In multicellular organisms, angiogenesis, the formation of new blood vessels from pre-existing ones, is an essential process for growth and development. Different mechanisms such as vasculogenesis, sprouting, intussusceptive, and coalescent angiogenesis, as well as vessel co-option, vasculogenic mimicry and lymphangiogenesis, underlie the formation of new vasculature. In many pathological conditions, such as cancer, atherosclerosis, arthritis, psoriasis, endometriosis, obesity and SARS-CoV-2(COVID-19), developmental angiogenic processes are recapitulated, but are often done so without the normal feedback mechanisms that regulate the ordinary spatial and temporal patterns of blood vessel formation. Thus, pathological angiogenesis presents new challenges yet new opportunities for the design of vascular-directed therapies. Here, we provide an overview of recent insights into blood vessel development and highlight novel therapeutic strategies that promote or inhibit the process of angiogenesis to stabilize, reverse, or even halt disease progression. In our review, we will also explore several additional aspects  that operate in parallel to canonical angiogenesis mechanisms and speculate how these processes may also be targeted with anti-angiogenic or vascular-directed therapies. The cardiovascular system is the first functional organ system that develops in the mammalian embryo. The blood vessels that comprise this organ initially originate by vasculogenesis, which involves the aggregation of endothelial precursor cells (angioblasts) into simple endothelial tubes . During 3 is already in need of new vasculature [3 sparks the formation of new blood vessels that support the proliferation of additional cancer cell clones while providing conduits for dissemination to distant sites [There is a limit to how much a tissue can expand without the generation of new vasculature to supply oxygen and nutrients. It has been estimated that tissue growth beyond the volume of one mmculature . To achiculature . Becauseculature . In tumoculature . Growth nt sites .In the eighteenth century, Joseph Priestly and Karl Wilhelm Scheele were among the first to discover the element oxygen, which they found to be important for combustion and burning of materials. Oxygen is of vital importance in cellular metabolism and energy production; oxygen also regulates vascularization thereby providing a feedback mechanism to prevent too low or too high oxygen pressure which can be detrimental. Discoveries in the early 1990s provided insight into the regulatory mechanisms of oxygen sensing which involves hypoxia-inducible factors (HIFs) and erythropoietin among others , 13. HIFThe key role of oxygen in the process of angiogenesis, together with the dependency of disease processes for angiogenesis has resulted in strategies to target hypoxia for the treatment of diseases, such as cancer, atherosclerosis, ischemia/reperfusion injury, eye diseases, arthritis, and endometriosis. For example, this can be directly done by applying oxygen to improve the effect of radio- or photodynamic therapy through the enhancement of reactive oxygen species , 19. In To overcome the time-distance constraints of diffusion, multicellular organisms have evolved mechanisms to generate blood vessels; thus, in most vertebrates, the vasculature is lined with ECs . There aThe process of vasculogenesis refers to the formation of blood vessels starting during early developmental stages, where endothelial precursor cells (angioblasts) derive from haemangioblasts and aggregate into simple endothelial tubes. Early during this process of vasculogenesis blood islands within the embryonic and extraembryonic mesoderm are formed. These islands contain haemangioblasts that differentiate into vascular precursor cells that express VEGFR2 (angioblasts) and eventually give rise to bona fide ECs that line the blood vessel wall , 30. A pA process termed “post-natal vasculogenesis” can also occur in adults. Circulating endothelial colony-forming cells (ECFCs), which have a stable phenotype and robust vessel-forming abilities, may be recruited to sites of ischemia. However, the numbers of circulating ECFCs are typically quite low in peripheral blood (constituting ~0.05–0.2 cells/mL of blood). This low frequency, coupled with variabilities in absolute numbers of ECFCs in patients with various pathological conditions (e.g. coronary artery disease or cancer) has made it challenging to understand the biology and anatomical origin(s) of these elusive cells in health and disease. Indeed, early studies identifying putative ECFCs in solid tumors and in sites of ischemia may have been confounded by large number of perivascular hematopoietic cells which closely resemble ECFCs in terms of marker expression and proximity to the vessel wall. However, circulating ECFC are definitively present in cord blood and, in the search for molecular markers to identify these ECFC, PROCR (protein C receptor) has emerged as a good candidate . In miceDespite the low numbers of ECFCs incorporating into blood vessels at active sites of angiogenesis, ECFCs play important auxiliary or paracrine roles through the release of growth factors that support, for example, mural cell or immune cell recruitment/survival. A good example is the unique high expression of neuregulin-1 (NRG-1) by ECFCs which provides anti-apoptotic and proliferative signals via activation of the PI3K/Akt pathway in stem cell-derived cardiomyocytes . SimilarIn contrast to vasculogenesis where blood vessels are de novo assembled by precursor cells, sprouting angiogenesis refers to the formation of blood vessels from a preexisting capillary bed . EndotheTip cell selection appears to be stochastic (perhaps related to heterogenous expression of VEGF receptors) and is dynamic in that tip/stalk cells can switch places during sprouting via a process that requires functional Notch and Dll4 ligand , 48. Spain vivo studies have used lineage tracing to identify transit amplifying ECs, differentiated ECs, and EVPs within blood vessel walls that express a suite of genes important for progenitor cell function . Notably, bone marrow chimeras have ruled out the participation of bone marrow as a source for EVPs in this setting. Several additional markers have been used to identify EVPs including CD157, ProcR and Sox9 [Notably, recent studies have identified so called “endovascular progenitors (EVPs)” with enhanced proliferative ability and superior capacity to form new blood vessels compared to otherwise “adult” ECs. EVPs may be poised to undergo multiple rounds of mitosis required during angiogenesis upon wound healing or other pathophysiological processes and could possess additional properties of tissue resident stem cells, including multipotency, self-renewal, and endothelial-to-mesenchymal transition (EndMT) . EVPs maand Sox9 . Vessel and Sox9 . Here, dand Sox9 , 66. Intand Sox9 . Similarand Sox9 . In sum,A variant of angiogenesis, different from sprouting, is intussusceptive angiogenesis. This process was first observed in post-natal remodeling of lung capillaries , 70, wheAngiogenesis reports on this form of angiogenesis describing it as”inverse intussusception” [It has also been recognized that blood vessels can remodel by the formation of functional vascular trees from the initial homogeneous capillary mesh; this takes place in preferential flow pathways of a capillary mesh, where these pathways enlarge and fuse while trans-vascular pillars are removed and less perfused capillaries regress. This form of angiogenesis, whereby the number of vessels decreases whereas the diameter of the resultant vessel is increased, is called coalescent angiogenesis  of brain capillaries . Deletio+ glioma that signaled through Wnt7 were more likely to undergo single cell migration similar to the spread of oligodendrocyte precursor cells during development. These Olig2+ cells were also enriched after anti-VEGF therapy suggesting that anti-angiogenic therapies may select for cancer cells with the ability to co-opt the vasculature. Single-cell vessel co-option also has important implications for BBB integrity and immune evasion. For example, preservation of the BBB has important consequences for therapeutic targeting of cancer cells; namely, inhibiting Wnt7-driven perivascular invasion enhanced the efficacy of temozolomide (TMZ) [+ and Olig2− cancers increased the number of microglia present compared to normal brain tissue; however, microglia in Olig2− (more angiogenic) tumors had a more activated (ameboid) morphology and an increased number of cells expressing genes related to macrophage infiltration.Griveau et al. demonstrated that Olig2de (TMZ) . In the Selection pressure driven by anti-angiogenic therapy may also drive vessel co-opting programs in cancer cells, or selectively alter the TME to promote vessel co-option . This no+ cells in a melanoma metastasis model described rare melanoma cells with functional markers of ECs including VE-cadherin and PECAM; these data are consistent with the identification of VE-cadherin+ melanoma cells in some human cells lines many years ago [+/cytokeratin+ cancer cells were found to incorporate into tumor vessels using patient explants, associate with worse overall survival, and contribute to drug resistance [Like vessel co-option, vasculogenic mimicry (VM) is a form of non-angiogenic tumor growth –93. In tears ago , 99. Intsistance . It has sistance . Howeversistance , 103. Insistance . Moreovesistance . Becausesistance . As proosistance . Taken tsistance –109The lymphatic vasculature is a circulatory system that contains lymph, a fluid similar to blood plasma, that is generated through capillary filtration and contains white blood cells, mainly lymphocytes. The lymph is circulated through lymph nodes and lymphoid organs and tissues, providing immunological defense against microorganisms. The lymph drains back into the blood circulation near the heart. The assembly of the lymphatic system occurs during embryonic development through coordinated mechanisms involving precursor cells  and epigThe key importance of blood vessel formation in development, normal physiology, and disease has made angiogenesis a broad field of study; thus, understanding the mechanisms of angiogenesis, for which a large array of available bioassays has been instrumental , 130, isIschemia is defined as the restriction of blood supply in a tissue leading to shortage of oxygen and tissue starvation due to lack of nutrients and incapacity to remove waste products. Ischemia is often caused by microvascular dysfunction, e.g. as associated with diabetes, hypotension, and sickle cell disease, or shortage of blood supply caused by vasoconstriction, vascular malformations, thrombosis, or embolism, (e.g. related to atherosclerosis); it can also be caused by trauma, pharmacological intervention, or by iatrogenic causes, such as radiotherapy or reductive surgery. Damage by ischemia is mediated by accumulation of waste products, inability to maintain mitochondrion function and cell membrane integrity, as well as the release of proteolytic