for constructing OECTs. Among of them, the PEDOT: PSS  is most The particular identification of OECTs is the doping reactions appear among the whole volume of the organic film, which is different from FETs occur on a thin interfacial region. Therefore, low-gate voltages can achieve large modulations in the drain current, herein, OECTs are effective switches, and powerful amplifiers. In addition, compared with the solid dielectric layers, the electrolyte solutions are more applicable for larger flexible devices and various substrates' integration  can be directly oxidized or reduced on the electrode. While the electro-inactive molecules  can be detected by electrochemical technique through bio-recognition methods. Guided by changes of the potential drop on the gate and channel, the biosensors can generally be subdivided into the enzymatic sensing, immune-sensing, and aptamer sensing  in biological system, can be directly oxidized or reduced on the electrodes. For example, DA can be oxidized to o-Dopamine quinone  at a cerm) linear calibration of DA without the interference from AA and UA, through changing the scan rates and gate voltages, in which case, the scan rate is an essential property for sensitivity. Classic electrochemical methods cooperate with OECT will promote the development of biosensing: fast scan cyclic voltammetry (FSCV) is widely used to detect electroactive chemicals, especially DA in neuro system,  compensating the anionic polymer , which leads to a change of the Vgeff, then a decrease of the channel current which is logarithmically proportional to concentration of enzymatic substrates.The sensing mechanism of the typical enzymatic OECTs sensors is shown in : EnzymesThe sensitivity of OECTs devices, lies on the nature of amplification provided by the optimized geometry and the sensitive enzymatic modification. After optimization using new materials and different modification technology, the detection levels of bioactive molecules can be promoted to a large extent. Apart from the mostly used p-type conjugated polymer, Pappa et al.  demonstrIn addition to the high sensitivity, selectivity is another key role for the biological sensors. So far, there are several methods to improve the selectivity by rationally designing the surface chemistry of gate electrode . For the amplification nature of organic electrochemical transistors, the biosensor that detect the cancer marker HER2 has the detection limit low to 10−14g mL−1, which is several magnitudes lower than original electrochemical methods. Moreover, the OECTs based HER2 biosensors have a detecting concentration range from 10−14 to 10−7g ml−1, which covers the detect amount of HER2 in normal and breast cancer cells. Additionally, these protein sensors can distinguish the cancer cells from the breast cancer cells attribute to the specificity of the modified antibody.Immunoassay is crucial in clinical analysis , toxins, and microbials in the environments . This LFP called the electrocorticography (ECoG) when using the electrode on the cortical surface, and when recording with the electrode inserted deep into the brain called stereoelectroencephalography (SEEG). The longer distance between the recording electrode and the location leads to useless signals for understanding the processes of neuropathology, hence the SEEG conduct the most informative signals. Conducting polymer-based electrode has extensively enhanced the electrical performances and biocompatibility of mental inserted intracortical electrodes. However, compared with the SEEG, the ECoG is non-invasive, the characteristics of ECoG have been significantly improved by using more stable polymers and rational building of devices , which has no obviously difference with the Pt electrodes, with the average threshold response of 84.5 nC (74.5 μC cm−2). Tunable channel thickness can result in tunable transconductance  Stability is an extremely valued performance for biosensing. The OECT has been proven to work stably in various electrolytes, such as cerebrospinal fluid, cell media, sweat, and tears. The durability in these complex electrolytes can last; (ii) Sensitivity could be largely improved by OECT. Since small changes on the input will lead to enormous changes on the output. OECTs display high transconductance values, characteristically high gain, and the time response can be improved by tuning the geometry and size of the channel; (iii) OECTs could be easily integrated with biological system, such as individual cells, tissues, and even whole organs. Moreover, the gate electrode and conducting polymers could be easily modified by various recognition molecules; (iv) Compatibility with photo lithographical techniques also promote the microscale devices construction, especially interesting for monitoring activities of In the past decade there has been noticeable progress on the advancing of OECTs for biological applications, however, numerous challenges still remain. These including: (i) The discovery and implementation of innovative active conducting polymers with enhanced properties with regard to conductivity, stability, patterning and restorability; (ii) The fabrication of devices for integrating multiplexed miniaturized arrays on versatile sensors, which may possibly power, record, or transmit the signals; (iii) The selectivity remains a big challenge for multi-chemicals and molecules sensing, especially for complicated samples ; (iv) The response time was slow for the OECT devices due to the ions of electrolyte moving into or out of the volume of channel. The solution of these questions would largely accelerate the wide application of OECT in biological system, especially for the development of wearable device and brain-machine interface.LB, CE, and WL collected the publications related to this review article, wrote the first draft of the manuscript and made subsequent corrections. PY, JF, and LM completed critical literature analysis and checked subsequent manuscript drafts.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.