enzymes. Reductions in blood flow and tissue oxygenation may trigger the formation of new capillaries in the periphery of a blockage or damaged vessel. These new capillaries provide an auxiliary source of blood, nutrients, and oxygen to the oxygen-starved tissue. In ischemic tissues where blood supply is restored by another mechanism, known as reperfusion injury, additional and different types of tissue damage may occur. Thus, restored oxygen levels in an ischemic tissue can cause toxicity due to inflammation and oxygen stress through the release of reactive oxygen species .In tissues where oxygen supply is diminished, the hypoxia-induced transcription factor HIF1-α is one of the major drivers of neovascularization due to transcriptional regulation of pro-angiogenic factors such as VEGFA . This reEngrafted tissues  frequently fail to thrive due to poor (neo)vascularization. The lack or impairment of anastomosis with host vasculature starves the engrafted tissue of oxygen and nutrients. Without adequate blood flow following anastomosis, tissue deterioration and necrosis will eventually lead to graft failure. Anastomoses of large vessels is typically followed by a burst of angiogenesis as new capillaries, stimulated by trophic signals from perivascular and immune cells, begin to form around the engrafted tissue. This period may be followed by vascular remodeling and vascular specialization at which time the engrafted ECs may acquire features of the host tissue microenvironment. It has been long-noted that there is a “window of opportunity” where a tissue graft must obtain a blood supply or will be doomed to nonperfusion/failure. Indeed, this observation was the precedent for some of the earliest attempts at tissue engraftment where skilled surgeons would suture skin flaps from, for example, a patient’s arm to support vascularization of a nose . However+ myeloid cells [Within the last few decades, it has become appreciated that providing engrafted tissues and organs with the building blocks that comprise blood vessels (e.g. ECs and pericytes) in addition to the growth factors (e.g. FGF2) that support their growth and survival, can improve the success of these grafts overall . Other sid cells . One surid cells . RecruitHealing wounds initiate angiogenesis through tissue response and repair mechanisms that generally depend on the type and extent of injury. In a simple wound or abrasion through the dermis, for example, the typical order of events includes rapid hemostasis, acute inflammation, proliferation, and finally maturation and scaring. Angiogenesis is initiated during the proliferation phase where ECs are activated by proinflammatory cytokines such as TNFα and IFNγ released by pro-inflammatory cells. These cytokines up-regulate cell adhesion molecules and chemokines that help to recruit and retain additional immune cells that aid in tissue repair or destruction of introduced pathogens. At first, neutrophils that express abundant matrix metalloproteinases (MMPs) are recruited to the wound site. MMPs such as MMP3 and MMP9 degrade the ECM including dense collagen fibers to create pathways for new vessels to sprout. Typically, these new vessels are leaky, disorganized, and highly abundant. Subsequent pruning of the neovasculature by PEDF and Sprouty2 is followed by vessel maturation and stabilization driven ultimately by the recruitment of pericytes and smooth muscle cells by factors such as TGFβ and PDGFBB , 145. Foin vitro angiogenesis [While wound healing in this simple example is a highly orchestrated process that resolves with scar formation, impaired angiogenesis underlies the failure for wounds to heal in chronic wounds such as diabetic ulcers. Even solid tumors are often described as “wounds that never heal” due to a smoldering, non-resolving inflammatory response . In diabogenesis . Thus, togenesis .The lymphatic system functions by allowing leukocytes to recirculate through the body and by supporting interstitial fluid back into the blood circulation. In conditions of a compromised lymphatic system, lymphedema can occur, which results in localized swelling of the tissue. Primary lymphedema is a rare congenital condition as seen in Turner syndrome, or it arises sporadically, often associated with other vascular abnormalities . SecondaThe concept that tumors cannot grow beyond a few millimeters without acquiring a new blood supply led to paradigm-shifting approaches to treat patients with different types of cancer. In essence, targeting the ECs lining tumor blood vessels, rather than cancer cells directly, was one of the first tumor microenvironment-centered strategies designed to thwart solid tumors. As is well-documented, anti-angiogenic therapy, while it produces robust inhibitory effects in pre-clinical models, has been less effective in human patients in clinical trials. However, anti-angiogenesis or perhaps vessel-targeted therapies remains a promising approach in combinatorial treatment regimens that include various chemotherapies and especially immunotherapies (see below) –158.Solid tumors acquire new blood vessels through diverse mechanisms; this includes intussusception, co-option and stimulation of vessel sprouting (highlighted above). In tumors, sprouting angiogenesis operates through the same mechanisms that control physiological angiogenesis, but these mechanisms may by hyper-activated without proper negative feedback Fig. . This reTumor angiogenesis is also an important mechanism through which metastasis formation is mediated , 161. Wh+ macrophages [Atherosclerotic lesions in large blood vessels develop due to genetic predisposition and a cholesterol-rich diet, high blood pressure and/or smoking, and are characterized by subendothelial accumulations of foamy macrophages (fatty streaks); these can later develop into fibroproliferative lesions by infiltration of myofibroblasts and deposition of layers of ECM. While in normal larger blood vessels the microvasculature is confined to the more peripheral layers of the adventitia and outer media, in vessels with atherosclerotic lesions, these microvessels are more abundant and infiltrate into the tunica intima  Fig. 4)4). Thus,rophages  and thatrophages  or throurophages .Fig. 4MiArthritis is a chronic autoimmune inflammatory disease that affects synovial joints. There are many types of arthritis, such as rheumatoid arthritis and osteoarthritis, but they have in common that autoimmunity is directed towards antigens in the cartilage and synovium, such as collagens, fibrinogen, and vimentin. The pathology of arthritis involves synovial hyperplasia, infiltration of immune cells, pannus formation and destruction of cartilage- and bone tissue . AngiogeApart from the role of angiogenesis in gynecological cancers , blood vEndometriosis—The presence of endometrial tissue outside the uterine cavity is called endometriosis  is one of the most vascularized tissues in the body with every adipocyte surrounded by one or more capillaries. Because of the metabolic nature of the tissue and the enormous growth capacity of adipocytes, a continuous expansion and remodeling of the vascular network is required . The molEarly research suggested that adipose tissue can be diminished by angiogenesis inhibitors. Treatment with anti-angiogenic agents, such as TNP-470, thalidomide, VEGF-A165b and endostatin mimics, resulted in initial reduction of adipose tissue and maintenance of body weight during aging –224. TheAngiogenesis is a hallmark of many ocular diseases with significant epidemiological and societal impact. These diseases involve aberrant neovascularization in the retina, choroid, iris and the cornea. Among the most prevalent conditions are diabetic retinopathy and age-related macular degeneration (AMD). The former pathology is induced by diabetes mellitus and it eventually leads to blindness caused by macular edema and abnormal retinal neovascularization. High glucose levels in the blood makes the microvasculature in the retina structurally and physiologically incompetent, resulting in hypoxia and subsequent VEGF production leading to neovascularization . VEGF alVascular malformations denote a broad spectrum of disorders characterized by dysfunctional endothelium and abnormalities in the basement membranes or perivascular pericytes. This also includes cancers of endothelial origin such as angiosarcoma or hemangioendothelioma. Abnormalities can occur throughout the vascular tree including large arteries and veins, venules, capillaries and lymphatics. While cancers or of vascular origin will not be covered here, these types of cancers can be benign (as in epitheloid hemangioma) or can be aggressive and difficult to diagnose (as in epitheloid angiosarcoma). In the later, a gene translocation between WWTR1  and CAMTA1 (a DNA binding protein expressed during development) drives the aberrant temporal expression of the chimeric WWTR1/CAMTA1 factor that results in EC transformation . We willin vivo without apparent side effects in mice [Infantile hemangioma (IH)—IH is a neoplasm that arises during infancy characterized by rapid initial growth and slow involution . Two pha in mice .Sporadic arteriovenous malformations (AVMs)—These typically present at birth and can be found anywhere in the body. AVMs may result in localized pain, bleeding and ulceration. Many AVMs arise due to activating mutations in genes critical for growth/proliferation. For example, EC expression of a mutant activating p.K57N missense Map2ki mutation is sufficient to produce vascular malformations in the brain, ear, and intestines in mice . A somatCerebral cavernous malformations (CCMs) – CCMs can be sporadic or inherited and the most common form are brain arteriovenous malformations. They typically present as three groups: sporadic  which are characterized venous abnormalities, familial, and radiation-induced . FamiliaSturge-Weber syndrome—In Sturge-Weber syndrome a somatic mutation in GNAQ  is found in ECs and this contributes to vessel pathogenesis such as enlarged blood vessels. Interestingly, GNAQ mutations drive constitutively active PLCB3 which increases ANGPT2—as a corollary, blocking ANGPT2 normalized enlarged vessels suggesting a potential treatment approach for Sturge-Weber syndrome . Recentlin vitro sprouting. Indeed, PIK3CA inhibitors have shown promising results in the treatment of PIK3CA-related lymphatic anomalies in a mouse model and in human patients [Lymphatic malformations – Similar to AVMs, lymphatic malformations (LMs) result in aberrant drainage and collection of fluid within cysts or channels. LMs may occur at any age but are most common in children and they typically present as bulging masses under the skin or clusters of small, reddish blisters. Hotspot mutations in PIK3CA and NRAS are frequent in LMs , 264. Inpatients . Mechanipatients . Notablypatients . Clonal patients , 269. Thpatients . Similarpatients . Other epatients –274.The COVID-19 pandemic revealed that SARS-CoV-2 is mainly a vascular pathology , 276. OnBeyond forming simple conduits for blood to flow, it is now well understood that ECs secrete paracrine factors  that signal to other cell types nearby and can therefore act as a source of paracrine mediators. Such perfusion-independent functions of ECs were noted some time ago when it was found that ECs induce expression of insulin from pancreatic endoderm thus providing inductive signals for organ development . Since tApart from growth factors, ECs may secrete miRNAs and other factors packaged in extracellular vesicles (EVs). These EVs may signal to neighboring cells in the nearby microenvironment or systemically by traveling through the circulation , 299. A In the years following the discovery of tumor angiogenesis as a cancer hallmark and important driver of tumor growth and metastasis, multiple angiogenesis inhibitors were developed with the goal of pruning the tumor-associated vasculature in the process starving tumors of nutrients and oxygen. Thousands of pre-clinical studies have validated that blocking pro-angiogenic pathways is an effective strategy for inhibiting solid tumor growth; many of these strategies were translatable into human patients . HoweverWithout a doubt, single cell RNA (scRNAseq) sequencing has revolutionized the field of vascular biology . While mIt is widely known that ongoing angiogenesis is associated with stimulation of pathways that promote immune suppression. Overexpression of angiogenic growth factors such as VEGF and FGF results in negative signaling in cytotoxic T-cells, while “pro-growth” signals are created for immune suppressive immune cells, such as regulatory T-cells and myeloid derived suppressor cells , 317. AnOne impact related to the discovery of endothelial cell anergy and the possibility to overcome it by angiogenesis inhibition relates to the successes of combinatorial immunotherapy and anti-angiogenic therapy. Clinical studies show that the success of immunotherapy, mainly immune checkpoint inhibition, is significantly enhanced by simultaneous treatment with angiogenesis inhibitors, mainly demonstrated with those targeting the VEGF signaling pathway . Seven oIn lymph nodes, including bronchus- and gut associated tissues, post-capillary venules can adopt a ‘high endothelial’ phenotype where ECs acquire a cuboidal or “plump and tall” morphology . These HECs are highly pliable and programmed to adapt to perturbations in metabolic/nutrient flux and changes in oxygen levels and flow. Some of these adaptations, for example changes in cell shape or tension (i.e. tensegrity), can occur rapidly which allows the vasculature to maintain hemostasis in the face of acute challenges. While inter-vessel, intra-vessel, and organotypic heterogeneity in ECs has been long appreciated, scRNAseq has allowed for unparalleled resolution in terms of understanding EC diversity and plasticity at the single cell level .in vitro cultured mature aortic vascular ECs lost EC characteristics such VE-cadherin expression and gained mesenchymal features reminiscent of smooth muscle cells through a process termed “endothelial-mesenchymal trans-differentiation”  [One of the best-characterized examples of endothelial plasticity is the transition of ECs into hematopoietic cells  which give rise to hematopoietic stem and progenitor cells during development . Similad EndMT) . The perd EndMT) . Especiad EndMT) . Interesd EndMT) .Numerous pathophysiological conditions are characterized by a process of EndMT where transitional or “hybrid” ECs have been identified. These hybrid ECs are suggested to be “maladapted” in that their conversion to a fibroblast or (myo)fibroblast-like state is associated with EC dysfunction . A good In myocardial infarction (MI), an elegant recent study found that 3–7 days after MI, ECs transiently acquire mesenchymal gene expression which the authors suggest is an adaptation to metabolic/hypoxic insult. These ECs underwent partial EndMT, indicated by reductions in most pan EC markers such as VE-cadherin and PECAM, while gaining mesenchymal markers including several collagens and the intermediate filament vimentin. Over time, it was found that partially differentiated ECs re-acquired their EC characteristics indicating the process is reversible to some extent . In atheAnti-angiogenic therapy was initially presented as a treatment modality in cancer that could potentially avoid drug resistance since the target of therapy is the endothelium. Unlike cancer cells, ECs are thought to be genetically stable and therefore are not expected to mutate or resist therapy. Furthermore, because most anti-angiogenic drugs are targeted towards angiogenic growth factors, their receptors, and their cellular signaling axes, cancer cells should not be directly affected  . VEGF haTargeting pathological angiogenesis has long been considered an anti-cancer treatment strategy. However, it is now apparent that many diseases and pathological conditions have underlying dysfunctional angiogenesis or maladapted ECs that contribute to disease progression. This raises the question of whether long-term clinical management of conditions such as arthritis or obesity can be managed by the judicious use of highly selective angiogenesis inhibitors. Anti-angiogenic approaches of course come with the caveat that physiological angiogenesis may also be affected  so this would have to be carefully considered. As has been shown in benign (non-cancer-associated) conditions such as macular degeneration, the targeted use of angiogenesis inhibitors can be used safely with high success. In the cancer setting, resistance to angiogenesis inhibitors presents a significant challenge to the continued development and use of this approach clinically. Compensation by upregulation of complementary growth factors, and switches to different modes of vascularization have made these types of therapies less effective. However, there has been some good success with using anti-angiogenic therapies in combination with chemotherapy or immunotherapy and we expect these approaches will continue to be optimized to produce better overall outcomes. In the future, new and exciting approaches applied to vascular biology such as artificial intelligence (AI) and machine learning could help to guide rational decision-making for novel drug combinations, aid with diagnosis, and help to identify biomarkers that predict responsiveness to anti-angiogenic therapies for multiple pathological settings –382.


Nanotechnology offers the possibility of revolutionizing cancer theranostics in the new era of precision oncology. Extracellular vesicles (EVs)-like biomimetic nanoparticles (EBPs) have recently emerged as a promising platform for targeted cancer drug delivery. Compared with conventional synthetic vehicles, EBPs have several advantages, such as lower immunogenicity, longer circulation time, and better targeting capability. Studies on EBPs as cancer therapeutics are rapidly progressing from in vitro experiments to in vivo animal models and early-stage clinical trials. Here, we describe engineering strategies to further improve EBPs as effective anticancer drug carriers, including genetic manipulation of original cells, fusion with synthetic nanomaterials, and direct modification of EVs. These engineering approaches can improve the anticancer performance of EBPs, especially in terms of tumor targeting effectiveness, stealth property, drug loading capacity, and integration with other therapeutic modalities. Finally, the current obstacles and future perspectives of engineered EBPs as the next-generation delivery platform for anticancer drugs are discussed. Cancer poses a serious threat to human health . ChemothExtracellular vesicles (EVs) , a new cTherefore, scientists have attempted to address the aforementioned limitations of EVs through various engineering approaches, including internal loading and external modification, fusion with other membrane components or various types of nanoparticles, and even the disruption of cell membrane structure and recombination of EVs analogues. These unnaturally engineered nanoparticles have undergone a series of manual operations and are no longer suitable for the definition of EVs. Therefore, it is necessary to define such EVs-based nanoparticles, which will facilitate subsequent analysis, research, and the formulation of corresponding standards and specifications. Based on the fact that these engineered nanoparticles have vesicle-like structures similar to EVs as well as biomimetic properties that mimic cell membranes, we refer to them as EV-like biomimetic nanoparticles (EBPs). All artificially engineered nanoparticles based on EVs or EV analogues, as well as those reconstituted with cell membrane components, are classified as EBPs. Table EBPs can be invoked as a substitute for EVs to exert their biological function, with simpler preparation methods, higher yield and production efficiency, and more effective combination therapy integration, showing better application prospects. Compared with traditional methods, artificial methods can significantly increase the yield and shorten the preparation time of EBPs . The mosThis review aims to summarize the utilization of EBPs as drug delivery vehicles in cancer treatment through different engineering strategies, including biological, physical, and chemical approaches, and discuss the challenges, potential solutions, and future prospects of EBPs in clinical applications. Figure To improve their scalability and therapeutic outcomes, a range of engineering strategies have been implemented to construct EBPs. The utilization of these strategies endow EBPs with multifunctionality and enhanced antitumor ability. Firstly, through genetic engineering of original cells, it becomes possible to alter the surface properties and confer specific targeting capabilities of EBPs, enabling their precise delivery to the site of the disease. Secondly, the fusion of EVs or cell membrane structures with synthetic nanoparticles can improve the drug loading capacity of the obtained EBPs, enhance their stability and biocompatibility, and thus improve their therapeutic effect. Thirdly, direct modification of EVs can also alter the intracellular delivery mechanism of nanoparticles, further augmenting their therapeutic impact. The integration of these engineering strategies not only expands the application scope of EBPs but also paves the way for the realization of precision medicine.Genetic engineering is one of the ways to obtain ideal EBPs. Genetic engineering techniques in EBPs display functional ligands on the membrane of donor cells by constructing plasmids and overexpressing proteins. Figure Genetic engineering approach requires a solid understanding of the physiological conformation of membrane proteins, since the function of many membrane proteins is associated with their complex tertiary structure. In addition to functional proteins, genetic engineering techniques can also introduce noncoding RNA sequences into EBPs, such as miRNA or small interfering RNA (siRNA) . ClusterDue to the properties of foreign substances, conventional nanoparticles are rapidly eliminated by the immune system after entering the bloodstream. To achieve superior anticancer effects, an ideal drug nanocarrier