For years, microbes have been widely applied as chassis in the construction of synthetic metabolic pathways. However, the lack of in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. In recent years, multiple methods have been applied to the construction of small metabolic clusters by spatial organization of heterologous metabolic enzymes. These methods mainly focused on using engineered molecules to bring the enzymes into close proximity via different interaction mechanisms among proteins and nucleotides and have been applied in various heterologous pathways with different degrees of success while facing numerous challenges. In this paper, we mainly reviewed some of those notable advances in designing and creating approaches to achieve spatial organization using different intermolecular interactions. Current challenges and future aspects in the further application of such approaches are also discussed in this paper. Corynebacterium glutamicum was used in the biosynthesis of medical materials such as l-amino acids  can precisely meet these requirements and provide clues for new spatial organization systems. To precisely edit genome, genome editing tools often rely on highly effective and sequence-specific DNA-binding. For example, zinc finger nuclease contains a engineerable zinc finger domain, which consists of three to six replaceable individual zinc finger repeats to bind desired DNA sequence . SimilarE. coli was also reported [Accordingly, recent studies have developed multiple enzyme clustering approaches with these DNA-binding proteins considering their potential for scalability and robustness. Currently, approaches inspired by ZFN and TALEN have already been used for the spatial organization of exogenous enzymes. In 2012, Conrado et al.  reportedreported . Comparereported . Using tMore interestingly, a recent study applied the CRISPER–Cas9 technique as a novel system to concentrate specific enzymes for metaplastic functions . In thisAs a new and rapidly advancing strategy for the spatial organization of pathway enzymes, the DNA-binding mechanism in genome editing tools provides a promising way to establish highly stable, extendable, and low-cost systems for such applications. On the other hand, the development of such methods also provides a new vision on how techniques such as genome editing could be used besides its current application in genome editing. In the meanwhile, it should be noted that, with further study on the structural basis and mechanisms of protein-nucleotide interactions, current DNA-binding domains can be further engineered to generate more robust DNA-binding proteins for new spatial organization systems in the near future.Since the metabolic pathways are basically a set of enzymes with different dynamics, finding ways for balancing the metabolic flow and reducing negative impact to the chassis is now an important field for synthetic biologists. Despite the strategies employed, the purpose of the spatial control and organization of pathway enzymes remains the same. As an important complementary and parallel approach along with traditional ways, such as rational design of expression systems and optimization of enzyme expression levels, the spatial organization of pathway enzymes aims to provide a highly predictable and stable control of the heterogeneous metabolic pathways and reduce the effect on the survival and growth of the chassis.However, since the detailed reason why spatial organization of pathway enzymes can enhance the heterologous pathways is yet to be understood, improving the predictability and stability of such scaffolds is still a challenge. Supported by both computational modeling and wet-lab experiments , the metIn addition, according to either numerical experiments or wet-lab results currently reported, the benefits of the spatial organization of pathway enzymes are affected by multiple factors such as spatial orientation of enzymes, distance between enzymes, and copy number of scaffold molecules. With multiple, convoluted unknown variables, the rational design and functional examination of scaffold systems are difficult. Hence, further study on structural biology, especially on the detailed mechanisms of the pathway-enhancing effect of spatial organization, is needed. An in-depth investigation on the substrate channeling process on natural existing enzyme clusters, such as the tryptophan synthesis complex, would be a great start. Knowledge on the influencing mechanisms of these factors will provide a sound foundation for the design and creation of new spatial organization methods, especially for highly tunable protein-nucleotide interaction strategies. Moreover, the rapid development and uncovering of new genome editing techniques such as the CRISPR-C2c2  and CRIS


Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, and it is one of the leading causes of cancer death in both men and women worldwide due to diagnosis in the advanced stage, rapid metastasis, and recurrence. At present, precision molecular targeted therapeutics directed toward NSCLC driven genes has made great progress and significantly improved the overall survival of patients with NSCLC, but can easily lead to acquired drug resistance. New methods are needed to develop real-time monitoring of drug efficacy and drug resistance, such as new molecular markers for more effective early detection and prediction of prognosis. Exosomes are nano-sized extracellular vesicles, containing proteins, nucleic acids and lipids, which are secreted by various cells, and they play an important role in the development of lung cancer by controlling a wide range of pathways. Tumor-derived exosomes are of great significance for guiding the targeted therapy of NSCLC and exosomes themselves can be a target for treatment. In this review, we describe the potential roles of tumor-derived exosomes and their clinical significance in NSCLC. Lung cancer is one of the leading causes of cancer-related death both in men and women  and remaThe diameter of exosomes which was first reported in the process of sheep reticulocytes maturation ranged from 40 to 100 nm, showing a characteristic cup-shaped morphology (after negative staining) or round well-delimited vesicles observed by transmission and cryo-electron microscope , 16. ExoThe immunological activities of exosomes affect the mechanisms of immunoregulation including immune activation, antigen expression regulation, intercellular communication, immunization surveillance, and immunosuppression . Tumor-dThe formation and development of NSCLC is influenced by many factors and mechanisms, which is a long-term and complex process. Exosomes secreted by lung cancer cells play a vital role in this process as mediators of intercellular communication.  in the tumor microenvironment can secrete exosomes containing lipids, amino acids, and TCA-cycle intermediates which can promote tumor growth under nutrient-stressed or nutrient-deprivation conditions . AngiogeMetastasis is one of the major causes of death in lung cancer patients, which is related to a variety of mechanisms and involves multiple steps . Tumor-dResistance to chemotherapy, radiotherapy and targeted therapy remains a major obstacle to cancer treatment . Drug reIncreasingly, studies suggest that exosomal miRNAs and proteins are positively associated with the stage and degree of tumor progression , indicatLiquid biopsy samples are becoming more frequently used in blood or other body fluids as biomarkers for NSCLC early diagnosis, treatment guidance and drug resistance monitoring . The speThe exosomes secreted by lung cancer cells, which enrich various proteins, such as EGFR, KRAS, claudins and RAB-family proteins, and promote the development of lung cancer, are effective biomarkers for early diagnosis of lung cancer  and the MicroRNAs (MiRNAs) are a class of small noncoding RNAs (the length of 18–25 nucleotides), post-transcriptional regulation molecules expressed in many organisms . The expIn summary, exosomes are becoming increasingly important in the diagnosis of NSCLC as biomarkers and the molecular basis of NSCLC targeted therapy.With the increasing understanding of molecular biology and genetics of tumors, the research and clinical application of targeted therapy has become a hot topic, which can improve prognosis and also guide therapeutic decision, thus reducing morbidity and mortality. On average, there are more than 300 mutations in each lung cancer, but only a few of these genes can promote or “drive” the lung tumorigenesis , mainly Studies have found that research based on exosomes and related components can provide new inspiration for the precision treatment of NSCLC.Exosomes have been developed as drug delivery vehicles for a variety of drugs, such as small molecular drugs, nucleic acid proteins and other drugs for cancer treatment, with low immunogenicity and toxicity , 107. LaExosomes play an important role in tumor development, which indicates reducing the contents of exosomes can help the targeted therapy of NSCLC. Exosomes may also serve as a direct target for NSCLC. Data shows that the prevention of exosomes production can inhibit tumorigenesis and a series of methods have been suggested to reduce the contents of exosomes : (1) BloThe diversity of exosomes components and functions associated with NSCLC provides multiple potential therapeutic targets for the treatment of NSCLC. Yang et al. found that the promotion of the expression of let-7 in exosomes was a potential target for the treatment of NSCLC because of its high tumor suppressor effect, great clinical significance and tissue specificity . Zhang eIn summary, studies on the treatment of lung cancer-related exosomes will provide a new idea for exploring precision treatment of NSCLC.  The specific mechanism of exosomes as an important part of the tumor microenvironment in the evolution of the NSCLC is not yet clear; (2) The sensitivity and specificity of exosomes application in the diagnosis and treatment of NSCLC still need improvement; (3) The acquisition of high purity exosomes is still an issue due to technical limitation and high cost; (4) The quantification, purification and preservation of exosomes has not yet been standardized; (5) The side effects of exosomes used in the targeted therapy cannot be completely determined. All of these problems restrict the application of exosomes in NSCLC. These reviews synthetically expound the multi-faceted nature of exosomes and potential value in the targeted therapy of NSCLC.