should have a long blood circulation time, as well as specific and efficient enrichment in tumor tissue . Using tInorganic materials such as gold, iron, and silica wrapped in the contents of cell membranes have unique physical, electrical, magnetic, and optical properties that can be used for diagnostic, imaging, and photothermal therapy (PTT) applications , 62. MosIn addition, direct modification is also an artificial engineering strategy. There are more concerns about direct modification of EBPs than liposomes because they must be modified without compromising the integrity of vesicles. “Click chemistry” reactions can form chemical bonds under very mild conditions and are utilized to covalently bind large biomolecules. This method can also anchor the targeted portion to the cell membrane, controlling the structure and density of the targeted ligand regardless of the cell of origin. In addition, the reaction can be performed during the purification of vesicles, so it is more suitable for clinical applications than the genetic engineering approach. Tian et al. adopted a copper-free click chemistry method to conjugate cyclic (Arg-Gly-Asp-d-Tyr-Lys) peptide [c(RGDyK)] into cerebrovascular endothelial cell-derived EBPs Fig. . EBPs-mePeptides as tumor cell targeting ligands have lower immunogenicity than antibodies, and researchers can use peptide libraries to screen for different tumor targets. For example, the small molecule polypeptide cNGQGEQc (CC8) that can bind to non-small cell lung cancer is a short peptide consisting of eight amino acid residues, which was first screened by Guo et al. using combinatorial chemical peptide library technology . They adWhile modifying the surface of EBPs, precautions must be taken to avoid membrane rupture, surface protein denaturation, and changes in osmotic pressure resulting from exposure to excessive temperature, pressure, solvents, or low or high salt concentrations. Moreover, chemical manipulation may lead to surface protein inactivation or nanoparticle aggregation. Apart from covalent modifications, there are promising noncovalent methods for stable biofilm modification, such as membrane insertion using lipid-binding ligands or hydrophobic drugs, receptor-ligand binding, and transient membrane permeability via electroporation. Figure It is a simple and direct method to modify functional molecules with lipophilic molecules and insert them into the cell membrane. DSPE-PEG, as a PEG phospholipid, has good biocompatibility and biodegradability and is often used as a nanoparticle carrier material to assist drug release, which can improve the biocompatibility and stability of the preparations , 96. FunIn addition, proteins or peptides can also be bound to the membrane surface of EBPs by protein ligases. In the study of Peng et al. the vesicles produced by erythrocytes were linked to the biotin-TRNGL enzyme, and biotin-TRNGL was sequentially coupled to a biotinylated linking peptide with streptavidin tetracer and biotinylated anti-EGFR nanoparticles (α-EGFR-VHH) to functionalize the surface of EBPs, which activated the innate immune system through the RIG-I receptor (RLR) pathway for anticancer treatment . Pham etGram-negative bacteria-derived outer-membrane vesicles (OMVs) hold significant potential as anticancer therapeutic agents in nanotechnology applications. However, OMVs’ intravenous injection can result in high toxicity and antibody-dependent clearance. To address these challenges, Qing et al. coated the OMVs’ surface with a pH-sensitive calcium phosphate (CaP) shell, which neutralizes the acidic tumor microenvironment . This ne6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB) can specifically kill HER2-positive cells [One common strategy to enhance the tumor targeting of nanoparticles is through modification with tumor-specific ligands. Human epidermal growth factor receptor-2 (HER2) is among the most widely examined breast cancer genes and is a prognostic indicator for clinical treatment monitoring that plays a crucial role in targeted therapy. Shi et al. successfully generated dual-targeted EBPs that targeted T-cell CD3 and tumor HER2 receptors via simultaneous overexpression of anti-human CD3 and anti-human HER2 antibodies Fig. . This teve cells .Fig. 7ScSuch therapeutic strategies are increasingly being applied to other common tumor targets. The urokinase plasminogen activator receptor (uPAR) is the main receptor of the plasminogen activation system, which is involved in the activation of plasminogen. EBPs overexpressing urokinase-type plasminogen activating peptide (uPA pep) can target uPAR and participate in specific tumor targeting . The comIn addition to large molecules such as proteins or antibodies, some small molecules can be used to decorate the surface of the EBPs in a more flexible way. Folic acid receptor (FR) is widely expressed on the surface of a variety of tumor cells. Compared with normal lung tissue, FR is highly expressed in tumor tissue (100 times) and only slightly expressed in normal cells. In addition to the method of binding the DSPE-PEG chain and then inserting it into the cell membrane, folic acid (FA) can also be bound to the cell surface by chemical reaction-mediated covalent binding. The mitochondrial heme synthesis pathway can efficiently convert 5-aminolevulinic acid (5-ALA) into the photosensitizer PpIX. By adopting this principle, Li et al. allowed cells to take up 5-ALA and then converted it into photosensitized agents for packaging into vesicles, which could not only overcome the low drug loading efficiency of the passive encapsulation method but also avoid the destruction of membrane integrity caused by the active encapsulation method . FA-modiLarge proteins such as antibodies can be displayed on the surface of vesicles without genetic engineering. As mentioned earlier, DSPE-PEG chains can not only modify small molecular compounds such as folic acid but also serve as links for proteins and peptides to be inserted into cell membranes. The terminal groups of PEG can be activated and linked to various targeting ligands, such as anti-PD-L1 antibodies, which are subsequently inserted into the outer membrane of EBPs through the lipid accumulation of DSPE . Since aIn addition to tumor cells, immune cells surrounding tumors are also popular targets for drug delivery. For example, mannose and mannose receptor (MR) pairs can recognize and capture antigen molecules, promote antigen presentation, and regulate the maturation and differentiation of immune cells. Given that both tumor cells and macrophages overexpress MR on their surfaces, mannose modification or mannosylation is an effective dual targeting strategy that can be involved in the design of specific nanomaterials. OMV comes from microorganisms, which are more easily recognized and phagocytosed by macrophages. In a study conducted by Guo et al. they demonstrated that OMVs modified with mannose exhibited excellent targeting ability towards a specific subtype of macrophages called M2-type macrophages. These M2-type macrophages are known to be present in the tumor microenvironment and have been implicated in promoting tumor progression . When OMMagnetic nanoparticles, together with an external magnetic field and/or magnetized implants, can deliver and anchor the particles to the target area, enabling local and sustained drug release. Hyperthermia refers to placing superparamagnetic ferric oxide in an AC electromagnetic field, which can make the magnetic direction randomly change between parallel and antiparallel so that magnetic energy can be transferred to particles in the form of heat, a property that can be utilized in the body to destroy sick cells. Iron oxide particles not only have magnetic responsiveness but also regulate the tumor microenvironment and trigger T-cell recruitment when combined with thermochemotherapy. Bose et al. successfully wrapped gold-iron oxide nanoparticles (GION) into EBPs derived from tumor cells to deliver therapeutic anti-Mir-21 and doxorubicin (DOX) drugs to cancer cells, achieving superior anticancer effects . DOX wasIn addition to enhancing targeting capabilities, the engineering strategies may also improve the anticancer effects of EBPs. Tumor necrosis factor-associated apoptosis inducing ligand (TRAIL) is a membrane protein that can induce tumor cell apoptosis with tumor targeting. Shi et al. developed EBPs using amphiphilic poly(lactic-co-glycolic acid) (PLGA) carrying oxaliplatin (OXA) and hydroxychloroquine as the core, and the cell outer membrane with TRAIL as the shell . These EThe antitumor properties of EBPs can be activated or promoted by the application of external light or magnetic fields. For example, thermosensitive and photosensitive EBPs are promising nanocarriers that effectively utilize the dual advantages of EBPs and thermotherapy/photodynamic therapy (PDT) to improve therapeutic efficacy and reduce side effects. In addition, the increase in temperature can be combined with chemodynamic therapy (CDT), which is an emerging therapeutic approach that utilizes Fenton-type reactions to produce highly cytotoxic hydroxyl radicals that can resist the anoxic environment of tumors. The hydrogen peroxide generated by the reaction of glucose oxidase (GOx) with glucose can enhance the Fenton reaction at the tumor site. Photothermally sensitive EBPs can be used to produce photothermal effects under irradiation, and the increase in temperature can further enhance the activity of GOx Fig. , while lEBPs carrying a photosensitizer also act as photosensitive nanocarriers. Ce6 is usually highly efficient at producing singlet oxygen because it is suitable for the development of PDT for tumors. However, like most photosensitizers to date, Ce6 is hydrophobic and tends to aggregate in solution, so its practical application has encountered some difficulties. Hydrophobic Ce6 loaded into the lipid bilayer can provide photoacoustic imaging and PDT functions. CPPO is another type of chemical cold light source with high brightness and high efficiency of chemiluminescence. Wang et al. used these two hydrophobic agents  to functionalize EBPs derived from M1 macrophages . The intSimilar to photosensitizers, gold nanoparticles are also photoresponsive. The MSC membrane encapsulated hollow gold nanoparticles with a near-infrared (NIR) response, which showed enhanced nanoparticle delivery efficiency and tumor killing ability compared to conventional metal nanoparticles. Compared with traditional metal semiconductor quantum dots and organic dyes, carbon quantum dots not only maintain excellent fluorescence properties but also overcome the shortcomings of poor optical stability, high toxicity, and complex preparation processes. Cao et al. produced ultrasmall two-dimensional vanadium carbide quantum dots to show a strong photothermal effect at NIR light, and the EBPs prepared by wrapping them in tumor cell membranes exhibited good fluorescence imaging ability and photothermal effects in cancer treatment. In addition to being wrapped in the interior of EBPs, gold nanoparticles can also gather on the surface of EBPs to achieve photothermal conversion and kill tumors . The useActivating the antitumor immune response at the tumor site and improving the immune tolerance microenvironment are also hot research directions in cancer treatment. EBPs derived from tumor cells overexpressing the high-affinity variant human PD-1 protein (havPD-1) can block the function of highly expressed PD-L1 in tumors, induce tumor cell apoptosis, and activate cytotoxic T cells in combination with low-dose PARP inhibitors, thereby effectively treating homotypic tumors . Liu et In addition to directly activating immune cells, the high molecular weight hyaluronic acid (HA) around tumor tissues is also a barrier to the infiltration of immune cells. As a membrane protein, hyaluronase itself has the inherent ability to be expressed on the surface of cell membranes and can be simply modified to express on the surface of cell membranes. Considering that hyaluronidase can degrade high molecular weight HA to low molecular weight HA (LMW-HA), some studies have used this feature to polarize macrophages into the M1 phenotype by LMW-HA and reduce the number of associated immunosuppressive cells, thus changing the immune microenvironment from immunosuppressive to immunosupportive, reshaping the tumor microenvironment, and improving the efficacy of chemotherapy . ProstagEBPs derived from bacteria have immunomodulatory activity, showing great potential for the development of anticancer drugs and nanotechnology applications. The outer membrane vesicles (OMVs) released by genetically engineered bacteria can serve as vaccine carriers to break autoimmune tolerance and induce the production of autoantibodies for cancer therapy. The BFGF antibody can effectively inhibit angiogenesis and tumor growth. The use of BFGF-modified OMVs (BFGF-OMVs) as an antitumor vaccine can induce high levels of anti-BFGF autoantibodies, which can inhibit angiogenesis, promote tumor cell apoptosis, and reverse tumor immunosuppression . Virus-dPTT induces immunogenic cell death (ICD) through NIR-mediated photothermal ablation and release of tumor-associated antigen (TAA), which subsequently activates the immune response in vivo. Tan et al. selected dipalmitoyl phosphatidylcholine (DPPC) as a thermosensitive lipid component to construct thermosensitive lipid nanoparticles, incorporated the PD-1/PD-L1 inhibitor BMS202 into NIR-responsive EVs, and finally constructed thermosensitive EBPs by membrane fusion that can target both primary tumors and lung metastases . Under lPolymeric nanoparticles are commonly used drug as carriers in cancer treatment. The utilization of pH, thermal, ultrasonic, enzyme, and light-sensitive block copolymers allows for controlled micellar dissociation, triggering drug release. This approach not only enhances the specificity and effectiveness of micellar drug delivery but also provides insights into the development of smart drug delivery systems. By integrating EBPs with these industrial products, drug delivery systems with improved stability, higher drug-carrying capacity, and slower release kinetics can be achieved.Chen et al. employed a novel polymerized non-ionic surfactant, Pluronic F-127, to encapsulate the drug Tegafol . The staDendrimers are capable of loading various types of drugs, including insoluble ones, in large quantities. Current research mainly focuses on nucleic acid and small molecule delivery. Both polymer types protect loaded therapeutic RNA, bind to the vesicle shell with targeted functionality, and exhibit effective tumor tissue killing effects. In the study by Wang et al. natural killer cell-derived exosomes (NKEXO) were utilized to encapsulate dendritic macromolecular cores for loading therapeutic miRNAs. Natural killer cells exhibit specific aggregation towards tumors without exerting cytotoxic effects on normal tissues. Simultaneously, the acidic tumor microenvironment facilitates the accumulation of these nanoparticles, thereby enhancing the biocompatibility and targeting capabilities of PAMAM dendrimers. Polyethylenimine (PEI), another widely used organic macromolecule, possesses a high cationic charge density and protonated amino nitrogen atoms. This facilitates the binding of negatively charged nucleic acid molecules, forming positively charged complex particles. Zhupanyn et al. developed an extracellular vesicle-modified PEI/siRNA complex. Upon intravenous administration in tumor-bearing mice, this complex showcased specific tumor inhibition through siRNA . The modIt is widely accepted that inorganic non-metallic nanoparticles are generally considered to have better biosafety compared to metal nanoparticles, along with higher drug loading capabilities. Among them, porous silicon nanoparticles exhibit excellent drug loading abilities, high biocompatibility, and biodegradability. In a study conducted by Wu et al. glutathione-responsive biodegradable silica nanoparticles were prepared to encapsulate catalase and ICG (Indocyanine Green). This fusion preparation not only enhances the efficacy of acousodynamic therapy but also maintains low toxicity to normal tissue . FurtherMetal–organic frameworks (MOFs) are a type of organic–inorganic hybrid material composed of organic ligands and metal ions or clusters, self-assembled through coordination bonds to form intramolecular pores. MOF-protein (MP) nanoparticles possess a large internal surface area, high non-covalent affinity, and excellent drug loading capabilities. The pH-responsive metal–ligand bonds within MP nanoparticles, upon cellular internalization, can release proteins in acidic endosomes and lysosomes. This protects the proteins from protease digestion and immune system clearance and enables selective targeting of homologous tumor sites .EBPs used as drug delivery systems in clinical trials need to fulfill the same basic requirements as traditional synthetic carriers, including simplicity, efficiency, and scalability . During It has been reported that the combination of tangential flow filtration (TFF) technology and ultra-high-speed centrifugation is a convenient and efficient method for enriching EVs. Lorenzini et al. have developed a large-scale cell culture method, in combination with TFF, that can support cell survival and EV production for at least 216 h in both EV-free human platelet lysate (hPL) and fetal calf serum (FCS) environments . This meCurrently, there are several approaches to increasing yield. One approach is to expand cell culture, such as by utilizing 3D culture or hollow fiber culture, to involve a larger number of cells in the production of vesicles. Another approach is to enhance the production of vesicles through chemical or physical stimulation of cells or direct disruption of reconstituted EV-like nanostructures . AchieviIn this review, various modification and engineering strategies of EBPs for efficient drug delivery in targeted cancer therapy are introduced. Through biomimetic technology, different cell membranes can be fused to produce hybrid EBPs that maintain the unique characteristics of the originating cells, thereby integrating the functions of various cell types into biomimetic nanoparticles. The coating of nanomaterials with cell membranes, such as erythrocyte-derived vesicles, is typically effective in prolonging blood circulation and reducing systemic clearance. Moreover, EBPs derived from immune cells can regulate and mediate immune responses to tumors, while inheriting the targeting abilities of the original cells to tumor tissues. Tumor cells, on the other hand, can be utilized to extract and produce EBPs due to their innate ability to target their own tumors while also possessing active immunity to the same.In recent years, combination therapy has emerged as a crucial trend in cancer intervention. Extensive research has demonstrated that EBPs represent a new and promising delivery platform for cancer combination therapy. Therefore, comprehending the various engineering strategies of EBPs is crucial for accelerating their clinical application and reducing obstacles in cancer combination therapy. As compared to traditional EVs, EBPs produced through artificial means offer greater efficiency and simplicity in drug delivery, as well as increased feasibility in combining EBPs with other therapeutic modalities to treat tumors. In this regard, some researchers are exploring the possibility of de novo synthesis to achieve a completely artificial synthesis of EBPs. As artificial products can achieve composition control and safety, they may better meet regulatory requirements and aid mass production. Hybrid EBPs based on cell membrane components may be a potential breakthrough in clinical applications. These hybrid EBPs may exhibit better delivery efficiency than conventional synthetic nanoparticles and greater stability than conventional EVs, thus demonstrating high applicability.Multiple clinical trials have demonstrated the potential efficacy of EBPs. However, several challenges must be addressed before their successful translation from the laboratory to clinical application. Research efforts must be directed towards a deeper understanding of the biological function of EBPs, scale-up production, the development of standardized purification protocols, and evaluation methods. Moreover, the quantitative determination of the physicochemical characterization of EBPs is essential to ensure drug loading, quality control, and stable storage. In order to achieve these goals, it is vital to develop large-scale bioreactors, standardized and reproducible culture conditions, and production programs. Optimization of extraction techniques to obtain sufficient cell membranes and identification and quantification of by-products from the production process are also necessary. To successfully translate EBPs from laboratory research to clinical application, it is essential to improve the purity and storage life of the final product. Manufacturing processes must be conducted under aseptic conditions to ensure high recovery rates of therapeutic EBPs under storage conditions without excessive damage to the biological content and particulate matter. The future of EBPs holds immense promise. These remarkable nanoparticles, inspired by the natural properties of extracellular vesicles (EVs) derived from cells, have the potential to revolutionize drug delivery and targeted therapy. By mimicking the surface characteristics and cargo-loading capabilities of EVs, EBPs can effectively navigate the complexities of the human body, reaching specific target sites with remarkable precision. The use of EBPs opens up new possibilities in personalized medicine, enabling tailored treatments that harness the body’s own natural processes. Furthermore, these nanoparticles demonstrate enhanced stability, biocompatibility, and prolonged circulation time, further augmenting their therapeutic potential. As research advances and technology evolves, we can eagerly anticipate the continued exploration and development of these innovative nanoparticles, bringing us closer to a future where precise and effective therapeutic interventions become a reality.