The current drug development practice lacks reliable and sensitive techniques to evaluate the immunotoxicity of drug candidates, i.e., their effect on the human immune system. This, in part, has resulted in a high attrition rate for novel drugs candidates. Organ-on-chip devices have emerged as key tools that permit the study of human physiology in controlled in vivo simulating environments. Furthermore, there has been a growing interest in developing the so called “body-on-chip” devices to better predict the systemic effects of drug candidates. This review describes existing biomimetic immune organs-on-chip, highlights their physiological relevance to drug development and discovery and emphasizes the need for developing comprehensive immune system-on-chip models. Such immune models can enhance the performance of novel drug candidates during clinical trials and contribute to reducing the high attrition rate as well as the high cost associated with drug development. The process of pharmaceutical drug development is typically long and expensive, taking up to twelve years of research effort and costing on average more than 1.7 billion US dollars per clinically applicable drug . For a nMechanistic studies of cellular systems are best-performed using experimental setups that closely mimic the native microenvironment of cells. Such controlled setups are possible with microfluidic technology, which has been widely employed in biological research to reduce cost of reagents, maximize information from precious samples and simultaneously provide spatio-temporal information about the dynamics of cells in their microenvironment . The latMicrofluidic organs-on-chip can be fabricated using various manufacturing techniques including micromolding, microetching, soft lithography and photo-polymerization. These technological processes have enabled the development of fine structures that closely resemble those in vivo ,20. FurtOver the past decade, many researchers have utilized organs-on-chips in order to evaluate the efficacy and toxicity of drugs in a more in vivo simulating environment . SpecifiThe immune system comprises a network of cells, tissues and organs that function together to protect the body against pathogens such as bacteria and viruses. Immune cells originate in the bone marrow where hematopoietic stem cells develop into common precursor cells that ultimately differentiate into immune cells. Common myeloid precursors give rise to different granulocytes such as neutrophils and basophils as well as to antigen presenting cells such as dendritic cells and macrophages which all circulate throughout the body and scan tissues for foreign antigens ,27. On tIt has been shown that the maintenance of immune homeostasis involves the coordination between immune components localized in various tissues besides lymphoid microenvironments . One sucAll types and all classes of pharmaceutical drugs perturb the immune system, but the mechanisms and immunological outcomes of these perturbations are not well defined . Lack ofThe skin is not only the largest organ of the human body but also the first organ of defense protecting against invading microorganisms and other pathogens. Besides acting as a physical barrier separating the internal organs of the body from the external environment, it is an active immune organ containing elements of both the innate and adaptive immune systems . ImmunitMany skin-on-chip platforms have been developed to aid in the process of drug development and testing. These vary in the level of complexity, the availability of vascular elements, and the types of cells they incorporate. For instance, O’Neill et al. developed a skin-on-chip model that simulates the outermost epidermal layer of the skin only. It consists of neonatal human epidermal keratinocytes cultured on collagen patches in microfluidic channels . This moTo achieve more physiological relevance, several studies have also incorporated vascularization into their skin-on-chip models utilizing primarily endothelial cells ,51,52. MAll the previously reported vascularized skin-on-chip models lack key immune cells such dendritic cells and T-lymphocytes, thus undermine the immunocompetence of skin. The only available model that incorporates immune elements is one developed by Ramadan and Ting . The modSimilar to the skin, the lining of the gastrointestinal (GI) tract is exposed to the external environment and therefore, it constantly encounters pathogens . This reAn efficient micro-engineered gut-on-chip should incorporate the key features of the GI tract which include: an epithelial barrier, a mucus layer, an immune component and a human-microbial interface. It should also support peristalsis like motion and the transport of nutrients or agents through the epithelium. Many of these features have been addressed by researchers in literature but to a variable extent