Pediatric neck infections and their complications, such as abscesses extending to deep neck compartments, are potentially life-threatening acute conditions. Medical imaging aims to verify abscesses and their extensions and exclude other complications. Magnetic resonance imaging (MRI) has proven to be a useful and highly accurate imaging method in acute neck infections in children. Children and adults differ in terms of the types of acute infections and the anatomy and function of the neck. This pictorial review summarizes typical findings in pediatric patients with neck infections and discusses some difficulties related to image interpretation. Most conventional neck infections can be managed conservatively. However, deep neck infections possess a high risk for serious complications, such as abscess formation, mediastinitis, airway compromise, and venous thrombosis due to the infection spreading along the fascial planes and potential face and neck spaces. These complications are potentially lethal . In chilPediatric acute neck infections arise from common infections originating in the ears, nose, or throat and have the potential to disseminate to the deep neck spaces through direct continuity or lymphatic drainage to the lymph nodes . When adIn clinical practice, these infections have typically been imaged with contrast-enhanced computed tomography (CECT) ,8, whichThe purpose of the current pictorial review is to present typical neck infection MRI findings in children, including the most useful edema patterns and typical abscesses, and underline the potential caveats and complications associated with deep neck infections. We focus on the soft tissue of the neck above and below the hyoid bone. Pediatric non-traumatic emergencies of the orbits, nose, and ear have recently been covered elsewhere .®; Guerbet, Villepinte, France) is routinely used. T2-weighted imaging is useful in assessing the anatomy: fat and water have a bright signal, lymphoid tissue has an intermediate signal, muscles have a low signal, and cortical bone has no signal. Specific edema patterns can be appreciated as bright areas in the fat-suppressed, T2-weighted images. We prefer the Dixon method for fat suppression because it reliably yields homogenous images even with large fields of view. The T1-weighted axial sequence is used to reference post-contrast Dixon sequences and to detect bone marrow fat obliteration in odontogenic neck infections. Pre-contrast T1-weighted sequences are also useful in assessing postoperative hematomas and chronic fluid collections, which may have a high T1 signal intensity. Together with diffusion-weighted imaging (DWI), contrast-enhanced sequences are used for diagnosing and characterizing abscesses. These sequences are important for assessing cystic masses, tumors, and necrotic lymph nodes [2. In the context of neck infections, the DWI serves two functions. First, it is used for demonstrating or excluding diffusion restriction related to purulence in abscesses, and second, it is used for detecting lymphoid tissue in tonsils and lymph nodes. Our MRI protocol is consistent with the protocol suggested by a recent multicenter international consensus paper [We utilized the Philips Ingenia 3 Tesla scanner . Our routine neck infection protocol comprises seven sequences and lasts approximately 30 min . We use ph nodes . The carus paper .MRI has been proven feasible in pediatric patients with suspected neck infections . In our Image quality can be deteriorated by artifacts induced by patient motion or metallic foreign bodies, but the proportion of non-diagnostic MRI scans is low (about 1%) even in the acute setting . EspeciaThe normal anatomy of the pediatric oropharynx is presented in Viral or bacterial infections are the most common causes of acute lymphadenopathy in children . The lymAs lateral lymphadenitis is common in children, the careful analysis of the lymph nodes is essential in looking for suppurative lymphadenitis  which may require surgery or drainage . ChildreThe thymus can often be seen in children as a homogenous mass with smooth margins in the upper mediastinum  and shouIn MRI, we define infection as (1) an abnormal, high signal indicating edema in fat-suppressed T2-weighted sequences or (2) a high signal in fat-suppressed post-contrast T1-weighted sequences indicating pathological tissue enhancement. Abscess MRI criteria include an abnormal isointense or hyperintense collection on T2-weighted sequences with low ADC values and no central enhancement, as well as enhancement surrounding this collection on T1-weighted Dixon post-contrast sequences . In diagReactive, non-suppurative soft tissue edema is common in acute neck infections . While aIn children, two of the most useful edema patterns are retropharyngeal edema (RPE) , and medME is seen in about one-quarter of patients with acute neck infections and can be divided into two categories: anterior and posterior. Anterior ME is commonly a continuum of edema from the visceral and/or anterior cervical spaces, whereas posterior ME is a continuation of RPE caudally. Similar to RPE, ME is encountered as often in children as in adults and is a significant predictor of the length of hospital stay .Tonsillitis (pharyngotonsillitis) is a common oropharyngeal infection. In our previous validation cohort, these types of infections were proportionally less common in children than in adults . MRI finStaphylococcus aureus infections tend to present with lateral lymphadenitis with large infectious mass-like lesions accompanied by widespread soft tissue edema, and careful assessment of both DWI and fat-saturated post-contrast T1-weighted sequences is crucial  in children may be bacterial or viral . In acutIn early lymphadenitis, the lymph node may be necrotic but not yet abscessed. It may be difficult to differentiate between a non-enhancing lymph node with restricted diffusion  and a lymph node with delayed enhancement accompanied by restricted diffusion  . Indeed,Cystic masses, such as branchial cleft cysts, thyroglossal duct cysts, and vascular malformations, comprise the majority of congenital neck masses in children . Some cyGas can be difficult to detect from MRI because the signal void may not be easily distinguished from other areas of low signal . All seqA mediastinal extension is related to a more severe course of illness in both children and adults  Figure . The infMRI findings of mediastinal extension include anterior or posterior edema and/or abscess formation and pleural fluid accumulation . Minor eLemierre’s syndrome has historically been used. With internal jugular vein thrombophlebitis, this syndrome also encompasses bacteremia  secondary to current oropharyngeal infection, resulting in septic emboli [When venous thrombosis in the context of deep neck infection is diagnosed, the eponym c emboli ,37. Lemic emboli  and thusc emboli . The sync emboli .Diagnosing thrombophlebitis is not always simple in MRI, as the thrombus may not be separable from a flow void in T1-weighted sequences . The lacEmergency MRI has been proven to be a feasible imaging method in children with acute deep neck infections. MRI has superior diagnostic accuracy compared to CECT, encouraging more widespread use, especially in younger individuals. The differences between children and adults in anatomic proportions and the distribution of infectious diseases in the neck result in distinctive MRI findings. The subtleties in MRI findings of pediatric deep neck infections require careful assessment and knowledge.To summarize the MRI findings: all acute neck infections cause varying degrees of edema, which is characterized as an abnormally high T2 signal on fat-suppressed images and enhancement shown as a high signal in fat-suppressed post-contrast T1-weighted sequences. For example, edema and post-contrast enhancement of the palatine tonsils and surrounding oropharyngeal mucosa are typical MRI findings in tonsillitis. Abscesses, such as those in peritonsillar, parapharyngeal, and retropharyngeal spaces, exhibit peripheral enhancement and a central, non-enhancing collection of pus, which can be effectively visualized with DWI as low ADC values. The location or extension of the abscess through various anatomical structures  can then be used for proper classification. Careful examination of all MRI sequences is important to avoid various pitfalls. For example, necrotic lymph nodes may be difficult to distinguish from purulent lymphadenitis. Early scans may confuse necrosis and delayed enhancement for non-enhancement, and both can show low ADC values, indicating high cellularity or purulence.


Among central nervous system (CNS) disorders, Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder and a major cause of dementia worldwide. The yet unclear etiology of AD and the high impenetrability of the blood–brain barrier (BBB) limit most therapeutic compounds from reaching the brain. Although many efforts have been made to effectively deliver drugs to the CNS, both invasive and noninvasive strategies employed often come with associated side effects. Nanotechnology-based approaches such as nanoparticles (NPs), which can act as multifunctional platforms in a single system, emerged as a potential solution for current AD theranostics. Among these, magnetic nanoparticles (MNPs) are an appealing strategy since they can act as contrast agents for magnetic resonance imaging (MRI) and as drug delivery systems. The nanocarrier functionalization with specific moieties, such as peptides, proteins, and antibodies, influences the particles’ interaction with brain endothelial cell constituents, facilitating transport across the BBB and possibly increasing brain penetration. In this review, we introduce MNP-based systems, combining surface modifications with the particles’ physical properties for molecular imaging, as a novel neuro-targeted strategy for AD theranostics. The main goal is to highlight the potential of multifunctional MNPs and their advances as a dual nanotechnological diagnosis and treatment platform for neurodegenerative disorders. CNS diseases including neurological disorders account for significant public health concerns and in 2019 were the leading cause of disability-adjusted life years (349 million) and the second leading cause of deaths (nearly 10 million) -flutemetamol (Vizamyl R®) -CONH2; g7) and conjugated to the PLGA polymer to obtain, through a nanoprecipitation procedure, an engineered NP (g7-NPs) able to enter the brain [Some categories of opioid peptides have been shown to penetrate the BBB and to be able to cross this barrier to a higher extent when glycosylated. Thus, an opioid peptide able to enter the brain was modified to eliminate the opioid activity . The newhe brain ,151. Thehe brain .To counteract the imbalance of zinc caused by Aβ plaques, Vilella et al.  designed10 was chosen as the AD model drug for the evaluation of the neuroprotective effects of TMC-modified PLGA NPs in APP/PS1 transgenic mice. Behavioral testing showed that the injection of coenzyme Q10-loaded TMC-modified PLGA-NP greatly improved memory impairment, restoring it to a normal level, and decreased the number of senile plaques when compared with unmodified NPs (Trimethylated chitosan (TMC) is a permanently quaternized chitosan (CS) derivative that is positively charged under physiological conditions. As a cationic ligand, TMC facilitates the brain transport of NPs through the AMT pathway to enhance drug delivery into the CNS. Coumarin-6-loaded PLGA-NP A and TMCfied NPs C,D. ThusDespite the fact that vesicles formed during AMT have a greater capacity  than those formed during RMT, drawbacks associated with AMT include its lack of selectivity  , which cAn outlook of different examples of nano-DDSs described in the literature for AD therapy and diagnosis are listed in In the context of AD, the development of targeted nanosystems holds great promise in overcoming the challenge of delivering therapeutic molecules across the BBB for an effective treatment of the disease. Since the BBB acts as a formidable obstacle to many drugs’ entrances into the brain, researchers have devised targeted nanosystems that utilize RMT and AMT transporters as potential pathways for transporting NPs to the specific affected area in the brain. The integration of targeted nanosystems with advanced imaging techniques, such as MRI, represents a promising approach to address the challenge of drug delivery across the BBB while allowing AD diagnosis and therapeutic monitoring.Iron oxide nanoparticles such as SPIONs have demonstrated their effectiveness as MRI contrast agents in visualizing and detecting AD-related pathological features, including amyloid plaques, in both in vitro and in vivo settings. Moreover, their functionalization with specific ligands, such as Aβ peptides or targeting moieties, has enhanced their affinity for those AD-related biomarkers and improved their ability to cross the BBB.Moreover, the biocompatibility and safety of SPIONs have been demonstrated with their nontoxic nature and biological tolerance. After injection, these NPs can be integrated into human natural processes of iron metabolism, further supporting their potential clinical application as diagnostic tools for AD, and ensuring safety during in vivo use. The ability to modify the surface charge and hydrophobic character of SPIONs is essential for their long-term diagnostic and therapeutic applications. These coatings prevent particle aggregation, degradation, and structural alterations, preserving the NPs’ magnetic and structural properties over time. Also, some coatings can respond to specific stimuli, such as changes in pH, temperature, or enzymes, triggering controlled drug release at the targeted site. This feature enhances the efficiency and efficacy of treatments, ensuring that therapeutic agents are delivered precisely where they are needed.Overall, the integration of diagnostic and therapeutic functions into a single system allows for a comprehensive approach to AD theranostics. Theranostics’ NPs offer the potential for personalized medicine, where diagnosis, targeted drug delivery, and real-time monitoring of treatment response can be achieved simultaneously. This could revolutionize the way AD is diagnosed and managed, potentially leading to more effective and tailored treatments for patients. Nevertheless, future research should focus on enhancing the selectivity of delivery systems, improving specificity in targeting ligands to minimize off-target effects, and increasing drug delivery efficiency. Additionally, exploring the use of external stimuli, such as magnetic fields, to trigger controlled drug release from nanosystems can further enhance their therapeutic potential.Despite the revised work in this study, researchers are continually working to address limitations associated with the in vivo use of magnetic nanoparticles. One of the main concerns is their potential for toxicity associated with the nanoparticle features that may affect their biocompatibility, biodistribution, and clearance. Addressing these challenges requires personalized nanoparticle synthesis and surface modifications to enhance safety and reduce size-dependent effects. Scaling up production, conducting rigorous clinical trials, and evaluating safety profiles are necessary for successful human implementation.In summary, targeted multifunctional magnetic nanosystems hold immense promise for advancing AD theranostics. These specialized nanoparticles offer a multifaceted approach to tackling the challenges associated with the ailment, ultimately aimed at enhancing the quality of life of patients in the fight against AD.


Bladder cancer (BCa) requires the investigation of alternative therapies. Prior to clinical testing, researchers require the use of animal models to thoroughly investigate their therapeutic efficacy. To appropriately mimic cancer response, the cancer must be developed and treated within the relevant organ. This creates an obstacle with BCa, as cancer presence and progression are difficult to evaluate due to its location. Therefore, non-invasive techniques have been developed that allow for visualization of the cancer from outside the bladder. These techniques include bioluminescence imaging (BLI), micro-ultrasound imaging (MUI), magnetic resonance imaging (MRI), and positron emission tomography (PET). This paper reviews these imaging techniques when used independently or in conjunction in an animal model where BCa is developed within the bladder.Bladder cancer (BCa) is the sixth most prevalent cancer in men and seventeenth most prevalent cancer in women worldwide. Current treatment paradigms have limited therapeutic impact, suggesting an urgent need for the investigation of novel therapies. To best emulate the progression of human BCa, a pre-clinical intravesical murine model is required in conjunction with existing non-invasive imaging modalities to detect and evaluate cancer progression. Non-invasive imaging modalities reduce the number of required experimental models while allowing for longitudinal studies of novel therapies to investigate long-term efficacy. In this review, we discuss the individual and multi-modal use of non-invasive imaging modalities; bioluminescence imaging (BLI), micro-ultrasound imaging (MUI), magnetic resonance imaging (MRI), and positron emission tomography (PET) in BCa evaluation. We also provide an update on the potential and the future directions of imaging modalities in relation to intravesical murine models of BCa. Bladder cancer (BCa) is the most common genitourinary malignancy and represents the sixth most prevalent cancer in men and seventeenth most prevalent cancer in women, with over 80,000 new cases and 17,000 associated deaths predicted for 2022 in the United States alone , lifetimAs indicated, the exploration of novel therapies for BCa requires an appropriate experimental model. In vitro models of BCa do not suffice as they are unable to recapitulate human BCa etiology and progression and lack many of the defining features of cancer . In vivoCurrent intravesical murine models of BCa include carcinogen-induced, orthotopic, transgenic, or humanized mice ,13,14,15As tumor formation and progression occur within the bladder of intravesical models, BCa is not readily detectable. However, an intravesical model is essential for a clinically relevant investigation of novel therapies. In earlier studies, intravesical BCa tumors were detected and monitored by physical examination such as bladder palpitation or clinical symptoms including hematuria, significant weight loss, and behavioral changes ,26. UponEstablished modalities that reveal and evaluate the occurrence of BCa within murine models include bioluminescence imaging (BLI), micro-ultrasound imaging (MUI), magnetic resonance imaging (MRI), and positron emission tomography (PET), as summarized in BLI allows for the visualization of BCa cells through the detection of light emission using an in vivo imaging system. BCa cell lines are first transfected with a luciferase vector and then inoculated into the bladder wall of a murine model. Subsequent intravascular or intraperitoneal administration with a D-luciferin substrate stimulates its oxidation by luciferase, converting D-luciferin to oxyluciferin, resulting in the emission of green light at 550–570 nm . ImportaAs genetic engineering of the cells is required, this imaging modality is limited to an orthotopic BCa model. In studies detecting intravesical BCa tumor presence in vivo, BLI confirmed tumor presence as early as 4 days post-inoculation  Table 1Table 1. 2) ranged from 0.95 to 0.99, which was corroborated by another study  Zr-DFO-panitumumab was investigated [89Zr] Zr-DFO-panitumumab injection resulted in tumor size-dependent accumulation, with a correlation coefficient of 0.99 exhibiting the quantitative capability of PET. Interestingly, these mice demonstrated inconsistencies in their EGFR protein expression and tumor sizes [Immuno-PET is another branch of PET imaging that utilizes antibodies for the specific targeting of molecules expressed by cancer cells and tumors. In an orthotopic xenograft model, detection of the epidermal growth factor receptor (EGFR)-expressing BCa cells by the radioimmunoconjugate [stigated  . This suggests that urinary analysis is a potential non-invasive approach to developing novel BCa therapeutics using intravesical murine models of BCa. A subsequent study demonstrated the use of urinary analysis for BCa diagnosis using activated protein kinase Cα [As discussed, common limitations of the above imaging modalities include the high cost and requirement of skilled operators and equipment availability. To combat these limitations, research has been conducted to detect and monitor BCa by urinary analysis. This emerging approach remains non-invasive as it utilizes cellular components of urine to provide information on intravesical BCa . Tan et inase Cα . However3. Although PET requires further investigation to establish a model suitable for BCa detection, recent studies have demonstrated this to be a very promising modality. Notwithstanding the limitations of the imaging modalities discussed, many modalities have complementary strengths, which suggests that their concurrent use may provide a more sensitive and thorough analysis of BCa detection and progression in an intravesical murine model. When limitations such as cost and availability exist, urinary analysis has the potential to be used. These modalities, either independently or in a multi-modal fashion, provide information that is essential to the production of successful BCa therapies.Current BCa treatment paradigms have limited therapeutic impact, illustrating a need for the investigation of novel therapies. Before testing the efficacy of these treatments in a clinical setting, a suitable animal model is required. This has necessitated extensive research with the aim of creating a clinically relevant intravesical murine model of BCa. Initial studies relied on sacrificing subsets of mice at varying timepoints to confirm the presence of BCa and monitor its progression. However, these few mice do not sufficiently represent what is occurring in the remainder of the cohort as it requires many experimental animals. In response to these limitations, imaging modalities such as BLI, MUI, MRI, and PET have been explored as a means of detecting the presence of BCa and longitudinally following tumor growth. These modalities allow for the repetitive, non-invasive imaging of mice, reducing the number of mice needed and optimizing the assessment of tumor response. Furthermore, they have demonstrated high sensitivity, detecting early-stage BCa prior to the onset of clinical signs, and high-resolution, imaging tumors as small as 0.4 mm