and for different applications. Intestine-on chip platforms vary in their complexity and functionality. The earliest and most simple model was developed by Kimura et al. to support long-term culture and monitoring of epithelial colorectal adenocarcinoma cells (Caco-2), which are common cellular models for examining small intestinal functions . It consKim et al.’s gut-on chip is not the only available on-chip model of the intestine; although it is the most physiologically relevant and the most versatile. Shim et al. similarly developed a gut-on chip but with villi-like structures fabricated as part of the microfluidic device rather than forming as a result of physiological cues . The resAlthough not usually perceived as an immune organ, the liver has been shown to possess key immunological properties such as the incorporation of a diverse population of resident immune cells including the body’s largest population of resident macrophages known as Kupffer cells as well as the production of acute phase proteins, complement components, cytokines and chemokines ,70. KupfMany liver-on-chip platforms have been developed to support the prolonged culture of liver cells and to facilitate investigations into the various functions of the liver including metabolism, detoxification and response to pharmaceutical drugs ,83,84,85Immune responses initiated at different locations throughout the body including the skin and gut are coordinated by secondary lymphoid organs, mainly the lymph nodes, which are situated at strategic locations throughout the body. Lymph nodes are cellular microenvironments for immune cells. They collect draining interstitial fluid that flows across all tissues accumulating foreign particles, distributed pharmaceutical drugs as well as immune cells that congregate there for intracellular communication purposes ,91. WithThe recreation of a lymph node in vitro is not an easy task, primarily because of its complex architecture and structural organization. Rosa and colleagues developed a microfluidic model of the paracortical region of the lymph node to examine the dynamics of interaction between dendritic cells and T-cells during flow at different shear stresses . The modThe above mentioned lymph node on chip devices successfully simulate several functions of the native lymph node, especially chemotaxis, the cellular response to chemokine gradients, where most of the lymph node studies were focused on this aspect. Surprisingly, only few of the existing studies assessed the lymph node’s ability to identify pathogens and fight infections. This aspect, in particular, is of vital importance in drug development and discovery to evaluate the toxicity of drug candidates to the immune components. Giese et al. developed an in vitro model of the lymph node and utilized it for immunological substance testing ,100. TheBesides the lymph node, the spleen, a small soft organ located in the upper left side of the abdomen, behind the rib cage and stomach, is also a lymphoid organ that is a key component of the immune system. It acts as a filter purifying blood from viruses, bacteria, worn out or damaged red blood cells (RBCs) and other pathogens. It has two main regions called the red pulp and white pulp that are separated by an interface called the marginal zone . The redDespite its importance in immunity, only few attempts have been made to model the spleen in vitro, perhaps because survival without a spleen is possible. Rigat-Brugarolas and colleagues developed a microfluidic device that mimics the physical properties and hydrodynamic forces of the splenon, the spleen’s basic functional unit . The devAll immune cells, including the lymphocytes of the adaptive immune system, originate in the bone marrow and some also mature there. Once ready, these cells move into the bloodstream and circulate through the lymph nodes and other lymphoid organs to perform their defensive functions. This makes the bone marrow an important component of the immune system, if not the most important. Any infection or disease that affects the bone marrow compromises the ability of the immune system to eradicate the body from pathogens, leading to serious diseases or death. The bone marrow is a complex organ containing both hematopoietic and non-hematopoietic cells surrounded by a shell of vascularized and innervated bone . HematopThe presence of various niches within the bone marrow, each having different components and different functional roles in hematopoiesis, make it difficult to reproduce the complex microenvironment of the bone marrow in vitro ,108. NevLymphatic vessels collect the substances that leak from the vascular system such as plasma, proteins, antigen presenting cells and pathogens and return them back to the circulatory system. Collectively, the substances picked up by the lymphatic vessels make up the lymph fluid. As lymph fluid flows through the lymphatic vessels, it passes through the lymph nodes, where its constituents are screened by lymphocytes. Once a foreign antigen is detected, lymphocytes proliferate and differentiate into effectors cells initiating an immune response. Thus, lymphatic vessels are crucial for immunity, as they mediate the communication between the tissues of the body and lymphoid organs. Recently, the view towards lymphatic vessels has shifted from simply regarding lymphatic vessels as passive conduits of lymph fluid and immune cells, to regarding them as active, tissue-specific participants in major physiological and pathophysiological processes . A lymphatic vessel on-chip platform should facilitate studies aimed at understanding the mechanism by which substances exit the circulatory system and enter the lymphatic system. More relevant to the immune context, lymphatic vessel on-chip should help in understanding how antigen-presenting cells and lymphocytes leave an inflamed tissue and enter into nearby afferent lymphatics. This process, in fact, is vital for the initiation and maintenance of an immune response . Sato etBesides the discussed immune organs-on-chip, other on-chip platforms have been developed to facilitate investigations into immune physiology and disease mechanisms as well as to aid in the process of drug development. These include inflammation-on-chip, cancer-on-chip and immunotherapy-on-chip ,115,116.The field of on-chip platforms for biological applications remains quite broad and contains many other organ-on-chip models that do not specifically examine immune functions such as adipose-on-chip, eye-on-chip and placenta-on-chip models ,126,127.Most existing organ-on chip platforms including those of the immune system replicate a single organ only. Although several multiorgan devices have been developed for systemic drug testing in vitro ,139,140,Despite the great potential of the organ-on-chip technology, key biological and technical challenges remain. These include achieving a proper scale in terms of organ size and cell number, attaining architectural complexity of the human tissues and organs in vitro and in a miniaturized manner, developing a universal perfusion medium suitable for multiple cell types within the same organ or within different organs connected together and utilizing primary human cells rather than cancerous cell lines ,141. FurBesides the challenges associated with developing and using organ-on chips, the adoption of such technology for drug development and testing presents another barrier. The technology needs to be extensively validated before it can be approved by the FDA as an efficient preclinical method of testing newly developed drugs. This would require strong collaboration between researchers, regulators and the industry.


Orthopedics is an important branch of medicine concerned with disorders of the bones, joints, spinal cord, peripheral nerves, muscles, connective tissue and cartilage . OrthopeOver the past decade, studies have revealed new classes of RNA molecules, including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), long noncoding RNAs (lncRNAs), PIWI-interacting RNAs (piRNAs) and circular RNAs (circRNAs). These noncoding RNAs (ncRNAs) not only regulate a variety of cell functions in the development of and physical processes involving skeletal muscle but also contribute to a number of orthopedic-related disorders . For exaTo facilitate investigations into the associations between orthopedic disorders and ncRNAs, we constructed a public database, the Orthopedic Diseases ncRNAome Atlas (ODNA), which contains manually curated data on orthopedic disease-associated, experimentally validated ncRNAs.We used Medical Subject Headings (MeSH) terminology to collect the disease names. To construct a high-quality database, we referred to the steps used by published databases such as NSDNA , Lnc2CanIn the process of obtaining the information, the following five steps were applied: the collection of the names of associated diseases and the ncRNA category, identification of relevant articles, exclusion of noneligible articles, extraction of useful data from the eligible articles and validation of the data by the third author. Electronic searches were performed independently by two reviewers in PubMed. The following were used as either keywords or MeSH terms in all combinations for the search: `each ncRNA symbol/ncRNA category name’ and `each orthopedic disease name/physical process’. The criteria for including ncRNAs in ODNA were as follows: (1) the involved disease was related to the musculoskeletal system; (2) the ncRNA was experimentally validated and has abnormal expression in subjects with the involved disease ; (3) the samples and species of the samples used in the validation were clearly stated; and (4) the experimental methods used to analyze the ncRNA analysis were clearly