The main functions of the testis, steroidogenesis and spermatogenesis, depend on the endocrine axis and systemic and local tolerance mechanisms. Infectious or non-infectious diseases may disturb testicular immune regulation causing infertility. Literature has illustrated that bacterial and viral infections lead to autoimmune infertility: either sperm antibodies or autoimmune epidydimo-orchitis. However, little is known about the association between non-infectious testicular pathologic diseases and autoimmunity. Here we review the novel aspect of varicocele and testicular cord torsion pathology linked to inflammation and discuss how immune factors could contribute to or modulate autoimmunity in ipsi- and contralateral testis. Infertility affects 10%–15% of couples, the male factor being present in around 50% of couples attempting to conceive. Moreover, global findings on sperm count strongly suggest a significant decline in male reproductive health, which seriously affects fertility .The causes of male infertility or subfertility can be categorized mainly as genetic (autoimmune regulator (AIRE) gene deficiency), acquired, or idiopathic. Some acquired conditions are infections, testis tumors, environmental factors, hormonal factors, lifestyle, and systemic diseases. Numerous bacteria and viruses infect epididymis and testis through an ascending canalicular or haematogenous via, respectively. Male infertility associated with immunological mechanisms may depend on the presence of: i) immune cells in the testis and excurrent ducts secreting cytokines and other inflammatory factors and/or ii) the presence of sperm antibodies. In the first case, immunopathological damage of the testis (and excurrent ducts) occurs through lymphomononuclear cell-mediated mechanisms triggered by antigens or pathogens that disrupt testicular immunoprivilege. In the second case, the antibodies generated may result in infertility by a variety of mechanisms .A severe reduction in the male fertility potential is also observed in varicocele (VC) and to lesser degree in testicular cord torsion (TCT), two pathologies originating in embryological/anatomical defects. Despite extensive literature on VC and TCT, mechanisms involved in damage to the contralateral testis and in long-term negative effects on fertility are not yet clear. With this review, we propose a different approach to this topic through the analysis and relevance of inflammation and immunologic mechanisms found in these two pathologies and discuss how immune factors may contribute to or modulate autoimmunity in ipsi- and contralateral testis.VC is a vascular lesion manifested as abnormal dilatation and tortuosity of the pampiniform plexus veins, which occur by a pathological reflux of blood into the internal spermatic vein causing the elevation of scrotal temperature. It is most commonly observed on the left side (85% of cases), although some men are affected bilaterally. Isolated right-sided VC is rare. Reflux of blood is most often found on the left side as the hydrostatic pressure is higher on this side owing to the perpendicular drainage of the left internal spermatic vein into the left renal vein. VC can be observed in 35%–44% of men with primary infertility and 45%–81% with secondary infertility. Although VC repair (varicocelectomy) is a good way to improve a patient’s semen quality, postoperative natural pregnancy outcomes are still being debated .A comprehensive review of the pathogenesis of VC is found elsewhere . CurrentHowever, some phenomena are not fully explained by the factors mentioned above; it is likely that other acquired risk factors and testicular immune microenvironment disorder contribute to irreversible testicular damage. Testicular biopsies in subfertile/infertile patients with left VC showed bilateral pathological alterations of seminiferous epithelium, sloughing of immature cells into the lumen of the tubules, and an increase in the interstitium tissue area . An elevImmune factors alter the normal function of the blood testis barrier (BTB) by changing the expression of cell junction adhesion molecule and increasing its permeability. BTB, also known as the Sertoli cell seminiferous epithelium barrier, is formed by cell junctions of adjacent Sertoli cells at the base of the seminiferous tubules. It is constituted of multiple cell junction types including tight junctions, basal ectoplasmic specializations, gap junctions, and desmosome-like junctions. Testicular biopsies of men with VC but unknown reproductive potential revealed intact functionality of BTB by intercellular tracer studies utilizing lanthanum nitrate . Neverthorchiopexy) are important to avoid irreversible ischemia-induced damage of the testis  and immediate surgery   whereas Systemic tolerance and local immune privilege are partners for complete immune protection of testis. Systemic tolerance involving antigen-specific regulatory T cells (Tregs) is maintained in peripheral lymphoid organs by continuously egressing germ cell antigens via transcytosis in Sertoli cells . TesticuHypoxia and oxidative stress in the ipsilateral testis in VC and TCT induce dysfunctional testis consistent with apoptotic germ cells observed in men and rat testis. As in autoimmune orchitis, a hypoxic and oxidative microenvironment also induces HIF-1α and NO, respectively. In long-term experimental VC, damage to seminiferous tubules is more severe, apoptosis of spermatogenic cells and expression of HIF-1α gradually increase whereas antiapoptotic proteins Bcl-2 decrease in ipsilateral testis . HIF-1α Cytokines are produced and secreted by immune cells and by testicular somatic cells in response to external stimuli. Increased levels of proinflammatory cytokines IL1, TNFα, IL6, IL18 may be responsible for disruption of BTB in VC. To our knowledge, no study has evaluated BTB in TCT. During testicular inflammation BTB integrity is impaired—denoted by increased permeability to tracers. Concomitantly, changes in expression of cell junction adhesion molecules were detected .Testicular injury via initial actions at the BTB to elicit subsequent damage to germ-cell adhesion, thereby leading to germ-cell loss, reduced sperm count, and male infertility or subfertility. In this context, there is an intricate relationship between the male gonad and the immune system resulting in production of antibodies against meiotic and postmeiotic germ cells. The presence of anti-sperm antibodies and the increase in the number of testicular immune cells in VC and TCT show that a humoral and cellular immune response occurs simultaneously with the histopathological damage, thereby suggesting involvement of an immunological mechanism that eventually impairs the contralateral testicular function. In fact, In early publications on TCT, an immunologic mechanism was also suggested as a possible mechanism involved in contralateral testis damage . Ozkan eAn early orchiectomy following TCT prevents the release of spermatic antigens, the formation of antibodies, and damage to the contralateral testis . Anti-lyIn this review, we emphasize that VC, TCT, and orchitis respond to common mechanisms of inflammation-related male infertility and theorize that prolonged VC and TCT negatively impact on contralateral testis through an autoimmune response. Consistent with this hypothesis, inhibition of inflammation can alleviate VC- and TCT-mediated pathogenesis .


Sleep homeostasis is a regulatory process that increases sleep time and/or intensity as a function of prior time spent awake. It is considered a ubiquitous feature of sleep and is often considered a requirement for identifying sleep in the animal kingdom. In mammals, the accumulation and discharge of sleep drive have traditionally been measured with changes in the non-rapid-eye-movement (NREM) sleep electroencephalograph (EEG). More specifically, NREM EEG slow waves (at least in some frequency bands) increase and decrease in proportion to the time spent awake or asleep, respectively. This marker of sleep homeostasis has been observed in all mammals and most bird species. In animals that lack comparable forebrain areas , sleep homeostasis is usually identified by compensatory changes in sleep time following sleep loss. Sleep homeostasis has therefore been reported in highly diverse animal species, including insects, worms, cephalopods, and even jellyfish  in mammals] may indeed overlap with sleep homeostasis, but not (so far) in an obvious way. It follows, therefore, that the more we know about different kinds of clocks, the better. In this vein, the opinion article byRotinen is important because it addresses the molecular mechanisms governing peripheral (non-SCN) clocks.All of this makes this Research Topic Jesús-Olmo et al. show the utility of this model by identifying the gene pum as a molecule that influences sleep homeostasis in flies. This is particularly interesting as pum mediates other homeostatic functions in the fly brain, including neuronal excitability. This Research Topic also includes new studies in rodent models. As shown by Choi et al., sex hormones influence sleep homeostasis as determined by classic methods of investigating these pathways . Hung et al. explored the role of a canonical clock gene  in basal sleep and wake expression in corticotropin-releasing factor (CRF) neurons. Previous studies have shown that CRF neurons communicate circadian signals, but the role of a canonical clock gene  in CRF neurons in basal sleep and wake had not been explored. Interestingly, the authors find that CRF neuron-specific Bmal1-deficient mice have normal sleep-wake organization, indicating that other mechanisms are likely involved in CRF-mediated effects on sleep expression. This Research Topic concludes with a study by Phillips et al., who provide new insights into interactions between signaling pathways implicated in sleep homeostasis  and circadian regulation. This was accomplished by investigating sleep, compensatory changes in sleep after sleep deprivation, and circadian organization in mice with variations in Val66Met polymorphism.As discussed above, invertebrate models have provided novel insights into sleep homeostasis. In conclusion, sleep homeostasis remains one of the (many) mysteries about sleep. However, scientists have made progress in identifying some of the putative, underlying mechanisms. This has come about through a combination of approaches, including molecular and circuit neurobiology in diverse animal species.The author confirms being the sole contributor of this work and has approved it for publication.