described. The exclusion criterion was repeated use of the same data.We downloaded all published studies and available supplementary files describing the associations between the diseases/physical processes and ncRNAs. Two authors independently extracted the data from the articles. We collected the following detailed information from the published studies: disease and ncRNA name, species, ncRNA expression pattern, experimental methods, target information, samples used, description of ncRNA function and references. The data were divided into data derived from high-throughput and low-throughput experiments, as previously described . High-thThe accuracy of the data entered was confirmed by a third reviewer. The retrieved results were last updated in February 2018. ODNA was built using MySQL and JSP. The scripts were written in Java. The web service is running on an Apache Tomcat web server.http://bio-bigdata.hrbmu.edu.cn/ODNA. The current ODNA database consists of 6207 miRNAs, 13 391 lncRNAs, 1011 circRNAs, 119 snoRNAs and 70 piRNAs and documents 175 orthopedic diseases and 62 physical processes across 11 species. The ncRNAs were curated from over 700 articles obtained from PubMed. The datasets in this article are available from the ODNA (http://bio-bigdata.hrbmu.edu.cn/ODNA/download.jsp) and Figshare repository record (https://figshare.com/s/519d4a09fa4e46feff99). The diseases and physical processes regulated by the included ncRNAs are displayed in The database is available at The ODNA provides a user-friendly web interface that facilitates easy database querying . On the Researchers making use of the database are encouraged to assess the validity and accuracy of the data and send us feedback through the website interface. The data will be checked and updated by our team. After verification, users will be able to upload data to the database. The ncRNA symbol, regulation pattern and reference title or PMID are the required fields. In the future, the intention is for the information for any given entry in the database to be curated on a voluntary basis.In recent years, with the rapid accumulation of ncRNA studies, several ncRNA-associated databases, such as miR2Disease , NSDNA , LncRNADSince the first profiling studies of ncRNAs during the stages of osteoblast and osteoclast differentiation, an increasing number of publications related to bone biology and pathologies have been published, as have many comprehensive reviews summarizing the characteristics of ncRNAs in skeletal cells ,16. A laTo the best of our knowledge, the ODNA is the first database focusing on orthopedic-associated ncRNAs. One strength of this database is that it includes almost all known orthopedic diseases, including arthritis, bone tumor/metastasis, spinal diseases, metabolic diseases, trauma, pediatric congenital malformations, muscle diseases, pain and sport-related injuries. In contrast, several published databases, such as LncRNADisease and miR2Disease, only document a limited number of orthopedic-related ncRNAs and only include one ncRNA category. Moreover, the ODNA includes the circRNAs associated with the musculoskeletal system, while the above-mentioned databases do not include circRNA-related data.Previously, most disease-related databases focused on the pathological mechanisms underlying disease progression, and the data regarding the physical functions of the ncRNAs were not well documented. Therefore, another strength of this database is that it includes data pertaining to the physiological processes of musculoskeletal cells, including the proliferation of bone cells, differentiation of stem cells, regeneration of nerve cells/muscular cells and development of the skeleton. As an integral part of orthopedics, biomaterials/implants influence the levels of ncRNAs, and this information is also included in the section on physical processes in the ODNA. Implants are always fixed to bone, but the mechanisms by which these biomaterials act on bone are unknown. Therefore, the ncRNA data related to implants may contribute to the understanding of the molecular mechanism by which implants affect bone regeneration.In the ODNA, the majority of the entries are associated with miRNAs and lncRNAs, suggesting that these categories of ncRNAs play key roles in the diagnosis and treatment of orthopedic-related diseases. The number of miRNA- and lncRNA-related studies has been steadily increasing over the past 10 years . These eIn summary, the ODNA is the first manually curated database of ncRNAs associated with the field of orthopedics, and it is a valuable resource for researchers seeking to understand the pathological mechanisms underlying musculoskeletal diseases. We hope the current version of the database and future updates will provide a context for the diagnosis and treatment of orthopedic-related disorders and can be used by different research communities.